Drug-drug Interaction Study Between Bictegravir/Emtricitabine/Tenofovir Alafenamide and Feminizing Hormones in Trans Women Living With HIV
Phase: PHASE4
Conditions: Hiv, Transgenderism
Sponsor: Maple Leaf Research

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Drug-drug interaction study between bictegravir/emtricitabine/tenofovir alafenamide and 
feminizing hormones in trans women living with HIV 
 
 
Principal Investigator: Dr. Mona Loutfy,  
 
Co-Principal Investigators: Dr. Kim Scarsi, Dr. Alice Tseng 
 
Community Principal Investigator: Ms. Yasmeen Persad 
 
Co-investigators: Dr. Ashley Lacombe-Duncan, Ms. Angela Underhill, Dr. Raymond Fung, Dr. Ian 
Armstrong, Dr. Amy Bourns, Dr. Louie Chan, Ms. Susan Hranilovic, Dr. Quang Nguyen, Dr. 
Hannah Kia, Dr. Thea Weisdorf, Ms. Wangari Tharao, Dr. Carmen Logie, Ms. Laura Warren, Ms. 
Tessa Senneker, Dr. Rupert Kaul, Ms. V. Logan Kennedy, Ms. Jennifer McCully, Ms. Roberta 
Halpenny  
 
Study Support/ Funding:  Gilead Sciences Inc. 
 
 
Date of Protocol:  November 10, 2022 
Protocol No.:  
CTN 331 
Version No. :         2.1 
 
 
GCP Statement 
 
This clinical study will be conducted in accordance with applicable Health Canada regulations, 
International Conference on Harmonisation of Technical Requirements for Registration of 
Pharmaceuticals for Human Use (ICH) guidelines on current Good Clinical Practice (GCP), and 
the Declaration of Helsinki. 
 
Confidentiality Statement 
 
This clinical study protocol contains information which is of a confidential, trade-secret or 
proprietary nature. The protocol is for the use of Sponsor-Investigator and his designated 
representatives participating in the investigational trial. It is not to be disclosed to any other 
person or party without the prior written approval of Sponsor-Investigator. 
 
 

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TABLE OF CONTENTS 
 
1 
INVESTIGATOR AGREEMENT ....................................................................................................5 
2 
ABSTRACT .................................................................................................................................6 
3 
INTRODUCTION ........................................................................................................................7 
3.1 
Issues facing trans women living with HIV ....................................................................... 7 
3.2 
Bictegravir (B) with tenofovir alafenamide (TAF) and emtricitabine (F) (B/F/TAF) as a 
good ART regimen for trans women living with HIV ................................................................... 7 
3.3 
DDI findings to date between ART drugs and feminizing hormones amongst trans 
women ........................................................................................................................................ 8 
4 
OBJECTIVES AND HYPOTHESES ..............................................................................................10 
5 
METHODS AND ANALYSIS ......................................................................................................11 
5.1 
Study Design and Setting................................................................................................ 11 
5.2 
Study Population ............................................................................................................ 12 
6 
INCLUSION AND EXCLUSION CRITERIA ..................................................................................13 
6.1 
Investigational Population (Group 1) ............................................................................. 13 
6.2 
Comparator Population (Group 2) ................................................................................. 14 
6.3 
Comparator Population (Group 3) ................................................................................. 15 
7 
DRUGS UNDER INVESTIGATION .............................................................................................16 
8 
STUDY PROCEDURES ..............................................................................................................19 
8.1 
Engagement with and screening of candidates/recruitment plan ................................ 19 
8.2 
Screening Visit ................................................................................................................ 20 
8.3 
Screening Visit Procedures ............................................................................................. 20 
8.4 
Baseline Visit Procedures................................................................................................ 22 
8.5 
Month 1 Visit Procedures (Group 1 and 2 who switched to B/F/TAF only) .................... 25 
8.6 
Month 2 Visit Procedures ............................................................................................... 25 
8.7 
Month 6 Visit Procedures (Group 1 & 2 only) ................................................................. 29 
9 
SCREEN FAILURES ...................................................................................................................30 
10 MANAGING POSITIVE HIV DIAGNOSTIC SEROLOGY ..............................................................30 
11 MANAGING HIV-1 RNA BLIPS .................................................................................................30 
12 STUDY WITHDRAWAL ............................................................................................................30 
13 EVALUATION, RECORDING, AND REPORTING OF ADVERSE EVENTS .....................................31 

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13.1 Definitions ...................................................................................................................... 31 
13.1.1 Adverse Event (AE) ..................................................................................................... 31 
13.1.2 Serious Adverse Events (SAE) .................................................................................... 31 
13.2 AE Description: Relationship to Study Treatment ............................................................. 32 
13.2 Follow-Up for Adverse Events ........................................................................................ 33 
13.3 SAE Reporting ................................................................................................................. 33 
13.4 Mandatory Reporting to Health Canada ........................................................................ 34 
14 POTENTIAL RISK TO HUMAN PARTICIPANTS TAKING BIKTARVY® .........................................34 
15 CLINICAL TRIAL DATA ON ADVERSE REACTIONS TO BIKTARVY® ...........................................34 
16 RISKS OF ESTRADIOL THERAPY ..............................................................................................35 
17 ADVERSE REACTIONS TO ESTRADIOL .....................................................................................35 
18 HANDLING, STORAGE, AND ACCOUNTABILITY OF B/F/TAF...................................................35 
19 RISK MANAGEMENT...............................................................................................................36 
20 PK SAMPLE COLLECTION AND ANALYSIS ...............................................................................36 
20.1 Antiretroviral PK Samples ............................................................................................... 36 
20.2 Estradiol Level Samples .................................................................................................. 37 
21 DATA MANAGEMENT .............................................................................................................37 
22 ADHERENCE TO PROTOCOL ...................................................................................................37 
22.1 Protocol Amendments .................................................................................................... 37 
22.2 Protocol Deviations ........................................................................................................ 38 
23 ENDPOINTS AND ADDITIONAL VARIABLES OF INTEREST.......................................................38 
23.1 Primary endpoints .......................................................................................................... 38 
23.2 Secondary Endpoints ...................................................................................................... 39 
24 STATICAL ANALYSES AND SAMPLE SIZE CALCULATION .........................................................43 
24.1 Statistical analyses ......................................................................................................... 43 
24.2 Sample size Justification ................................................................................................. 44 
25 ETHICS AND DISSEMINATION ................................................................................................44 
25.1 Ethical considerations .................................................................................................... 44 
25.2 Informed consent ........................................................................................................... 44 
25.3 Compensation................................................................................................................. 45 
25.4 Confidentiality ................................................................................................................ 45 

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25.5 Research Ethics Board (REB) .......................................................................................... 46 
25.6 Dissemination plan ......................................................................................................... 46 
26 CONCLUSION AND IMPACT ....................................................................................................46 
27 REFERENCES ...........................................................................................................................48 
APPENDIX A – Study Schematic .....................................................................................................51 
APPENDIX B1 – List of Prohibited Medications .............................................................................52 
APPENDIX B2 – Drug Interactions and recommendations on management .................................55 
APPENDIX C – Schedule of Events..................................................................................................56 
APPENDIX D – NIAID standardized toxicity grading system ..........................................................59 
APPENDIX E1 – PROTOCOL REQUIRED LAB ASSESSMENTS ...........................................................60 
APPENDIX E2 – REQUIRED BLOOD VOLUMES................................................................................61 
 
 
 
 

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1 
INVESTIGATOR AGREEMENT 
 
Title:    
Drug-drug interaction study between bictegravir/emtricitabine/tenofovir 
 
alafenamide and feminizing hormones in trans women living with HIV 
 
Protocol No.: 
CTN 331 
Version No.: 
2.1 
 
Date:  
November 10, 2022 
 
 
This clinical study will be conducted in accordance with applicable Health Canada regulations, 
ICH guidelines on current GCP, and the Declaration of Helsinki. 
 
I confirm that I have read and understand this protocol and I agree to conduct this clinical study 
in accordance with the design and specific provisions of the protocol, with the exception of a 
change intended to eliminate an immediate hazard to participants.  
 
I agree to promptly report to the applicable ethics boards any changes in the research activity 
and all unanticipated problems involving risks to human participants or others. Additionally, I 
will not make any changes in the research without prior ethics and sponsor approval, except 
where necessary to ensure the safety of study participants. 
 
 
 
 
 
 
 
 
Name 
 
Signature 
 
Date (dd-mmm-yyyy) 
 
 
 
 
 
 
 
 
 
 
 

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2 
ABSTRACT 
Introduction: Transgender (trans) women have been found to be at higher risk of, and to be 
disproportionally affected by, HIV for multiple biologic and social reasons. Trans women living 
with HIV have also been found to have low usage and adherence rates to antiretroviral therapy 
(ART). Both healthcare providers and trans women, themselves, have expressed concerns of 
drug-drug interactions (DDIs) between ART drugs and feminizing hormones, which have in turn 
been shown to contribute to low rates of ART usage amongst trans women living with HIV. The 
objective of this DDI study is to investigate the pharmacokinetic effects of the common feminizing 
hormone regimens (oral estradiol [Estrace® or Lupin-estradiol] with an anti-androgen 
(pharmaceutical and/or surgical and/or medical) on the antiretroviral combination 
bictegravir/emtricitabine/tenofovir alafenamide (B/F/TAF;Biktarvy®] and vice versa.  
 
Methods and analysis: Participants will be assigned to three groups: group 1 will include 15 trans 
women living with HIV who are taking feminizing hormones and ART (investigational group); 
group 2 will include 15 premenopausal cisgender women living with HIV taking ART (ART control 
group); group 3 will include 15 trans women living without HIV taking feminizing hormones 
(hormone control group). Women living with HIV will have to be virally suppressed for at least 
three months and they will have to already be taking B/F/TAF or have their current ART regimen 
switched to B/F/TAF at baseline. Trans women participants will be required to be on 2 mg oral 
estradiol or higher and an anti-androgen (pharmaceutical, medical or surgical). Plasma ART drug 
concentrations will be sampled at the 2-month visit and compared among trans women living 
with HIV on feminizing hormones and premenopausal cis women living with HIV. Serum estradiol 
and total testosterone concentrations will be sampled at the baseline and month 2 visits and 
compared among trans women living with and without HIV. The primary endpoints are B/F/TAF 
pharmacokinetic parameters (Cmin, Cmax and AUC) between trans and cis women living with HIV 
at month 2 and the estradiol concentrations (Cmin, C4h, Cmax and AUC) and the proportions of the 
month 2 C4h that are within target (200 to 735 pmol/L) between trans women living with and 
without HIV at month 2. Other endpoints will include the mean estradiol and testosterone 
concentrations at baseline and the difference between baseline and month 2 estradiol pre-dose 
and C4h, HIV viral load, adherence, adverse events, patient-reported outcomes (i.e., symptoms), 
satisfaction with feminizing hormones, satisfaction with ART regimen, and health status. This trial 
will serve to provide empirical evidence regarding a lack of, or presence of DDIs between B/F/TAF 
and feminizing hormones. 
 
Dissemination: The findings will be disseminated through publication in peer-reviewed journals 
as well as presented at national and international conferences and community groups.   
 
Trial registration number: Will be registered once Research Ethics Board (REB) approval is 
granted. 
 
 
 

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3 
INTRODUCTION  
3.1 
Issues facing trans women living with HIV 
Transgender (trans) women have been found to be at higher risk of, and to be disproportionally 
affected by, HIV for multiple biologic (e.g., increased risk of biologic transmission via sexual 
practices) and social reasons (e.g., gender-based stigma and discrimination).1 Globally, trans 
women experience disproportionately high rates of HIV compared to cisgender (cis) adults.1 
Canadian archival research estimated that 54% of trans women deaths in Montreal pre-2000 
were HIV-related.2 A study of trans people living in Ontario estimated an HIV prevalence of 2.9%, 
lower than global estimates, yet ten times higher than the provincial prevalence of 0.23%.3  
 
While access to HIV-related healthcare – including initiation of and adherence to antiretroviral 
treatment (ART) – is essential for the survival and wellbeing of people living with HIV,4,5 a 
significant amount of research has described lower ART use and adherence among trans women 
living with HIV.6-10 One of the main contributing reasons identified by trans women living with 
HIV for this discordance in access and adherence is the fear of drug-drug interactions (DDIs) 
between ART drugs and feminizing hormones.11 Trans women consider the use of feminizing 
hormones so vital to their being and lives (e.g., safety) that many trans women living with HIV 
would rather take feminizing hormones and not ART drugs, if they perceive they have to make a 
choice.11 Studies show that many healthcare providers will still withhold feminizing hormones if 
a trans woman living with HIV is taking ART.11 Also, trans women living with HIV who believe ART 
may have negative effects on their feminizing therapy are three times more likely to take higher 
than prescribed doses of hormones.12  These concerns can lead to trans women choosing to 
either not take ART at all, or to take ART and buy their feminizing hormones from the street/black 
market and be medically unsupervised. Neither of these options are suitable or ideal, and can 
contribute to potentially severe side effects (e.g., stroke).13 
 
The ideal scenario is a shared decision-making medical care model where both the healthcare 
provider and the trans woman understand the science behind DDIs between ART drugs and 
feminizing hormones, allowing both important therapies to be prescribed and monitored to meet 
the needs of the trans woman with medically safe dosing and sustained viral suppression. The 
objective for this clinical study is just that: to investigate bidirectional DDIs between the most 
commonly prescribed feminizing hormone regimens (oral estradiol [Estrace® or Lupin-estradiol] 
with an anti-androgen [e.g., spironolactone, cyproterone, finasteride, leuprolide, bicalutamide, 
dutasteride, and/or orchiectomy, and/or hypogonadism]) and the antiretroviral combination of 
bictegravir/emtricitabine/tenofovir alafenamide (B/F/TAF; Biktarvy®).  
 
3.2 
Bictegravir (B) with tenofovir alafenamide (TAF) and emtricitabine (F) (B/F/TAF) as a 
good ART regimen for trans women living with HIV 
The current Canadian standard of care for HIV is an ART regimen with two to three active 
antiretroviral drugs – but more commonly three, typically two from the nucleoside reverse 
transcriptase inhibitor class along with a third agent.14 The latest American 2021 DHHS guidelines 
indicate that the recommended second or third agent for most ART-naïve patients should be an 
integrase strand transfer inhibitor (INSTI).15 In today’s world, an individual who is newly 

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diagnosed with HIV will likely be started on a single tablet regimen (STR) to maximize tolerability 
and adherence. Prior to 2018, there were three commercially available three-drug INSTI-based 
STRs in Canada: Stribild, Genvoya and Triumeq. Each of these STRs, while potent and highly 
effective, have limitations. Currently in Canada, most patients who were taking Stribild have been 
changed to the newer formulation of Genvoya, which contains tenofovir alafenamide (TAF) 
rather than tenofovir disoproxil fumarate (TDF). TAF has demonstrated significantly lower renal 
and bone toxicity in clinical trials, which is believed to be related to differences in the disposition 
of tenofovir (TFV) between the two prodrugs while maintaining high intracellular concentrations 
at the site of action for HIV. While both are well tolerated, Genvoya and Stribild have the 
disadvantage of being formulated with a booster, cobicistat (COBI), and DDIs are a concern with 
co-administered drugs such as hormone drugs. Triumeq is an STR that contains a combination of 
abacavir (ABC), lamivudine (3TC) and dolutegravir (DTG). Triumeq has the disadvantages of 
requiring an HLA-B*5701 test prior to administration and is associated with life threatening 
hypersensitivity reactions, which limit its use in HLA-B*5701-positive patients or when an HLA-
B*5701test is unavailable. Triumeq is also associated with central nervous system side effects, 
most significantly that of insomnia thought to be related to DTG and perhaps the co-
administration with ABC.16 Furthermore, many studies and guidelines advise about the concern 
of a potential increase in cardiovascular disease events (CVE) including myocardial infarctions 
with the use of ABC.13 The potential increased risk of CVE is of particular importance in trans 
women taking hormone therapy, as feminizing hormones have also been associated with an 
increased risk of CVE.17,18  Biktarvy® (B/F/TAF) was approved and has been available in Canada as 
an STR since 2018 and we believe that it represents a generally well tolerated,  convenient, 
pharmacologically favorable, and highly effective ART strategy for trans women living with HIV. 
Also, B/F/TAF has been found to be pharmacologically favorable with no DDIs between B/F/TAF 
and an ethinyl estradiol/norgestimate containing oral contraceptive in healthy adults.19 However, 
it is unclear if this is generalizable to feminizing hormones used by trans women, which typically 
contain higher doses of 17β-estradiol. Therefore, for these many reasons, we believe that it is 
important to assess the DDIs between B/F/TAF and feminizing hormones among trans women 
living with HIV. Demonstrating a lack of detrimental DDIs between B/F/TAF and common 
feminizing hormone therapy will provide an evidenced-based approach for the management 
of HIV among trans women. 
 
