A Multi-Compartment, Single and Multiple Dose Pharmacokinetic Study of the Vaginal Candidate Microbicide 1% Tenofovir Gel

Background Tenofovir (TFV) gel is being evaluated as a microbicide with pericoital and daily regimens. To inhibit viral replication locally, an adequate concentration in the genital tract is critical. Methods and Findings Forty-nine participants entered a two-phase study: single-dose (SD) and multi-dose (MD), were randomized to collection of genital tract samples (endocervical cells [ECC], cervicovaginal aspirate and vaginal biopsies) at one of seven time points [0.5, 1, 2, 4, 6, 8, or 24 hr(s)] post-dose following SD exposure of 4 mL 1% TFV gel and received a single dose. Forty-seven were randomized to once (QD) or twice daily (BID) dosing for 2 weeks and to collection of genital tract samples at 4, 8 or 24 hrs after the final dose, but two discontinued prior to gel application. Blood was collected during both phases at the seven times post-dose. TFV exposure was low in blood plasma for SD and MD; median Cmax was 4.0 and 3.4 ng/mL, respectively (C≤29 ng/mL). TFV concentrations were high in aspirates and tissue after SD and MD, ranging from 1.2×104 to 9.9×106 ng/mL and 2.1×102 to 1.4×106 ng/mL, respectively, and did not noticeably differ between proximal and distal tissue. TFV diphosphate (TFV-DP), the intracellular active metabolite, was high in ECC, ranging from 7.1×103 to 8.8×106 ng/mL. TFV-DP was detectable in approximately 40% of the tissue samples, ranging from 1.8×102 to 3.5×104 ng/mL. AUC for tissue TFV-DP was two logs higher after MD compared to SD, with no noticeable differences when comparing QD and BID. Conclusions Single-dose and multiple-dose TFV gel exposure resulted in high genital tract concentrations for at least 24 hours post-dose with minimal systemic absorption. These results support further study of TFV gel for HIV prevention. Trial registration ClinicalTrials.gov NCT00561496


I. INTRODUCTION
Tenofovir (the generic name for the active compound, interchangeable with its chemical name 9-[(R)-2-(phosphonomethoxy)propyl] adenine or PMPA) is currently being evaluated as a potential vaginal microbicide. A prodrug of tenofovir is currently approved for antiretroviral therapy (Viread ® or tenofovir disoproxil fumarate [DF]). The prodrug, tenofovir DF, has substantially greater oral bioavailability than tenofovir. Tenofovir is a nucleotide analog that blocks viral replication by inhibiting reverse transcriptase; therefore, it can block local viral replication once infection has occurred. The nucleotide class of drugs bypasses the initial phosphorylation step necessary for activation of the nucleoside class of drugs (such as Retrovir ® ). Therefore, nucleotide analogs may possess greater activity in a broader range of activated or resting cell types and their lack of requirement for virus specific activation may decrease the propensity for the development of antiviral resistance. Tenofovir has shown activity in target immune cells: lymphocytes (T cells), monocyte/macrophages and dendritic (Langerhans) cells. Therefore, tenofovir gel is a vaginal microbicide candidate designed to prevent the sexual transmission of HIV.
Tenofovir 1% gel was well tolerated in rabbit vaginal toxicity tests. Six separate studies of vaginal transmission inhibition performed in non-human primates provided evidence for some degree of efficacy of tenofovir gel [1]. An intravaginal pharmacokinetic study using 1% tenofovir (14C labeled) gel in macaques showed penetration into the vaginal epithelium with peak tissue concentration at 8 hours (20.64 mg-eq/g, 6.6-fold greater than 15 minute tissue concentration) after exposure. No significant radioactivity was detected in whole blood or plasma. Tenofovir has been shown to block HIV-1 transmission across the mucosa in a human cervical tissue-derived organ culture model [2].
Rabbit vaginal tissue biopsies were evaluated for total tenofovir content 8 hours following intravaginal gel and intravenous single dose and 4 hours following intravaginal dosing (single dose, twice daily for one week and twice daily for two weeks). Intraveneous and intravaginally administrated tenofovir was detected in vaginal rinse, tissue biopsies as well as blood samples. Surprisingly, mean vaginal tissue concentration of tenofovir collected 8 hours post dose were similar following single intravenous and intravaginal administration (950 ng/mL and 940 ng/mL, respectively). The mean vaginal tissue concentrations of tenofovir collected 4 hours post intravaginal dose (single dose, twice daily for one week and twice daily for two weeks) were higher than those at 8 hours (2,817 ng/mL, 3,146 ng/mL and 11,409 ng/mL, respectively). Systemic absorption following single intravaginal administration of 1% tenofovir gel was barely detectable and only so within the first 30 minutes. Multiple intravaginal dosing (twice daily for 1 week and twice daily for 2 weeks) resulted in low systemic levels of tenofovir (Cmax =239 ng/mL and 71 ng/mL, respectively, vs. 10,211 ng/mL following single intravenous dose). Tenofovir AUC 0-4hr following a single IV dose was ten times greater than following the last daily dose for either intravaginal multiple dosing regimen. No vaginal irritation was observed following intravenous tenofovir. Minimal irritation was reported following a single intravaginal dosing or twice daily for a week. Mild vaginal irritation was reported following twice daily intravaginal dosing for two weeks.
Tenofovir (0.3% and 1%) gel has recently been tested in a Phase I study conducted by the HIV Prevention Trials Network (HPTN) assessing the safety and acceptability of tenofovir gel used vaginally either once or twice daily for 14 days among sexually abstinent and sexually active HIVuninfected, and HIV-infected women. Twenty-five of the women in the study had PK testing with the limit of quantitation: 3.0 ng/mL. Fourteen of 25 women (56%) had low, but detectable, serum tenofovir levels at some point in the 12 hours after dosing on either Day 0 (following the first dose) or on Day 13 (after daily dosing); three of the 14 had detectable levels on both days (Figure 1) [3]. The maximum tenofovir concentrations (Cmax) ranged from 3.1 to 25.8 ng/mL, with no clear dose-concentration relationship identified. For the participant with the 25.8 ng/mL level, this peak level occurred 2 hours following the dose; the level rapidly declined to 10.8 ng/mL at 4 hours and was undetectable at 12 hours following the dose. Besides this outlier with the highest tenofovir level, the next highest Cmax was 7.1 ng/mL. Considering all women in the PK cohort, the median tenofovir Cmax was 3.4 ng/mL (interquartile range: below limit of quantitation [3.0 ng/mL] to 4.7 ng/mL) which corresponds to approximately 1% of the maximum and 7% of the minimum blood concentrations following multiple oral doses of tenofovir DF 300 mg [3]. The serum levels fell below the detection limit in approximately 87% of all samples taken between 0.5 and 24 hours. Figure 1 demonstrates tenofovir blood concentration following vaginal administration of 1% tenofovir gel. All levels for all women with measurable tenofovir levels in the blood are shown. (14 of 25; lower limit of quantitation (LLOQ) approximately 3.0 ng/mL [dotted line]). The legend indicates "cohort" -"ID" -"study day". For reference the tenofovir level associated with the median 24 hour post-dose blood concentration following an oral 300 mg tenofovir dose is indicated with dashed line [4]. No clinically significant systemic toxicity was detected in this study. Therefore, it can be concluded that tenofovir 1% vaginal gel used twice daily was well tolerated in abstinent and sexually active HIV uninfected and HIV infected women, with limited systemic absorption. Extended safety and effectiveness studies are warranted based on these initial data.
A phase II study to determine the safety of tenofovir 1% intravaginal gel used for 24 weeks, and to gain additional information regarding the product's acceptability is ongoing: it is a four arm, randomized controlled trial, comparing two frequencies of use (daily and coitally dependent), and corresponding placebo gel arms.
One of the concerns related to the use of tenofovir as a microbicide in the gel form is the uncertainty regarding the timeframe during which it will be converted to the active antiviral metabolite and be expected to potentially provide protection during coitus. Currently we do not know how long it will take the immune cells in the vaginal mucosa to convert tenofovir to its di-phosphorylated antiretroviral metabolite. What we do know is that the activation of tenofovir is dependent upon anabolic phosphorylation by intracellular nucleoside kinases, whose activity and availability are dependent upon the activation state of the cell. Thus it is possible that the absolute rate of tenofovir activation will be dependent upon a wide range of cervico-vaginal factors, including inflammatory state and hormonal influences, as well as the physiochemical properties of tenofovir gel in the vagina.
A number of in vitro studies have indicated that the uptake and metabolism of tenofovir to its active metabolite by immune cells is a rapid process, giving rise to potentially antiretroviral levels of tenofovir diphosphate with intracellular half-lives in the range of 10 to 50 hours [5]. Radiolabeled (3H) tenofovir is rapidly taken up by resting and activated lymphocytes, suggesting that cellular uptake is via endocytosis. Subsequent metabolism of tenofovir appears to proceed quickly. The mono-and di-phosphate metabolites accumulate rapidly, reaching approximately 0.1 micromolar and approximately 0.3 micromolar, respectively, at 6 hours after exposure, whereas the original drug is not detectable (within 0.5 to 24 hours). This suggests that tenofovir is rapidly processed upon entry to the cell to the mono-and then to the di-phosphate metabolite. The metabolic properties of tenofovir may facilitate the formation and maintenance of a barrier to virus replication that could be effective for coital-dissociated microbicide applications.
Although information about the uptake and metabolism of tenofovir in the local cervicovaginal tissues of humans after vaginal application is needed, the long intracellular half-life of tenofovir supports a daily or twice-daily clinical dosing regimen. If uptake and metabolism to its active form in human cervicovaginal tissue is rapid, then coitally-associated application may also be feasible. The optimal anti-retroviral vaginal microbicide would work locally at the cellular level to prevent HIV transmission, would not accumulate in the cervicovaginal tissues, and would not be absorbed systemically through the blood or lymphatic system.
Since the hypothesized key mechanism of action of intravaginal tenofovir is inhibition of viral replication locally, adequate tissue concentration in the genital tract is crucial. The current study will assess the local and systemic absorption and concentration of tenofovir in 49 women in a single-dose phase followed by a once or twice-daily two week phase to determine genital tract concentration and to develop an evidence based strategy to select a gel dosing strategy.

II. STUDY OBJECTIVES
• To assess the local absorption and concentration of tenofovir after a single dose and after 14 days of once or twice-daily dosing.
• To assess the systemic absorption of tenofovir after a single dose and after 14 days of once or twice-daily dosing.

III. ENDPOINTS
• Concentration of tenofovir after single tenofovir gel vaginal dose in local genital tract compartments (intraluminal, mononuclear cells and vaginal tissue levels).
• Concentration (Cmax) and time of maximum concentration (Tmax) of tenofovir after single tenofovir gel vaginal dose in the systemic compartment.
• Concentration of tenofovir after two weeks of once and twice-daily vaginal administration of tenofovir gel in local genital tract compartments (intraluminal, mononuclear cells and vaginal tissue levels).
• Concentration (Cmax), time of maximum concentration (Tmax), and time to one half concentration (t½) of tenofovir after two weeks of once and twice-daily vaginal administration of tenofovir gel in the systemic compartment.

IV. TEST ARTICLES
Tenofovir gel is a clear, transparent, viscous gel packaged in epoxy inner-lined aluminum tubes with a white polyethylene screw cap equipped with a puncture tip. Each tube contains 7 grams of 1% tenofovir gel (weight/weight) formulated in purified water with edetate disodium, citric acid, glycerin, methylparaben, propylparaben, hydroxyethylcellulose, and pH adjusted to four to five. The gel is applied with a polyethylene applicator capable of administering a 4mL (equal to four gram) dose of gel. All study products will be stored at room temperature (15º-30º C).

V. STUDY DESIGN
This is a multi-center, pharmacokinetic study involving a single-dose phase, a wash out phase and a two week once or twice-daily dosing phase for each of 49 participants. University of Pittsburgh and Advances in Health will each enroll 14 participants and PROFAMILIA will enroll 21 participants. To construct a PK curve after a single vaginal administration of tenofovir gel, repeated biopsies over time are necessary; however, since repeat biopsies over time in the same individual is not feasible, participants will be randomly assigned to one of the seven time-points and there will be seven volunteers for each time-point.
In the single-dose phase, each volunteer will undergo a screening exam and if she is determined to be eligible and has given informed consent, she will apply the single dose in the clinic and will ambulate for approximately 15 minutes to distribute the gel after which she may ambulate as desired. Cervicovaginal samples and biopsies will be collected at one of seven time-points [0.5, 1, 2, 4, 6, 8, and 24 hour(s)] after the single dose. Blood samples will be drawn at 0.5, 1, 2, 4, 6, 8, and 24 hour(s) Pharmacokinetic Study of Tenofovir Gel: Protocol #A04-095 June 18, 2007 FINAL Version 4.0 4 after the single dose as clinically feasible. An interim analysis of approximately 21 participants (ideally 7 at each site) measuring tenofovir in plasma, vaginal biopsies, and cervicovaginal fluid following the initial dose will be performed.
There will be at least a one month wash out period between the one week phase and the two week phase.
In the two week phase, the study supplies will be distributed and the participants will be randomized to vaginally administer each dose either once or twice-daily for two weeks. At the one week followup visit, a blood sample will be drawn prior to the morning dose to obtain a trough value and cervicovaginal samples will be collected four hours after the morning dose. At the two week followup visit, blood samples will be drawn prior to the morning dose to obtain a trough value and at 0.5, 1, 2, 4, 6, 8, and 24 hour(s) after the final morning dose. Cervicovaginal samples and biopsies will be collected at 4, 8 or 24 hours after the final morning dose. It is expected that after two weeks of dosing, a steady state should be reached.
Up to 10 participants at the University of Pittsburgh site, who have completed the first two phases of the study, will be asked to participate in a third phase to have cervicovaginal samples, biopsies and blood samples collected 12 hours after a single-dose. This substudy is necessary because it would be logistically difficult to collect samples at 12 hour time-points in the main study (they would have to be collected during the night).