3.3 
DDI findings to date between ART drugs and feminizing hormones amongst trans 
women  
Recent studies20-23 with trans women living without HIV who were taking TDF/FTC (Truvada) for 
pre-exposure prophylaxis (PrEP) revealed potential reduced plasma TFV-diphosphate (TFV-DP) 
and FTC-triphosphate (FTC-TP) concentrations. Two of the studies also showed reduced TFV-TP 
and FTC concentrations in rectal tissues.20,23 
 
The iFACT (Interaction between the use of Feminizing Hormone Therapy and Antiretroviral 
agents Concomitantly among Transwomen) study22, done in Thailand, aimed to do a two-way 
analysis of DDIs to show the effect of PrEP on feminizing hormones and vice versa. This was 
achieved by giving 20 participants an initial 3-week period of the feminizing hormones, E2 

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valerate and cyproterone, and then a 2-week period of both the combined feminizing hormone 
regimen and daily TDF/FTC as PrEP and then a final 3-week of TDF/FTC as PrEP only. Intensive E2 
pharmacokinetic (PK) parameters [plasma was collected at 0 (pre-dose), 1, 2, 4, 6, 8, 10, 12 and 
24 hours after directly observed medication ingestion] and testosterone concentration at 24 
hours (C24) were measured at weeks 3 (without PrEP) and 5 (with PrEP), and intensive TFV PK 
parameters were measured at weeks 5 (with feminizing hormones) and 8 (without feminizing 
hormone). The PK parameters revealed a reduction by 12% of TFV area under the curve [AUC]0-
24 (p = 0.03) and 18% of C24 of TFV (p < 0.001) when participants were on feminizing hormones. 
There were no significant changes in E2 PK parameters on PrEP.  A proposed theory for the 
reduction involves alteration of drug clearance of TFV by E2.  
 
Similar reductions were noted in another study done by Shieh et al (2018)20, where plasma TFV 
and FTC C24 (trough) concentrations in 8 trans women on feminizing hormones (on the regimen 
they were taking) were lower by 32% (p = 0.010) and 32% (p = 0.038) respectively, when 
compared to 8 cis men. Both groups of participants were on TDF/FTC as PrEP for 7 days and at 
the last dose, the PK parameters were measured. Plasma TFV and FTC 24-hr AUC-time curve 
among the trans women trended toward and were significantly lower by 27% (p = 0.065) and 
24% (p = 0.028), respectively. Estradiol concentrations did not significantly change by TDF/FTC. 
Rectal concentrations of TFVdp concentrations were 68% lower in trans women when compared 
to cisgender men, which resonated with another study’s finding where rectal TFVdp:dATP, was 
7-fold lower in transgender versus cisgender participants (p< 0.05) and inversely correlated with 
female sex hormones (p< 0.05)23. In this study (2019), patients living with and without HIV who 
were taking TDF/FTC (as ART or PrEP, respectively) as part of clinical care were enrolled and 
divided into 3 cohorts: trans women taking feminizing hormones, cis men, and postmenopausal 
cis women.  Blood and rectal tissue were collected at a sampling visit 16–24 hours after the last 
self-reported TDF/FTC dose. A follow-up visit was conducted within 14 days. Ten participants 
were taking TDF/FTC as part of ART and 2 were taking PrEP. In blood and rectal tissue, 
TFVdp/FTCtp and dATP/dCTP concentrations were similar across groups; however, rectal 
TFVdp:dATP, which is an important effector of PrEP potency, was 7-fold lower in transgender 
versus cisgender participants (P < 0.05) and inversely correlated with female sex hormones (P < 
0.05) while FTCtp:dCTP did not differ between transgender and cisgender participants. 
 
In January 2020, the first DDI study assessing the impact of feminizing hormones on the drug 
concentrations of an ART regimen and vice versa was published.24 This DDI study conducted in 
Thailand enrolled 20 trans women who were newly diagnosed with HIV. The study was done in a 
similar fashion as the iFACT study with trans women taking PrEP: the feminizing hormone and 
ART regimens were tightly controlled during the study and then intensive PK parameters were 
collected. Participants were given the feminizing hormones, estradiol 2 mg and cyproterone 25 
mg once a day only, then the feminizing hormones plus ART (the STR TDF/FTC/efavirenz), then 
ART only in a chronological order.  Intensive E2 PK were measured at weeks 3 (without ART) and 
5 (with ART), and intensive TFV and EFV PK were measured at weeks 5 (with feminizing 
hormones) and 8 (without feminizing hormones). The differences in the mean ratio of E2 AUC, 
Cmax, and C24h post-ART vs. pre-ART were 0.72 (p<0.001), 0.81 (p=0.006), and 0.64 (p=0.004), 

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respectively while the differences in the mean ratio of TFV AUC, C24h, and EFV C24h on vs. off 
feminizing hormones were 0.86 (p=0.002), 0.83 (p=0.006), and 0.91 (p=0.02), respectively. This 
implied that E2 PK values were significantly lowered in the presence of an ART regimen consisting 
of TFD/FTC/efavirenz and vice versa. As such, since DDIs have been demonstrated between ART 
drugs and feminizing hormones and since there is a paucity of DDI studies between ART and 
feminizing hormones amongst trans women, our study is of high importance.  
 
4 
OBJECTIVES AND HYPOTHESES 
Primary Objective #1: To compare month 2 PK parameters (Cmin, Cmax, AUC) of the ART drugs 
(B/F/TAF), among trans women living with HIV who are taking B/F/TAF and feminizing hormones 
(group 1) to pre-menopausal cis women living with HIV who are taking B/F/TAF (group 2); and  
 
Primary Objective #2: To compare the month 2 estradiol concentrations (Cmin, C4h, Cmax, AUC) and 
the proportions of the month 2 C4h that are within target (200 to 735 pmol/L) between trans 
women living with HIV taking feminizing hormones and B/F/TAF (group 1) and trans women living 
without HIV taking feminizing hormones and not B/F/TAF (group 3). 
 
Hypotheses: Our first primary hypothesis is that there will be no difference in month 2 B/F/TAF 
pharmacokinetic (PK) parameters (Cmin, Cmax, AUC) between trans women living with HIV and 
premenopausal cis women living with HIV taking B/F/TAF. Our second primary hypothesis is that 
there will be no difference in the month 2 estradiol concentrations (Cmin, C4h, Cmax, AUC) between 
trans women living with HIV who are taking B/F/TAF and feminizing hormones and trans women 
living without HIV who are only taking feminizing hormones. 
 
Secondary Objectives: 
1. To determine the baseline, and the difference between baseline and month 2, estradiol pre-
dose and C4h and testosterone concentrations compared between groups 1 and 3 and the 
proportions within the target; 
2. To determine satisfaction with medical gender affirmation for trans women living with HIV 
(group 1) and living without HIV (group 3) at baseline and month 2, and at the 6 month 
timepoint for group 1 only;  
3. To determine the proportion of participants with an undetectable HIV-RNA (<40 copies/mL) 
at all follow-up visits (months 1, 2 and 6) for trans and cis women participants living with HIV 
as a whole cohort and for those who switched to B/F/TAF; 
4. To determine adherence for trans and cis women participants living with HIV at baseline and 
all follow-up visits (months 1, 2 and 6 as a whole cohort and for those who switched to 
B/F/TAF); 
5. To determine the frequency and severity of B/F/TAF-related adverse effects at all follow-up 
visits (months 1, 2 and 6) for trans and cis women participants living with HIV as a whole 
cohort and for those who switched to B/F/TAF; 

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6. To determine the patient-reported outcomes (PROs) at baseline and months 2 and 6for trans 
and cis women participants living with HIV as a whole cohort and for those who switched to 
B/F/TAF; as well as those in group 3 at baseline and month 2 as an exploratory analysis;  
7. To determine patient satisfaction with ART at baseline and months 2 and 6for trans and cis 
women participants living with HIV as a whole cohort and for those who switched to B/F/TAF; 
and  
8. To determine participants’ health status in all groups at baseline and month 2 and additionally 
at month 6 for trans and cis women participants living with HIV. 
 
5 
METHODS AND ANALYSIS 
5.1 
Study Design and Setting 
This will be a three-armed parallel group, longitudinal, 6-month DDI study. The study will be 
conducted through Maple Leaf Research (MLR) in Toronto and participants will be recruited 
through the affiliated clinic and other recruiting clinics.  All study visits will be conducted through 
MLR, either in person or virtually except for the Month 2 visit which must be conducted in person 
at MLR in Toronto due to the peripheral blood mononuclear cells (PBMC) processing 
requirement.  The study schematic is presented in Appendix A. Unlike the Thai DDI studies22,24, 
this study is relatively pragmatic in nature as trans women will be on varying estradiol doses (2 
mg or higher) and taking an anti-androgen (pharmaceutical [e.g., spironolactone, cyproterone, 
finasteride, leuprolide, bicalutamide, dutasteride] and/or surgical [orchiectomy] and/or medical 
[hypogonadism]) reason. In the Canadian context, a pragmatic study is the only one possible due 
to the limited number of trans women in Canada. While this can be seen as a limitation, it is also 
an advantage as it allows us to capture data on varying scenarios.   
 
The maximum duration of enrolment from screening to the 6 month visit is 9 months (up to 90 
days allowed from screening to baseline visit) for cis and trans women living with HIV to complete 
(group 1, 2). For Group 3 (trans women without HIV) their enrolment will be a maximum duration 
of 5 months. 
 
An important aspect of this study is the use of community-based research (CBR) principles.25-30 
Research with trans women would not be possible if not done using CBR and working with trans 
women champions and leaders in the field. This population has a lack of trust for the system 
including researchers and academic institutions and can often be difficult to enroll into research 
studies. Our team has been conducting CBR for over a decade and most recently launched the 
CBR-driven Trans Women HIV Research Initiative (TWIRI) with and for trans women living with 
HIV. TWIRI is led by Ms. Yasmeen Persad, a trans woman community leader in Toronto and the 
TWIRI community research coordinator (see www.transwomenHIVresearch.com). Also, the 
conception of the idea of this study came from the community, and more specifically from the 
Canadian Institutes of Health Research (CIHR) Canadian HIV Trials Network (CTN) Trans Working 
Group, made up of mainly trans community members. Also, for this study we will engage the 
support of a trans woman as a community research participant assistant who will help recruit 
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(e.g., by helping participants get to lab for blood draws and help participants fill out surveys (if 
needed). The community research participant assistant will also ensure that all matters are taken 
care of, to the best of their ability, to increase trust and ensure a safe environment. Participants 
may also choose to bring their own support person and this person would receive an honorarium 
for their time. Furthermore, a TWIRI community advisory board (CAB) has been formed which 
will review and advise on the study and enrollment. 
 
5.2 
Study Population 
Participants will be assigned to three groups: group 1 will include 15 trans women living with HIV 
who are taking feminizing hormones (oral estradiol [2 mg or higher] with an anti-androgen 
(pharmaceutical [e.g., spironolactone, cyproterone, finasteride, leuprolide, bicalutamide, 
dutasteride] and/or surgical [orchiectomy] and/or medical [hypogonadism]) and suppressive ART 
(investigational group); group 2 will include 15 pre-menopausal cis women living with HIV taking 
suppressive ART (ART control group); and group 3 will include 15 trans women living without HIV 
taking feminizing hormones (oral estradiol [2 mg or higher] with an anti-androgen 
(pharmaceutical [e.g., spironolactone, cyproterone, finasteride, leuprolide, bicalutamide, 
dutasteride] and/or surgical [orchiectomy] and/or medical [hypogonadism]) (hormone control 
group). Women living with HIV (groups 1 and 2) will have to be virally suppressed for at least 
three months on a combination ART regimen with two suppressed viral loads and no clinical 
suspicion of prior important ART drug resistance. As mentioned above, one unique feature of this 
study is that a community research participant assistant (a trans woman leader in the Toronto 
community) will be contracted to assist with recruitment, and visit attendance at Baseline and 
Month 2 procedures; this will be important as participants will be enrolled from six Toronto sites 
in addition to Maple Leaf Medical Clinic: 790 Bay Street Clinic in Toronto, Ontario, Canada; 
Sherbourne Community Health Centre in Toronto, Ontario, Canada; Dr. Raymond Fung’s 
endocrinology clinic in Toronto, Ontario, Canada; Church-Wellesley Clinic in Toronto, Ontario, 
Canada; St. Michael’s Department of Family and Community Medicine at Unity Health in Toronto, 
Ontario, Canada; and Women's Health in Women's Hands Community Health Centre in Toronto, 
Ontario, Canada. If recruitment is poor in Toronto, the Montreal site will be added and recruiting 
primarily from one clinic, Clinique Quartier Latin, which is the clinic that cares for the most trans 
women living with and without HIV in Montreal. A second clinic in Montreal may also be added 
to recruit participants, Clinique Médicale Quorum, under the leadership of Dr. Andrew Bui-
Nguyen.  There is a possibility of adding other sites across Canada, as this study will be advertised 
through National Trans Networks. Interested participants can also call the sites directly to screen 
for the trial.   
 
A formative retrospective chart review study has been completed entitled “The Montreal 
Toronto Trans HIV Study – Characterizing the Trans Women Population living with and without 
HIV in Montreal and Toronto”. This study informed us of the most commonly prescribed 
feminizing hormones and ART regimens in these populations. Preliminary results reveal that we 
captured data from the charts of nearly 1500 trans women living with and without HIV cared for 
in Toronto and Montreal. This high number of trans patients is likely the case because Canada 
and particularly Toronto are considered trans-friendly urban settings, especially by immigrant 

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racialized trans people. Of the approximate 1500 patients, 74 in Toronto and 12 in Montreal were 
identified as living with HIV. Based on this formative study, which involved collaborations with 
seven clinics serving the majority of trans women accessing feminizing hormones in Toronto and 
Montreal, we will be ideally positioned for recruitment in the current study. If recruitment is poor 
in Toronto, the Montreal site will be added and recruiting primarily from one clinic, Clinique 
Quartier Latin, which is the clinic that cares for the most trans women living with and without 
HIV in Montreal. 
 
From the preliminary findings from this chart review, we have found that oral estradiol is the 
most common form of estrogen replacement used, at varying doses. We have also identified that 
the most common androgen-blocking agents used are spironolactone or cyproterone, split 
evenly between the two. This has informed the development of the inclusion/exclusion criteria. 
 
6 
INCLUSION AND EXCLUSION CRITERIA 
6.1 
Investigational Population (Group 1)  
Inclusion Criteria 
1. Is a trans woman or person with transfeminine experience (individual assigned male sex 
at birth who currently identifies as a woman or person with transfeminine experience 
and is at any stage of medical, social, or legal transition); 
2. Is 18 years of age or older; 
3. Is living with HIV;  
4. Is currently taking combination ART and has been taking these medications for at least 3 
months prior to the screening visit; 
5. Is willing to adjust their ART dosing to the morning if they are currently taking it at night 
for at least 28 days before the baseline visit until the completion of the Month 2 visit; 
6. Has had an undetectable viral load for at least 3 months on at least two occasions, where 
most recent (within 12 months including screening) VL<40 copies/mL and second (within 
24 months) VL<200 copies/mL with no suggestion of relevant ART drug resistance prior 
to screening; 
7. Is willing to switch to or stay on B/F/TAF for the duration of the study (i.e., 6 months); 
8. Is currently taking oral estradiol (2 mg or higher) and an anti-androgen (pharmaceutical 
[e.g., spironolactone, cyproterone, finasteride, leuprolide, bicalutamide, dutasteride] 
and/or surgical [orchiectomy] and/or medical [hypogonadism]) and has been taking 
these medications together at the current dose for at least 3 months prior to the 
screening visit; AND: 
a. Is willing to adjust their oral estradiol dosing to twice a day if they are taking > 2 
mg per day (which is currently recommended as best practice) for at least 28 
days before the baseline visit until the completion of the Month 2 visit, with the 
morning dose set at 2 mg (remaining dose can be taken at night), OR;   
b. If their estradiol dosing is 2 mg per day and they are currently taking their 
estradiol at night, they must be willing to switch their dosing to the morning for 

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at least 28 days before the baseline visit until the completion of the Month 2 
visit; 
c. Is willing to swallow their oral estradiol if they are currently taking it sublingually 
or crushing it for at least 28 days before the baseline visit and until the 
completion of the Month 2 visit; 
d. Is willing to keep their anti-androgen (if applicable) medication(s) unchanged 
until the completion of the Month2 visit; 
9. If taking progesterone, is willing to keep the same medication/dose until the completion 
of the Month 2 visit and must have been taking that dose for at least 3 months prior to 
the screening visit;  
10. Is willing and able to provide full consent for their participation. 
Exclusion Criteria 
1. Is clinically unable to switch ART to B/F/TAF based on their physician’s opinion; 
2. Is taking hormonal non-prescription or natural health products in addition to feminizing 
hormone therapy; 
3. Has significant underlying diseases which could worsen due to their participation, or 
affect their ability to follow the study procedures; 
4. Has kidney or liver impairment (ALT > 5X normal; serum creatinine [eGFR] < 30 mL per 
minute); 
5. Is taking medications known to interact with feminizing hormones, B/F/TAF, or its 
individual components or has taken such medications within the past 28 days prior to 
baseline (List of prohibited medications in Appendix B1).  
6. Has a known hypersensitivity to bictegravir (BIC), emtricitabine (FTC), tenofovir 
alafenamide (TAF) or to any ingredient in the formulation.  
 
6.2 
Comparator Population (Group 2) 
Inclusion Criteria  
1. Is a cis woman (individual assigned female sex at birth who currently identifies as a 
woman); 
2. Is of reproductive age and 18 years of age or older; 
3. Is living with HIV;  
4. Has a regular period (every 24 – 35 days), or if period is irregular or absent, it is due to 
the administration of a progesterone only hormone contraceptive (see Appendix B1); or 
if uterus removed (hysterectomy), ovarie(s) remain functional (FSH level<27.0 IU/L);  
5. Is currently taking combination ART and has been taking these medications for at least 3 
months prior to screening; 
6. Is willing to adjust their ART dosing to the morning if they are currently taking it at night 
for at least 28 days before the baseline visit until the completion of the Month 2 visit; 
7. Has had an undetectable viral load for at least 3 months on at least two occasions, 
where most recent (within 12 months including screening) VL<40 copies/mL and second 
(within 24 months) VL<200 copies/mL with no suggestion of relevant ART drug 
resistance prior to screening; 
8. Is willing to switch to or stay on B/F/TAF for the duration of the study (i.e., 6 months);  

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9. Is willing and able to provide full consent for their participation. 
 
Exclusion Criteria 
1. Is clinically unable to switch B/F/TAF based on their physician’s opinion; 
2. Is pregnant or is planning on getting pregnant for the duration of the study; 
3. Has undergone surgery to remove their ovaries (i.e. oophorectomy) or has a condition in 
which their ovaries produce little or no estrogen (i.e. primary ovarian insufficiency, 
hypogonadism) or does not have ovaries naturally (i.e. unilateral ovarian absence); total 
hysterectomy is allowed as long as ovaries are intact and functional; 
4. Is taking an estrogen containing or conjugated estrogens hormonal therapies; IUD and 
progesterone only contraception are allowed (See Appendix B1/2 for list for both 
prohibited and allowed contraceptives); 
5. Has significant underlying diseases which could worsen due to their participation, or 
affect their ability to follow the study procedures; 
6. Has kidney or liver impairment (ALT > 5X normal; serum creatinine [eGFR] < 30 mL per 
minute); 
7. Has a known hypersensitivity to bictegravir (BIC), emtricitabine (FTC), tenofovir 
alafenamide (TAF) or to any ingredient in the formulation.  
8. Is taking medications known to interact with B/F/TAF, or its individual components or 
has taken such medications within the past 28 days prior to baseline (List of prohibited 
medications in Appendix B1/2). 
 
6.3 
Comparator Population (Group 3) 
Inclusion Criteria 
1. Is a trans woman or person with transfeminine experience (defined as individual 
assigned male sex at birth who currently identifies as a woman or person with 
transfeminine experience irrespective of medical, social, or legal transition); 
2. Is 18 years of age or older; 
3. Is HIV negative at screening;   
4. Is currently taking oral estradiol (2mg or higher) and an anti-androgen (pharmaceutical 
[i.e., spironolactone, cyproterone, finasteride, leuprolide, bicalutamide, dutasteride] 
and/or surgical [orchiectomy] and/or medical [hypogonadism]) and has been taking 
these medications at the current dose for at least 3 months prior to the screening visit, 
AND: 
a. Is willing to adjust their oral estradiol dosing to twice a day if they are taking > 2 
mg per day (which is currently recommended as best practice) for at least 28 
days before the baseline visit until the completion of the Month 2 visit, with the 
morning dose set at 2 mg (remaining dose can be taken at night) OR;   
b. If their estradiol dosing is 2 mg per day and they are currently taking their 
estradiol at night, they must be willing to switch their dosing to the morning for 
at least 28 days before the baseline visit until the completion of the Month 2 
visit; 

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c. Is willing to swallow their oral estradiol if they are currently taking it sublingually 
or crushing it for at least 28 days before the baseline visit until the completion of 
the Month 2 visit; 
d. Is willing to keep their anti-androgen (if applicable) medication(s) unchanged 
until the completion of the Month2 visit; 
5. If taking progesterone, is willing to keep their dose the same until the Month 2 visit and 
must have been taking that dose for at least 3 months prior to the screening visit;  
6. Is willing and able to provide full consent for their participation. 
Exclusion Criteria 
1. Is taking hormonal non-prescription or natural health products in addition to feminizing 
hormone therapy; 
2. Has taken HIV pre-exposure prophylaxis (PrEP) (i.e, TDF/FTC or TAF/FTC) or HIV post-
exposure prophylaxis (PEP) in the prior 28 days from baseline and plans to continue 
during the study;  
3. Has significant underlying diseases which could worsen due to their participation, or 
affect their ability to follow the study procedures; 
4. Has kidney or liver impairment (ALT > 5X normal; serum creatinine [eGFR] < 30 mL per 
minute); 
5. Is taking medications known to interact with feminizing hormones or has taken such 
medications within the past 28 days prior to baseline (List of prohibited medications in 
Appendix B1). 
 
7 
DRUGS UNDER INVESTIGATION 
In this DDI study two groups of drugs are being assessed: 
1. As the HIV ART, the combination of B/F/TAF, known as Biktarvy®, which is a widely available 
STR approved by Health Canada is the drug under investigation. Biktarvy is only available in a 
single formulation of 50 mg bictegravir (as bictegravir sodium) / 200 mg emtricitabine / 25 
mg tenofovir alafenamide (as tenofovir alafenamide hemifumarate). Thus, there is no 
alternative dosage. Participants in groups 1 and 2 will either have to already be taking B/F/TAF 
or will be required to switch to it at baseline and they will self-administer B/F/TAF once daily 
(in the morning) with or without food.  
● For those who switch to B/F/TAF from another ART regimen, there will be no washout 
period between therapies in order to maintain virologic control. For those participants 
who switch at baseline to B/F/TAF (switch participants), the drug B/F/TAF (Biktarvy®) 
will be supplied at baseline at no cost by Gilead Sciences for the duration of the study 
(i.e., 6 months). Switching ART participants must take B/F/TAF in the morning until at 
least the Month 2 visit, if they prefer, they can switch from taking their Biktarvy® in 
the morning to taking it later in the day after the Month 2 visit. 
● For those who are already taking B/F/TAF, they must take B/F/TAF in the morning 
from the day after the screening until at least the Month 2 visit, if they prefer, they 

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can switch from taking their Biktarvy® in the morning to taking it later in the day after 
the Month 2 visit.  If required for any reason, B/F/TAF (Biktarvy®) can be supplied at 
baseline at no cost by Gilead Sciences for the duration of the study (i.e., 6 months). 
 
2. As the feminizing hormone therapy, the drugs under investigation are oral 17β-estradiol 
(estradiol) [Estrace® or Lupin-estradiol®] as the estrogen and an anti-androgen 
(pharmaceutical [e.g., spironolactone, cyproterone, finasteride, leuprolide, bicalutamide, 
dutasteride] and/or surgical [orchiectomy] and/or medical [hypogonadism]). In trans women, 
the administration of exogenous feminizing hormones is necessary to induce physical 
changes, and also improves social and emotional adjustment, often in combination with 
gender affirming surgery.17,18 While there are different estrogen preparations that are 
possible, in many parts of Canada oral estradiol is used as it is the only estrogen therapy 
covered by provincial drug coverage systems. Similarly, while the anti-androgens most 
commonly used in Canada are spironolactone and cyproterone for the same reason, gender 
affirming surgeries are becoming more common and thus many trans women may not need 
an anti-androgen (as they have had an orchiectomy or have a condition such as 
hypogonadism, for example). Estrogen therapy with 17β-estradiol is recommended over 
conjugated estrogens, as it can be easily monitored through laboratory testing and 
conjugated estrogen is thought to be more thrombogenic. In general, as per the 
Endocrinology Society Clinical Practice Guideline,18 estrogen therapy aims to achieve serum 
estradiol concentrations within the normal range for pre-menopausal women, approximately 
370 to 735 pmol/L, but is often variable depending on the assay. Doses of estradiol range 
from 1 mg to a maximum of 6 mg per day.  
● The University of California, San Francisco (USCF) guidelines recommend splitting the 
dose to twice a day when the dose is > 2 mg per day. From our formative chart review, 
we identified that 4 mg once a day (OD) is a common dose and therefore, participants 
must take 2 mg BID until the Month 2 Visit. Therefore, at the screening visit, trans women 
participants in both groups 1 and 3 must divide their dose to twice a day (BID) if they are 
taking > 2 mg per day and take 2 mg in the morning and the rest of the dosing in the 
evening Participants who are only taking 2mg of estradiol once daily at night must switch 
their dosing to the morning.  This will be beneficial from a PK perspective as then all 
participants will be taking 2 mg of estradiol in the morning and the heterogeneity of 
interparticipant estradiol PK will then be reduced.  
● Also, participants who either crush or take the oral estradiol sublingually must switch to 
swallowing the tablet orally from the day after the screening visit until the Month 2 visit, 
otherwise the estradiol concentrations will be markedly higher.  
● Participants in groups 1 and 3 will be asked to refrain from the use of estrogenic therapies 
other than their prescribed feminizing hormone regimen, and that any use of concomitant 
over-the-counter medications, prescription medications, or natural health products will 
be reported to study investigators. 

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The anti-androgen therapies spironolactone and cyproterone both work to prevent the action 
of testosterone but have vastly different effects on the suppression of testosterone levels. 
Spironolactone acts mainly as an androgen receptor blocker to directly prevent testosterone 
from binding and exerting its effects; the suppression of testosterone production is a much 
weaker effect. Cyproterone also blocks the androgen receptor but also effectively suppresses 
testosterone production through inhibition of luteinizing hormone secretion.31,32 
Spironolactone is rapidly and extensively metabolized by non-cytochrome p450 mechanisms 
to numerous active metabolites. These active metabolites have a t1/2 of approximately 15 
hours and accumulate following multiple doses. Spironolactone and its metabolites are 
approximately 90% bound to plasma proteins and undergo urinary and biliary elimination.33 
Cyproterone is rapidly and completely absorbed following oral administration with a Tmax of 
3 to 4 hours. The t1/2 is approximately 38 hours with biliary (60%) and urinary (33%) excretion 
of the unchanged drug. Cyproterone is metabolized by CYP3A4 and therefore clinically 
relevant interactions with CYP3A4 inducers or inhibitors are possible.33 The effects of 
antiandrogens cannot be directly evaluated through total testosterone measurement as 
estrogen supplementation also acts to decrease testosterone, and because these drugs work 
by multiple mechanisms and do not exclusively reduce total testosterone production. Less 
common anti-androgen therapies include finasteride, leuprolide, bicalutamide, dutasteride. 
Finasteride and dutasteride are both 4-aza steroid competitive inhibitors of 5α-reductase 
(5AR) which is responsible for converting testosterone to dihydrotestosterone (DHT); they 
are often used by cis men to support hair growth for male pattern baldness and similarly 
contribute to hair growth for trans women. Bicalutamide is a member of the nonsteroidal 
antiandrogen (NSAA) group of medications. It works by blocking the androgen receptor, the 
biologic target of the androgen sex hormones, testosterone and DHT. Like spironolactone, 
bicalutamide does not lower testosterone levels. Leuprolide is a gonadotropin-releasing 
hormone (GnRH) analogue acting as an agonist at pituitary GnRH receptors. Agonism of GnRH 
receptors initially results in the stimulation of LH and FSH by the anterior pituitary initially 
leading to increased serum estradiol and testosterone levels. However, because propagation 
of the hypothalamic-pituitary-gonadal (HPG axis) is incumbent upon pulsatile hypothalamic 
GnRH secretion, pituitary GnRH receptors become desensitised after several weeks of 
continuous therapy. This protracted downregulation of GnRH receptor activity is the targeted 
objective of leuprolide therapy and ultimately results in decreased LH and FSH secretion, 
leading to a dramatic reduction in estradiol and testosterone levels regardless of sex. It is 
most commonly used by adolescents or trans folks in puberty due to its mechanism of action. 
Both bicalutamide and leuprolide are used to treat prostate cancer. If participants have a 
condition in which their testes produce little or no testosterone (i.e. hypogonadism) then the 
participant will not be required to take an anti-androgen. Finally, orchiectomy is the surgical 
anti-androgen method that some trans women choose. The testosterone measurement is a 
secondary outcome and participants are able to enroll regardless of the anti-androgen they 
are 
taking. 
Participants 
must 
agree 
to 
remain 
on 
the 
same 
antiandrogen 
(pharmaceutical/surgical/medical) until after the Month 2 visit. 
 

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8 
STUDY PROCEDURES 
8.1 
Engagement with and screening of candidates/recruitment plan 
Trans women as well as cis women living with HIV are a sensitive patient population necessitating 
trauma-informed and participatory approaches. As a research team, we have been practicing CBR 
in partnership with trans women community leaders for the past 11 years.34 We have launched 
TWIRI with a trans woman community leader in Toronto, Yasmeen Persad. Yasmeen is a co-
principal investigator of this study and is assisting with its guidance. Yasmeen will also contribute 
to the study’s advertisement through the Toronto community and nationally, which will aid with 
recruitment. TWIRI has already put together a CAB as well, who have reviewed some of the study 
documents and heard a presentation about the study; and have shown interest in the study. The 
final study protocol, documents and the recruitment plan and advertisement will be reviewed 
with the CAB members, partly facilitating the recruitment. As previously mentioned, a 
community research participant assistant will be hired to connect with potential candidates, 
engage them, assist them with the Baseline and Month 2 visits (if preferred by participant, they 
can also bring their own support person) and to ensure the study is safe for them. This approach 
will help ensure that research practices are informed, empathetic, and trauma-aware; all of 
which are necessary for the success of the project.  
 
In addition to the CAB and through Yasmeen and the community research participant assistant’s 
connections, participants will be recruited from the six participating recruitment sites who 
participated in the aforementioned retrospective Montreal Toronto chart review study. These 
sites include: Maple Leaf Medical Clinic, Church-Wellesley Clinic, Sherbourne Health Centre, Dr. 
Raymond Fung’s office (the busiest trans endocrinologist in Toronto), and St. Michael’s 
Department of Family and Community Medicine at Unity Health. Since the formative chart review 
was conducted, we have a sense of the sample of candidates from each site. Site physicians, 
nurses, and staff have been engaged over the past two years as part of the formative chart review 
and will be keen to reach out to potential candidates as a member of the circle of care. We will 
also recruit cis women living with HIV from 790 Bay Street Clinic and Women's Health in Women's 
Hands Community Health Centre.  
 
If needed, Montreal will have two participating recruitment sites. The central study site, Clinique 
Quartier Latin, has captured 48 trans women candidates from the chart review. The care 
providers there will be able to inform potential candidates about the study for potential 
enrolment. This clinic also provides HIV care and will be able to enroll cis women living with HIV. 
A second Montreal site has been added, Clinique Médicale Quorum, and one of their physicians 
has a significant number of trans patients and he is keen to be involved in research. Therefore, 
recruitment will occur there as well. Recruitment flyers will also be produced and distributed to 
other clinic centers across Canada for referrals of potential interested candidates.  
 
If recruiting site care providers have interested patients, they will (with verbal consent from their 
patient) send a referral (for research purposes) to Dr. Mona Loutfy (in Toronto) noting the 
person’s interest, eligibility, and HIV viral loads within the prior 24 months history for those that 
are living with HIV.  

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The study will be advertised through the Maple Leaf Medical Clinic website 
(www.mlmedical.com), Groups of Trans Women, Women Living with HIV and potential study 
participants can also contact Maple Leaf Research directly to participate.  Also, the study has 
been approved by the CIHR Canadian HIV Trials Network (CTN) and the flyers will be distributed 
through their networks. 
 
The study procedures to be conducted for each subject are presented in tabular form in Appendix 
C and described in the text below. Appendix E1/2 also provides additional details about study 
procedures. For the purpose of this study: one month is equivalent to 28 days.  
 
All the questionnaires to be completed can be found in the DDI Main Study Surveys document.  
 
8.2 
Screening Visit 
Interested candidates will be screened, consented, and enrolled at MLR in Toronto. All potential 
candidates will have a screening visit to confirm eligibility.  The approved informed consent must 
be reviewed prior to any study procedures. All participant questions must be answered, and the 
consent form must be signed before screening study activities can begin. A copy of the signed 
consent form must be offered / provided to the participant, the original is kept in their study 
source.  This visit can be conducted virtually. 
 
8.3 
Screening Visit Procedures 
● Written informed consent completed prior to any other assessments (can be completed 
through eSIGN (e.g ADOBE sign). 
● Demographic data (questionnaire), important medical history (including HIV status, 
gender dysphoria history, surgical history, CIS women- menses history (cycle frequency-
see questionnaire), and medications used by the subject to treat or to prevent HIV, 
feminizing hormones or hormonal contraception, herbal/over-the counter medication, 
and any other medication used by the subject in the 28 days prior to screening are 
collected to confirm eligibility. Appendix B1 will be referred to, to confirm eligibility. A 
doctor visit and/or physical exam is only required if deemed medically necessary by the 
study personnel and physician; it will be completed by one of the study physicians 
virtually/ by phone/ or in person (i.e if the participant is experiencing an adverse event or 
wants to discuss the dosage change for medication).  
 
● Screening Blood Sample Collection: 
o Routine bloodwork: serum creatinine [eGFR] and ALT only for all groups. 
o Hepatitis B Surface antigen testing and Hepatitis C antibody testing for all groups. 
o For groups 1 and 2: a urine protein test will be done  
o For groups 1 and 2: an HIV-1 RNA Viral Load will be drawn to confirm viral 
suppression. 