VI. SELECTION OF PARTICIPANTS
The volunteers for this study will be recruited through existing databases, as protected health information permits, and, if necessary, through advertisements in local newspapers and radio which have been approved by the site's IRB.
Only women will be recruited since intravaginal gel can only be used by women. Although the NIH has mandated that children (defined as anyone under the age of 21) be included in research trials when appropriate, this study will enroll "children," aged 18 and older who are able to give informed consent. This study fits one of the "Justifications for Exclusion" as set forth by the NIH for excluding younger children. Specifically, "insufficient data are available in adults to judge potential risk in children" and "children should not be the initial group to be involved in research studies." Efforts will be made by the sites to recruit subjects so that the racial and ethnic characteristics of the subject population will reflect the demographics of the study sites. No exclusion criteria shall be based on race or ethnicity.

A. Inclusion Criteria
• Must be between 18-45 years old, inclusive; • Must be in general good health by volunteer history without any clinically significant systemic disease (including liver disease/hepatitis, gastrointestinal disease, kidney disease, thyroid disease, osteoporosis or bone disease); • Must have regular menstrual cycles (minimum of 26 and maximum of 38 days); • Must be at least 2 months since last pregnancy outcome and have had at least two spontaneous menses; • Must abstain from sexual activity and use of intravaginal products for 72 hours prior to the start of the single-dose phase and multi-dose phase, for at least one week following vaginal biopsies and for the duration of the study phases; • Willingness to give voluntary consent, sign an informed consent form and comply with study procedures as required by the protocol;

B. Exclusion Criteria
• Currently pregnant or at risk for pregnancy (may be using Paragard ® IUD, effective barrier method, female sterilization or abstinence, or be sexually active with a vasectomized partner); • Currently breastfeeding or planning to breastfeed during the course of the study; • Use of any hormonal contraceptives within 30 days of enrollment; • Use of Depo-Provera within 120 days of enrollment; • Positive wet mount for Trichomonas vaginalis at the time of screening or enrollment; • Deep epithelial genital findings such as abrasions, ulcerations, and lacerations, or vesicles suspicious for a sexually transmitted infection; • Current or past use of any anti-retroviral therapies including but not limited to systemic tenofovir (Viread®); • Current or recent drug or alcohol abuse; • Current or anticipated use of drugs on a daily basis that may reduce renal function (e.g. acyclovir or ibuprofen) or liver function (e.g. tylenol); • HIV positive at the time of screening; • Hepatitis B surface antigen (HBsAg) positive at the time of screening; • Grade 1 or higher serum chemistry or complete blood count abnormality in accordance with DAIDS toxicity • Abnormal finding on laboratory or physical examination or a medical condition which, in the opinion of the investigator, would make participation in the study unsafe or would complicate interpretation of data; • Participation in any other research study in the 30 days prior to screening and/or plans to participate in any other research study during entire study duration.

C. Post-Admission Withdrawal Criteria
• Failure to follow protocol requirements that are judged severe enough by the investigator to

A. Visit 1: Screening Visit
Volunteers will be given information about and requirements of the study. Volunteers will be asked if they are willing to participate in both phases of the study. All volunteer questions regarding the study will be answered. If the volunteer is eligible and wishes to participate in the study, she will be asked to sign an informed consent and the following will be performed: • an interview to obtain medical history and demographic information; • pre-test HIV counseling; • a urine sample will be obtained for a urine pregnancy test to confirm that the volunteer is not pregnant and to perform a dipstick urinalysis (urine microscopy and culture will be performed as clinically indicated); • baseline blood sample will be collected for complete blood count (CBC), serum chemistries, Hepatitis B surface antigen (HBsAg) test and HIV testing; and • height and weight will be measured and recorded; • screening gynecological examination: o a vaginal sample will be collected for wet mount/pH, o cervical swabs will be taken to test for Neisseria gonorrhea (NG) and Chlamydia trachomatis (CT), o a bimanual pelvic examination will be performed.
Women who test HIV positive at screening are not eligible for enrollment. They will be counseled and given referrals for medical and social services. If symptomatic yeast, BV, or a UTI is clinically diagnosed at this visit, the volunteer will be treated, preferably with oral medication and, if she is otherwise eligible, the enrollment visit will be scheduled after completion of treatment and resolution of symptoms. If Neisseria gonorrhoea, Chlamydia trachomatis, or Trichomonas vaginalis is diagnosed the volunteer will be treated or referred for treatment and will not be eligible for enrollment. If deep epithelial disruptions (such as abrasions, ulcerations, or lacerations) or vesicles suspicious for a sexually transmitted infection are observed on the female genitalia the participant will not be eligible for enrollment and testing should be performed as clinically indicated or she should be referred for treatment, as appropriate. If deep epithelial disruption is identified that is either iatrogenic or unlikely to have been caused by infection and likely to resolve quickly, the appointment can be rescheduled, and the participant may be enrolled in the study once the finding has resolved. Eligible volunteers will be scheduled for an enrollment visit when they are not menstruating and an attempt will be made to schedule the visit in the second half of the menstrual cycle, ideally menstrual cycle day 19-24. The volunteer will abstain from sexual activity and use of intravaginal products for 72 hours prior to the enrollment visit (start of the single-dose phase) and for the duration of the study phase and will be instructed to abstain from the following activities during this time: • Use of any vaginal product other than the study gel including spermicides, lubricants, vaginal hygiene products, diaphragms, and cervical caps.

B. Visit 2: Enrollment Visit
The enrollment visit should take place within 8 weeks of the screening visit. The results from the screening visit will be reviewed. Volunteers with abnormal blood screening test results will be advised to seek advice and/or care from their primary care provider or referrals to appropriate resources will be given. A urine sample will be obtained for a urine pregnancy test to confirm that the volunteer is not pregnant. A pelvic examination will be performed in which the vulva, vagina and cervix will be visually inspected for non-intact epithelium or other findings. If deep epithelial disruption is identified, the participant will not be enrolled until the finding has resolved.
If eligible, the volunteer will be enrolled and randomized to one of seven time-points [0.5, 1, 2, 4, 6, 8, and 24 hour(s)] after the single dose for collection of cervicovaginal samples and biopsies. A baseline blood sample for systemic tenofovir level will be drawn. The participant will be instructed to void prior to the first dose. The participant will apply the single-dose in the research unit and will ambulate for approximately 15 minutes to distribute the gel, after which she may ambulate as desired. The participant will be instructed to avoid emptying her bladder or bowel within the first hour after application, if possible.
Blood samples will be collected at 0.5, 1, 2, 4, 6, 8, and 24 hour (s) after the single-dose for plasma measurement of tenofovir. See protocol section XI.B for protocol adherence definitions. The participant will leave the research unit after the 8 hour blood draw and will return the following day for the 24 hour blood draw (as well as cervicovaginal samples and biopsies for those assigned to the 24 hour time-point); therefore, this visit will occur over a 24 hour time period. The participant will be asked about vaginal activity and adverse events that occur between the 8 hour and the 24 hour visit. Each participant will undergo the following pelvic examination procedures at her randomly assigned time-point: • The volunteer should be supine for 15 minutes before the pelvic examination to allow for pooling to occur.
• Direct aspiration of the cervicovaginal fluid pooling (avoiding tenofovir gel when possible) in the posterior fornix with a vaginal aspirator syringe will be performed to measure intraluminal concentration of tenofovir.
• Endocervical specimen will be collected with two 360º turns of a cytobrush to measure local intracellular tenofovir in mononuclear cells.
• The areas of the planned biopsies will be gently wiped with a small cotton swab moistened with warm saline followed by a small cotton swab with betadine.
• Topical 20% benzocaine gel will be applied to the areas of the planned biopsies for pain control. Women can opt to have the biopsy without anesthesia by preference or if they are allergic to benzocaine or can be given non-steroidal anti-inflammatory drugs prior to the procedure for pain relief as per local site standards.
• Full-thickness biopsies measuring approximately 5mm by 3mm will be obtained from an area located on the upper one-third of the vagina (at around 7-8 o'clock), and from the lower Pharmacokinetic Study of Tenofovir Gel: Protocol #A04-095 June 18, 2007 FINAL Version 4.0 one-third of the vagina (at around 7-8 o'clock) with cervical biopsy forceps to measure tissue concentration of tenofovir.
• If adequate hemostasis is not obtained with gentle pressure, Monsel's solution or silver nitrate can be applied, as needed, to the biopsy site.
The volunteer will be given a Participant Guidebook (Appendix II) that will give detailed instructions on how to insert the tenofovir, as well as what she can expect during each visit. The guidebook will include post-biopsy instructions that include avoiding sexual activity, douching or tampon use for at least a week following the biopsy to allow time for the biopsy site to heal.
The volunteer will also abstain from sexual activity and use of intravaginal products for 72 hours prior to Visit 3 (start of the two-week phase) and for the duration of the study phase and should be instructed to abstain from the following activities during this time: • Masturbation; • Oral contact with the female genitalia; • Vaginal and anal intercourse; • Douching; • Penetration of the vagina by fingers, tampons, sex toys, or any other objects • And use of any vaginal product other than the study gel including spermicides, lubricants, vaginal feminine hygiene products, diaphragms, and cervical caps.
There will be at least 30 days between this visit and the beginning of the two week phase to ensure total elimination of the drug from the participant and healing of the biopsy sites.

C. Visit 3: (Two-week Phase of once or twice-daily use)
This visit will be scheduled for when the volunteer is not menstruating and should be scheduled early in the menstrual cycle so that the last dose at the time of the two week follow-up will occur in the second half of the menstrual cycle, ideally menstrual cycle day 19-24.
A urine pregnancy test will be performed on each volunteer to confirm that she is not pregnant. Blood will be drawn to obtain systemic levels of tenofovir. A pelvic examination will be performed in which the vulva, vagina and cervix will be visually inspected for non-intact epithelium or other findings, and to verify healing from the single-dose phase. If deep epithelial disruption is identified, the participant will not continue in the study until the finding has resolved.
Participants will be randomly assigned to either once-daily or twice-daily application and will receive study supplies. Participants assigned to once-daily use will be instructed to insert the gel in the mornings at approximately 24-hour intervals. Participants assigned to twice-daily use will be instructed to insert the gel in the mornings and evenings at approximately 12-hour intervals. All participants will be instructed to begin dosing on the day after Visit 3.
Participants will be instructed to void prior to applying each dose and to ambulate for approximately 15 minutes after each application, after which they may ambulate as desired, and, to avoid emptying their bladders or bowels within the first hour after application, if possible.
If a participant misses a dose, she should insert it when she remembers, if possible, and then resume her regular schedule. If, at the time that she becomes aware that she missed a dose, it is within 6 hours of the next scheduled dose she should skip the missed dose and resume her regular schedule. If the participant misses two or more doses she should contact the study site for further instructions. The participant will be given instructions for gel use and a diary card to complete the date and time of all product applications and symptoms that occur during the study.
The participant will be instructed to return for a one week follow-up and will be asked to wait to insert her morning dose on the day of the scheduled visit as it will be applied at the clinic.

D. Visit 4: (One week follow-up)
A urine pregnancy test will be performed to confirm that the participant is not pregnant. The diary card will be reviewed and participants will be asked about any adverse events and will be evaluated, if necessary, as described in Section VII. H. A blood sample will be drawn prior to the morning dose to obtain a trough plasma measurement of tenofovir. The morning dose will be applied in the research unit.
Each participant will undergo the following pelvic examination procedures at 4 hours after the morning dose: • Participants should be supine for 15 minutes before the pelvic examination to allow for pooling to occur.
• Direct aspiration of the cervicovaginal fluid pooling in the posterior fornix with a vaginal aspirator syringe will be performed to measure intraluminal concentration of tenofovir.
• Endocervical cytobrush specimen will be collected with two 360° turns of a cytobrush to measure local intracellular tenofovir in mononuclear cells.
The participant will be instructed to resume her assigned dosing schedule and to return for a two week follow-up and will be asked to wait to insert her final morning dose as it will be applied at the clinic.

E. Visit 5: (Two week follow-up)
A urine pregnancy test will be performed to confirm that the participant is not pregnant. The diary card will be reviewed and collected and participants will be asked about any adverse events and will be evaluated, if necessary, as described in Section VII. H. A copy of the back of their diary card will be given to the participant to record any symptoms and medications between Visit 5 and Visit 6. Each participant will be randomized to one of three time-points (4, 8 or 24 hours after the final dose) to provide cervicovaginal samples and biopsies. A blood sample will be drawn prior to the morning dose to obtain a trough plasma measurement of tenofovir. The final morning dose (14 th or 27 th ) will be applied in the research unit. Blood samples will be collected at 0.5, 1, 2, 4, 6, 8, and 24 hour (s) after the dose for plasma measurement of tenofovir. Complete blood count and serum chemistries will also be drawn with the final collection. Each participant will undergo the following pelvic examination procedures at her randomly assigned time-point: • The volunteer should be supine for 15 minutes before the pelvic examination to allow for pooling to occur. • Direct aspiration of the cervicovaginal fluid pooling (avoiding tenofovir gel when possible) in the posterior fornix with a vaginal aspirator syringe will be performed to measure intraluminal concentration of tenofovir.
• Endocervical specimen will be collected with two 360º turns of a cytobrush to measure local intracellular tenofovir in mononuclear cells.
• The areas of the planned biopsies will be gently wiped with a small cotton swab moistened with warm saline followed by a small cotton swab with betadine.
• Topical 20% benzocaine gel will be applied to the areas of the planned biopsies for pain control. Women can opt to have the biopsy without anesthesia by preference or if they are allergic to benzocaine or can be given non-steroidal anti-inflammatory drugs prior to the procedure for pain relief as per local site standards.
• Full-thickness biopsies measuring approximately 5mm by 3mm will be obtained from an area located on the upper one-third of the vagina (at around 4-5 o'clock) and from the lower one-third of the vagina (at around 4-5 o'clock) with cervical biopsy forceps to measure tissue concentration of tenofovir.
• If adequate hemostasis is not obtained with gentle pressure, Monsel's solution or silver nitrate can be applied, as needed, to the biopsy site.
If the participant will not be taking part in the substudy, Visit 6 will be scheduled for 1-2 weeks from this visit to allow time for the biopsy sites to heal. Substudy Participants only: If the participant will be taking part in the substudy, she will not have a Visit 6 in the main study, but instead will be scheduled for Substudy Visit 1. This visit will be scheduled for when the participant will not be menstruating and an attempt will be made to schedule the visit in the second half of the menstrual cycle, ideally menstrual cycle day 19-24. There will be at least 30 days between this visit and the beginning of the substudy to ensure total elimination of the drug from the participant and healing of the biopsy sites.
The volunteer will also abstain from sexual activity and use of intravaginal products for 72 hours prior to Substudy Visit 1 (start of the substudy) and for the duration of the substudy. These activities include: • Masturbation; • Oral contact with the female genitalia; • Vaginal and anal intercourse; • Douching; • Penetration of the vagina by fingers, tampons, sex toys, or any other objects • And use of any vaginal product including spermicides, lubricants, vaginal feminine hygiene products, diaphragms, and cervical caps.