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▪ 
If a HIV-1 RNA Viral Load for groups 1 and 2 is available from their doctors’ 
offices within two weeks prior to the screening visit, these results can be 
used instead with consent from the participant.   
o For group 3: an HIV-1 RNA Diagnostic Serology will be done to confirm that they 
are HIV negative. 
▪ 
For a positive HIV-1 RNA Diagnostic Serology, the participant will be 
excluded, and they will be immediately linked to the rapid HIV care 
programs developed at the Maple Leaf Medical Clinic or their referring 
physician site.   
o For group 2: serum Beta-HCG test is completed; they will be excluded if positive. 
o For group 2: FSH test will be completed 
▪ 
For a FSH level ≥27.0 IU/L, the participant will be excluded and deemed 
post-menopausal. 
 
● Estradiol Dosing: 
o Trans women taking an oral estradiol dose > 2 mg per day must divide their dose 
so that they are taking 2 mg in the morning and the rest at night (e.g. 3 mg of 
estradiol would be divided into 2 mg qAM and 1 mg qPM). There are no known 
side effects related to this change in dosing.  
o If they need to switch to a divided dose and need a prescription, the participant 
can meet with a study physician (by phone, video, or in-person) and they will be 
asked to switch immediately (i.e., the next day).  
o Participants taking an estradiol dose of 2 mg per day at night must switch their 
dosing to the morning and they will be asked to switch immediately (i.e., the next 
day). 
o Participants crushing or taking their oral estradiol sublingually must switch to 
swallowing the tablets and they will be asked to switch immediately (i.e., the next 
day). 
o Once confirmed or switched to the required oral estradiol dose (as explained 
above), participants must remain on this same regimen until the completion of 
the month 2 visit.  
 
● ART Dosing: 
o Women in groups 1 and 2 taking their ART medication at night must switch their 
dosing to morning and must remain on this same regimen until the completion of 
the month 2 visit. They will be asked to switch immediately (i.e., the next day).  If 
they are taking Atripla, which is qHS dosing; the participant can switch to morning 
dosing after baseline with the switch to Biktarvy®. 
 
● Screening window (from the screening to baseline visit):  
o Baseline visit for participants that switch their oral estradiol dosing from the night 
to the morning or split their dosing or change to swallowing a tablet, must be 
booked > 28 days to allow for estradiol concentration stabilization. 

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o Baseline visit for participants that switch their ART dosing from the night to the 
morning, must be booked > 28 days to allow for ART concentration stabilization.  
o Otherwise, the baseline visit can be scheduled after the HIV-1 RNA Viral Load or 
HIV-1 RNA Diagnostic Serology results are received around 14 days. 
o The maximum screening visit window is 90 days; participants whose baseline visit 
date will fall past 90 days can be rescreened.  
 
● Questionnaires (to be completed with the coordinator): 
o All groups: Demographic questionnaire 
o Group 2: Menses History 
 
● Participant Instructions: 
o Participant instruction sheet for baseline visit is reviewed and given to 
participants. 
o Participants will  be reminded to avoid drinking grapefruit juice for the duration of 
the study due to the possibility of interaction with Estrace.  
 
● Follow-Up 
o Study personnel will contact participants who switched their oral estradiol dosing 
from the night to the morning or split their dosing or changed to swallowing a 
tablet within a week after their screening visit to confirm that participants have 
started the new regimen and document the start date. 
8.4 
Baseline Visit Procedures 
● The Baseline procedures may occur as soon as all eligibility criteria are confirmed. The 
baseline visit must occur within 90 days after the screening visit. This visit can be 
conducted in person or virtually. 
● Important medical history (including HIV status, gender dysphoria history, surgical 
history, CIS-women menses history (cycle frequency-see questionnaire), and medications 
used by the subject to treat or to prevent HIV, feminizing hormones or hormonal 
contraception, herbal/over-the counter medication, and any other medication used by 
the subject is reviewed to confirm eligibility as per Appendix B1.  
● Adverse events will be reviewed and documented in the source document, only AEs 
related to either estradiol or B/F/TAF determined by the principal investigator will be 
entered in the CRF.  
● Height and weight will be collected for all groups when visit is done in person. If visit is 
done virtually, it will be done at Month 2. 
● A doctor visit and/or physical exam is only required if deemed medically necessary by the 
study personnel and physician; it will be completed by one of the study physicians 
virtually/ by phone/ or in person.  
● For groups 1 and 2: a urine protein test will be done  
 
 
 

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● Fasting: 
o All participants are required to fast (no food and drinks [except water]) for 10 
hours. 
 
● Dosing Times: 
o Participants that split their oral estradiol into AM & PM dosing need to document 
the time of their evening doses (the day prior to and the evening of the baseline 
visit). 
o Participants need to document the time of oral estradiol and ART (if applicable) 
the day before and the day of the visit. 
 
● Breakfast:  
o A Breakfast of 350-500 calories must be consumed within 30 minutes of starting. 
The start time must be documented.   
 
● Blood Sample Collection: 
o Blood Draw Group 1  
▪ 
Pre-dose serum estradiol and testosterone samples are collected 5-25 
minutes after breakfast start time. Time must be recorded. 
▪ 
Routine bloodwork (CBC, electrolytes [sodium, chloride, potassium], 
serum creatinine [eGFR], ALT, total bilirubin, glucose, HbA1C, and lipids). 
▪ 
HIV-1 RNA Viral Load & Lymphocyte Subsets (CD4 and CD8). 
● If there is an HIV-1 RNA viral load blip (i.e., > 40 and < 500 
copies/mL), the participant needs to repeat the HIV-1 RNA Viral 
Load in 2-3 weeks; if the repeat viral load is still > 200 copies/mL, 
they will be withdrawn from the study. 
●  If the baseline HIV-1 RNA viral load is detectable at > 500 
copies/mL, they will be withdrawn from the study; and replaced 
with another participant. 
▪ 
Feminizing hormones and current ART are taken 30 minutes (±5 mins) after 
the start of breakfast.  Time must be documented. 
▪ 
One (1) serum estradiol sample is collected 4 hours (±10 mins) after 
feminizing hormones are taken. Time must be documented. If the visit is 
conducted virtually, this sample will be optional.  
 
o Blood Draw Group 2:  
▪ 
Serum estradiol and testosterone samples are collected 5-25 minutes after 
breakfast start time. Time must be recorded.   
▪ 
Routine bloodwork (CBC, electrolytes [sodium, chloride, potassium], 
serum creatinine [eGFR], ALT, total bilirubin, glucose, HbA1C, and lipids) 
and serum Beta-HCG.  
▪ 
HIV-1 RNA Viral Load & Lymphocyte Subsets (CD4 and CD8). 

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● If there is an HIV-1 RNA viral load blip (i.e., > 40 and < 500 
copies/mL), the participant needs to repeat the HIV-1 RNA Viral 
Load in 2-3 weeks; if the repeat viral load is still > 200 copies/mL, 
they will be withdrawn from the study. 
●  If the baseline HIV-1 RNA viral load is detectable at > 500 
copies/mL, they will be withdrawn from the study; and replaced 
with another participant. 
▪ 
The participants in group 2 do not have a C4h estradiol sample collected. 
▪ 
The pregnancy test must be negative, if positive, the participant will be 
withdrawn and considered a screen failure. 
 
o Blood Draw Group 3:  
▪ 
Pre-dose serum estradiol and testosterone samples are collected 5-25 
minutes after breakfast start time. Time must be recorded. 
▪ 
Routine bloodwork (CBC, electrolytes [sodium, chloride, potassium], 
serum creatinine  [eGFR], ALT, total bilirubin, glucose, HbA1C, and lipids). 
▪ 
HIV-1 RNA Diagnostic Serology  
● If an HIV-1 RNA Diagnostic Serology returns positive, the 
participant will be immediately linked to the rapid HIV care 
programs developed at the Maple Leaf Medical Clinic or their 
referring physician site.  
▪ 
Feminizing hormones are taken 30 minutes (±5 mins) after the start of 
breakfast. Time must be documented. 
▪ 
One (1) serum estradiol sample is collected 4 hours (±10 mins) after 
feminizing hormones are taken. Time must be documented.  If the visit is 
done virtually, this sample is optional. 
 
● B/F/TAF Dosing: 
o Participants in Groups 1 and 2 not already taking B/F/TAF as their ART regimen, 
must switch to B/F/TAF at baseline visit (they will take their first dose of B/F/TAF 
the next day after the baseline visit). They speak with a study physician (virtually/ 
by phone/ or in person) to discuss possible side effects and adherence; the study 
physician is to enter the B/F/TAF prescription in the chart. Only Participants that 
switch their ART regimen to B/F/TAF at baseline are booked for a virtual month 1 
standard of care visit.   
o Document start date of B/F/TAF. 
o Switch participants in group 1 and 2 are dispensed 2 bottles of B/F/TAF at baseline 
as part of the study.  If participants taking B/F/TAF at start of study need assistance 
with medication, please contact the study team for further instructions.  
 
● Questionnaires: 
o Groups 1: FEM-SQ, Adherence, HSIDM PRO, HIVTSQs, SF-36. 
o Groups 2: Adherence, HSIDM PRO, HIVTSQs, SF-36, Menses History (to be 
completed with a coordinator). 

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o Groups 3: FEM-SQ, HSIDM PRO, SF-36.  
 
● Participant Instructions: 
o Participant Instruction Sheet for month 2 visit is given & reviewed for day before 
and day of visit instructions. 
 
● Follow-Up 
o Study personnel will contact group 1 and 2 participants who switched to B/F/TAF 
within a week after their baseline visit to confirm that participants have started 
the new regimen and document the start date and any side effects (if any). 
 
8.5 
Month 1 Visit Procedures (Group 1 and 2 who switched to B/F/TAF only) 
● The Month 1 standard of care visit can be conducted virtually / by phone with the study 
physician (as per clinical procedures). 
● This study visit is scheduled relative to the day that participants started B/F/TAF (i.e., Start 
Date + 28 days [± 5 days]). 
● Review and document any changes in medical history (including, gender dysphoria 
history, surgical history, and medications used by the subject to treat HIV, feminizing 
hormones or hormonal contraception, herbal/over-the counter medication, and any 
other medication used by the subject. This will be reviewed to confirm eligibility as per 
Appendix B1. 
● Adverse events will be reviewed and documented in the source document, only AEs 
related to either estradiol or B/F/TAF determined by the principal investigator will be 
entered in the CRF.  
 
8.6 
Month 2 Visit Procedures 
● Participants must come to Maple Leaf Research in Toronto to complete the Month 2, due 
to the PBMC processing requirements.  Additional travel support will be provided for 
participants travel and the stay in Toronto (see #22 Ethics & Dissemination for more 
details). 
● Month 2 visit is scheduled 8 weeks [± 5 days] after the baseline visit, with exception to 
participants who switched to B/F/TAF, month 2 visit is scheduled 8 weeks after the 
B/F/TAF start date [± 5 days]. 
● Review and document any changes in medical history (including HIV status, gender 
dysphoria history, surgical history, CIS-women menses history (questionnaire) and 
medications used by the subject to treat or to prevent HIV, feminizing hormones or 
hormonal contraception, herbal/over-the counter medication, and any other medication 
used by the subject. This will be reviewed to confirm eligibility as per Appendix B1. 
● Adverse events will be reviewed and documented in the source document, only AEs 
related to either estradiol or B/F/TAF determined by the principal investigator will be 
entered in the CRF.  
● For participants that started the study already taking B/F/TAF - A doctor visit and/or 
physical exam is only required if deemed medically necessary by the study personnel and 

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physician; it will be completed by one of the study physicians virtually/ by phone/ or in 
person.  
● For participants that switched to B/F/TAF at baseline- A standard of care visit is required, 
it can be conducted virtually / by phone or in person with the study physician (as per 
clinical procedures) to review and discuss possible side effects and adherence.   
 
● Fasting 
o All participants are required to fast (no food and drinks (except water) for 10 
hours. 
 
● Dosing Times: 
o Participants that split their oral estradiol into AM & PM dosing need to document 
the time of their evening doses (the day prior to and the evening of the Month 2 
visit). 
o Participants need to document the time of oral estradiol and ART (if applicable) 
the day before and the day of the visit. 
 
● Breakfast  
o A Breakfast of 350-500 calories is given upon arrival to all participants. 
o The start time for breakfast must be documented. 
● Weight will be collected for all groups. 
 
● Blood Sample Collection: 
o A catheter can be inserted for the day to perform the required laboratory work 
otherwise a regular butterfly needle or multi-sample blood collection needle can 
be used (decision is up to participant and lab tech based on comfort and safety). 
 
o Blood Draw Group 1: 
▪ 
Pre-dose samples: 
● serum estradiol and testosterone and pre-dose whole blood 
B/F/TAF PK samples are collected 5-25 minutes after breakfast start 
time (see lab manual for processing instructions). Time must be 
recorded. 
● Routine 
bloodwork 
(CBC, 
electrolytes 
[sodium, 
chloride, 
potassium], serum creatinine [eGFR], ALT, total bilirubin, glucose, 
HbA1C, and lipids) 
● HIV-1 RNA Viral Load  
o If there is an HIV-1 RNA viral load blip <200, participant 
needs to repeat in 2-3 weeks.  
▪ 
Feminizing hormones and B/F/TAF are taken 30 minutes (±5 mins) after 
the start of breakfast.  Time must be documented. 
▪ 
Post-dose samples: 
● Seven (7) post-dose serum estradiol samples are collected 1H, 2H, 
3H, 4H, 6H, 8H, 24H (±10 mins) after feminizing hormones are 

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taken on morning of month 2 visit (see lab manual for processing 
instructions).  All times must be documented.   
● Nine (9) post-dose whole blood B/F/TAF PK plasma samples are 
collected 0.5H, 1H, 1.5H, 2H, 3H, 4H, 6H, 8H, 24H  (±10 mins) after 
B/F/TAF is taken on morning of month 2 visit (see lab manual for 
processing instructions).  All times must be documented.   
● If any timepoints for estradiol and B/F/TAF PK sample collection fall 
out of the window, blood sample will be drawn regardless, and 
time must be documented.  
▪ 
DBS whole blood sample is collected 2H after B/F/TAF is taken (±10 mins) 
(see lab manual for processing instructions).   
▪ 
PBMC whole blood samples are collected 2H after B/F/TAF is taken (±10 
mins).  The processing of samples must start within 30 minutes at the Dr. 
Rupert Kaul’s University of Toronto lab.  (see lab manual for processing 
instructions). 
▪ 
Participants can take their AM oral estradiol and B/F/TAF after the 24H 
samples are collected. 
 
o Blood Draw Group 2  
▪ 
A urine pregnancy test must be negative, if positive, the participant must 
be withdrawn. 
▪ 
Predose samples: 
● Serum estradiol and testosterone samples and B/F/TAF pre-dose 
whole blood samples are collected 5-25 minutes after breakfast 
start time (see lab manual for processing instructions). Time must 
be recorded. 
● Routine 
bloodwork 
(CBC, 
electrolytes 
[sodium, 
chloride, 
potassium], serum creatinine [eGFR], ALT, total bilirubin, glucose, 
HbA1C, and lipids) and serum Beta-HCG. 
● HIV-1 RNA Viral Load,  
o If there is an HIV-1 RNA viral load Blip <200, participant 
needs to repeat in 2-3 weeks.  
▪ 
B/F/TAF medication is taken 30 minutes (±5 mins) after the start of 
breakfast.  Time must be documented. 
▪ 
Post-dose samples: 
● Nine (9) post-dose whole blood B/F/TAF PK plasma samples are 
collected 0.5H, 1H, 1.5H, 2H, 3H, 4H, 6H, 8H, 24H (±10 mins) after 
B/F/TAF is taken on morning of month 2 visit (see lab manual for 
processing instructions).  All times must be documented.   
● If any timepoints for B/F/TAF PK sample collection falls out of the 
window, blood sample will be drawn regardless, and time will be 
documented  
▪ 
DBS whole blood sample is collected 2H after B/F/TAF is taken (±10 mins) 
(see lab manual for processing instructions).   

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▪ 
PBMC whole blood samples are collected 2H after B/F/TAF is taken (±10 
mins).  The processing of samples must start within 30 minutes at the 
Rupert Kaul lab.  (see lab manual for processing instructions).  
▪ 
Participants can take their AM B/F/TAF after the 24H samples are 
collected. 
 
o Blood Draw Group 3  
▪ 
Pre-dose samples: 
● Pre-dose serum estradiol and testosterone samples are collected 
5-25 minutes after breakfast start time. Time must be recorded. 
● HIV-1 RNA Diagnostic Serology 
o If an HIV-1 RNA Diagnostic Serology returns positive, they 
will be immediately linked to the rapid HIV care programs 
developed at the Maple Leaf Medical or their referring 
physician site. The participant will be withdrawn. 
● Routine 
bloodwork 
(CBC, 
electrolytes 
[sodium, 
chloride, 
potassium], serum creatinine[eGFR], ALT, total bilirubin, glucose, 
HbA1C, and lipids). 
▪ 
Feminizing hormones are taken 30 minutes (±5 mins) after the start of 
breakfast. Time must be documented. 
▪ 
Post-dose samples: 
● Seven (7) serum estradiol samples are collected 1H, 2H, 3H, 4H, 6H, 
8H, 24H (±10 mins) after feminizing hormones are taken on 
morning of month 2 visit (see lab manual for processing 
instructions).  All times must be documented.   
● If any timepoints for estradiol PK sample collection falls out of the 
window, blood samples will be drawn regardless, and time will be 
documented  
▪ 
Participants can take their AM oral estradiol after the 24H samples are 
collected.   
 
o If the participant does not complete all of the required estradiol and/or PK time 
points or any estradiol values are abnormal the principal investigator needs to be 
consulted to determine if Month 2 procedures should be repeated. 
 
● Questionnaires: 
o Groups 1: FEM-SQ, Adherence, HSIDM PRO, HIVTSQs, SF-36. 
o Groups 2: Adherence, HSIDM PRO, HIVTSQs, SF-36, Menses History (to be 
completed with a coordinator). 
o Groups 3: FEM-SQ, HSIDM PRO, SF-36. 
 
● B/F/TAF Dosing: 
o Dispense 4 bottles of B/F/TAF to group 1 and 2 participants (If required). 

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o If B/F/TAF was switched to the morning, it can be switched back to the evening 
after this visit. 
o Participants are not required to keep empty bottles. 
 
● Next steps: 
o The Month 2 visit will be the last study visit for Group 3 participants. 
o After completion of this visit participants can change back to their original 
estradiol dosing if they prefer. If needed, a new prescription can be requested 
from the study physician.  
There will a Month 6 visit for participants in Group 1 and 2.  Remind participants 
to return any leftover study medication at month 6, empty bottles are not 
returned. 
 
8.7 
Month 6 Visit Procedures (Group 1 & 2 only) 
● Month 6 is scheduled 24 weeks [± 5 days] after the baseline visit.  This visit can be 
conducted virtually. 
● Important medical history (including HIV status, gender dysphoria history, surgical 
history, CIS-women menses history) and medications used by the subject to treat HIV, 
feminizing hormones or hormonal contraception, herbal/over-the counter medication, 
and any other medication used by the subject are reviewed and updated as required. This 
will be reviewed to confirm eligibility as per Appendix B1. 
● Adverse events will be reviewed and documented in the source document, only AEs 
related to either estradiol or B/F/TAF determined by the principal investigator will be 
entered in the CRF.  
● For participants that started the study on B/F/TAF- A doctor visit and/or physical exam is 
only required if deemed medically necessary by the study personnel and physician; it will 
be completed by one of the study physicians virtually/ by phone/ or in person.  
● For participants that switched to B/F/TAF at baseline- A standard of care visit is required, 
it can be conducted virtually / by phone or in person with the study physician (as per 
clinical procedures) to review and discuss possible side effects and adherence.   
● Blood Sample Collection: 
o Routine bloodwork (CBC, electrolytes [sodium, chloride, potassium], serum 
creatinine [eGFR], ALT, total bilirubin, glucose, HbA1C, and lipids(non-fasting).  
o HIV-1 RNA Viral Load  
▪ 
If there is an HIV-1 RNA viral load blip or viremia (i.e., > 40 copies/mL), the 
participant needs to repeat the HIV-1 RNA Viral Load in 2-3 weeks. 
o Lymphocyte Subsets (CD4 and CD8). 
 
B/F/TAF Dosing: 
o Participants that switched to B/F/TAF as their ART regimen, can either switch back 
to their old regimen or continue taking B/F/TAF after this visit. The study physician 
can provide them with a prescription. 

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o Document the last dose of B/F/TAF in the study. Participants need to return any 
leftover study medication.  Participants are not required to keep empty bottles. 
 
● Questionnaires: 
o Group 1: 
▪ 
FEM-SQ, Adherence, HSIDM PRO, HIVTSQs, HIVTSQc, SF-36. 
o Group 2: 
▪ 
Adherence, HSIDM PRO, HIVTSQs, HIVTSQc, SF-36, Menses History (to be 
completed with a coordinator). 
9 
SCREEN FAILURES  
● A screen failure is an individual who consents to participate in this study but does not 
complete the baseline visit because they do not meet inclusion / exclusion criteria. 
● Trans women who test positive for HIV at screening are considered screen failures. 
● Cis women who have a positive pregnancy test at screening are considered screen 
failures. 
● Any screen failure must be replaced with another participant until the group number is 
met. 
10 MANAGING POSITIVE HIV DIAGNOSTIC SEROLOGY 
● If an HIV-1 RNA Diagnostic Serology returns positive, the participants will be 
immediately linked to the rapid HIV care programs developed at the Maple Leaf Medical 
or their referring physician site. The participant will be withdrawn. 
11 MANAGING HIV-1 RNA BLIPS  
● If there is an HIV-1 RNA viral load blip (i.e., > 40 and < 500 copies/mL), the participant 
needs to repeat HIV-1 RNA Viral Load in 2-3 weeks; if the repeat viral load is still > 200 
copies/mL, at baseline or month 2, they will be withdrawn from the study and replaced 
with another participant from the same group. 
● If the baseline or month 2 HIV viral load is detectable at > 500 copies/mL, they will be 
withdrawn from the study; and replaced with another participant. 
12 STUDY WITHDRAWAL 
If a participant cannot or does not complete the study for any reason or is lost to FU they need 
to be replaced with a participant from the same group.  Please contact the study principal 
investigator to discuss. 
● If a participant has a high-risk HIV exposure and HIV post-exposure prophylaxis (PEP) is 
required for a month-participants, needs to be withdrawn but can be rescreened after 30 
days. 

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● For a positive HIV-1 RNA Diagnostic Serology, the participant will be excluded/withdrawn, 
and they will be immediately linked to the rapid HIV care programs developed at the 
Maple Leaf Medical Clinic or their referring physician site.   
● If there is an HIV-1 RNA viral load blip (i.e., > 40 and < 500 copies/mL), the participant 
needs to repeat HIV-1 RNA Viral Load in 2-3 weeks; if the repeat viral load is still > 200 
copies/mL, at baseline or month 2, they will be withdrawn from the study and replaced 
with another participant from the same group. 
● If the baseline or month 2 HIV viral load is detectable at > 500 copies/mL, they will be 
withdrawn from the study; and replaced with another participant. 
● If CIS woman becomes pregnant between screening and month 2 they will be 
withdrawn and replaced with a participant. 
● Participants are free to withdraw from participation in the study at any time upon 
request. 
● The participant will be withdrawn if any clinical adverse event (AE), laboratory 
abnormality, or other medical condition or situation occurs such that continued 
participation in the study would not be in the best interest of the participant.  
● The participant will be withdrawn if they meet an exclusion criterion (either newly 
developed or not previously recognized) that precludes further study participation.  
 
13 EVALUATION, RECORDING, AND REPORTING OF ADVERSE EVENTS  
 
13.1 Definitions  
13.1.1 Adverse Event (AE)  
In this study, an adverse event (AE) is any untoward medical occurrence in a patient or clinical 
investigation participant, administered a study medication/intervention, which has a causal 
relationship with this treatment. An AE can therefore be any unfavorable and unintended sign 
(including an abnormal laboratory finding), symptom, or disease temporally associated with the 
use of an investigational intervention. 
 
During each visit with the participant, information on AEs will be gathered and documented 
accordingly. AEs will be graded as mild, moderate, severe, or life-threatening and assessed by 
causality as related to the study intervention.  
 
In this study, any other new conditions that are not related to study drugs will be captured in 
the Medical History and do not need to be graded. Stable chronic conditions which are present 
prior to clinical trial entry and any worsening of these pre-existing conditions will also be 
accounted for in the participant’s medical history.  
 
13.1.2 Serious Adverse Events (SAE)  
In this study, an SAE is defined as an AE meeting one of the following criteria at any dose: 
• Results in death during the period of protocol-defined surveillance  

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• Is a life-threatening event (defined as a participant at immediate risk of death at the time 
of the event)  
• Results in in-patient hospitalization or prolongation of existing hospitalization during the 
protocol-defined surveillance  
• Results in persistent or significant disability or incapacity (disability is defined as a 
substantial disruption of a person’s ability to conduct normal life functions)  
• Is a congenital anomaly or birth defect  
Any other important medical event that may not result in one of the above outcomes, may be 
considered a SAE when, based upon appropriate medical judgment, the event may jeopardize 
the participant and may require medical or surgical intervention to prevent one of the outcomes 
listed above. Examples of such medical events include allergic bronchospasm requiring intensive 
treatment in an emergency room or at home, blood dyscrasias or convulsions that do not result 
in in-patient hospitalization, or the development of drug dependency or drug abuse. 
 
Participants will be monitored during the study period for SAEs. If an SAE is ongoing at the time 
a participant discontinues/completes the study, the SAE will be followed until the Investigator 
agrees that the event is satisfactorily resolved, becomes chronic, or that no further follow-up is 
required. 
 
13.2 AE Description: Relationship to Study Treatment  
For all collected AEs (including SAEs), the clinician who examines and evaluates the participant 
will determine the AE’s causality based on temporal relationship and his/her clinical judgment. 
The degree of certainty about causality will be graded using the categories below: 
 
Definitely Related: There is clear evidence to suggest a causal relationship, and other possible 
contributing factors can be ruled out. The clinical event, including an abnormal laboratory test 
result, occurs in a plausible time relationship to Investigational Product administration and 
cannot be explained by concurrent disease or other products or chemicals. The response to 
withdrawal of the product (de-challenge) should be clinically plausible. The event must be 
pharmacologically or phenomenologically definitive, with use of a satisfactory re-challenge 
procedure if necessary. 
 
Probably Related: There is evidence to suggest a causal relationship, and the influence of other 
factors is unlikely. The clinical event, including an abnormal laboratory test result, occurs within 
a reasonable time sequence to administration of the Investigational Product, is unlikely to be 
attributed to concurrent disease or other products or chemicals, and follows a clinically 
reasonable response on withdrawal (de-challenge). Re-challenge information is not required to 
fulfill this definition. 
 
Possibly Related: There is some evidence to suggest a causal relationship (e.g., the event 
occurred within a reasonable time after administration of the trial medication). However, the 
influence of other factors may have contributed to the event (e.g., the participant’s clinical 

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condition, other concomitant events). Although an adverse drug event may rate only as 
“possibly related” soon after discovery, it can be flagged as requiring more information and 
later be upgraded to “probably related” or “definitely related”, as appropriate. 
 
Unlikely: A clinical event, including an abnormal laboratory test result, whose temporal 
relationship to Investigational Product administration makes a causal relationship improbable 
(e.g., the event did not occur within a reasonable time after administration of the trial 
medication) and in which other products or chemicals or underlying disease provides plausible 
explanations (e.g., the participant’s clinical condition, other concomitant treatments). 
 
Not related: The AE is completely independent of Investigational Product administration, 
and/or evidence exists that the event is definitely related to another etiology. There must be an 
alternative, definitive etiology documented by the clinician. 
 
13.2 Follow-Up for Adverse Events  
Any AE that occurs between the time that a study participant commences the study and the time 
that the participant departs the study at the end of the final study visit (or at the time of early 
discontinuation of the participant from the study for any reason) will be captured and recorded. 
At each contact with the participant, the investigator (or designate) must seek information on 
AEs by specific questioning and, as appropriate, by examination. 
 
AEs that had previously been reported by the study participant will also be reassessed for 
duration, intensity and possible reoccurrence. Assessment of safety will include clinical 
observation and monitoring of study related lab results.  
 
All AEs (including SAEs) will be followed until resolution or until the investigator and the 
clinical/medical monitor are in agreement that the AE has resolved, stabilized or become chronic 
and no further follow-up is required. 
 
13.3 SAE Reporting 
All SAEs which occur during the course of the study must be reported to the Sponsor-Investigator 
within 24 hours of the site becoming aware of the event.  
 
SAEs will be reported to: 
 
Dr. Mona Loufty  
Phone:  
416-725-9566 
Fax:  
416-465-0502 
E-mail:  
mona.loufty@wchospital.ca 
 

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13.4 Mandatory Reporting to Health Canada 
Per section C.05.014 of the Canadian Food and Drug Regulations, SAEs meeting Health Canada 
expedited reporting criteria (serious and unexpected adverse drug reactions) will be submitted 
to Health Canada. Expedited reporting of reactions that are serious but expected is not required. 
Expedited reporting is not required for serious events from clinical investigations that are 
considered unrelated to the study treatment, whether or not the event is expected. 
  
During the trial, the Sponsor-investigator will inform Health Canada in an expedited manner of 
any serious, unexpected adverse drug reaction in response to the use of the study treatment that 
has occurred during the study, as follows: 
 
a) Where it is neither fatal nor life-threatening, within 15 days after becoming aware of the 
information;  
b) Where it is fatal or life-threatening, within 7 days after becoming aware of the 
information.  Within 8 days after having initially informed Health Canada of the fatal or 
life-threatening SAE, submit as complete a report as possible.  Follow-up reports of fatal 
or life-threatening reactions must include an assessment of the importance and 
implication of the findings, including relevant previous experience with the same or 
similar drugs.  
  
The Investigator is responsible for informing the local Research Ethics Board (REB) of SAEs that 
occur at the site in compliance with local requirements. 
 
 
14 POTENTIAL RISK TO HUMAN PARTICIPANTS TAKING BIKTARVY® 
 
A few risks are associated with taking Biktarvy® for people living with HIV-1. The only absolute 
contraindication to the drug is a known hypersensitivity to the drug itself. Severe acute 
exacerbations of hepatitis B have been reported in patients co-infected with HIV-1 and HBV, and 
it may occur with discontinuation of Biktarvy®. Therefore, patients co-infected with HIV-1 and 
HBV who discontinue Biktarvy® should be closely monitored with clinical and laboratory follow-
up for at least several months after stopping treatment. 
 
 
15 CLINICAL TRIAL DATA ON ADVERSE REACTIONS TO BIKTARVY® 
 
The adverse event profile of Biktarvy® is very favorable. The primary safety assessment of 
Biktarvy® was based on Weeks 48, 96, and 144 pooled data from 1274 patients in two 
randomized, double-blind, active-controlled trials (Study 1489 and Study 1490). The most 
common adverse reactions (all grades) reported in at least 5% of patients in the Biktarvy® group 
in study 1489 were diarrhea, nausea, and headache. No adverse events in at least 5% of patients 
in the Biktarvy® group were reported in Study 1490. The safety profile of Biktarvy® was consistent 
through week 144 in both studies.  

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Additional adverse reactions (all grades) occurring in less than 2% of patients in Study 1489 and 
Study 1490 included:  
• Gastrointestinal disorder: abdominal pain, dyspepsia, flatulence, vomiting. 
• Psychiatric Disorders: depression. 
• Skin and subcutaneous tissue disorders: rash. 
• Suicidal ideation or suicide attempt (in patients with a pre-existing history of depression 
or psychiatric illness)  
 
The majority of adverse reactions were Grade 1. Only 0.9% of patients in both the 1489 Study 
and the 1490 Study randomized to Biktarvy® discontinued due to an adverse event, compared to 
the 1.6% and 1.8% in the abacavir [ABC]/DTG/lamivudine [3TC] and DTG + FTC/TAF arm 
respectively.  
 
 
 
16 RISKS OF ESTRADIOL THERAPY 
 
A few risks are associated with taking estrogen therapy for transwomen. These risks include 
venous thrombosis/thromboembolism, increased risk of cardiovascular disease, weight gain, 
decreased libido, hypertriglyceridemia, elevated blood pressure, decreased glucose tolerance, 
gallbladder disease, benign pituitary prolactinoma, mental health effects and infertility. Since 
participants in this study will be already taking estradiol prior to entering the study, these risks 
have been being monitored by their primary prescribing physician and the ongoing risk in the 
study are considered low.  
 
17 ADVERSE REACTIONS TO ESTRADIOL  
 
The adverse event profile of estradiol is very favorable. Women rarely have severe side effects 
from taking estradiol. The most common adverse reactions are abdominal pain, nausea and 
vomiting. Other possible adverse reactions are depression, nervousness, and/or irritability, 
allergic reaction and rash, increased blood sugar levels, change in blood pressure, acne, change 
in cholesterol and/or triglyceride levels, change in weight. As study participants will be already 
taking estradiol prior to entering the study and are to be on a stable dose, side effects are likely 
to have already occurred and be resolved and managed by their primary prescribing physician. 
Any adverse event that occurs related to estradiol and any lab results obtained from this study 
will be sent to the primary physician, with the permission of the participant to help monitor the 
risks stated above. 
 
18 HANDLING, STORAGE, AND ACCOUNTABILITY OF B/F/TAF  
 
B/F/TAF must be stored in a secured place within the temperature range specified on the label. 
The storage temperature should be continuously monitored and recorded with a calibrated (if 

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not validated) temperature monitoring device(s). Only authorized study personnel should be 
allowed access to the study interventions. Storage conditions will be assessed during pre-study 
activities. B/F/TAF will be dispensed in their original marketed packaging containing 30 tablets in 
each bottle. Empty bottles are not required to be returned to study sites; however, participants 
should return any leftover study medication at the end of study, or a note written in chart 
explaining otherwise.  
 