F. Visit 6: Final follow-up
The participant will return 1-2 weeks after Visit 5. A urine pregnancy test will be performed. The participant will be asked about any adverse events since her last visit and the copy of the symptoms and medications portion of her Diary Card will be collected. A gynecological examination will be Pharmacokinetic Study of Tenofovir Gel: Protocol #A04-095 June 18, 2007 FINAL Version 4.0 13 performed in which the vulva, vagina and cervix will be visually inspected for non-intact epithelium or other findings, and to verify healing from the biopsies. If the examination is normal the participant will be exited from the study. Otherwise the participant will be followed until the exam is normal or she is referred for follow-up care, as clinically indicated.

Substudy (Visit 1)
Up to 10 participants at the University of Pittsburgh site who have completed the first two phases of the study will be asked to participate in a third phase. This substudy is necessary because it would be logistically difficult to collect samples at 12 hour time-points in the main study (they would have to be collected during the night).
A baseline blood sample for systemic tenofovir level will be drawn. A urine sample will be obtained for a urine pregnancy test to confirm that the volunteer is not pregnant. A pelvic examination will be performed in which the vulva, vagina and cervix will be visually inspected for non-intact epithelium or other findings. If deep epithelial disruption is identified, the participant may continue in the study when the finding has resolved. The participant will be given a single-dose to use at home in the evening at a specified time, along with a Substudy Diary Card and instructions. The participant will be instructed to void prior to inserting the dose. The participant will apply the single-dose at home (at a time that will fall approximately 12 hours before her next visit) and will ambulate for approximately 15 minutes to distribute the gel, after which she may ambulate as desired. She will be scheduled to return to the research clinic the following morning.

Substudy (Visit 2)
Blood samples will be collected 12 hours after the single dose for plasma measurement of tenofovir. The diary card will be reviewed and collected and participants will be asked about any adverse events and will be evaluated, if necessary, as described in Section VII. H. A copy of the Diary Card will be given to the participant to record any symptoms and medications between Substudy Visit 2 and Substudy Visit 3. Each participant will also undergo the following pelvic examination procedures at the 12 hour time-point: • The volunteer should be supine for 15 minutes before the pelvic examination to allow for pooling to occur. • Direct aspiration of the cervicovaginal fluid pooling (avoiding tenofovir gel when possible) in the posterior fornix with a vaginal aspirator syringe will be performed to measure intraluminal concentration of tenofovir.
• Endocervical specimen will be collected with two 360º turns of a cytobrush to measure local intracellular tenofovir in mononuclear cells.
• The areas of the planned biopsies will be gently wiped with a small cotton swab moistened with warm saline followed by a small cotton swab with betadine.
• Topical 20% benzocaine will be applied to the areas of the planned biopsies for pain control.
Women can opt to have the biopsy without anesthesia by preference or if they are allergic to Pharmacokinetic Study of Tenofovir Gel: Protocol #A04-095 June 18, 2007 FINAL Version 4.0 14 benzocaine or can be given non-steroidal anti-inflammatory drugs prior to the procedure for pain relief as per local site standards.
• Full-thickness biopsies measuring approximately 5mm by 3mm will be obtained from an area located on the upper one-third of the vagina (at around 7-8 o'clock), and on the lower one-third of the vagina (at around 7-8 o'clock) with cervical biopsy forceps to measure tissue concentration of tenofovir.
• If adequate hemostasis is not obtained with gentle pressure, Monsel's solution or silver nitrate can be applied, as needed, to the biopsy site.
Post-biopsy instructions will be given to the participant that include avoiding sexual activity, douching or tampon use for at least a week following the biopsy to allow time for the biopsy site to heal.

Substudy (Visit 3)
The participant will return 1-2 weeks after substudy Visit 3. A urine pregnancy test will be performed on all participants. The participant will be asked about any adverse events since her last visit and the copy of the Substudy Diary Card will be collected. A gynecological examination will be performed in which the vulva, vagina and cervix will be visually inspected for non-intact epithelium or other findings, and to verify healing from the biopsies. If the examination is normal the participant will be exited from the study. Otherwise the participant will be followed until the exam is normal or she is referred for follow-up care, as clinically indicated.

H. Adverse Event Follow-up/Unscheduled Visit
If the participant experiences moderate to severe symptoms (Appendix VI) including vaginal burning, itching, pain, spotting or bleeding at any time during product usage, she will be instructed to contact the clinic and may be evaluated with laboratory testing, physical examination and visual and colposcopic inspection as clinically indicated. If product is visible, an attempt should be made to rinse it off using normal saline. If a wet mount and/or STI testing are performed and positive for infection, the subject will be treated and discontinued from the study. The subject may continue in the protocol if the wet prep is negative, there is no STI diagnosed, there are no findings of deep epithelial disruption on the genitalia severe enough to make further application of the product inadvisable, the investigator feels that the participant's health is not significantly jeopardized, and the participant wishes to continue.
If a participant experiences severe irritation and cannot get to the clinic the same day, she will be instructed to stop product use, soak in a sitz bath, and douche with plain warm water. The volunteer will be encouraged to come to the clinic as soon as possible for evaluation.
In addition, she will be given post-biopsy instructions to contact the clinic if she experiences vaginal bleeding (if more than spotting), foul-smelling vaginal discharge, fever and/or chills, or severe lower abdominal pain and will be evaluated as clinically indicated.
If the participant reports symptoms of a urinary tract infection (UTI) at an unscheduled visit, a cleancatch urine dipstick and other urine testing as indicated will be performed. If a UTI is diagnosed the participant will be treated and discontinued from the study.

I. Early Discontinuation
If a participant is discontinued or withdrawn from the study early, final discontinuation procedures including urine pregnancy test, blood sample for systemic levels of tenofovir, complete blood count, and serum biochemistries should be performed. In addition, cervicovaginal specimens including vaginal biopsies should be obtained, if possible, and if relevant to study endpoints.

J. Laboratory Procedures
Vaginal wet mount/pH At the screening visit, a sample of fluid taken with a cotton swab from the lateral fornix will be evaluated for signs of BV, trichomonas and/or candida. Vaginal pH will also be measured. If vaginal symptoms develop during the study, additional wet mounts will be done as indicated. Neisseria gonorrhea (NG) and Chlamydia trachomatis (CT) At the screening visit, an endocervical specimen will be testing for N. gonorrhoeae and C. trachomatis using Nucleic acid hybridization test or PCR Urine pregnancy test A urine specimen will be collected and tested according to the specifications of the test kit manufacturer at each scheduled visit and as indicated.
Dipstick urinalysis At the screening visit, a clean catch urine sample will be tested via Multistix 10 SG Reagent Strips (Bayer) and as indicated at other visits.

CBC & Serum biochemistries, Hepatitis B, and HIV
Venous blood sample will be drawn and the serum will be tested for HBsAg and HIV antibodies at the screening visit. Samples will be processed according to the specifications of the laboratory (See Appendix IV). CBC and serum biochemistries will be measured at screening and at the 2-week follow-up visit.
RPR or VDRL and HSV If a genital ulcer is present, screening for syphilis using the venereal disease research laboratory (VDRL) or rapid plasma regain (RPR) test may be performed and testing for Herpes simplex virus may be performed by obtaining a specimen for culture from the lesion. A positive screening test for syphilis will be confirmed using the FTA or TPPA test.

VIII. CRITERIA FOR DISCONTINUATION OF STUDY
The study may be stopped if, in the opinion of CONRAD: 1. Review of AEs shows an unexpected, significant or unacceptable risk to the participants enrolled in the study;

2.
The site has failed to enroll subjects at an acceptable rate; 3. The protocol requirements have not been adhered to; and/or 4. Administrative reasons.

IX. STUDY MATERIALS
The sites will manage the distribution of the study product according to the protocol. Each site will be provided with a quantity of demonstration samples and study product kits equivalent to the number of patients they are to enroll. Supplies will be distributed by Bilcare Inc. (Phoenixville, PA) under CONRAD's supervision.
See section "IV. Test Articles" for a description of 1% tenofovir gel.
Each study tube will be labeled with the study number and lot number and the following information: "1% Tenofovir Gel, For Vaginal Use Only, Keep out of Reach of Children--Caution: New Drug: Limited by Federal Law to Investigational Use". See Appendix II for a copy of the participant instructions.
Single Use Kits: The single-dose kit will contain a labeled tube of 1% tenofovir gel (7 gram tube), an empty applicator and patient instruction sheet. See Appendix II for a copy of the participant instructions. The outside of the single-dose kit box will have a two-part, tear-off label, with the information listed below. Multiple Dose Kits: There are two different multiple dose kits for once and twice-daily dosing for 14days.
Once-a-day for 14-day kits will each contain 14 labeled tubes of 1% tenofovir gel (7 gram tube), 14 empty applicators and 1 patient instruction sheet. See Appendix II for a copy of the participant instructions. The outside of the multiple dose kit box will have a two-part, tear-off label, with the information listed below. Twice-a-day for 14-day kits will each contain 27 labeled tubes of 1% tenofovir gel (7 gram tube), 27 empty applicators and 1 patient instruction sheet. See Appendix II for a copy of the participant instructions. The outside of the multiple dose kit box will have a two-part, tear-off label, with the information listed below. All used and unused study product at the end of the study will be recorded and disposed of by the clinical site.
Study product should be stored at room temperature (15°-30° C) in a locked cabinet or secure area in the clinic.

A. Sample Size Justification
Statistical considerations were not the primary basis for choosing to enroll 49 women in this Phase I study; the study size was based primarily on large part on feasibility considerations (e.g., cost and time). Nonetheless, the chosen size should be sufficient to characterize relevant pharmacokinetic profiles. For example, in the previous HPTN vaginal dosing study that evaluated 25 women, maximum likelihood estimates of the standard deviation of logged plasma concentrations ranged from 0.41 to 0.82 at various time-points between hours 1 and 12 following initial dosing (these estimates account for censoring below the analytic limit of detection LOD through the use of log-normal likelihood methods). Based on these data, the expected half-width of 95% confidence intervals for mean (logged) plasma concentration in the proposed study, with forty nine women at each blood draw time-point, is 0.1 to 0.2. This compares favorably with expected mean plasma levels of about 0.5 logs. The use of nonlinear pharmacokinetic (PK) models to account for repeated observations on each participant should provide improved precision of predicted concentration curve time-points in the current study.
Although discussing the expected precision of vaginal tissue concentrations is rather speculative prior to performing this original study, we can conclude that by obtaining measurements from seven women at each time-point, and assuming similar standard deviations as observed in the plasma data, the half-width of confidence intervals should be about 0.5 logs (compared to mean tissue levels that should be substantially larger than 0.5 for at least some of the sampled time-points).

Randomization, Allocation Concealment, and Blinding
A random permuted blocks method will be used to generate three random allocation sequences. The first sequence will be used to randomize women to one of seven biopsy time-points following initial dosing of the gel, stratified by site. The second sequence will be used to randomize women to either once-or twice-daily dosing in the 14 day follow-up period, stratified by site. The third sequence will be used to randomize women to one of the three concluding biopsy time-points (i.e., 4, 8 or 24 hours following the final, in-clinic, dosing), stratified by site and dosing arm within site. The random sequences will be created using a SAS (SAS Institute, Cary, NC) program validated by FHI.
It is not possible to blind clinic staff or study participants to the biopsy time-point assignments or dosing schedules once allocation has taken place. In order to conceal the allocation procedure, however, random assignments will be contained within sequentially numbered, sealed opaque envelopes. Participants will be randomized to biopsy time-points and a dose schedule by giving the next available randomization number to the next eligible participant. The randomization envelopes will be maintained in a secure location at the clinic. Each envelope will be used only once. For documentation purposes, a sealed copy of the allocation list will be sent to CONRAD and a second copy will be maintained at FHI. CONRAD and FHI staff working on the trial will not have access to the sealed copies nor any electronic data identifying assignments prior to the scheduled un-blinding of data analysts and other study staff not working at the clinic.

Statistical Considerations
A detailed analysis plan will be developed and approved prior to the end of data collection. The following is a summary of the planned analyses. Any changes made from this summary with respect to primary study outcomes will be documented in the detailed analysis plan.