19 RISK MANAGEMENT  
 
Risk minimization, management, and assessment procedures have been implemented in the 
study to minimize and assess potential risks to participants who participate in this clinical study 
with Estrace® or Lupin-estradiol and Biktarvy®. Components include: (1) specific study entry and 
exclusion criteria to ensure that participants who have underlying characteristics that potentially 
increase their risk for an adverse outcome are excluded; (2) protocol-specific procedures for 
minimizing and managing certain AEs, such as diarrhea, nausea, headache, etc. related to 
Biktarvy® and Estrace® or Lupin-estradiol; (3) ongoing follow-up at Month 1 for participants who 
switched to Biktarvy® at baseline and at Month 6 for participants living with HIV.  
 
Any participants switched to Biktarvy® who do not tolerate the medication will be returned 
(rescued) to their prior antiretroviral therapy (ART) regimen and withdrawn from the study. 
 
20 PK SAMPLE COLLECTION AND ANALYSIS  
 
20.1 
Antiretroviral PK Samples 
 
Whole blood samples for storage of ART plasma PK concentrations will be collected from the 
participants as mentioned above. Similarly, PBMC samples will be collected to assess TFV-DP and 
FTC-TP concentrations at one time point (2h) during the study visit. These samples will be 
processed at Dr. Rupert Kaul’s University of Toronto’s research laboratory.  
 
In addition, dry blood spots (DBS) will be collected to assess TFV-DP and FTC-TP. While PBMC is 
the preferred sample for intracellular drug concentration testing, there is an increasing body of 
literature supporting the use of DBS which has shown correlation with the PBMC values and 
adherence.35 Due to the ease of taking this sample, a C2h sample will be collected at Month 2 visit. 
The advantage of doing DBS testing is the ease of collection and processing.  
 
Bioanalysis of all samples (minus the plasma TAF concentrations) will be performed under the 
direction of Dr. Kim Scarsi at the Antiviral Pharmacology Laboratory at the University of Nebraska 
Medical Center, Omaha, Nebraska, United Stated of America (USA). Dr. Scarsi is one of the top 
PK and DDI researchers in the USA and has particular expertise in studying DDIs between ART 
drugs and hormonal contraceptive medications. She has carried out such work for the US AIDS 
Clinical Trial Group studies over the years; all assays in her lab are validated according to Federal 

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Drug Association (FDA) guidance and evaluated in the NIH DAIDS Clinical Pharmacology Quality 
Assurance Program.  
The assay ranges are as follows: 
● BIC plasma 20-10,000 ng/mL 
● FTC plasma 10-1,500 ng/mL 
● TFV-DP PBMC and DBS 50-10,000 fmol/sample 
● FTC-TP PBMC and DBS 1,000-200,000 fmol/sample 
 
Plasma TAF concentration analyses and reporting will be performed at QPS LLC, Delaware 
Technology Park, 3 Innovation Way, Suite 240, Newark, Delaware, 19711, USA. 
The TAF plasma assay is: 
● TAF method QPS 60-1578 with assay range of 1-1000 ng/mL. 
Concentrations of antiretrovirals will be determined in plasma samples using the currently 
approved bioanalytical methodology; no human DNA analysis will be performed on these 
samples.  
 
20.2 
Estradiol Level Samples 
Serum estradiol samples will be collected from the participants as mentioned above. Estradiol 
bioanalysis will be performed under the control of Life Labs. 
 
21 DATA MANAGEMENT 
 
Visit data collection forms (eCRFs) are available electronically using a secure electronic system 
and will be created and managed in REDCap through the CIHR Canadian HIV Trials Network (CTN) 
in Vancouver, BC; data will be kept on their institutional secure server. Research site study 
personnel can access the electronic eCRFs in real-timeParticipant questionnaires are available 
through the electronic system REDCap; they will be self-administered by participants 
electronically in real-time at each visit. If the participant prefers, a research coordinator can assist 
them completing the questionnaires.  Paper questionnaires are available as backup.  Data entry 
verification will include algorithms that automatically check completed forms for missing, out-of-
range, or inconsistent values. Aberrant entries will be checked according to the data 
management agreement and reported promptly to the original interviewer for immediate 
reconciliation. An audit trail will be created for all data changes. The database will be maintained 
on a secure server and in a password-protected partitioned drive and backed up daily.  
 
22 ADHERENCE TO PROTOCOL  
 
22.1 Protocol Amendments  
All protocol amendments will be reviewed and approved and if applicable submitted to the 
applicable regulatory agencies for prior approval or notification. The Investigator must sign and 
date the amendment prior to implementation. All protocol amendments must also be submitted 
to the ethics committee.  
 

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22.2 Protocol Deviations  
No deviations from this protocol will be permitted without the prior written approval of the 
Sponsor-Investigator, except when the modification is needed to eliminate an immediate hazard 
or hazards to participants. Any deviations that may affect a participant’s treatment or informed 
consent, especially those increasing potential risks, must receive prior approval from the REB 
unless performed to remove an immediate safety risk to the participants. In this case it will be 
reported to the REB and the Sponsor immediately thereafter. Any departures from the protocol 
must be documented. 
 
23 ENDPOINTS AND ADDITIONAL VARIABLES OF INTEREST  
 
23.1 Primary endpoints 
Primary endpoints #1:  relates to the comparison of the PK parameters of interest, AUC, 
maximum (Cmax) and minimum (Cmin) plasma concentration of BIC, FTC, and TAF, and PBMC and 
DBS TFV-DP and FTC-TP concentrations will be compared between trans women living with HIV 
(group 1) and premenopausal cis women living with HIV (group 2). 
 
These are the standard PK endpoints for DDI studies.36 The FDA Guidance document on topic 
indicates that AUC and Cmax are the standard change in drug exposure parameters to be 
measured.37 We will also be reporting Cmin which is standard in the field. The FDA indicates that 
the PK results of DDI studies should be presented as the geometric mean ratio of the observed 
PK exposure measures with and without the perpetrator drug and include the associated 90% 
confidence interval (CI). It is also recommended to report on the measures of the observed 
variability of the interaction.  
 
Primary endpoints #2: relates to the hormone concentrations routinely tested when trans 
women are taking feminizing hormones, namely estradiol concentrations. The primary endpoints 
for estradiol concentrations will be Cmin, C4h, Cmax, and AUC and the proportions of the month 2 
C4h that are within target (200 to 735 pmol/L) and will be compared between groups 1 and 3. 
Testosterone concentration will not be assessed as a primary endpoint due to its high variability. 
 
In general, estrogen therapy aims to achieve serum estradiol concentrations within the normal 
range for pre-menopausal women; approximately 200 to 735 pmol/L.18,31 Oral micronized 17β-
estradiol is rapidly absorbed and is metabolized in both the gastrointestinal (GI) tract and liver to 
estrone (E1), which is further metabolized to estrone sulfate (E1S) and other conjugates.32,38 The 
oral bioavailability of 17β-estradiol is approximately 5% due to a large first pass effect39 and the 
half-life (t1/2) of oral estradiol is often reported to be 0.5-1h.40 Conversely, when measured in PK 
studies, the elimination t1/2 has been found to range from approximately 12-20 hours.32 This 
extended t1/2 is likely the result of enterohepatic recirculation and  continuous interconversion 
between estradiol, E1, and E1S.32,39 At 2 mg dosages of 17β-estradiol administered once daily 
acutely, the Cmax of estradiol was 489.6 pmol/L (at 8 hours), which reduced to a 24h Cmin of 146.8-
220.2 pmol/L/mL. As can be seen, the Cmin is below the desired estradiol concentration target in 
these studies and therefore, is the reason that most clinicians do not use the estradiol Cmin as the 

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concentration used to monitor estradiol dosing clinically. In another study, Cmax was C4h. It is 
predicted that the Cmax will be at 4 hours; however, this is variable and not well-established. 
Therefore, a full series of estradiol concentration samples will be taken from 1 hour to 8 hours 
after administration. Clinicians indicate to their trans patients taking estradiol to take the pill at 
night and go to the laboratory in the morning to capture the estradiol level at 8-12 hours post-
dose. We have chosen 8 hours post dosing as our final peak sample as 12 hours will not be 
feasible with laboratory and research facility hours. Since the timing of the estradiol 
concentration is tricky, we are currently undertaking a supplemental pilot study to draw the full 
series of estradiol concentrations from 8 trans women living with HIV on ART and 8 trans women 
living without HIV who meet our inclusion and exclusion criteria to inform the final endpoints for 
this study. 
 
23.2 Secondary Endpoints 
Secondary endpoints #1: are the baseline estradiol pre-dose and C4h and total testosterone 
concentrations, and these will be compared between groups 1 and 3 and assessed to see the 
proportion that meets target. The baseline estradiol pre-dose and C4h and total testosterone 
concentrations will be taken on variable ART regimens for the trans women living with HIV; the 
month 2 estradiol pre-dose and C4h and total testosterone concentrations will be taken from the 
trans women living with HIV who are taking B/F/TAF; and the change in estradiol pre-dose and 
C4h and total testosterone concentrations from baseline to month 2 in those who switched to 
B/F/TAF can be compared to those who did not and to those in group 3. Group 3 values also 
provide information on the degree of variability of estradiol concentrations. The desired 
concentrations of estradiol are between 200-735 pmol/L and the desired concentration for 
testosterone is < 2 nmol/L. The frequency and proportion of those within the target of 200-735 
pmol/L for estradiol concentration and < 2 nmol/L for testosterone concentrations will be 
reported as well as the frequency and proportion that are below and above target.  
 
Testosterone concentrations are routinely measured in the care of trans women after initial start 
of feminizing hormones and are easier to test than estradiol as the concentration is less impacted 
by the timing of its sampling. For ease, the testosterone concentration in our study will be 
measured at the same time as the estradiol. Free testosterone represents the portion of 
testosterone unbound to serum proteins and depends on levels of sex hormone binding globulin 
(SHBG).40 While free testosterone can be measured, assays are unreliable. Consensus is lacking 
on the role of free vs. total testosterone concentrations. Total testosterone concentrations are 
reliable and readily available; however, they do not describe the actual bioavailable testosterone 
level. Bioavailable testosterone is free testosterone plus testosterone weakly bound to albumin. 
SHBG is elevated in the presence of estrogen, and in particular with exogenous estrogen 
supplementation, more so with oral estrogen than with parenteral routes due to first pass 
hepatic activity.18 The Endocrine Society recommends monitoring of the total testosterone level, 
with a target range of < 2 nmol/L and for that reason, we will be measuring total testosterone 
concentration.  
 

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Secondary endpoint #2: Total score on the FEM-SQ, a questionnaire for measuring satisfaction 
with medical gender affirmation, will be compared between groups 1 and 3 throughout the study 
period. The FEM-SQ is a new questionnaire developed by our team through mixed-methods 
survey development including expert consultation and focus groups with 23 trans women 
(version 1), semi-structured interviews with over 10 trans women (version 2), and quantitative 
data collection (survey administration) with 300 trans women (version 3). While hormone 
concentration measurement is numerically easier to capture, understanding how satisfaction 
with gender affirmation differs between trans women taking feminizing hormones and ART and 
those only taking feminizing hormones without ART is of critical importance for understanding 
and reducing barriers to ART uptake.  
 
Secondary endpoint #3: Proportion of patients with an undetectable HIV-RNA (<40 copies/mL) 
will be compared between groups 1 and 2 throughout the study period. 
 
As all participants living with HIV will have an undetectable HIV-1 RNA viral load at baseline, the 
number and proportion of those maintaining virologic suppression at month 1 (for those who 
switched to B/F/TAF at baseline), month 2 and month 6 will be reported for those in group 1 and 
group 2 as a whole cohort and for those who switched to B/F/TAF. 
Secondary endpoint #4: ART adherence will be assessed at each visit for groups 1 and 2 using a 
validated visual analogue scale (VAS) of ART use over the prior month at each visit.41 This VAS is 
known as the Medication Adherence Self-Reported Inventory (MASRI) and was developed by 
Walsh, Mandalia and Gazzard and validated and published in AIDS in 2002. It starts with a 
permissive statement prefaced in order to encourage accurate responses. This reads as `We 
understand that many people on anti-HIV medication find it very difficult to take it regularly and 
often miss doses. We won't be surprised if you have missed lots of doses as well. We need to 
know how many doses you have missed'. The MASRI VAS is for the proportion of doses taken in 
the preceding month. The instructions for the VAS read: `put a cross on the line below at the 
point showing your best guess about how much medication you have taken in the last month. 
We would be surprised if this was 100% for most people, e.g., 0% means you have taken no 
medication; 50% means you have taken half your medication; 100% means you have taken every 
single dose of medication'. The VAS ranges from 0 to 100% in 10% intervals. For validation, the 
VAS was compared to the gold standards of MEMS Caps (MC) and pill count (PC). The mean self-
reported adherence by VAS over the preceding month was 93.3% (SE, 1.2%) and was strongly 
associated with both MC (r. 0.63; P, 0.001) and PC (r. 0.75; P, 0.001). In multivariable analysis, 
the strongest association between a MASRI item and MC was for the VAS. Also, the VAS items 
were inversely associated with viral load (p=0.01) confirming clinical importance. 
 
Secondary endpoint #5: Frequency and severity of B/F/TAF related adverse effect for participants 
in groups 1 and 2 who have changed to B/F/TAF at each follow-up visit. 
 

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Adverse effects will be recorded using the NIAID standardized toxicity grading system (Appendix 
D) and will be reported as a frequency and proportion of with any adverse effect, any grade 2 or 
higher and any requiring stopping the treatment.   
 
Secondary endpoint #6: PROs will be assessed for groups 1 and 2 at all study visits using the HIV 
Symptom Index Distress Module (HSIDM) at baseline, months 2 and 6. However, since the scale 
does not mention HIV and asks about symptoms solely, group 3 participants will be asked to 
complete it at baseline and month 2. 
 
The HSIDM, also referred to as the HIV Symptom Index (HSI), is a validated patient-reported 
outcome (PRO) instrument that assesses the burden of 20 common symptoms associated with 
HIV treatment or disease. The instrument has been validated repeatedly and is available in French 
and English. It is considered to be the gold standard in HIV symptom research. Participants are 
asked about their experience with each of 20 symptoms during the past 4 weeks using a 5-point, 
Likert-type scale. Response options and scores are as follows: (0) “I don’t have this symptom;” 
(1) “I have this symptom and it doesn’t bother me;” (2) “I have this symptom and it bothers me 
a little;” (3) “I have this symptom and it bothers me;” (4) “I have this symptom and it bothers me 
a lot.” The 20 symptoms comprise of fatigue/loss of energy, difficulty sleeping, nervous/anxious, 
diarrhea/loose 
stools, 
changes 
in 
body 
composition, 
feeling 
sad/down/depressed, 
bloating/pain/gas in stomach, muscle aches/joint pain, problems with sex, trouble remembering, 
headaches, pain/numbness/tingling in hands/feet, skin problems/rash/itching, cough/trouble 
breathing, fever/chills/sweats, dizzy/light-headedness, weight loss/wasting, nausea/vomiting, 
hair loss/changes, and loss of appetite/food taste. The score is most commonly dichotomized 
symptoms being not bothersome (scores of 0 or 1) or bothersome (scores of 2, 3 and 4). The 
overall bothersome symptom counts at baseline and at months 2 and 6 are generated by 
counting the number of individual symptoms scored as bothersome per participant and can 
therefore be reported as a frequency and proportion of those with no or any bothersome 
symptoms and a median (IQR) of the number of bothersome symptoms. 
 
Secondary endpoint #7: Since taking ART regimens is a big part of this study, we will be asking 
about satisfaction with ART regimen using the HIV Treatment Satisfaction Questionnaire 
(HIVTSQ). There are two versions of the HIVTSQ that have been developed. At the baseline, 
month 2 and month 6 visits, the 10-item HIVTSQ status version (HIVTSQs) will be used. The 
developers, Woodcock and Bradley, found that nine of the 10 items in the HIVTSQ performed 
well, but one of the items, the one on “demands” did not.42 So, they developed a revised 
questionnaire called the HIVTSQ new change version (HIVTSQc).43 The HIVTSQc will be used in 
addition to the HIVTSQs at month 6. The HIVTSQs was validated in 2001 with 150 people living 
with HIV with principal components analyses suggesting that participant ratings of nine items 
could be summed to compute the total satisfaction scale (Cronbach's alpha 0.82), and/or divided 
into the subscales of 1) general satisfaction/clinical (alpha 0.80) and 2) lifestyle/ease (alpha 0.74). 
The HIVTSQs also showed construct validity: with negative correlation to viral load (Spearman's 
r - 0.33 p < 0.01). The HIVTSQc was validated with 97 participants and confirmatory factor analysis 
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lifestyle/ease change with an alpha 0.88) and the 10‐item treatment satisfaction change scale 
with an alpha 0.92 which also met construct validity. Since these studies, the HIVTSQ has been 
expanded to include 12 items and is superior and we have purchased the license for the use of 
the 12-item HIVTSQs and HIVTSQc in both French and English. 
 
The questionnaires are both scored out of 66. The HIVTSQs score is calculated by summing the 
answers to questions 1 to 11 which have a range from 0 to 6 making a score that has a range 
from 0 to 66 with a higher score meaning that one is more satisfied with their treatment. Item 
number 12 is not included in the composite score, it is stand alone. The assessed change or an 
improvement in medication satisfaction, the total HIVTSQs score from the baseline is subtracted 
from the follow up score. For the HIVTSQc, which is the same as the HIVTSQs except for minor 
word alterations in question 9, answers from questions 1 to 11 are added up but this time the 
answers range from -3 to + 3 to assess deterioration and improvement in satisfaction compared 
to before. The total score ranges from -33 to +33.  
 
Secondary endpoint #8: Participant health status in all groups will be assessed using the SF-36 at 
baseline and month 2, which allows us to convert to a utility and conduct cost-effectiveness 
analyses in the future, if desired. The SF-36 will be repeated for Groups 1 and 2 at the 6-month 
visit. 
The SF-36 is a 36-item, patient-reported survey of patient’s health status, or quality of life, and is 
one of the most commonly used scales to measure health status as it can be converted to a utility 
and used in health economics.44 The SF-36 consists of eight scale scores (vitality, physical 
functioning, bodily pain, general health perceptions, physical role functioning, emotional role 
functioning, social role functioning, and mental health). These eight scale scores are the weighted 
sums of the questions in their section and then each scale is directly transformed into a 0-100 
scale on the assumption that each question carries equal weight. SF-36 is interpreted in 
ascending fashion with lower scores equating to more disability and higher scores meaning less 
disability. The individual scale scores and overall scale score are most often reported as a mean 
and standard deviation (SD). We are choosing the SF-36 as the health status measure as it is the 
most robust, it is available in French and English, and it is free of charge. 
 
Additional variables: Basic sociodemographic variables will be collected such as age [to be 
reported as a median with interquartile range (IQR)] and race/ethnicity [White, African, 
Caribbean Black (ACB), Indigenous or Other (N/%)]. Clinical variables will include weight 
(median/IQR), height (median/IQR), body mass index (median/IQR), and hepatitis B or C infection 
(N/%). For the trans women participants, dose of estradiol will be recorded (N/% and 
median/IQR), which anti-androgen/orchiectomy/medical is used (N/%) and its dose (N/%), and 
whether gender affirming surgery has taken place, more specifically orchiectomy which can 
impact testosterone concentrations. For participants living with HIV, duration of HIV infection 
(median/IQR), duration of ART (median/IQR), which ART regimen they are currently taking, 
baseline VL (median/IQR), baseline CD4 count (median/IQR) will be collected. For cis women, a 
menstrual history will be recorded including if their cycles are regular (N/%).  

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24 STATICAL ANALYSES AND SAMPLE SIZE CALCULATION 
 
24.1 Statistical analyses 
Baseline sociodemographic and clinical variables (including age, city of care, race/ethnicity, 
baseline ART drugs, CD4 count, etc.) will be summarized using medians and IQRs and means and 
SDs for continuous variable, when appropriate, and frequencies and proportions for categorical 
variables and presented in table format.  
 
For the ART drugs’ PK parameters, we will be determining the 2-month PK data of plasma BIC, 
plasma FTC, plasma TAF, and PBMC and DBS TFV-DP and FTC-TP for trans and cis women living 
with HIV. For each woman, plasma AUC, Cmax and Cmin will be reported. Analyses will take place 
on logarithmically transformed AUC, Cmax and Cmin data. The geometric mean ratio (GMR) will be 
calculated by comparing the trans women (group 1) to cis women (group 2) and the associated 
90% CIs will be reported. The two groups will be considered similar if the observed GMR is not 
less than 0.7 when comparing group 1 to group 2. A 30% lower exposure was chosen as an 
acceptable difference for this secondary objective, as 30% is a conventional reduction in exposure 
that is often considered acceptable and does not require consideration of a dose adjustment. To 
establish bioequivalence between groups would require a significantly higher sample size, which 
is not likely to be necessary given what we know about the metabolism/elimination of the 
proposed ART and feminizing hormones.  
 
Since a full series of estradiol concentrations will be done over time at month 2 for groups 1 and 
3, for each woman, serum AUC, Cmax, C4h, and Cmin for estradiol will be reported. As above, 
analyses will take place on logarithmically transformed AUC, Cmax, C4h, and Cmin data. The 
geometric mean ratio (GMR) will be calculated by comparing the trans women living with HIV 
(group 1) to trans women living without HIV (group 3) and the associated 90% Cis will be 
reported. The two groups will be considered similar if the observed GMR is not less than 0.7 when 
comparing group 1 to group 3. A 30% lower exposure was chosen as an acceptable difference for 
this secondary objective, as 30% is a conventional reduction in exposure that is often considered 
acceptable and does not require consideration of a dose adjustment.  
 
Another estradiol endpoint is determining the proportion of estradiol C4h concentrations that are 
within range or out of range at month 2. The target range for estradiol is 370 to 735 pmol/L for 
the Endocrine Society and 200 to 500 pmol/L for Sherbourne Health and therefore our target 
range will be from 200 to 735 pmol/L. These proportions will be compared between groups 1 and 
3 using the Chi-square test. The proportions of below, within and above target at baseline and 
month 2 as a bar graph with 3 colours, one for each target (within, below, above) and the change 
in categories compared from month 2 to baseline will also be presented using chi-square testing.  
 
The secondary hormone endpoints to be assessed are the mean estradiol Cmin and C4h and total 
testosterone concentrations at baseline and month 2 in groups 1 and 3. The hormone 
concentrations will be reported using means/SD and compared using the independent t-test. 

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Furthermore, for trans women living with HIV who were on a different former ART regimen prior 
to baseline and were switched to B/F/TAF, the mean change in estradiol C4h and total 
testosterone concentration from baseline to month 2 can be assessed using the paired t-test.  
Similarly, the hormone concentrations (estradiol and total testosterone) for group 2 will also be 
assessed and can be compared in a similar fashion as an exploratory analysis.  
 
Other secondary outcomes that will be reported include medical gender affirming satisfaction 
using a scale which will be summarized using a mean and SD for each visit and using a repeated 
measures diagram and compared between groups using the independent t-test. Viral response 
which will be reported using frequency and proportion of participants who maintain viral 
suppression at months 1, 2 and 6. Adherence will be reported using the VAS,41 which is a Likert 
scale and will be summarized using a mean and SD for each visit and using a repeated measures 
diagram. Adverse event rate to B/F/TAF will be reported for groups 1 and 2 using frequencies and 
proportions. The PRO scale used is most commonly dichotomized and each symptom will be 
reported as frequencies and proportions for the various symptoms and compared from baseline 
to month 2 and 6 for both groups 1 and 2. An exploratory analysis will be conducted to compare 
to group 3. Finally, drug regimen satisfaction will be reported as a means (SDs) and compared 
between groups 1 and 2 at baseline and month 2 and 6 using a t-test. Overall, SF-36 scores will 
be reported as means (SDs) compared between groups at baseline and month 2 for all groups 
and additionally at month 6 for groups 1 and 2.  
 
24.2 Sample size Justification 
The sample size is being determined primarily for the primary hypothesis of no difference 
between ART plasma AUC, if we assume coefficient of variation (CV) [AUC] for each ART drug of 
0.33 (based on Gallant, 2017 Lancet for BIC; AUC=96 mcg*h/mL, SD=32), the proposed sample 
size of 15 participants in group 1 and 2 provides 80% statistical power to detect 30% lower 
exposure during feminizing hormone therapy use based on an alpha of 0.1, consistent with the 
FDA guidance to report GMR + 90% confidence intervals between each group.  
 
25 ETHICS AND DISSEMINATION 
 
25.1 Ethical considerations  
This study will be conducted in accordance with the ethical principles for human research 
outlined in the Declaration of Helsinki, the Tri-council Policy Statement: Ethical Conduct for 
Research Involving Humans – TCPS 2 (2018), and the International Council for Harmonisation of 
Technical Requirements for Pharmaceuticals for Human Use (ICH)’s Good Clinical Practice 
Standards which are all followed in Canada.  
 
25.2 Informed consent 
All participants will be given detailed oral and written information about the study. Consent 
forms describing in detail the study medication/intervention(s) study procedures and risks will 
be given to each participant and written documentation of informed consent is required prior to 
starting any study procedures. Participants must sign an informed consent document that has 

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been approved by a participating centre’s REB prior to any procedures being done specifically for 
the trial. Each participant should have sufficient opportunity to discuss the study, have all of their 
questions addressed and consider the information in the consent process prior to agreeing to 
participate.  Participants may withdraw consent at any time during the course of the study 
without prejudice. The informed consent form will be signed and dated by the participant and 
the investigator or delegate. The original signed informed consent form will be retained in the 
participant’s study files and a copy will be provided to the participant. 
 
The informed consent process must be conducted, and form signed before the participant 
undergoes any screening procedures that are performed solely for the purpose of determining 
eligibility for the study. 
 
The informed consent will be available in English and revisions to the consent form will be made 
according to requirements of the REB. Annual REB renewals will be made to the REB and all 
required amendments will be made in a timely fashion. 
 
25.3 Compensation 
All participants are provided the following to account for food, travel, childcare, time, and other 
expenses: 
 
Screening  
Baseline  
Month 1 
Month 2 
(Day 1) 
Month 2 
(Day 2) 
Month 6 
Group 1 
$40 
$80 
$20 
$160   
$40 
$40 
Group 2 
$40 
$50       
$20 
$160   
$40 
$40 
Group 3 
$40 
$80 
 
$160  
$40 
 
 
For participants that need to travel from outside of the Greater Toronto Area for the study visits, 
study related travel expenses will be covered on a case-to-case basis.  Expenses must be pre-
approved with study team. Meal allowance will be calculated based on current CIHR standards 
for meals not provided during study visits (Breakfast $19.10, Lunch $18.90, Dinner $47.35). 
 
25.4 Confidentiality 
All participant-related information including CRFs, laboratory specimens, evaluation forms, 
reports, etc. will be kept strictly confidential. All records will be kept securely at study sites behind 
2 locked doors and will be only accessible to research staff. Participants will be identified only by 
means of a coded number specific to each participant. All computerized databases will identify 
participants by numeric codes only and will be password protected. All digital files will be 
restricted to study personnel and will be kept on secure servers at Maple Leaf Medical Clinic. 
 

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Due to questionnaires being sent to the participant’s email when visits are conducted virtually, 
email addresses for each participant will be collected in the eCRF. However, during data export 
this identifying information will be removed to maintain confidentiality. 
 
Upon request, and in the presence of the investigator or their representative, participant records 
will be made available to the study sponsor, monitoring groups representative of the study 
sponsor, representatives of funding groups, and applicable regulatory agencies for the purpose 
of verification of clinical trial procedures and/or data, as is permissible by local regulations. 
 
All study materials will be kept for 15 years before destruction as per Health Canada Guidelines. 
 
25.5 Research Ethics Board (REB) 
Research ethics approval for the conductance of this study will be obtained from Veritas IRB. 
Ethics reviews will also be sought at Unity Health Toronto. Informed consent will be obtained 
prior to study activities at the screening visit. 
 
The REB will review all appropriate study documentation to safeguard the rights, safety, and well-
being of the participants. The study will be conducted only at sites where ethics approval has 
been obtained. A copy of the protocol (including protocol amendments), all versions of informed 
consent forms, other information to be completed by participants such as survey instruments or 
questionnaires, and any proposed advertising/recruitment materials must be reviewed and 
approved by the REB prior to implementation of the trial. The investigator will be responsible for 
obtaining REB approval of the annual Continuing Review throughout the duration of the study. 
The investigator will notify the REB of serious adverse events as applicable. The investigator will 
seek prior ethics approval for any protocol deviations except when the change is intended to 
eliminate an immediate hazard to participants. In this case, the protocol deviation will be 
promptly reported. 
 
25.6 Dissemination plan 
Data analyses and then dissemination will begin when all study participants have completed the 
month 2 study visit and the data up to that point has been collected and verified. The results will 
be published and presented at national and international conferences, on the Maple Leaf Medical 
Clinic website, and to community groups as appropriate. Also, other knowledge translation tools 
useful for community members such as infographics, fact sheets and community reports will be 
produced in consultation with the CAB aimed to support trans women living with HIV to 
understand the findings and issues with DDIs between ART drugs and feminizing hormones. Also, 
results in the form of infographics will be provided to all recruitment sites and study participants 
if desired. 
 
26 CONCLUSION AND IMPACT 
 
This study is of global importance for trans women living with HIV, an underserved population, 
and their care providers. If our hypotheses are correct (i.e., that there are no clinically significant 

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DDIs and impact of feminizing hormones on B/F/TAF and vice versa), trans women living with HIV 
that live in jurisdictions where care providers are still restricting feminizing hormones due to 
potential DDIs with ART can be reassured and provided the therapies that they need. 
Subsequently, trans women who are not taking ART due to fear from such restrictive care from 
care providers and are purchasing hormones illegally could be reassured and access the care they 
need in an informed manner. Furthermore, the results of this study are important in light of the 
reduced TDV and FTC concentrations found amongst trans women taking TDF/FTC as PrEP and 
feminizing hormones. As we will be reporting on plasma TAF and FTC and intracellular PBMC and 
DBS TDV-DP and FTC-TP levels, this is likely crucial information. 
  
 
 

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27 REFERENCES 
 
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17. World Professional Association for Transgender Health [WPATH]. Standards of care for the 
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22. Hiransuthikul A, Janamnuaysook R, Himmad K, et al. Drug-drug interactions between 
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23. Cottrell ML, Prince HMA, Schauer AP, et al. Decreased Tenofovir Diphosphate concentrations 
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24. Hiransuthikul A, Himmad L, Kerr SJ, et al. Drug-drug interactions among Thai HIV-positive 
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25. Israel BA, Schulz AJ, Parker EA, Becker AB. Review of community-based research: assessing 
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26. De Las Nueces D, Hacker K, DiGirolamo A, Hicks LS. A systematic review of community- based 
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27. Carter A, Greene S, Nicholson V, O’Brien N, Sanchez M, de Pokomandy A, Loutfy M, Kaida A, 
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28. Travers R, Wilson MG, Flicker S, Guta A, Bereket T, McKay A, van der Muelen A, Cleverly S, 
Dickel M, Globerman J, Rourke SB. The greater involvement of people living with AIDS 
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AIDS Care. 2008; 20(6): 615-624. 
29. Scheim AI, Appenroth MN, Beckham SW, Goldstein Z, Grinspan MC, Keatley JG, Radix A. 
Transgender HIV research: Nothing about us without us. Lancet. 2019;19. 
30. Bauer G, Devor A, Heinz m, Marshall Z, Pullen SA, Pyne J; CPATH Research Committee. CPATH 
Ethical Guidelines for Research Involving Transgender People & Communities. Canada: 
Canadian Professional Association for Transgender Health; 2019. 
31. Bourns A. Guidelines and protocols for hormone therapy and primary care for trans clients 
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32. Lobo RA, Cassidenti DL. Pharmacokinetics of oral 17 beta-estradiol. J Reproduct Med 1992; 
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33. AA PHARMA INC. Cyproterone Product Monograph. In: INC. AP, editor.; August 28th, 2013. 

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34. Loutfy M, Greene S, Kennedy VL, et al. Establishing the Canadian HIV Women’s Sexual and 
Reproductive Health Cohort Study (CHIWOS): Operationalizing Community-based Research 
in a Large National Quantitative Study. BMC Medical Research Methodology. 2016;16(1):101. 
35. Schauer AP, Sykes, C, Cottrell ML, Prince H, Kashuba ADM. Validation of an LC-MS/MS assay 
to simultaneously monitor the intracellular active metabolites of tenofovir, emtricitabine, 
and lamivudine in dried blood spots. J Pharm Biomed Anal. 2018;149:40-45. 
36. U.S. Department of Health and Human Services. Clinical drug interaction studies – 
Cytochrome P450 Enzyme- and Transporter-mediated drug interactions. Food and Drug 
Administration. 2020. https://www.fda.gov/media/134581/download 
37. Bourns A. Guidelines for gender-affirming primary care with trans and non-binary patients. 
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38. Mattison DR, Karyakina N Fau - Goodman M, Goodman M Fau - LaKind JS, LaKind JS. 
Pharmaco- and toxicokinetics of selected exogenous and endogenous estrogens: a review of 
the data and identification of knowledge gaps. Crit Rev Toxicol 2014; 44(8): 696-724. 
39. Stanczyk FZ, Archer Df Fau - Bhavnani BR, Bhavnani BR. Ethinyl estradiol and 17beta-estradiol 
in combined oral contraceptives: pharmacokinetics, pharmacodynamics and risk assessment. 
Contraception 2013; 87(6): 706-27. 
40. Soldin OP, Mattison DR. Sex differences in pharmacokinetics and pharmacodynamics. 
Pharmakokinet 2013; 48(3): 143-157. 
41.  Walsh JC, Mandalia S, Gazzard BG. Responses to a 1 month self-report on adherence to antiretroviral 
therapy are consistent with electronic data and virological treatment outcome. AIDS. 2002 Jan 
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42. Woodcock A, Bradley C. Validation of the HIV treatment satisfaction questionnaire (HIVTSQ). 
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43. Woodcock A, Bradley C. Validation of the revised 10-item HIV treatment satisfaction 
questionnaire status version and new change version. Value Health. 2006;9(5):320-333. 
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1992;305(6846):160-164. 
 