Analysis Populations
A number of different analysis populations will be employed in this study. Although all study data will be summarized, the primary analysis set will exclude data collected from women who failed to comply with their assigned dosing schedule. Data points that fall outside of the allowed collection window (as specified in the protocol, Section XI.B) will also be excluded from analyses that depend on specific sampling times, unless the observed sampling time falls in an allowable (alternative) window. Further details regarding the analysis sets used for each primary and secondary analysis will be provided in the expanded analysis plan.

Censored Data
Based on the HPTN vaginal dosing study, it is anticipated that a large percentage of plasma tenovofir measurements will fall below the analytic limit of detection. Likewise, some censoring of data from the genital tract compartments is expected. Consequently, the plasma and genital tract concentration data will be described in two different ways: using medians, inter-quartile ranges, and ranges (in which case censored data contribute as percentiles of the distributions); and by employing appropriate empirical PK models (e.g., one-compartmental models) that incorporate data censored below the analytic limit of detection (LOD) using appropriate likelihood techniques.

Analysis of Primary Endpoints
Two vaginal biopsy samples will be collected from each woman at each of their assigned time points (i.e. within 24 hours of their initial, single-dose and then, following a washout period, within 24 hours after their 14-day dosing regimen). The geometric mean concentration of tenofovir following the initial (day zero) dosing will be plotted for the maximum of 7 subjects per time period. Cmax and Tmax values will be estimated based on the median response values across time. The lack of repeat sampling times from each subject (i.e., biopsy samples are only taken at one time point for each woman in each phase of the study) invalidates the formal use of PK modeling for these data. However, nonlinear models (e.g., one-compartmental models) will be employed in an empirical exercise to obtain complimentary estimates of Cmax, Tmax, and their standard errors. The models will be validated to the extent possible given the available data.
Intraluminal tenofovir concentrations and cytobrush specimen results will be summarized in a similar fashion to biopsy samples. The concentration of tenofovir in blood following the initial (single) dosing will also be plotted over time for each subject. Cmax, Tmax, and T½ values will be obtained for each subject and summarized across subjects using medians (Cmax and T½ values will be imputed for those subjects whose measurements all fall below the LOD.). Cmax, Tmax, and T½ values will also be estimated using a one-compartment PK model that accounts for repeated observations from each subject. Censored data will be handled by incorporating appropriate likelihood methods in the estimation procedure.
Similar exercises will be performed to estimate Cmax, Tmax, and T½ values following 14 days of daily dosing. Qualitative differences in PK curves between the once-and twice-daily dosing schedule, and between the single dose and daily dosing results, will be evaluated both graphically and through modeling exercises. Where reasonable, data collected from the same woman (i.e., following the initial dose and following 14 days of daily dosing) will be incorporated in these supporting analyses.
It is possible that the act of taking biopsy samples could influence systemic blood tenofovir concentrations at later time points. Although it is unlikely that the study size is sufficient to identify such effects, these potential processes will be explored, as follows: The median tenofovir blood concentration at time point t+1 will be estimated for each group of 7 women having a biopsy at the previous time point (t). This median value will be compared to the median blood concentration for all remaining women who have their biopsy after time t. For example, the median blood concentration at 2 hours will be computed for the 7 women who had their biopsy at the 1 hour time point. This will be compared to the median 2-hour blood concentration for the remaining 35 women who have their biopsy at 2 or more hours after initial dosing. These differences will be summarized for each of the 6 possible time point comparisons. The effects of certain baseline factors (e.g., age, ethnicity, BMI) on Cmax, Tmax, and T½ values will be evaluated using analysis of variance techniques. These analyses will be exploratory and descriptive in nature.

Substudy analysis
Exercises of the single-dose phase will be repeated for up to 10 women at the Pittsburgh site who continue to the third phase of the study. The exercise will explore within-subject effects of this subsample after the single-dose application at 8 time points including the additional time point of 12-hour following the single dose.

Interim Analysis.
An interim analysis of approximately first 21 participants (ideally 7 at each site) completing the single-dose phase of the trial measuring tenofovir in plasma, vaginal biopsies, and cervicovaginal fluid following the initial dose will be performed. Genital compartment and blood concentration values will be summarized and plotted across sampling time points. Likewise, adverse event summaries will be provided to the project team. However no formal statistical tests will be conducted, and therefore no stopping rules will be applied. But it is possible that the results of this interim analysis could lead to an adjustment in the sampling times for the second phase of the trial.

A. Loss to Follow-up
If a participant fails to appear for a scheduled visit, at least three attempts to contact her should be made over the subsequent 30 days. These attempts should be documented in the participant's study file. The final attempt must be a certified letter with return-receipt requested to the participant, or an outreach attempt. A copy of this letter should be in her file. After these three attempts, no further efforts need be made to find her, but her file should remain open until study closeout.
If the participant does not return to the clinic before the study is closed, the Final Disposition (FINAL) Form will be completed at the time of study close-out. The form should indicate that the participant was lost to follow-up. The "lost to follow-up" designation cannot be made for any participant until the closing date of the study.

B. Protocol Adherence
Participants in the two-week phase will be considered compliant with the study regimen if they have used at least 80% of scheduled doses (at least 11 out of 14 of the scheduled doses in the once-daily group and at least 21 out of 27 in the twice-daily group) and as long as they have used at least two doses prior to PK sampling. The study site should carefully record the date and time of each collection of PK blood and cervicovaginal sampling. The time of sampling will be considered a protocol deviation if it falls ± 15 minutes outside of the 30 minute and 1 hour time-points, ± 30 minutes outside of the 2, 4, 6 or 8 hour time-points or ± 2 hours outside of the 12 or 24 hour timepoints.

C. Adverse Events
An adverse event or adverse experience (AE) is any untoward medical occurrence in a clinical investigation subject administered a pharmaceutical product and which does not necessarily have 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 product, whether or not considered related to the investigational product. Preexisting events that increase in frequency or severity in nature during or as a consequence of use of a drug in human clinical trials will also be considered as adverse events. Any medical condition or clinically significant laboratory abnormality with an onset date before the first date of study drug administration is considered to be pre-existing, and should be documented in the source document for the participant.
According to 21 CFR 312.32 "IND Safety Reports," the following definitions of terms apply to adverse events occurring in clinical studies involving drugs.
"Associated with the use of the drug" means: "There is a reasonable possibility that the experience may have been caused by the drug." "Serious adverse drug experience" (SAE) means: "Any adverse drug experience occurring at any dose that results in any of the following outcomes: Death, a life-threatening adverse drug experience, inpatient hospitalization or prolongation of existing hospitalization, a persistent or significant disability/incapacity, or a congenital anomaly/birth defect. Important medical events that may not result in death, be life-threatening, or require hospitalization may be considered a serious adverse drug experience when, based upon appropriate medical judgment, they may jeopardize the patient or subject and may require medical or surgical intervention to prevent one of the outcomes listed in this definition." "Unexpected adverse drug experience" means: "Any adverse drug experience, the specificity or severity of which is not consistent with the current investigator brochure; or, if an investigator brochure is not required or available, the specificity or severity of which is not consistent with the risk information described in the general investigational plan or elsewhere in the current application, as amended." Any adverse events that are serious (whether expected or unexpected) and possibly associated with use of the drug must be immediately reported to CONRAD by telephone (21 CFR 312.64). If there is any question whether the adverse event meets any of these criteria, it should be reported by telephone anyway. In addition, a completed Adverse Event (AE) Form must be faxed to CONRAD as soon as possible.
CONRAD will notify FHI (the IND holder) who will notify the FDA by telephone or fax of any unexpected serious AEs associated with the use of the drug as soon as possible, but no later than 7 calendar days after initial receipt of the information from the investigator. In addition, FHI will notify all participating investigators and the FDA in writing. The written notification will be made as soon as possible and in no event later than 15 calendar days after initial receipt of the information.
Any serious adverse event that is associated with treatment and is unexpected must be reported promptly to the Institutional Review Board.
CONRAD will follow all serious adverse events with the cooperation of the investigator.
Adverse events that do not fall into the categories of associated with drug, serious, and unexpected may include illnesses, discomfort, pain, or any medical problem such as cold symptoms or other complaints. All adverse events, whether serious or non-serious and whether or not related to the study drug, should be recorded on the Adverse Event (AE) Form.
Each adverse event should be graded for severity using the DAIDS Table for Grading the Severity of Adverse Events (Appendix VI). Note: In Appendix IV, the table of Lab Normal Values indicates any modifications by CONRAD to DAIDS' normal accepted values; For each adverse event, an assessment of the relatedness to the test agent should be made using the following scale: • Unrelated: Onset of the AE had no reasonable temporal relationship to administration of the study product or a causal relationship to administration of the study product is biologically implausible or the event is attributed to an alternative etiology.
• Possibly Related: Onset of the AE has a reasonable temporal relationship to study product administration and a causal relationship is not biologically implausible.
• Probably Related: Onset of the AE has a strong temporal relationship to administration of the study product that cannot be explained by the subject's clinical state or other factors and a causal relationship is not biologically implausible.
• Definitely Related: Onset of the AE shows a distinct temporal relationship to administration of the study product that cannot be explained by the subject's clinical state or other factors or the AE occurs on rechallenge or the AE is a known reaction to the product or chemical group or can be predicted by the product's pharmacology.
The study agents that must be used in determining relationships of AEs are tenofovir gel and the study gel delivery applicator.

D. Management/Replacement of Participants Who Discontinue
Participants who discontinue the study may not re-enroll and will not be replaced.

E. Pregnancy
If a pregnancy is diagnosed, the site should notify CONRAD as soon as possible by telephone. The course of the pregnancy should be followed until it has an outcome. If the subject seeks care outside the site, every effort should be made to obtain her consent for the site to receive a copy of her medical records related to the pregnancy, its outcome and the health of the neonate, if applicable. The participant will be discontinued from the study and exit procedures followed.

F. Protocol Violations
If a protocol violation is required to protect the life or physical well being of a participant in an emergency, it may be carried out without prior approval from CONRAD or the IRB. The investigator must, however, report the violation to CONRAD and the IRB as soon as possible, no later than five working days after the emergency occurred. CONRAD will notify the FDA if indicated. The investigator will also keep a record of all such violations, with documents showing the dates of and reasons for each violation.
Other violations from the protocol may not be carried out without prior approval from the IRB if the change involves the rights, safety, or welfare of participants and without prior approval of the FDA if the change involves the study's scientific soundness or the rights, safety, or welfare of participants.
If an inadvertent protocol violation has occurred, CONRAD should be notified immediately to determine what steps should be taken.
Protocol violations may be documented on Protocol Violation Forms (PVF), a generic form developed by FHI for any study in which it manages data.

G. Modification of Protocol
No modification of this protocol may be made without the approval of CONRAD.

A. Study Initiation
At study initiation, the following forms and records will be required: o Name of laboratories to be used to process study specimens o CV of laboratory director(s) o Copy of laboratory licenses with expiration dates o Copy of normal values for tests done by each laboratory for this study

B. Study Conduct
During the study, a monthly report of participants screened, enrolled, completing each visit, and discontinued, as well as any adverse events related to product use will be sent to CONRAD.
The following forms and records will be maintained at the study site: o Approval letters of protocol and any protocol amendments o Approval letters for original and any revised consent forms o Annual approval letter o Other IRB correspondence • Updates on CVs and laboratory information • Current Investigator Brochure • IND Safety Reports • General correspondence Annual progress reports and a final report will be submitted by the site to the IRB. Copies of all documents submitted to the IRB should be sent to CONRAD.

XIII. MONITORING
A member of the CONRAD staff will monitor the site approximately every 6 to 8 weeks and/or on an as-needed basis. All clinical records and case report forms for the participants enrolled in this study will be made available for review by authorized individuals. A Visit Log will be maintained at the site in which all site monitoring visits will be recorded.

XIV. USE OF INFORMATION AND PUBLISHING
All information concerning the drug supplied by CONRAD to the investigator and not previously published is considered confidential.
No data collected in this study will be published without prior written approval from CONRAD.

XV. ANTICIPATED LENGTH OF STUDY
Recruitment for the study is expected to take 6-8 months. Each subject's participation will last approximately 4-6 months, making the clinical portion of the study last about 12 months. Data analysis will take 3 months and the Final Report an additional month.

XVI. CASE REPORT FORMS (CRFs)
All data will be recorded on 3-ply CRFs supplied by CONRAD. Each CRF must be initialed by the study staff member who completed it. The principal investigator, however, is responsible for the accuracy of data entered on the CRF and will sign a statement on the discontinuation form for each participant stating that he or she has reviewed all CRFs for that participant and certifies that all information on them is correct.
The source documents including signed informed consent forms, laboratory reports, patient records, and participant diary cards should be maintained in the participant's medical chart or study file, and should be available for review during monitoring visits.
The original and yellow copies of each CRF will be submitted to FHI after review by the CONRAD Clinical Research Monitor. The pink copy will be retained in the participant's clinic study file. It is imperative that the original and all copies match in every detail. Before a CRF is separated, corrections may be made on the CRF itself, so that they are recorded identically for all three copies. The person making the change must date and initial each change. Once the copies of a CRF are separated, however, changes may not be made on the copies themselves; they must be recorded on paper data discrepancy report forms. Once the correction has been made and the discrepancy form has been signed, a copy of the form should then be attached to the relevant CRF at the site, and a copy should be submitted to FHI. Use of opaque correction fluid is not permitted for any corrections to CRFs at any time.
All data queries sent to the investigator not in conjunction with a monitoring visit should be answered by the investigator in a timely fashion. The data relevant to the query will be updated based on the investigators' written response. Investigators must keep copies of all queries stapled to the CRFs in the participant's study file.
A detailed data management plan will be written before the data is collected for this study.

XVII. RECORDS RETENTION
Original and yellow copies of all CRFs will be kept at FHI. Photocopies of CRFs and pertinent study documentation (such as IRB correspondence, participant master list, etc.) will be kept by CONRAD.
The signed original informed consent documents for each participant, pink copies of CRFs, and originals of all study documentation (e.g. drug inventory forms, participant clinic records, original laboratory reports, diaries, etc.) will be retained by the investigator for a minimum of two years after U.S. Food and Drug Administration approval or withdrawal of a New Drug Application (NDA). If an NDA is not submitted within six years of the last follow-up visit, the center may request permission in writing from CONRAD to destroy the records. No records may be destroyed without written permission from CONRAD.
The investigator may be subject to a field audit by FDA inspectors to validate the participation of study participants and to verify the data reported on the Case Report Forms. This audit could occur while the study is in progress, several years after the study is completed, or when the data are under review by the FDA as part of the new drug approval (NDA) process. All of the participants' records and other study documentation must be filed and accessible on short notice (3-5 days) during the study and subsequent retention period.

A. Institutional Review Board (IRB) Review and Approval
Before initiating the study, the study must be approved in writing by the local IRB or Ethical Review Committee, in accordance with FDA regulations (21 CFR Part 56). The study will be conducted in accordance with all conditions of approval by the IRBs. The site will obtain written re-approval of the research by the IRB annually.

B. Informed Consent
No participant may be admitted into this study until the investigator has obtained her legally effective informed consent. An investigator shall seek such consent only under circumstances that provide the prospective participant with sufficient opportunity to consider whether or not to participate in the study. Informed consent must be obtained without coercion, undue influence, or misrepresentation of the potential benefits or risks that might be associated with participation in the study.
Informed consent encompasses all oral or written information given to the participant about the study and the study materials. This includes the consent form signed by the participant, the instructions for use of study materials that are provided to the participant, recruitment advertising, and any other information provided to the participant. All such information that is given to the participant will be in a language that is understandable to her. The information will not include any language in which the participant is made to waive any of her rights or which releases or appears to release the investigator, the investigator's institution, or CONRAD from liability for negligence.
Informed consent will be documented by the use of a written consent form that is signed by the participant and the investigator (or designee). A copy of the signed consent form will be given to each participant. The original signed consent form for each participant will be kept in the participant's study file. The consent form must include each of the basic and additional elements of informed consent described in 21 CFR Part 50.25 and must describe each of the risks or discomforts to the participant that have been identified by CONRAD as reasonably foreseeable. CONRAD will provide a sample consent form that meets these requirements (Appendix VII). If the investigator revises the sample consent form or develops a new one, the new or revised consent form must be submitted to CONRAD for review at least one week before it is submitted to the local IRB.

C. Subject Confidentiality
The confidentiality of all subjects enrolled into this study will be protected to the fullest extent possible. Subjects' clinic records may be audited by CONRAD staff, U.S. FDA personnel, or other individuals authorized in writing by CONRAD to audit the study. However, study subjects should not be identified by name on any Case Record Form or on any other documentation sent to CONRAD and will not be reported by name in any report or publication resulting from data collected in this study.

XIX. ADDITIONAL INVESTIGATOR RESPONSIBILITIES
In addition to the previously described responsibilities, the principal investigator is responsible for signing and dating the cover page of this study protocol. The signed and dated original must be submitted to CONRAD, and a copy must be maintained by the principal investigator at the site with the study files.
All protocol amendments must be signed and dated by the principal investigator. The signed and dated original must be submitted to CONRAD, and a copy must be maintained by the principal investigator at the site. Amendments must be approved by CONRAD and the IRB before implementation.
The principal investigator will provide CONRAD with a Curriculum Vitae (CV) for herself/himself showing the education, training, and experience that qualifies her or him as an expert in the area of clinical investigation specific to the product under investigation and her/his affiliation with the site at which the study is being conducted. CVs also must be provided for all individuals listed as subinvestigators on the 1572 form showing the education, training, and experience that qualify them for their role in the study, and their affiliation with the study site.
The principal investigator will supply CONRAD with copies of the lab director's CV, current license and/or laboratory certification (such as the Clinical Laboratory Improvement Act [CLIA] certification) of any laboratory used for the study. The principal investigator is responsible for obtaining any updates to these documents and sending them to CONRAD in a timely fashion. This includes documentation of the normal ranges of the laboratory tests used by the laboratory. The principal investigator will ensure that appropriate health care or referral is provided for the study participants throughout the study. Copies of all these documents must be maintained at the site.
The principal investigator will follow guidelines set forth in the Declaration of Helsinki and will follow U.S. Department of Health and Human Services regulations regarding the Health Information Portability and Accountability Act (HIPAA -45, CFR 164). In addition, the principal investigator should follow Good Clinical Practice (GCP) guidelines.
Throughout the course of the study, the principal investigator will prepare and submit to CONRAD whatever reports are required and detailed in the Subcontract Agreement to be signed prior to initiating the study. These reports summarize the accomplishments of the assignment and usually include the following: • The total number of participants admitted; • The number of and reasons for discontinuations due to adverse events; Pharmacokinetic Study of Tenofovir Gel: Protocol #A04-095 June 18, 2007 FINAL Version 4.0 • A description of all serious adverse events; • The number of participants completing the study; • All changes in the research activity; and • All unanticipated problems involving risks to participants or others.
The final technical report will be submitted within 45 days after completion of the study unless this period is extended in writing by the CONRAD Clinical Director.

IF YOU HAVE A MEDICAL EMERGENCY
Seek emergency care immediately.
Ask your emergency provider to contact the study clinician at the phone number listed above. 3. Make sure the plunger is pushed all the way in. Attach the applicator to the tube by screwing it onto the threaded end.
4. Starting from the bottom of the tube, squeeze the tenofovir gel out of the tube until the plunger is fully extended (it will not come all the way out).
5. Unscrew the applicator from the tube and set the used tube aside.
6. Stand, sit, or lie down in whatever position you find most comfortable for inserting tampons or other vaginal products.
7. Hold the applicator in the middle and insert the open end of the applicator into your vagina until your fingers touch your vagina.
If the applicator does not go in easily, change the angle of insertion, or lubricate the tip with a small amount of tenofovir gel. Do not use any other lubricant. Do not push the applicator in against resistance.
8. Once the applicator is inserted, push the plunger all the way in to dispense the gel.
9. Remove the applicator and throw it away.
10. Put the white plastic cap back on the tube. Put the tube in the zip-lock bag provided for used tubes.
11. Please remember to bring your entire box of study supplies including all used and unused gel tubes to your next visit.

HOW TO FILL OUT YOUR DIARY CARD (ONCE-DAILY DOSING)
Each reporting day begins at 12:00am (midnight) and ends at 11:59pm. Please insert the tenofovir gel in the morning, at approximately the same time each morning.
Fill out one row -in blue or black ink -each day (every day) that you use the gel. You will fill out only the first AM/PM box as you will only insert the tenofovir gel once a day. Always write the date and time you actually insert the gel. If you forget to insert the gel at your normal time, even if you forget until the next day, insert it as soon as your remember. HOWEVER, if you remember when there is only 6 hours until you are supposed to insert the next dose, skip the forgotten dose and continue with your regular schedule. If you miss two or more doses, contact the clinic immediately to let them know.
Please use the Symptoms and Medication section (on the back of your Diary Card) to record any medications you use while you are in the study. Also, you will record in this section your experience with tenofovir gel use. This includes any itching, burning, irritation, pain, discharge, etc. you experience when using the gel.
When you fill out the Symptom column, please BRIEFLY describe the symptom and your experience. When reporting on possible symptoms in your genital area, please refer to Figure A below to explain where you experience the symptom. All of your comments are very important to us.
If you experience any vaginal spotting or bleeding or have any moderate symptoms (where you cannot perform some of your normal activities) or severe symptoms (where you cannot perform most or any of your normal activities), please call the study clinic immediately to schedule a visit. If it is an emergency, seek emergency care immediately, and as your emergency care provider to contact the clinic.
We will review your Diary Card with you at each visit. If you have any questions at any other time, please feel free to call the study clinic.

HOW TO FILL OUT YOUR DIARY CARD (TWICE-DAILY DOSING)
Each reporting day begins at 12:00am (midnight) and ends at 11:59pm. Please insert the tenofovir gel once in the morning and once in the evening, at approximately the same times each day.
Fill out one row -in blue or black ink -each day (every day) that you use the gel. You will fill out two AM/PM columns each day, one for the morning dose and one for the evening dose. The third column is in case you insert an evening dose after midnight. (Example: Let's say you usually insert the gel at 9am and 9pm every day. On 7/15/06, you insert the morning dose at 9am, so you fill in the first column. Then you forget to insert your evening dose until 12:01am (after midnight). Now you'd go to the next line for 7/16/06 and fill in 12:01am in the first column, 9am in the second column and 9pm in the third column.) REMEMBER: always write the date and time you actually inserted the gel. If you forget to insert the gel at your normal time, even if you forget until the next day, insert it as soon as your remember. HOWEVER, if you remember when there is only 6 hours until you are supposed to insert it the next time, skip the forgotten dose and continue with your regular schedule. If you miss two or more doses, contact the clinic immediately to let them know.
Please use the Symptoms and Medication section (on the back of your Diary Card) to record any medications you use while you are in the study. Also, you will record in this section your experience with tenofovir gel use. This includes any itching, burning, irritation, pain, discharge, etc you experience when using the gel.
When you fill out the Symptom column, please BRIEFLY describe the symptom and your experience. When reporting on possible symptoms in your genital area, please refer to Figure A below to explain where you experience the symptom. All of your comments are very important to us.
If you experience any moderate symptoms (where you cannot perform some of your normal activities) or severe symptoms (where you cannot perform most or any of your normal activities) including vaginal burning, itching, pain, spotting or bleeding, please call the study clinic immediately to schedule a visit. If it is an emergency, seek emergency care immediately, and as your emergency care provider to contact the clinic.
We will review your Diary Card with you at each visit. If you have any questions at any other time, please feel free to call the study clinic.

POST-BIOPSY INSTRUCTIONS
For ONE WEEK following the biopsy, do not: • Douche • Masturbate • Use tampons • Have vaginal or anal sex • Put anything in your vagina, including fingers, sex toys or any other objects After your vaginal biopsy, feel free to rest for a few minutes before going home.
It is normal to have some mild pain or spotting for several days after your procedure. Dark/blackcolored discharge may also be normal if you had medication applied to your vagina to control bleeding. You may wear a sanitary pad for any bleeding.
You may take a pain reliever for mild pain as recommended by your study clinician. Be sure to take only recommended medications.
You may immediately resume your normal diet.
Notify your study clinician immediately if you have any of the following: • Bleeding (if more than spotting) • Foul-smelling discharge from your vagina • Fever and/or chills • Severe lower abdominal pain Your study clinician may give you additional or alternate instructions after the procedure, depending on your particular situation.

AFTER VISIT 2 (ENROLLMENT VISIT -part 2)
Today, you completed the last part of Visit 2.
Your next visit, Visit 3, will begin the two-week phase and should be scheduled at least one month from today when you are not expected to menstruate for at least the next 15 days: Date: _________________________________________ Time:___________________ Between today and your next visit: • You will have about a one month "wash-out" period • For at least ONE WEEK after this visit, follow Post-biopsy Instructions.
• You may continue normal sexual activity from the period that lasts from one week after this visit, until 72 hours before your next visit, unless you are experiencing severe abdominal pain, bleeding (if more than spotting), foul-smelling discharge, or fever and/or chills.
• Starting 72 hours before your next visit, please do not: At your next visit, you will: • Give a urine specimen • Be asked if you had any health problems since your last visit • Have a pelvic exam • Be assigned to use the tenofovir gel either once or twice daily for two (2)

AFTER VISIT 3 (BEGINNING OF PHASE 2)
Today, you completed Visit 3, and started the second study phase.
Your next visit will be your One-week Follow Up (in one week): Date: _________________________________________ Time:___________________ Between today and your next visit: • Depending on which group you were assigned, follow the appropriate instructions to insert tenofovir gel ____________ a day, every day at about the same time(s). • Some instructions to keep in mind: Empty your bladder and bowels before you insert each dose and try not to empty them again for at least one hour after you insert each dose. Walk around for at least 15 minutes after each dose If you miss a dose, insert it as soon as you remember and then resume your regular schedule. However, if you remember when it is 6 hours or less before your next scheduled dose, then skip the forgotten dose and resume your regular schedule. If you miss two or more doses, call the study site for further instructions.
• After you insert the gel each time, fill out your Diary Card with the information required.
Also record any symptoms and medications on the back of the Diary Card. At your next visit, you will: • Give a urine specimen • Review your Diary Card with the clinic staff and be asked if you had any health problems since your last visit • Return your used gel tubes from the past week.

AFTER VISIT 4 (ONE-WEEK FOLLOW UP)
Today, you completed Visit 4, your One-week Follow Up.
Your next visit will be your Two-week Follow Up (in one week): Date: _________________________________________ Time:___________________ Between today and your next visit: • Depending on which group you were assigned, continue using the tenofovir gel as often as you did last week, ____________ a day, every day.
• After you insert the gel each time, fill out your Diary Card with the information required.
Also record any symptoms and medications on the back of the Diary Card.
• Until the end of the study, please do NOT: At your next visit, you will: • Give a urine specimen • Review your Diary Card with the clinic staff and be asked if you had any health problems since your last visit. Your Diary Card will be collected but you will be given a copy of the back of it, to record any symptoms and medications until the end of the study. • Return your used gel tubes from the past week.
• Have a pelvic exam • Have a blood sample taken before you insert the final dose of gel • Stay at the clinic for 8 hours for repeated blood draws at different times • Provide vaginal fluids and biopsy samples if you were selected for the 4-hour or 8-hour groups Bring this guidebook and your entire box of study supplies with you to your next visit. • Give a urine specimen • Review the copy of your Symptoms and Medications form with the clinic staff and be asked if you had any health problems since your last visit. The copy of your Symptoms and Medications portion of your diary card will be collected at this visit. • Have a pelvic exam Be exited from the study -THANK YOU! Bring this guidebook with you to your last visit.

If you ARE participating in the substudy:
You will be given a Substudy Participant Guidebook today (Visit 5)

IF YOU HAVE A MEDICAL EMERGENCY
Seek emergency care immediately.
Ask your emergency provider to contact the study clinician at the phone number listed above. 8. Once the applicator is inserted, push the plunger all the way in to dispense the gel.
9. Remove the applicator and throw it away.
10. Put the white plastic cap back on the tube. Put the tube in the zip-lock bag provided for used tubes.
11. Please remember to bring all study supplies, including your used gel tube, to your next appointment.

INSTRUCTIONS:
1. If you have any symptoms that occur during the study, please record them below.
2. If you take any medication to treat your symptoms, please record the type of medication below.

HOW TO FILL OUT YOUR DIARY CARD
The reporting day begins at 12:00am (midnight) and ends at 11:59pm.
Using blue or black ink, fill out the date and time that you actually inserted the gel in the evening. (This should be the same day you received the gel, and approximately 12 hours before your next scheduled appointment.) If you forget to insert the gel until after 2 hours after the assigned time, call the clinic immediately for further instructions.
Please use the Symptoms and Medication section of your Substudy Diary Card to record any medications you use while you are in the study. Also, you will record in this section your experience with tenofovir gel use. This includes any itching, burning, irritation, pain, discharge, etc. you experience when using the gel.
When you fill out the Symptom column, please BRIEFLY describe the symptom and your experience. When reporting on possible symptoms in your genital area, please refer to Figure A below to explain where you are experiencing the symptom. All of your comments are very important to us.
If you experience moderate symptoms (where you cannot perform some of your normal activities) or severe symptoms (where you cannot perform most or any of your normal activities) including vaginal burning, itching, pain, spotting or bleeding at any time during product usage, please call the study clinic immediately to schedule a visit. If it is an emergency, seek emergency care immediately, and as your emergency care provider to contact the clinic.
We will review your Diary Card with you at the next visit. If you have any questions at any other time, please feel free to call the study clinic.

POST-BIOPSY INSTRUCTIONS
For ONE WEEK following the biopsy, do not: • Douche • Masturbate • Use tampons • Have vaginal or anal sex • Put anything in your vagina, including fingers, sex toys or any other objects After your vaginal biopsy, feel free to rest for a few minutes before going home.
It is normal to have some mild pain or spotting for several days after your procedure. Dark/blackcolored discharge may also be normal if you had medication applied to your vagina to control bleeding. You may wear a sanitary pad for any bleeding.
You may take a pain reliever for mild pain as recommended by your study clinician. Be sure to take only recommended medications.

You may immediately resume your normal diet
Notify your study clinician immediately if you have any of the following: • Bleeding (if more than spotting) • Foul-smelling discharge from your vagina • Fever and/or chills • Severe lower abdominal pain Your study clinician may give you additional or alternate instructions after the procedure, depending on your particular situation.

AFTER VISIT 5 OF THE MAIN STUDY
Today, you completed the main study. You have volunteered to participate in the Substudy.
Your next visit, Substudy Visit 1, will begin the third and last phase and should be scheduled at least one month from today when you are not menstruating, ideally between days 19-24 of your cycle: Date: _________________________________________ Time:___________________ Between today and your next visit: • You will have about a one month "wash-out" period • For at least ONE WEEK after this visit, follow Post-biopsy Instructions.
• You may continue normal sexual activity from the period that lasts from one week after this visit, until 72 hours before your next visit, unless you are experiencing severe abdominal pain, bleeding (if more than spotting), foul-smelling discharge, or fever and/or chills.
• Starting 72 hours before your next visit, please do not: • You will insert the single dose of tenofovir gel this evening at _________ PM (approximately 12 hours before your scheduled visit time tomorrow).
• Some instructions to keep in mind: Empty your bladder and bowels before you insert each dose and try not to empty them for at least one hour after you insert each dose. Walk around for at least 15 minutes after each dose • After you insert the gel, fill out your Substudy Diary Card with the information required. Also record any symptoms and medications on the bottom portion of the Diary Card.
• Until the end of the study, please do not: At your next visit, you will: • Give a urine specimen • Review your Diary Card with the clinic staff and be asked if you had any health problems since your last visit. Your diary card will be collected, but you will receive a copy of it to record any symptoms and medications until the end of the study. • Have a pelvic exam • Provide blood, vaginal, and biopsy samples 12 hours after your evening dose Bring this guidebook and your used gel tube with you to your next visit.
Tenofovir levels (see appendix V) • Blood Plasma assay IV Antibody/Antigen testing • HIV • Hepatitis (HBsAg) * A full CBC with differential or metabolic panel with additional blood parameter may be drawn depending on the laboratory panels available at the sites. However, only blood parameters indicated on this form will be used for entry criteria and for grading of adverse events.

Blood plasma:
Blood for PK analysis will be collected at the appointed times. A Vacutainer Access Device which locks into a heparin lock may be used for the first seven blood collections baseline and (0.5, 1, 2, 4, 6, and 8 hours) after the single dose and after two weeks of dosing in order to facilitate sample collection. Approximately 0.5 mL of blood should be removed prior to drawing a sample into the vacutainer. This Vacutainer Access Device will be removed after the 8 hour draw and the 7 th blood collection will be obtained at 24 hours. For each blood sample the following procedure applies: • Collect 3 ml of whole blood in a purple top tube with K-EDTA.
• Within 30 minutes of collection (or up to 1 hour on ice) spin whole blood at 4º C @ 3000 RPM x 10 minutes • Pipette 1mL of plasma into a labeled cryogenic vial 1 • Cap vial and freeze at -80º C.
• The specimens will be stored at the site pending shipment in batches on dry ice to the central laboratory (UNC).

Cervicovaginal fluid:
Direct aspirates of cervicovaginal fluid pooling will be collected at the enrollment visit, one week follow-up visit and two week follow-up visit. For each cervicovaginal fluid sample the following procedure applies: • Use vaginal aspirator syringes (2.5 mL) supplied by laboratory to collect direct aspirates.
• For maximum volume collection, the study subject should be supine for 15 minutes prior to collection.
• Place fluid into a labeled cryogenic vial.
• Cap vial and freeze at -80º C.
• Store at the site pending shipment in batches on dry ice to the central laboratory (UNC).

Cytobrush specimens:
Cytobrush specimens will be collected at the enrollment visit, the one week follow-up visit and the two week follow-up visit. These samples will be stored, frozen, and analyzed by Gilead if vaginal mononuclear cells are of a sufficient quantity to measure intracellular tenofovir diphosphate. For each cytobrush sample the following procedure applies: • After sample collection, place cytobrush in 5 mL of phosphate-buffered saline (PBS) solution in a 15 mL falcon tube.
• Vigorously mix the tube and brush to remove all of the cells from the cytobrush.
• Remove the cytobrush from the tube and spin the falcon tube at 4º C @ 400 RCF x 15 minutes.
Pharmacokinetic Study of Tenofovir Gel: Protocol #A04-095 June 18, 2007 FINAL Version 4.0 • Carefully pour off or pipette and discard the supernatant, being careful not to disturb the cell pellet.
• Resuspend the pellet in 1 mL PBS with gentle agitation.
• Mix 10 uL of cell suspension sample with 10 uL of 0.4% Trypan Blue Stain in a small conical tube to count mononuclear cells. See box below for instructions.
• Spin the remainder of the 1 mL sample at 4º C @ 400 RCF x 15 minutes.
• Carefully pour or pipette off the supernatant and discard, being careful not to disturb the cell pellet.
• Add 1.0 mL of well-mixed, ice cold 70% methanol solution (prepared by separately measuring with graduated cylinders 70 mL of MeOH with 30 mL of H2O) to the 15 mL falcon tube containing the pellet. Vortex lightly to lyse the cells.
• The specimens will be stored at -80 º C at the site pending shipment in batches on dry ice to the central laboratory (Gilead).
• Use a Cell Counting Chamber or hemocytometer to count the number of viable mononuclear cells per ml of suspension. Viable cells exclude Trypan Blue and remain clear under microscopy, while dead cells stain blue due to Trypan blue uptake. Mononuclear cells appear small and round under the microscope. Do not count red blood cells or epithelial cells which will appear larger with distinctive margins.
• To prepare the counting chamber, carefully clean the mirror-like polished surface and coverslip with lens paper. Place the coverslip over the counting surface prior to putting on the cell suspension.
• Introduce about 10 uL of the Trypan blue-cell mixture into one of the V-shaped wells with a pasteur pipette. The area under the coverslip will fill by capillary action. Introduce enough liquid so that the mirrored surface is just covered. Place the charged counting chamber on the microscope stage and focus the counting grid at low power.
• One entire grid on standard hemocytometers with Neubauer rulings can be seen at 40x (4x objective). The main divisions separate the grid into 9 large squares (like a tic-tac-toe grid). Each square has a surface area of one square mm, and the depth of the chamber is 0.1 mm. The entire counting grid lies under a volume of 0.9 mm-cubed.
• To estimate the total number of viable mononuclear cells/ml (T and B lymphocytes) of the suspension, systematically count the four large corner squares. For cells that overlap a ruling, count a cell as "in" if it overlaps the top or right ruling, and "out" if it overlaps the bottom or left ruling.
• To get the final count in cells/ml, multiply this total count by 10,000. Then divide this result by 4 (the total surface area counted). For example suppose you count 100 cells (total) in the four large corner squares. This is the number of cells/mL (eg. 100 cells x 10,000 ÷ 4 = 250,000 cells/mL).
• Finally, multiply the number above by 2 to get the total number of cells (since you diluted the cells 1:1 with Trypan blue before counting). In this example, 250,000 cells/mL x 2 = 500,000 cells/mL (or 500,000 cells total, since you are working with 1mL of cell suspension). A summary of this equation is below:

Vaginal Biopsies:
Biopsies will be collected at the enrollment visit and two-week follow-up visit and will be stored under specified conditions pending shipment in batches on dry ice to the central laboratory. For each biopsy sample the following procedure applies: • Weigh labeled cryogenic vial prior to specimen placement.
• Place biopsy specimen in vial and weigh biopsy and vial.
• Snap freeze either in a liquid nitrogen bath or in an ethanol or acetone bath containing dry ice.
• The specimens will be stored at the site pending shipment in batches on dry ice to the central laboratory (UNC).

General Instructions Estimating Severity Grade
If the need arises to grade a clinical AE that is not identified in the DAIDS AE grading table, use the category "Estimating Severity Grade" located at the top of Page 3. For AEs that are not listed in the table but will be collected systematically for a study/trial, protocol teams are highly encouraged to define study-specific severity scales within the protocol or an appendix to the protocol. (Please see "Template Wording for the Expedited Adverse Event Reporting Section of DAIDS-sponsored Protocols".) This is particularly important for laboratory values because the "Estimating Severity Grade" category only applies to clinical symptoms.

Grading Adult and Pediatric AEs
The DAIDS AE grading table includes parameters for grading both Adult and Pediatric AEs. When a single set of parameters is not appropriate for grading specific types of AEs for both Adult and Pediatric populations, separate sets of parameters for Adult and/or Pediatric populations (with specified respective age ranges) are given in the table. If there is no distinction in the table between Adult and Pediatric values for a type of AE, then the single set of parameters listed is to be used for grading the severity of both Adult and Pediatric events of that type.

Determining Severity Grade
If the severity of an AE could fall under either one of two grades (e.g., the severity of an AE could be either Grade 2 or Grade 3), select the higher of the two grades for the AE.

Basic Self-care Functions Adult
Activities such as bathing, dressing, toileting, transfer/movement, continence, and feeding.

Young Children
Activities that are age and culturally appropriate (e.g., feeding self with culturally appropriate eating implement). Life-threatening reduction in oral intake Basic Self-care Functions -Adult: Activities such as bathing, dressing, toileting, transfer/movement, continence, and feeding.

LLN Lower limit of normal
Basic Self-care Functions -Young Children: Activities that are age and culturally appropriate (e.g., feeding self with culturally appropriate eating implement).

Usual Social & Functional Activities -Adult:
Adaptive tasks and desirable activities, such as going to work, shopping, cooking, use of transportation, pursuing a hobby, etc.
Usual Social & Functional Activities -Young Children: Activities that are age and culturally appropriate (e.g., social interactions, play activities, learning tasks, etc. Life-threatening consequences (e.g., hypotensive shock) Basic Self-care Functions -Adult: Activities such as bathing, dressing, toileting, transfer/movement, continence, and feeding.
Basic Self-care Functions -Young Children: Activities that are age and culturally appropriate (e.g., feeding self with culturally appropriate eating implement).

Usual Social & Functional Activities -Adult:
Adaptive tasks and desirable activities, such as going to work, shopping, cooking, use of transportation, pursuing a hobby, etc.
Usual Social & Functional Activities -Young Children: Activities that are age and culturally appropriate (e.g., social interactions, play activities, learning tasks, etc. Cerebral vascular accident (CVA, stroke) with neurological deficit Basic Self-care Functions -Adult: Activities such as bathing, dressing, toileting, transfer/movement, continence, and feeding.
Basic Self-care Functions -Young Children: Activities that are age and culturally appropriate (e.g., feeding self with culturally appropriate eating implement).

Usual Social & Functional Activities -Adult:
Adaptive tasks and desirable activities, such as going to work, shopping, cooking, use of transportation, pursuing a hobby, etc.
Usual Social & Functional Activities -Young Children: Activities that are age and culturally appropriate (e.g., social interactions, play activities, learning tasks, etc. Symptoms causing inability to perform basic self-care functions OR Hospitalization indicated (other than emergency room visit) OR Headache with significant impairment of alertness or other neurologic function Insomnia Basic Self-care Functions -Adult: Activities such as bathing, dressing, toileting, transfer/movement, continence, and feeding.
Basic Self-care Functions -Young Children: Activities that are age and culturally appropriate (e.g., feeding self with culturally appropriate eating implement).

Usual Social & Functional Activities -Adult:
Adaptive tasks and desirable activities, such as going to work, shopping, cooking, use of transportation, pursuing a hobby, etc.
Usual Social & Functional Activities -Young Children: Activities that are age and culturally appropriate (e.g., social interactions, play activities, learning tasks, etc. Dyspnea or respiratory distress Basic Self-care Functions -Adult: Activities such as bathing, dressing, toileting, transfer/movement, continence, and feeding. Basic Self-care Functions -Young Children: Activities that are age and culturally appropriate (e.g., feeding self with culturally appropriate eating implement).

Usual Social & Functional Activities -Adult:
Adaptive tasks and desirable activities, such as going to work, shopping, cooking, use of transportation, pursuing a hobby, etc.
Usual Social & Functional Activities -Young Children: Activities that are age and culturally appropriate (e.g., social interactions, play activities, learning tasks, etc. Disabling bone pain with radiographic findings causing inability to perform basic self-care functions Basic Self-care Functions -Adult: Activities such as bathing, dressing, toileting, transfer/movement, continence, and feeding. Basic Self-care Functions -Young Children: Activities that are age and culturally appropriate (e.g., feeding self with culturally appropriate eating implement).

Usual Social & Functional Activities -Adult:
Adaptive tasks and desirable activities, such as going to work, shopping, cooking, use of transportation, pursuing a hobby, etc.
Usual Social & Functional Activities -Young Children: Activities that are age and culturally appropriate (e.g., social interactions, play activities, learning tasks, etc.     over the next 24 hours. You will also be randomly assigned (like the flip of a coin) to one of seven different time groups for the collection of samples of vaginal fluids, cells and tissues after using a single dose of tenofovir gel. Then there will be a rest period of about one month. For the second part of the study, you will be randomly assigned (like the flip of a coin) to use the gel either once a day or twice a day for two weeks. After one week of gel use, you will provide vaginal and cervical samples. After two weeks of gel use, you will provide blood samples over the next 24 hours and there will be 3 different time groups that you will be randomly assigned (like the flip of a coin) to for the collection of samples of vaginal fluids, cells and tissues. You cannot choose your groups, and the study staff cannot choose your groups for you. You have an equal chance of being placed in each one of the groups. If enrolled, you would have at least 6 study visits and your participation in this study would last about 4 months.
Up to 10 participants at one of the sites, who have completed the first two phases of the study, will be asked to participate in a third phase to have blood samples, vaginal fluids, cells and tissues collected 12 hours after a single dose of tenofovir gel.

Exclusions
You will not be allowed to participate in the study if you: • Have irregular menstrual cycles • Are pregnant, are less than 2 months from your last pregnancy or are breastfeeding • Are positive for HIV infection or Hepatitis B • Have gonorrhea, Chlamydia or Trichomonas infection • Have a history of abnormal Pap smear that has not been evaluated and treated, if indicated, according to standard guidelines • Have used tenofovir or other anti-HIV medication • Are currently or have recently used drugs or alcohol • Are using or plan to use on a daily basis medications that may affect your kidneys or liver (like acyclovir, ibuprofen or tylenol) • Have used hormonal contraceptives in the month prior to enrollment or Depo-Provera in the four (4) months prior to enrollment.
• Have any abnormal findings on laboratory tests or during physical examination or a medical condition which, in the opinion of the study investigator, would make the study unsafe for you or would make it hard for the researchers to interpret the results of the study • Have participated in any other investigational trial within 30 days prior to screening.
While in this study, you must be protected from pregnancy. Acceptable methods of birth control while in the study include: non-hormonal intrauterine device (IUD), female sterilization (tubes tied), vasectomized partner, effective barrier method or abstinence (no intercourse). You will also be required not to have intercourse or any vaginal activity during the study phases. However, you will be allowed to continue your normal sexual activities between the study phases.

Study Procedures
Visit 1 (Screening) Procedures to determine if you are eligible to take part in a research study are called "screening procedures". For this research study, the screening procedures include the following: 1. The study procedures and potential risks will be explained to you. You will read the consent form. After all of your questions have been answered, you will decide if you want to participate in the study. If you decide to participate, you will sign the consent form and you will receive a copy of the signed consent form.
2. Once you have signed the consent form, you will be asked questions to collect medical information to make certain you are eligible for the study. You will also be asked questions about your background (i.e., age, education, etc.) as well as questions about your sexual history and use of birth control methods.
3. Your height and weight will be measured and recorded. 5. You will be counseled about HIV testing.
6. About six teaspoons of your blood will be taken from your arm for laboratory tests of your serum chemistries, blood sugar, complete blood counts, hepatitis B and HIV testing. These tests are being done to make sure that these blood tests are normal at the start of the study.
7. You will be given a pelvic examination. o During the pelvic exam, the study clinician will use a speculum (an instrument placed into your vagina and expanded), to help the clinician see your vagina and cervix (the opening to the uterus or womb). o The clinician will use a cotton swab ("Q-tip") to take fluid from your vagina and cervix. Tests for gonorrhea, Chlamydia and vaginal infections (yeast, trichomonas and bacterial vaginosis) will be done. o An examination of your internal pelvic organs will be performed by the clinician who will place one or two gloved fingers into your vagina while gently pressing on the abdomen with the other hand. o If you have an infection with yeast or bacterial vaginosis, you will get a prescription for treatment. You will be eligible to participate in the study once you have finished the medication. o If you test positive for Trichomonas you will receive a prescription for treatment, but you are not eligible to participate in the study.
8. If you are eligible to participate in the study, there will be some limits on you that will begin 72 hours before the next visit and continue until the end of the first part of the study. You will also have these same limits 72 hours before Visit 3 that will continue until after Visit 6 (the Final Visit). Please do not: 1. This visit should take place within 2 months of your screening visit and will be scheduled when you are not menstruating. Your test results from the previous visit will be reviewed. You will not be enrolled in the study if the tests of your blood chemistries, liver and kidney functions, blood sugar, blood counts, or tests for gonorrhea, Chlamydia are abnormal or if you are positive for HIV or Hepatitis B. You will be told to see your own doctor for treatment. If you do not have a place to go for treatment, the research staff will help you find a doctor.
2. Although it is unlikely that you will have tenofovir in your blood, a blood sample will be drawn to make sure that you start the study without tenofovir in your blood. Research staff may insert one needle in your arm that will most likely remain for the next six blood collections to reduce the number of times that you will have the needle inserted.
3. A urine sample will be collected to test for pregnancy. o If your pregnancy test is positive, you will not be enrolled in the study. o If you have symptoms of a bladder infection, the urine sample will be tested for infection.
4. If you have vaginal or genital symptoms or an abnormal exam, you may be tested for gonorrhea, Chlamydia, vaginal infections, syphilis, and/or herpes.

5.
A pelvic examination will be performed in which the vulva, vagina and cervix will be examined by the clinician for cuts in the skin. If a deep cut is seen, you will not be enrolled until it has healed.
6. If you are eligible for the study, you will be enrolled and randomly assigned to one of seven time-points for collection of specimens from your cervix and vagina. You will be asked to empty your bladder and bowels and then will be given the first dose to insert in the clinic. After inserting the gel, you will be asked to walk around the examination room for 15 minutes to help spread the gel, after which time you may walk around as you like.
7. You will be asked to try not to empty your bladder or bowel within the first hour after application.
8. Research staff will collect your blood 7 times (at 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours and 24 hours after you spread the gel) to measure how much, if any, tenofovir gel was absorbed into your blood. You will stay in the clinic until after your 8 hour blood sample is collected.
9. You will return the next morning for the 24 hour blood sample collection, as well as your 24 hour vaginal biopsy (sample of your vaginal tissue) if you were assigned to the 24 hour group. At that time you will be asked if you had any health problems since you left the research center.
10. You will undergo pelvic examination procedures and collection of vaginal samples at your assigned time (this may be when you return the next morning if you were assigned to the 24 hour group). The study clinician will take fluid from the back of your vagina and will brush the endocervix (opening of your cervix) to obtain specimens which will be evaluated to see how much, if any, tenofovir gel is detected in the vaginal fluid and in the endocervix. The study clinician will gently wipe a small area of your vagina with a small cotton swab of warm saline, and then again with a cotton swab of antiseptic to clean it. The clinician will also apply a numbing gel to the area. The study clinician will then remove two small biopsies, about the size of a grain of rice. These samples will be stored until they are evaluated to see how much, if any, tenofovir can be detected in the tissue.
11. You will be told to avoid sexual activity, douching or tampon use for at least one week following the biopsy to allow time for the biopsy sites to heal.
12. You will receive a Participant Guidebook that will give detailed instructions on how to insert the tenofovir, as well as what you can expect during each visit 13. Your next visit will be in approximately one month. There will be some limits on you that will begin 72 hours before the next visit and continue until the end of the study. 4. A blood sample will be drawn to measure tenofovir in your blood.

5.
A pelvic examination will be performed in which the vulva, vagina and cervix will be examined by the clinician for cuts in the skin and to make sure the biopsy areas have healed. If a deep cut is seen, you will not continue in the study until it has healed.
6. You will be randomly assigned to either once a day or twice a day gel use and will receive study supplies. If you are assigned to once-daily use, you will be instructed to insert the gel in the morning at approximately the same time every day, starting the following day. If you are assigned to twice-daily use, you will be instructed to insert the gel in the morning and evening at approximately the same time every day, starting the following day. In addition, you will be asked to walk around for at least 15 minutes after insertion of the gel and to try not to empty your bladder or bowels within the first hour after application.
7. You will be given a diary card and instructions. You will record the time(s) that you apply the gel each day. If you experience any burning, irritation, pain, itching, etc. while using the gel, you will record that information on the diary card. If you experience any moderate symptoms (you have to stop some normal activities) or severe symptoms (you are unable to perform most normal activities) please call the clinic. If you have these symptoms at any time during this study you may undergo evaluation including laboratory testing, physical examination, and/or visual inspection of your genital area with a colposcope (magnifying lens) as the clinician determines. You will also record any medication that you use while you are in the study on the diary card.
8. Your next visit will be scheduled in one week. You will be told to bring all used gel tubes to this visit. You will be asked to wait until your visit to the research center to insert your morning dose for that day.
Visit 4 (One Week follow-up) 3. The study staff will review your diary card and you will be asked if you had any medical problems since your last visit.

4.
A blood sample will be taken to measure how much, if any, tenofovir gel was absorbed into your blood after which you will insert the gel.
5. You will undergo pelvic examination procedures and collection of vaginal samples 4 hours after you insert the gel. The study clinician will take fluid from the back of your vagina and will brush the endocervix (opening of your cervix) to obtain samples which will be evaluated to see how much, if any, tenofovir can be detected in the vaginal fluid and the endocervix.
6. You will be instructed to continue using the study gel as assigned and will be scheduled for your two week follow-up visit. You will be told to bring all used and unused gel tubes to this visit. You will be asked to wait until your visit to the research center to insert your final dose.
Visit 5 (Two Week follow-up) 3. You will be randomly assigned to one of three times (either 4, 8 or 24 hours after you insert the gel), and at your assigned time you will undergo pelvic examination procedures and collection of vaginal samples.
4. The study staff will review and collect your diary card and you will be asked if you had any medical problems since your last visit. If you are not participating in the substudy, you will be given a copy of the back of your diary card to record any symptoms and medications between this visit and Visit 6.

5.
A blood sample will be taken to measure how much, if any, tenofovir gel was absorbed into your blood after which you will insert the morning dose of gel. In addition, research staff will collect your blood 7 times (at 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours and 24 hours after you insert the gel) to measure how much, if any, tenofovir gel was absorbed into your blood. Research staff will insert one needle in your arm that will most likely remain for the first six blood collections to reduce the number of times that you will have the needle inserted. Your blood will also be tested for serum chemistries, blood sugar and complete blood counts to make sure that they have not changed since the start of the study.
6. When you return the following morning for the 24 hour blood draw (and your 24 hour biopsy, if you were assigned to the 24 hour time) you will be asked if you had any health problems since you left the research center.
7. At your assigned pelvis examination time (either 4, 8 or 24 hours after you insert the gel), the study clinician will take fluid from the back of your vagina and will brush the endocervix (opening of your cervix) to obtain samples which will be stored and evaluated to see how much, if any, tenofovir can be detected in the vaginal fluid and in the endocervix. The study clinician will gently wipe a small area of your vagina with a small cotton swab of warm saline, and then again with a cotton swab of antiseptic to clean it. The clinician will also apply a numbing gel to the area. The study clinician will then remove two small biopsies, or samples of the vaginal tissue, about the size of a grain of rice. These samples will be stored until they are evaluated to see how much, if any, tenofovir can be detected in the tissue.
8. If you will not be taking part in the substudy, you will be told to avoid sexual activity, douching or tampon use for at least one week following the biopsy to allow time for the biopsy site to heal and will schedule an appointment to return within 1-2 weeks for the final visit.
9. Substudy Participants only: You will not have a Visit 6 in the main study, but instead will be scheduled for Substudy Visit 1. This visit will be scheduled at least 30 days from this visit for when you will not be menstruating and an attempt will be made to schedule the visit in the second half of your menstrual cycle, ideally menstrual cycle day 19-24.
10. You will be told to avoid sexual activity, douching or tampon use for at least one week following the biopsy to allow time for the biopsy sites to heal.
11. You will receive a substudy Participant Guidebook that will give detailed instructions on how to insert the tenofovir, as well as what you can expect during each visit 12. You may continue normal sexual activity from the period that lasts from one week after this visit, until 72 hours before Substudy Visit 1, unless you are experiencing severe abdominal pain, bleeding (if more than spotting), foul-smelling discharge, or fever and/or chills.
13. There will be some limits on you that will begin 72 hours before the next visit and continue until the end of the study. Visit 6 (Final Visit) 1. You will be asked if you had any medical problems since your last visit. The copy of the symptoms and medications portion of your diary card will be collected.

2.
A urine sample will be collected to test for pregnancy o If your pregnancy test is positive, you will be followed until the pregnancy reaches outcome. o If you have symptoms of a bladder infection, the urine sample will be tested for infection 3. If you have vaginal or genital symptoms or abnormal exam, you may be tested for gonorrhea, Chlamydia, vaginal infections, syphilis, and/or herpes.

4.
A pelvic examination will be performed in which your genital area including the vulva, vagina and cervix will be examined by the clinician to make sure the biopsy areas have healed. If a deep cut is seen, you will be followed until it has healed.
5. You will be exited from the study. If you had any medical problems related to the use of the tenofovir gel, the problem(s) will be followed until resolved.

Substudy: 12 hour time-point
Up to 10 participants at the University of Pittsburgh site who have completed the first two phases of the study will be asked to participate in a third phase, a substudy. If you decide to participate, there will be three visits in this phase.
Substudy Visit 1 1. You will be asked if you had any medical problems since your last visit.

2.
A urine sample will be collected to test for pregnancy o If your pregnancy test is positive, you will be followed until the pregnancy reaches outcome. o If you have symptoms of a bladder infection, the urine sample will be tested for infection 3. If you have vaginal or genital symptoms or an abnormal examination, you may be tested for gonorrhea, Chlamydia, vaginal infections, syphilis, and/or herpes.

4.
A pelvic examination will be performed in which your genital area including the vulva, vagina and cervix will be examined by the clinician to make sure the biopsy areas have healed. If a deep cut is seen, you will be rescheduled until the cut has healed.
Pharmacokinetic Study of Tenofovir Gel: Protocol #A04-095 June 18, 2007 FINAL Version 4.0 91 5. A blood sample will be drawn to measure tenofovir in your blood 6. You will be given a single-dose to use at home that evening at a specified time, along with a Substudy Diary Card and instructions. You will be instructed to empty your bladder and bowels prior to inserting the dose. You will insert the dose at home and will walk around for 15 minutes to spread the gel, and then may walk around as you like.
7. You will be scheduled to return to the research clinic the following morning. 3. The study staff will review and collect your diary card and you will be asked if you had any medical problems and/or vaginal activity since your last visit. You will be given a copy of the Diary Card to record any symptoms and medications between this visit and your final substudy visit.
4. Blood samples will be collected approximately 12 hours after you inserted the tenofovir gel the night before to see how much, if any, tenofovir gel was absorbed into your blood.
5. Each participant will also undergo the following pelvic examination procedures at the 12 hour time-point: a. The study clinician will take fluid from the back of your vagina and will brush the endocervix (opening of your cervix) to obtain samples which will be stored and evaluated to see how much, if any, tenofovir can be detected in the vaginal fluid and in the endocervix. The study clinician will gently wipe a small area of your vagina with a small cotton swab of warm saline, and then again with a cotton swab of antiseptic to clean it. The clinician will also apply a numbing gel to the area. The study clinician will then remove two small biopsies, or samples of the vaginal tissue, about the size of a grain of rice. These samples will be stored until they are evaluated to see how much, if any, tenofovir can be detected in the tissue.
6. You will be told to avoid sexual activity, douching or tampon use for at least one week following the biopsy to allow time for the biopsy sites to heal and will schedule an appointment to return within 1-2 weeks for the final visit.
Substudy Visit 3 1. You will be asked if you had any medical problems since your last visit. The copy of your Diary Card will be collected.

2.
A urine sample will be collected to test for pregnancy o If your pregnancy test is positive, you will be followed until the pregnancy reaches outcome. o If you have symptoms of a bladder infection, the urine sample will be tested for infection 3. If you have vaginal or genital symptoms or abnormal exam, you may be tested for gonorrhea, Chlamydia, vaginal infections, syphilis, and/or herpes.

4.
A pelvic examination will be performed in which your genital area including the vulva, vagina and cervix will be examined by the clinician to make sure the biopsy areas have healed. If a deep cut is seen, you will be followed until it has healed.
5. You will be exited from the study. If you had any medical problems related to the use of the tenofovir gel, the problem(s) will be followed until resolved.
Medical records for examinations and/or treatments at this clinic that are related to this study will become part of your study record. Research staff may access your medical records at this clinic for study related information. You may be asked to participate in additional study procedures but you will be asked for your consent at that time. We may try to contact you in person at your home if we are unable to reach you by telephone or mail.

Risks
Risks of the study may include: 1. Genital Exams: You may experience some discomfort or pressure during the exam of your genital area and inside your vagina. You may have mild vaginal spotting/bleeding which should stop shortly after the examination.
2. Blood draw: You may experience temporary discomfort from the needle stick when your blood is taken. You may feel dizzy, faint or lightheaded. You may have bruising, swelling, or infection where the needle goes into your arm.
3. Vaginal Biopsy: You may feel a pinch and/or have some mild pain when the sample is taken. You might also have some bleeding. Bleeding can usually be stopped by applying an iron-containing substance (Monsel's solution) or silver-containing compound (silver nitrate) to the biopsy area which might lead to dark-colored discharge. In rare cases, a stitch might need to be placed to stop the bleeding. You may feel slight soreness or discomfort in your vagina for a day or two after the test. You may experience some vaginal spotting/bleeding and a slight discharge for up to a week after a biopsy. Rarely, a biopsy can cause an infection or prolonged bleeding. If needed, you can use a sanitary pad for the bleeding. You must avoid having sex, douching, or using tampons for at least a week, and avoid rigorous activity for at least a day after your biopsy to allow time for the biopsy site to heal. You will be given an instruction sheet and should notify the study clinician if you experience vaginal bleeding (if more than spotting), foul-smelling vaginal discharge, fever and/or chills, or severe lower abdominal pain.
4. Tenofovir Gel: It is important to use the study gel as instructed by the staff. You will be putting the gel in your vagina. The gel should not be put in the mouth. The gel used in this study may have side effects. We are still learning about the gel and some of the effects are still unknown. Some possible, more common, side effects that you may experience are dryness, burning, itching, pain, etc. of your genital area during the product use. You may also have discharge if the gel comes out of your vagina or you may experience lower abdominal pain. If the symptoms are moderate (you must stop some normal activities) or severe (you are unable to perform most normal activities), you should call the research site and an appointment will be set up, if necessary. If you cannot come to the clinic on the same day, you will be allowed to stop using the product, wash your vagina with plain warm water, apply medication (if needed) and come to the clinic as soon as possible for evaluation. Some women who used the gel had redness and slight bruising of the genital area seen during examination.
It is possible that tenofovir gel could be absorbed from the vagina into the blood. In an earlier study of tenofovir gel in women, a very small amount of tenofovir gel was absorbed from the vagina into the blood in about half of the women tested. If the gel is absorbed into the blood, it is not known whether it will cause any bad effects.
The following side effects have been reported with the use of oral tenofovir pills taken by people who were sick. These side effects may have been because of other medicines that patients were taking or because of their health: • Upset stomach, vomiting, gas, loose or watery stools.
• Lack of energy.
• Kidney damage or failure. • Inflammation or swelling and possible damage to the pancreas.
• Rash • Low phosphate, a chemical in the blood.
• Allergic reaction, which may include fever, rash, upset stomach, vomiting, loose or watery stools, abdominal pain, achiness, shortness of breath or a general feeling of illness. • Decreased bone mineral density.
The study gel used in this study may not protect you against sexually transmitted infections ( with the HIV virus during the study, the study clinician will provide you a referral for counseling and clinical care.
It is not known if tenofovir gel poses a risk to an embryo or fetus. You will be instructed not to have sexual intercourse with women or men, even with a condom, during the study phases. If you do become pregnant while using tenofovir gel, the study clinician will ask for your consent to receive a copy of medical records related to the pregnancy, its outcome and the health of the newborn infant, if applicable. 5. Inconvenience: Participation in the study may cause an inconvenience to your daily schedule because you will need to come to the clinic for at least 6 different visits and 2 additional blood draws.
There may be other risks not yet identified.

Benefits
There are no direct benefits to you for participating in this study. This study will, however, provide information that may lead to the availability of a new vaginal product for prevention of the transmission of HIV.
You will receive pelvic exams and counseling and testing for HIV and STIs. You will also have tests to check the overall health of your liver, kidneys and blood cells. This study will not provide you with medical care, but study staff will refer you to other available sources of care. If any of your tests show anything that is not normal, you will be referred for follow-up care.

Alternative Treatments
You are not participating in this study because you have a condition requiring treatment. Rather, you have volunteered to participate in order for the investigators to learn more about tenofovir gel. You do not have to participate in this study, if you choose not to.

Costs and Payments
There is no cost to you for participating in this study. You will be compensated for travel, parking, time off work, inconvenience, etc as described in the table below. If you leave the study early, you will be paid for each completed visit. The study center will process the request for your payment as quickly as possible, but there may be a delay of two to three weeks after the end of participation before you receive the check.

Visit Time Needed Compensation
The cost of this study, including administrative fees, payments to volunteers, and investigator support for the visits and tests, is being paid for by CONRAD which is receiving financial support from the International Partnership for Microbicides (IPM). CONRAD also receives financial support from the United States Agency for International Development and from private foundations.

New Information
Any new information obtained during the course of this research that may affect your health, welfare and/or willingness to continue participation in the study will be provided to you in a timely manner.

Confidentiality
Study records that identify you will be kept confidential as required by law. Federal Privacy Regulations provide safeguards for privacy, security, and authorized access to your records. Except when required by law, you will not be identified by name, social security number, address, telephone number, or any other direct personal identifier in study records disclosed outside of the research site. For records disclosed outside of the research site, you will be assigned a unique code number. The results of this research may be presented at meetings or in publications. Your identity will not be disclosed in those presentations.
In conducting this research study, members of the research team will report the results of your studyrelated health information, medical history, physical examinations and laboratory tests, as described in this consent, to CONRAD, the manager of the study. This information may be further disclosed to Family Health International (FHI) and other contracted organizations.
All health information will be maintained in strict confidence as required by law. However, your health information may be disclosed if required by law. Once your health information is disclosed for research, such as to the financial sponsor, federal privacy laws may no longer protect the information.
By signing this consent form to participate in the study, you are authorizing the research team to share your health information, as described in this consent form.
You have the right to withdraw your authorization for the sharing of your health information but you must do so in writing. Authorization for sharing data that have already been sent to CONRAD or Family Health International (the Data Coordinating Center) cannot be withdrawn. Your authorization for the sharing of your health information for this study will have no expiration date. You have the right to review your records, once the study has concluded. The study results and informed consent will be retained in your research record for at least six years or until after the study is completed, whichever is longer. At that time either the research information not already in your medical record will be destroyed or information identifying you will be removed from such study results. Any research information in our medical record will become a permanent part of that document and will be kept indefinitely.
Your records may be reviewed and/or copied in order to meet state and/or federal regulations. Reviewers may include, for example, representatives from the Food and Drug Administration, representatives of CONRAD and the Institutional Review Board that oversees this research.
In addition, if your research record is contained in your medical record, you authorize the investigators and representatives from CONRAD to have access to the records.

Ending Your Participation
Your participation is voluntary and you may refuse to participate in or withdraw from the research study at any time. If you choose to withdraw from the study, you must contact the study center, but you may still come to this clinic for health care and will receive exactly the same health care as you would normally receive had you stayed in the study. It may be necessary for research staff to withdraw you from the study if you are unable or unwilling to follow the study instructions, if you develop or are found to have any condition which would make further participation possibly hazardous to your health, or which would interfere with interpretation of the results of the study, or if the investigator determines that continuing in the study would not be in your best interest. If you do withdraw, or are withdrawn, you will be asked to undergo all evaluations necessary for your safety and well-being as determined by clinic staff. You will also be asked to return any unused product and the diary card. At that time we will ask your permission to continue using all information about you that has already been collected as part of the study prior to your withdrawal.

Compensation for Illness or Injury
If you suffer a physical injury or illness as a direct result of participating in this research study, shortterm medical treatment may be provided at no cost to you. No money has been set aside for long-term medical care, lost wages, disability, or pain and/or inconvenience due to a research related injury or illness. However, you do not waive any of your legal rights by signing this consent form.

If You Have a Problem or Question
IF YOU HAVE A MEDICAL EMERGENCY: In the case of a medical emergency, you are advised to call your primary care provider or immediately go to the nearest emergency room. Please report this event to the study center as soon as possible but NOT before you have received the necessary medical care.
IF YOU HAVE A HEALTH PROBLEM: If you have a health problem that does NOT require emergency medical care and you think it might be related to your being in this research study, please call the study center.

Voluntary Consent
I certify that I have read all of this consent form and that I understand it. A copy of this signed consent form will be given to me. My signature below means that I freely agree to participate in this investigational study.

___________________ ___________________________________________ Date
Signature of Participant

___________________________________________ Printed Name of Participant
Investigator's Statement: I certify that I have explained to the above individual the nature and purpose of the study, potential benefits, and possible risks associated with participation in this study. I have answered any questions that have been raised and have witnessed the above signature. I have explained the above to the volunteer on the date stated on this consent form.

Time that gel was inserted
Circle AM or PM for each. Note: Depending on your assigned group, you should only insert the gel once or twice a day. The third column below is in case you insert an evening dose after midnight.