 
 
 
 
 
 
 
 
 

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APPENDIX A – Study Schematic 

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APPENDIX B1 – List of Prohibited Medications 
Additional Hormonal Therapy:  
Estrogen containing products and 
conjugated estrogens 
Combined Oral Contraceptives:  
Ethinyl estradiol/levonogestrel, ethinyl 
estradiol/desogestrel, ethinyl estradiol/norethindrone, 
ethinyl estradiol/norethindrone/ethinyl estradiol, 
ethinyl estradiol/norgestimate, ethinyl 
estradiol/drosperinone,  ethinyl estradiol/norgestrel 
Patch: norelgestromin/ethinyl estradiol  
Ring: etonogestrel/ethinyl estradiol  
Conjugated estrogens  
Allowed Contraceptives 
Copper IUD 
Levonorgestrel IUDs (e.g. Mirena) 
Progesterone- only contraception incl. Depo-Provera 
 
Prohibited medications with Biktarvy 
(bictegravir, emtricitabine, tenofovir alafenamide): 
 
*Refer to table in Appendix C for DDIs which can be managed  
 
Anticonvulsants  
Carbamazepine  
Fosphenytoin- Phenytoin 
Oxcarbazepine  
Primidone  
Anti-HBV  
Adefovir  
Anti-infectives 
 
Clarithromycin  
Erythromycin 
Rifabutin 
Rifampin 
Antiarrhythmics 
Dofetilide 
Verapamil   
Antidepressants   
Nefazodone 
St. John’s Wort  
Antifungals  
Fluconazole  
Itraconazole  
Ketoconazole 
Posaconazole  
Voriconazole  
Antihypertensive 
Bosentan  
Antineoplastic agents  
Apalutamide  
Enzalutamide  

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Antiprogesterone 
Mifepristone  
Barbiturate 
Phenobarbital 
CNS Stimulant 
Modafinil  
 
 
Prohibited medications with Estradiol: 
 
Anti-infectives  
Clarithromycin  
Erythromycin  
Rifabutin 
Rifampin  
Antiarrhythmic 
Verapamil  
Anticonvulsants  
Carbamazepine 
Fosphenytoin- Phenytoin 
Oxcarbazepine 
Antidepressants  
Nefazodone 
St. John’s Wort 
Antifungals  
Fluconazole  
Itraconazole 
Ketoconazole  
Posaconazole  
Voriconazole  
Antihyperlipidemic  
Gemfibrozil 
Antihypertensive  
Bosentan  
Antineoplastic Agents  
Apalutamide 
Enzalutamide 
Tamoxifen 
Antiprogesterone  
Mifepristone  
Barbiturate  
Phenobarbital 
CNS Stimulant  
Modafinil  
Immunosuppressant  
Cyclosporine (systemic)  
 
 
Prohibited medications with Cyproterone: 
 
Anti-infectives 
 
Clarithromycin 
Erythromycin  
Rifabutin 
Rifampin  
Antiarrhythmic 
Verapamil  
Anticonvulsants  
Carbamazepine  
Fosphenytoin- Phenytoin 
Oxcarbazepine  
Antidepressant  
Nefazodone  
St. John’s Wort 

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Prohibited medications with Cyproterone: 
 
Antifungals 
Fluconazole  
Itraconazole  
Ketoconazole 
Posaconazole  
Voriconazole  
Antihypertensive  
Bosentan  
Antineoplastic Agents  
Apalutamide  
Enzalutamide  
Antiprogesterone  
Mifepristone  
Barbiturate  
Phenobarbital 
CNS Stimulant  
Modafinil  
 
 
Prohibited medications with Spironolactone: 
 
Antihypertensive 
Eplerenone 
Diuretics   
Amiloride 
Triamterene 
Electrolyte Supplements  
Potassium salts  
Immunosuppressants 
Cyclosporine (systemic)  
Tacrolimus (systemic)  
 
 
Prohibited medications with Estradiol Testing Assay: 
 
Antineoplastics Aromatase Inhibitor 
exemestane (Aromasin)  
Estrogen receptor antagonists 
fulvestrant (Faslodex) 
 
 
 

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APPENDIX B2 – Drug Interactions and recommendations on management 
 
Medications interacting with Biktarvy 
(bictegravir, emtricitabine, tenofovir alafenamide): 
Agents    
Mechanism of DDI   
Management 
Polyvalent cation containing 
products (e.g. Mg, Al, Ca, Fe)  
Calcium or iron 
supplements, cation 
containing antacids or 
laxatives, sucralfate, 
buffered medications 
 
May decrease the 
concentration of 
Bictegravir.  
 
 
Administer Biktarvy 2 hours before or 2 hours 
after taking medications or oral supplements 
containing polyvalent cations. 
Biktarvy and medications or oral supplements 
containing polyvalent cations can be taken 
together with food.  
*taken from Biktarvy drug monograph 
 
 

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APPENDIX C – Schedule of Events 
Protocol Activity 
Screening 
Baseline 
Month 1* 
Month 2 
Month 6 
Notes 
Informed Consent  
W 
 
 
 
 
 
Inclusion & Exclusion 
W 
 
 
 
 
 
Enrollment & Information 
W 
 
 
 
 
 
Demographic Data (questionnaire – Screening 
Only), Medical History, Concurrent Medication 
History, Menses Questionnaire 
W 
W 
X 
W 
X 
Demographics (e.g. age, race, education, 
etc.(questionnaire) screening only; medical history 
(e.g. FHT history, HIV history, menses history 
questionnaire, etc.) and medication history (e.g. 
gender affirming surgery, chronic ongoing conditions, 
psychiatric, major medical conditions) ; feminizing 
hormones, hormonal contraception, medications used 
to treat or prevent HIV, herbal/over-the counter 
medication, and any other medication used in the 30 
days prior to screening).  The medical and medication 
history is updated at each visit as required. 
Height and weight 
 
W 
 
W 
 
Height will only be collected at Baseline.  
Doctor Visit & Physical Exam 
O 
O 
X 
O 
O 
A doctor visit and/or physical exam is only required if 
deemed medically necessary by the study personnel 
and physician. If a participant switches to B/F/TAF, 
they will be seen by a physician at each visit to allow 
for follow-up of side effects and adherence. 
AE related to B/F/TAF or oral estradiol 
 
X 
X 
X 
X 
 
BLOOD WORK 
 
 
 
 
 
 
Routine blood work 
W 
W 
 
W 
X 
Routine blood work includes CBC, electrolytes 
[sodium, potassium, chloride], serum 
creatinine[eGFR], ALT, total bilirubin, glucose, HbA1C, 
and lipids.  
For screening serum ALT & serum creatinine[eGFR] 
only. 

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Protocol Activity 
Screening 
Baseline 
Month 1* 
Month 2 
Month 6 
Notes 
Hepatitis B Surface antigen & Hepatitis C antibody  
W 
 
 
 
 
 
FSH Test  
C 
 
 
 
 
 
Urine Pregnancy and/or Serum Beta-HCG 
C 
C 
 
C 
C 
Urine pregnancy test is only required at month 2. 
Urine Protein Test  
X 
X 
 
 
 
 
HIV-1 RNA Diagnostic Serology 
Z 
Z 
 
Z 
 
 
HIV-1 RNA viral load 
X 
X 
 
X 
X 
 
Lymphocyte Subsets CD4 + CD8 
 
X 
 
 
X 
 
Serum testosterone and estradiol levels  
 
W/T 
 
W 
 
All participants: pre-dose estradiol and testosterone 
concentrations are collected at baseline & month 2.  
Trans women have an additional estradiol C4H level at 
baseline (optional if visit is virtual). 
Seven (7) post-dose serum estradiol levels 
 (1h, 2h, 3h, 4h, 6h, 8h, 24h)   
 
 
 
T 
 
 
Ten (10) ART PK plasma samples  
(pre-dose, 0.5h, 1h, 1.5h, 2h, 3h, 4h, 6h, 8h, 24h)  
 
 
 
X 
 
 
PBMC (2h) & DBS (2H) for TFV-DP and FTC-TP  
 
 
 
X 
 
 
QUESTIONNAIRES 
 
 
 
 
 
 
FEM-SQ 
 
T 
 
T 
T 
 
Adherence  
 
X 
X 
X 
X 
 
HSIDM PRO 
 
W 
 
W 
X 
 
HIVTSQs 
 
X 
 
X 
X 
 
HIVTSQc 
 
 
 
 
X 
 
SF-36 
 
W 
 
W 
X 
 
W: All to complete (Group 1, 2, 3)                       
X: Cis and trans women living with HIV to complete (group 1, 2)         
O: If deemed necessary 
T: Trans women to complete (Group 1, 3)          
Z: Women living without HIV to complete (group 3)        
C: Cis women living with HIV to complete (group 2)1 

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Abbreviations: BIC, bictegravir; F or FTC, emtricitabine; TAF, tenofovir alafenomide; PK, pharmacokinetics; DBS, dried blood spot; TFV-DP, tenofovir diphosphate; FTC-TP, emtricitabine 
triphosphate; FEM-SQ, Feminizing Medical Gender Affirmation Satisfaction Scale; HSIDM, HIV Symptom Index Distress Module; PRO, patient-reported outcomes; HIVTSQ, HIV 
Treatment Satisfaction Questionnaire; SF-36, Short Form 36. 
 
Study visits:  
● The baseline visit for participants that do not need to change their ART and/or estradiol dose/timing can be booked as soon as screening results are available (approximately 
14 days due to HIV-1 RNA Diagnostic Serology and HIV-1 RNA viral load results). 
● The baseline visit for participants required to change their estradiol dosing and/or ART dose/timing must be scheduled at least 28 days after the changed dose. 
● Participants requiring a switch to B/F/TAF will be switched at baseline, preferably as soon as screening results are received but they will be allowed to complete their current 
ART prescriptions (which could take as long as 90 days) before switching to B/F/TAF.  
● The maximum screening visit window is 90 days; participants whose baseline visit date will fall past 90 days can be rescreened.  
* Month 1 is required for participants who change their ART regimen to B/F/TAF; it must be scheduled at least 28 days after the B/F/TAF start date . Standard of Care visit, can be 
done virtually.   
● Month 2 is scheduled 8 weeks after baseline, except for the participants who change their ART regimen to B/F/TAF, month 2 is scheduled 8 weeks after the date B/F/TAF was 
started. 
● Month 6 is scheduled 24 weeks after baseline except for the participants who change their ART regimen to B/F/TAF, month 6 is scheduled 24 weeks after the date B/F/TAF was 
started. 
● Study visits will have a buffer window of ± 5 business days.

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APPENDIX D – NIAID standardized toxicity grading system 
 
Item 
Grade 1 Toxicity 
Grade 2 Toxicity 
Grade 3 Toxicity 
Grade 4 Toxicity 
1.2 Hematology 
 
Hemoglobin (g/dL) 
9.5 - 10.5 
8.0 - 9.4 
6.5 - 7.9 
< 6.5 
Absolute Neutrophil Count (x 
109/L 
1.0 - 1.5 
0.750 - 0.999 
0.500 - 0.749 
<0.500 
Platelets (x 109/L) 
75 - 99 
50 - 74.9 
20 - 49.9 
<20 or diffuse petechiae 
WBC ( cells/L) 
2 x 109 – 2.499 x 
109 
50 x 109 – 100 x 109 
1 x 109  - 1.499 x 109 
< 1 x 109 
1.3 Enzymes 
 
AST (SGOT) 
1.25 - 2.5 x upper 
normal limit 
2.6 - 5 x upper normal 
limit 
5.1 - 10 x upper normal 
limit 
> 10 x upper normal limit 
Alkaline phosphatase 
1.25 - 2.5 x upper 
normal limit 
2.6 - 5 x upper normal 
limit 
5.1 - 10 x upper normal 
limit 
> 10 x upper normal limit 
Amylase 
1.1 - 1.5 x upper 
normal limit 
1.6 - 2.0 x upper normal 
limit 
2.1 - 5.0 x upper normal 
limit 
> 10 x upper normal limit 
1.4 Chemistries 
 
ALT 
1.25 – 2.5 x upper 
normal limit  
2.5 - 5 x upper normal 
limit  
5 - 10 x upper normal 
limit  
> 10 x upper normal limit 
Cholesterol (mmol/L) 
5.18 – 6.19 
6.19 – 7.77 
> 7.77 
NA 
Glucose (mmol/L) 
Fasting  
6.11 – 6.95 
6.95 – 13.89 
13.89 – 27.75 
> 27.75 
Glucose (mmol/L) 
Nonfasting 
6.44 – 8.89 
8.89 – 13.89 
13.89 – 27.75 
> 27.75 
Hyponatremia (mmol/L) 
130 - 135 
123 - 129 
116 - 122 
< 116 or mental status 
changes or seizures 
Hypernatremia 
(mmol/L) 
146-150 
151-157 
158-165 
> 165 or mental status 
changes or seizures 
Hypokalemia (mmol/L) 
3.0 - 3.4 
2.5 - 2.9 or replacement 
Rx required 
2.0 - 2.4 or intensive 
replacement Rx req. or 
hospitalization req. 
< 2.0 or paresis or ileus or 
life-threatening 
arrhythmia 
Hyperkalemia (mmol/L) 
5.6 - 6.0 
6.1 - 6.5 
6.6 - 7.0 
> 7.0 or life threatening 
arrhythmias 
Hypoglycemia (mmol/L) 
3.0 - 3.5 
2.2 - 2.9 
1.7 - 2.1 
< 1.7 or mental status 
changes or coma 
Hyperglycemia (mmol/L) 
(note if fasting) 
6.4 - 8.9 
9.0 - 13.9 
14.0 - 28.0 
> 28 or ketoacidosis 
Hyperbilirubinemia (µmol/L) 24 - 33 
34 - 55 
56 - 110  
> 110 
LDL (mmol/L) 
3.37 – 4.12 
4.12 – 4.90 
> 4.90 
NA 
Potassium, High (mmol/L) 
5.6 – 6.0 
6.0 – 6.5 
6.5 – 7.0 
> 7.0 
Potassium, Low (mmol/L) 
3.0 – 3.4 
2.5 – 3.0  
2.0 – 2.5 
< 2.0 
Total Bilirubin  
1.1 – 1.6 x upper 
normal limit 
1.6 – 2.6 x upper normal 
limit 
2.6 – 5.0 x upper normal 
limit 
> 5.0 x upper normal limit  
Sodium, High (mmol/L) 
146 – 150 
150 – 154 
154 – 160 
>160 
Sodium, Low (mmol/L) 
130 - 135 
125 - 130 
121 - 125 
> 160 
Triglycerides (mmol/L) 
1.71 – 3.42 
3.42 – 5.7 
5.7 – 11.4 
> 11.4 
Urea 
1.25 - 2.5 x upper 
normal limit 
2.6 - 5 x upper normal 
limit 
5.1 - 10 x upper normal 
limit 
> 10 x upper normal limit 
Creatinine (µmol/L) 
130 - 180 
181 - 360  
361 - 720 
> 720 or dialysis required 
1.5 Lymphocyte Subsets CD4  
Absolute CD4+ Count (cells/L) 300 - 400 
200 - 300  
100 - 200 
> 100 
 

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APPENDIX E1 – PROTOCOL REQUIRED LAB ASSESSMENTS 
Laboratory Assessments 
Parameters 
Hematology  
Complete blood count (CBC)  
- 
White blood cell count (WBC) 
- 
Hemoglobin (Hb) 
- 
Mean corpuscular volume (MCV)  
- 
Platelet Count  
Chemistry  
Total Bilirubin 
Lipids  
- 
Cholesterol 
- 
Triglycerides (TGs) 
- 
High-density lipoprotein (HDL – C) 
- 
Low – density lipoprotein (LDL-C) 
- 
Non-HDL – C 
- 
Total Cholesterol (TC)/HDL – C 
Electrolytes 
- 
Sodium 
- 
Chloride 
- 
Potassium  
Alanine Aminotransferase 
(ALT) 
 
HbA1c 
Glucose  
 
 
 Hepatitis screening (PHL) 
Hepatitis B virus surface 
antigen (HBsAg) 
Hepatitis C antibody (if positive 
perform plasma hepatitis C virus 
quantitative test) 
Hormone 
Estradiol – 17 Beta  
Total Testosterone  
Follitropin (FSH) 
Lymphocyte Subsets CD4 + 
CD8  
CD4+ Percent   
CD4+ Absolute Count  
CD8+ Percent 
CD8+ Absolute Count    
CD4/CD8 ratio  
Pregnancy testing  
Serum β human chorionic 
gonadotropin (β – hCG) 
pregnancy test 
Urine β – hCG pregnancy test  
Urine 
Protein test 
Human immune deficiency 
virus-1 (HIV – 1) serology 
(PHL) 
HIV Ab/Ag CMIA Screen 
Geenius™ Confirmation Assay  
p24 Ag ELFA 
HIV – 1 RNA Viral Load 
(PHL) 
Roche cobas® HIV-1 assay or current Public Health Ontario testing 
platform  
Pharmacokinetics (PK) 
Investigational antiretroviral therapy (ART) PK samples will be 
collected for both Group #1 and Group #2  
Dried blood spot (DBS) for Group #1 and Group #2  
Human peripheral blood mononuclear cells (PBMC) for Group #1 and 
Group #2  

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APPENDIX E2 – REQUIRED BLOOD VOLUMES 
Group 1: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Group 2: 
 
 
 
 
 
 

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Group 3: