Guidance  for Industry 
 
Vaginal Microbicides: 

Development for the 

Prevention of HIV Infection
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 

Food and Drug Administration 

Center for Drug Evaluation and Research (CDER)
  
 
November 2014 

Clinical/Antimicrobial 
 
 
   
Guidance  for Industry 
 
Vaginal Microbicides: 

Development for the 

Prevention of HIV Infection
 
 
 
 
 
Additional copies are available from: 

 
Office of Communications, Division  of Drug Information 

Center for Drug Evaluation  and Research 

Food and  Drug Administration 
 
10903 New Hampshire Ave., Bldg. 51, rm. 2201 

Silver Spring, MD   20993-0002 

Tel:  301-796-3400; Fax: 301-847-8714; Email:  druginfo@fda.hhs.gov 

http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 

Food and Drug Administration 

Center for Drug Evaluation and Research (CDER)
  
 
November 2014 

Clinical/Antimicrobial 
 
 
   
TABLE OF CONTENTS 
 
 
I.   INTRODUCTION............................................................................................................. 1
  
II.   BACKGROUND ............................................................................................................... 2
  
III.   DEVELOPMENT PROGRAM ....................................................................................... 3
  
A.   General Considerations ................................................................................................................. 3
  
1.  Nonclinical Considerations ............................................................................................................. 3
  
a.   Nonclinical safety...................................................................................................................... 3
  
b.   Nonclinical virology ................................................................................................................. 4
  
c.   Other nonclinical studies........................................................................................................... 7
  
2.  Drug Development Population ........................................................................................................ 8
  
3.  Early Phase Clinical Considerations ............................................................................................... 8
  
a.   Safety and pharmacokinetic considerations .............................................................................. 8
  
b.   Drug product characteristics that affect end-user acceptability ................................................ 9 
 
c.   Dose selection ........................................................................................................................... 9
  
4.  Efficacy Considerations................................................................................................................. 10 
 
5.  Safety Considerations .................................................................................................................... 11 
 
a.   Adequacy of the safety database ............................................................................................. 11 
 
b.   General safety considerations ................................................................................................. 11 
 
c.   Safety in specific populations ................................................................................................. 13 
 
d.   Other safety considerations ..................................................................................................... 15 
 
B.   Specific Considerations for Efficacy Trials ............................................................................... 17 
 
1.  Trial Design, Randomization, and Endpoints ................................................................................ 17 
 
2.  Choice of Comparator ................................................................................................................... 17 
 
3.  Enrollment Criteria ........................................................................................................................ 18 
 
4.  Trial Procedures ............................................................................................................................ 18 
 
5.  Trial Duration................................................................................................................................ 19 
 
6.  Statistical Considerations .............................................................................................................. 19 
 
a.   Endpoint analysis  .................................................................................................................... 19 
 
b.   Strength of evidence ............................................................................................................... 19 
 
c.   Adherence ............................................................................................................................... 20 
 
d.   Interim analysis and data monitoring committee .................................................................... 21 
 
e.   Missing data and sensitivity analysis ...................................................................................... 21 
 
7.  Combination Products ................................................................................................................... 21 
 
a.   Vaginal microbicide plus device ............................................................................................. 22 
 
b.   Combination product intended for multiple indications ......................................................... 22 
 
8.  Risk-Benefit Considerations........................................................................................................... 23 
 
C.   Other Considerations ................................................................................................................... 23 
 
1.  Clinical Virology ............................................................................................................................ 23 
 
2.  Additional Clinical Pharmacology  ................................................................................................ 23 
 
3.  Chemistry, Manufacturing, and Controls  ...................................................................................... 24 
 
4.  Condom/Device Function Studies.................................................................................................. 25
  
5.  Labeling Considerations................................................................................................................ 25 
 
REFERENCES............................................................................................................................ 27 
 
 
 
  Contains Nonbinding Recommendations 
Guidance for Industry1
 
 
Vaginal Microbicides:   Development for the  

Prevention of HIV Infection 
 
 
 
 
 
This guidance represents the Food and Drug Administrations (FDAs) current thinking on this topic.  It 
does not create or confer any rights for or on any  person and does not operate to bind FDA or the public.  
You can use an alternative approach if the approach satisfies the requirements of the applicable statutes 
and regulations.  If you want to discuss an alternative approach, contact the FDA staff responsible for 
implementing this guidance.  If you cannot identify the appropriate FDA staff, call the appropriate 
number listed on the title page of this guidance.  
 
 
 
 
I.  INTRODUCTION  
 
This guidance provides recommendations for the development of vaginal microbicides regulated 
within the Center for Drug Evaluation and Research (CDER) at the Food and Drug 
Administration (FDA) for the prevention of human immunodeficiency virus (HIV) infection.  
Specifically, this guidance addresses the FDAs current thinking regarding the overall 
development program and clinical trial designs to support the development of vaginal 
microbicide drug products.2    
 
Information in this guidance is also generally relevant for developing vaginal microbicides that 
are part of a drug-device combination product.  Guidance on development and testing of devices 
can be obtained from the Center for Devices and Radiological Health (CDRH).3   
 
For the purposes of this guidance, we define vaginal microbicides as intravaginal drug products 
that reduce the risk of HIV acquisition.  Vaginal microbicides are designed to be self-
administered products.  Microbicides can be developed as vaginal formulations including gels, 
                                                 
1 This  guidance has been  prepared by the Division of  Antiviral Products in the Center for Drug Evaluation and  
Research (CDER) in cooperation with the Division of Bone, Reproductive, and Urologic Products in CDER at the  
Food and Drug Administration.  
 
2 For the purposes of this guidance, all references to drugs  include both human  drugs and therapeutic biological 
products  unless otherwise specified.  
 
3 See the guidance for industry Latex Condoms for Men  Information for 510(k) Premarket Notifications:  Use of  
Consensus Standards for Abbreviated  Submissions and the Class II special controls guidance document Labeling for 
Natural Rubber Latex Condoms Classified  Under 21 CFR 884.5300. We update guidances periodically.  To make  
sure you have the most recent  versions of these guidances, check the FDA Medical Devices guidance Web page at 
http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocuments/default.htm.  
1 
 Contains Nonbinding Recommendations 
creams, tablets, films, drug-impregnated sponges, and drug-impregnated vaginal rings.  
Microbicides therefore offer a women-initiated HIV prevention method, which would form a 
useful addition to the existing prevention interventions.  Sponsors can choose to develop drug 
products with coitally dependent dosing (pre-coital dosing, or post-coital dosing, or pre-coital 
plus post-coital dosing) or a coitally independent dosing scheme (e.g., daily dosing, intermittent 
dosing, or sustained release formulation such as vaginal ring). 
 
This guidance does not address other forms of HIV prevention such as prophylactic vaccination 
or prevention mediated exclusively by mechanical barrier devices (e.g., male condoms).  
Inquiries regarding vaccines should be addressed to the Center for Biologics Evaluation and 
Research. Inquiries regarding mechanical barrier devices should be addressed to CDRH.  
Prevention of sexually transmitted infections (STI) other than HIV also is not addressed in this 
guidance. Additionally, general issues of statistical analyses or clinical trial design are not 
addressed in this guidance. Those topics are addressed in the ICH guidances for industry E9  
Statistical Principles for Clinical Trials and E10 Choice of Control Group and Related Issues in 
Clinical Trials.4     
 
Sponsors considering development of vaginal microbicides are encouraged to consult this 
guidance and to communicate with the FDA through the pre-investigational new drug application 
consultation program and throughout drug development.  
 
FDAs guidance documents, including this guidance, do not establish legally enforceable 
responsibilities. Instead, guidances describe the Agencys current thinking on a topic and should 
be viewed only as recommendations, unless specific regulatory or statutory requirements are 
cited. The use of the word should in Agency guidances means that something is suggested or 
recommended, but not required. 
 
 
II.  BACKGROUND 
 
The natural history of HIV infection includes a brief symptomatic period characterized by 
intense viral replication or acute HIV infection.  This acute phase is followed by a clinically 
latent period and eventual progression to a state of profound immunodeficiency known as 
acquired immunodeficiency syndrome (AIDS).   
 
Sexual transmission accounts for the majority of HIV infections both in the United States and 
globally. Behavior change through counseling, male and female condoms, antiretroviral therapy 
for the infected partner, and treatment of STIs can reduce the risk of HIV acquisition.  However, 
despite these prevention methods, HIV incidence in the United States has not declined and 
remains stable at 56,300 new infections annually (Hall, Song, et al. 2008).  Worldwide, the 
annual incidence is estimated to be about 2.7 million infections (UNAIDS 2011).  
 
                                                 
4  We  update  guidances periodically.  To make sure you  have the most recent version of a guidance, check the FDA  
Drugs guidance Web page  at  
http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm.  
2 
 Contains Nonbinding Recommendations 
In 2012 the first drug product for oral pre-exposure prophylaxis (PrEP), tenofovir/emtricitabine, 
was approved to reduce the risk of sexually acquired HIV infection in adults at high risk.  Data 
from trials in both men who have sex with men and heterosexual populations supported this 
approval. Despite its efficacy, this intervention poses several challenges including adherence to 
daily pill intake, the potential for renal and bone toxicities, the potential for decreased condom  
use, and the development of resistance to drugs that are also used for HIV treatment.   
 
With respect to women and the HIV epidemic, present trends indicate that women account for 23 
percent of new infections in the United States (CDC 2010) and 50 percent of all new infections 
globally. Heterosexual transmission plays a major role in HIV infection in women, and among 
the available options for reducing heterosexual male-to-female transmission, virtually all require 
male partner agreement to be effective.  For example, using male condoms, which are widely 
available through condom promotion programs, depends on the male partners level of condom  
acceptance (Kulczycki, Kim, et al. 2004).  In many settings, women are unable to insist on or 
negotiate use of condoms or another available prevention method. In these situations, gender 
inequality, reliance on men for economic security, and compromised relationship dynamics may 
contribute to the prevention challenge (Aziz and Smith 2011).  Taken together, these limitations 
emphasize the need for novel prevention approaches that allow women to independently control 
their HIV acquisition risk. Lastly, a womans product preference may change over time  
depending on factors including the desire to conceive, partners product preference, perceived 
risk for other STIs, or dosing convenience.  At-risk women need multiple prevention options to 
meet their needs.  In this context, vaginal microbicides offer the potential of a unique female-
controlled method.   
 
 
III.  DEVELOPMENT PROGRAM 
 
A.  General Considerations 
 
1.  Nonclinical Considerations 
 
a.  Nonclinical safety  
 
General recommendations for supportive nonclinical safety studies including their design and 
timing are addressed in other FDA and ICH guidances for industry, such as the ICH guidance for 
industry M3(R2)  Nonclinical Safety Studies for the Conduct of Human Clinical Trials and 
Marketing Authorization for Pharmaceuticals:  Questions and Answers. For a drug product 
currently approved as a vaginal formulation for another indication, additional toxicology studies 
usually are not needed unless the approved vaginal drug product is reformulated.  
Recommendations for biologically derived products are discussed in the ICH guidance for 
industry S6(R1) Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals.  
Nonclinical considerations specific to vaginal microbicide development are discussed in this 
guidance. 
 
Sponsors should assess a candidate vaginal microbicide for the potential to cause cervicovaginal 
inflammation or epithelial breakdown.  Local safety and tolerability can be evaluated alone or as 
3 
 Contains Nonbinding Recommendations 
part of repeat-dose toxicology studies.  These studies should include scoring systems to quantify 
the extent of erythema, edema, leukocyte infiltration, and ulceration or disruption of epithelium,  
as well as detailed histopathologic assessments of vaginal tissue. A positive control such as 4 
percent nonoxynol-9 should be included for comparison.  For drug products containing an entity 
originally approved as a nonvaginal formulation, sponsors should conduct bridging toxicology 
and vaginal irritation studies. 
 
Sponsors should evaluate systemic drug absorption following vaginal administration because the 
extent of plasma exposures is a key determinant of systemic toxicity.  When lower systemic 
exposures are achieved following vaginal administration in animals compared to humans, 
nonclinical studies using an alternate route of administration may be needed to attain the 
appropriate exposure. In addition to cervicovaginal and systemic toxicity, sponsors should 
assess local tolerance via rectal application in animal studies before evaluating rectal safety in 
humans.  
 
Reproductive toxicology studies should be completed and submitted for FDA review before 
dosing pregnant participants in clinical trials (also see section III.A.5.c., Safety in specific 
populations (Pregnant women)).  Sponsors should measure systemic drug exposures in these 
studies. Similar to repeat-dose toxicology studies discussed previously, if animal exposures are 
lower than human exposures, then sponsors should conduct animal studies with other routes of 
administration to yield relevant systemic exposures.  In circumstances where systemic drug 
exposures in both humans and animals are below limits of detection, sponsors should conduct an 
embryo/fetal development study with vaginal drug administration to evaluate potential effects 
mediated through local and/or regional drug distribution.   
 
Sponsors should conduct carcinogenicity studies in rats and mice, including one study with 
vaginal drug administration to evaluate for local tumorigenic potential.  For detailed information 
for animal carcinogenicity studies, sponsors should refer to the ICH guidances for industry S1A 
The Need for Long-Term Rodent Carcinogenicity Studies of Pharmaceuticals, S1B Testing for 
Carcinogenicity of Pharmaceuticals, and S1C(R2) Dose Selection for Carcinogenicity Studies. 
The oral carcinogenicity studies may not be needed if systemic drug exposures in both animals 
and humans are below the limits of detection.  In addition, the local carcinogenicity study may 
not be needed for drug products with systemic exposures below the limits of detection and with 
adequate clinical data supporting absence of vaginal irritation with long-term use.  Sponsors are 
strongly encouraged to propose their plans for nonclinical carcinogenicity assessments during 
phase 2 of development to allow sufficient time for study conduct and completion in advance of 
a new drug application (NDA) submission.  If sponsors determine these studies are not needed 
for their drug product, they should initiate discussions in phase 2 to seek FDA concurrence.  
 
b.  Nonclinical virology 
 
The biology of HIV vaginal transmission is not fully understood and the following 
recommendations may be modified as the field evolves.  In addition to identifying the  
mechanism of action, nonclinical virology studies should address the following:    
 
4 
 Contains Nonbinding Recommendations 
   Quantification of antiviral activity and the potential for cellular toxicity  
   Selection and characterization of resistant HIV 
   Effects on other sexually transmitted pathogens and the assays used to detect them 
   Effects on normal vaginal microflora 
 
Validated animal models for predicting efficacy in humans do not exist.  Sponsors often use 
animal models to obtain supportive activity data; however, they are not needed to support 
approval of an indication.  Generally, animal model studies are designed to show a protective 
effect against vaginal challenge with simian immunodeficiency virus or chimeric simian/human 
immunodeficiency virus in nonhuman primates.  While animal models can be useful for 
providing proof of concept of antiviral activity and for informing drug product development 
decisions, at present these models lack sufficient validation to support regulatory approval.  
Humanized mouse models also may be useful in evaluating activity against vaginal challenge of  
HIV; however, the limited availability of human hematopoietic progenitor cells may restrict the 
use of these models (Denton, Estes, et al. 2008; Berges, Akkina, et al. 2008).  
 
Quantification of antiviral activity and cellular toxicity 
 
Because the principal objective of the microbicide is to prevent HIV transmission, studies should 
demonstrate inhibition of HIV replication in cell culture.  Dose-response curves should be 
generated to determine the range of antiviral activity.  These results should reflect the 
concentration of the drug required to reduce HIV replication by 50 percent (i.e., 50 percent 
effective concentration (EC ) value). 
50
 
Sponsors should use well-characterized laboratory strains of HIV-1 in initial evaluations to 
validate antiviral activity.  After demonstrating antiviral activity against standard laboratory 
strains, evaluation should be expanded to cover a broad range of clinically relevant viruses 
including those isolated from the reproductive tract, multiple isolates representing each of the 
HIV-1 clades, CCR5 and dual CCR5/CXCR4 co-receptor tropic strains, and HIV-2.  The 
candidate microbicide should also have demonstrable antiviral activity against several (greater 
than or equal to 20) temporally and geographically distinct isolates, including U.S. strains and 
strains endemic to regions where clinical trials will be conducted.  Additionally, sponsors should  
assess antiviral activity against multiple isolates from each HIV-1 clade and HIV-2.  The median 
and range of EC  values should be provided. 
50
 
Sponsors should assess antiviral activity in peripheral blood mononuclear cells, primary 
macrophage and dendritic cell cultures, and cervicovaginal explants because these cultures 
represent the cell types likely involved in sexual transmission of HIV (Wu and Kewalramani 
2006). Assays including continuous cell lines such as ME-180 cervical epithelial cells (CD4-
transformed cell line) or GHOST X4/R5 human osteosarcoma cells may provide supporting data; 
however, these cannot be substituted for primary cultures because their biological relevance to 
human infection is unclear at this time.  Primary cultures should be harvested from several 
different donors to verify antiviral activity across a genetically diverse population of subjects.  
These studies are particularly important for drugs targeting host proteins (e.g., CCR5 or CD4) 
because polymorphisms may affect antiviral activity.  
 
5 
 Contains Nonbinding Recommendations 
In addition to the diversity of viral variants and cellular targets, other variables associated with 
transmission may affect activity.  Sponsors should perform assays under conditions consistent  
with drug product use to determine: 
 
   Antiviral activity across a range of multiplicities of infections  
   Antiviral activity following pH transition from an approximate pH 4 to pH 7 
   Protection against infection by cell-free and cell-associated HIV-1 virus  
   Antiviral activity in the presence of seminal plasma and cervicovaginal lavage fluid 
   Virucidal kinetics (in seconds or minutes) for nonspecific agents inactivating virus  
 
Furthermore, sponsors should verify antiviral activity of the proposed microbicide formulation to 
identify excipient effects. The results for the effect of each variable should include the fold-shift 
in EC  value relative to infection under standard conditions. 
50
 
Studies determining range of antiviral activity should be conducted under biologically relevant 
conditions and results should demonstrate that antiviral activity is not a result of damage to host 
cells. Cytotoxic compounds may reduce capacity of the host cell to support HIV replication that 
may be misinterpreted as direct antiviral activity.  To differentiate direct antiviral activity from  
negative effects on the host cell, cytotoxicity should be quantified as the cytotoxicity 
concentration (CC ) value, the drug concentration required to reduce culture metabolism or 
50
viability by 50 percent.   
 
Additionally, the therapeutic index (TI) should be calculated as the ratio of the CC  and EC  
50 50 
values (TI = CC /EC ).  Generally, a higher TI value indicates a more specific antiviral effect 
50 50
and greater likelihood that an effective concentration of the drug can be achieved without 
undesired off-target effects on the host cell.  Further, the TI can be useful in comparing different 
candidate microbicides or in quantifying relative activity of a particular microbicide against 
different viral variants or different cell types.  In general, we do not recommend development of 
a drug with a TI less than or equal to 10; sponsors interested in pursuing development of a drug 
with TI less than or equal to 10 should discuss the basis for pursuing development with the FDA.  
Sponsors should refer to the National Institute of Allergy and Infectious Diseases/Division of 
Acquired Immunodeficiency Syndrome (DAIDS)/National Institutes of Health nonclinical 
resources for assistance in developing their microbicide.5  
 
Selection and characterization of resistant virus 
 
HIV seroconversion during microbicide use may result in resistant virus and affect HIV 
treatment options.  Understanding the pathway to resistance development (i.e., the amino acid 
substitutions that confer reduced susceptibility to an antiviral drug) is useful in understanding the 
potential risks of microbicide failure and in guiding resistance monitoring plans in clinical trials.  
Characterization of resistant isolates also can provide supporting evidence for the proposed 
mechanism of action.  
 
                                                 
5 See http://www.niaid.nih.gov/labsandresources/resources/atrg/pages/microbicidesprevhivtrans.aspx.  
6 
 Contains Nonbinding Recommendations 
Because the genetic barrier to resistance may vary as a function of drug concentration, resistant 
variants should be selected in cell-culture at different microbicide concentrations.  Several 
independent resistant isolates should undergo genotypic and phenotypic testing.  Resistance-
associated substitutions should be verified by phenotypic characterization of site-directed 
recombinant viruses expressing the mutant protein.  If substitutions arise in viral proteins or 
protein complexes targeted by approved antiretroviral drugs, cross-resistance should be assessed 
including: 
 
   Assessment of the susceptibility of approved drugs to microbicide-resistant variants  
   Assessment of susceptibility of the microbicide to variants resistant to approved 
antiretroviral drugs 
 
Effect on STIs and normal human microflora 
 
Local pathologic changes associated with STIs as well as vaginal microflora alterations may 
affect the risk for vaginal HIV transmission. Sponsors should test for antimicrobial activity 
against common STI pathogens, such as Neisseria gonorrhoeae, Chlamydia trachomatis, herpes 
simplex virus type-2, and trichomonas.  Testing antimicrobial activity is of particular importance 
for drug products with a nonspecific mechanism of action and/or a low TI.  Testing also should 
be performed to assess static and cidal activity on normal resident microflora such as 
Lactobacilli. Sponsors should be aware that the microbicide may contain components inhibitory 
to STI assays (e.g., sulfated polysaccharides can affect certain polymerase chain reaction assays) 
that may interfere with STI diagnosis.  Hence, microbicide effects on sensitivity of STI 
diagnostic assays used in clinical trials should be evaluated using concentrations consistent with 
drug product use. 
 
c.  Other nonclinical studies 
 
Condom compatibility studies are nonclinical studies intended to evaluate effects of a 
microbicide on the physical properties of condoms.  Concomitant use of microbicide and 
condoms is expected both in clinical trials as well as real-world use following microbicide 
approval, regardless of whether the microbicide is labeled for use with the condom.  Condom  
compatibility studies are needed to determine whether use of microbicide with condoms affects 
the rate of condom failure compared to condoms used alone.  
 
Sponsors should refer to the American Society for Testing and Materials (ASTM) D7661-10, 
Standard Test Method for Determining Compatibility of Personal Lubricants With Natural 
Rubber Latex Condoms, for study design and methodology.  Studies should include both male 
and female condoms composed of a variety of materials including natural rubber latex, 
polyisoprene, polyurethane, and nitrile.  In addition to baseline, conditioned, and positive 
controls, concurrent evaluation of the effects of microbicide and placebo on each condom type 
should be performed.  Data analyses and presentation should follow recommendations outlined 
in the above-mentioned ASTM standard.  
 
In addition to condom compatibility testing, sponsors should perform viral penetration testing to 
assess the drugs effects on the barrier properties of male and female condoms.  Sponsors should 
7 
 Contains Nonbinding Recommendations 
use the method described by Lytle et al. (1992) to evaluate viral penetration.  Sponsors are 
strongly encouraged to provide study proposals for FDA review and comment before initiating 
nonclinical compatibility studies.  
 
Positive or equivocal findings in nonclinical compatibility studies may prompt the need for 
clinical data to fully understand effects of microbicide on condom function.  Clinical data 
obtained from condom function studies are discussed further in section III.C.4., Condom 
Device/Function Studies. 
 
Sponsors should also evaluate the effect of a microbicide on the physical properties of other 
commonly used barrier contraceptive devices (e.g., diaphragms).  Before initiating these studies, 
sponsors should contact the FDA to obtain feedback for their proposed plan and/or protocol. 
 
2.  Drug Development Population  
 
Clinical development should be pursued in women at risk of acquiring HIV through sexual 
transmission.  Microbicide efficacy trials generally are conducted in high HIV prevalence areas 
or populations because the HIV seroconversion rate is a critical factor in determining sample 
size. Because the largest number of new HIV infections occurs outside the United States, 
clinical efficacy data supporting an indication likely will be obtained from trials conducted at 
foreign sites.  FDA regulations permit accepting foreign trial data in support of a marketing 
application, provided the foreign studies meet the same requirements in 21 CFR part 312 as are 
applicable to U.S. studies conducted under an investigational new drug application.6  The clinical  
development program should include U.S. subjects to ensure applicability of data to the U.S. 
population. Additionally, for certain vaginal formulations, data from a label comprehension 
study conducted in U.S. subjects should be submitted with the NDA (see section III.C.5., 
Labeling Considerations). 
 
3.  Early Phase Clinical Considerations 
 
Because HIV seroconversion is an infrequent event even in high prevalence areas, proof of 
concept can be measured only in trials with relatively large sample sizes.  As a result, vaginal 
microbicide development generally proceeds directly from phase 1 to large-scale phase 2b or 
phase 3 trials. The objective of early phase clinical development is to provide sufficient data for 
preliminary safety, tolerability, acceptability, and pharmacokinetics to support rational drug 
development and choice of dose or doses to take forth into late-phase trials. 
 
a.  Safety and pharmacokinetic considerations 
 
Initial safety should be evaluated in sexually active healthy women at low risk for potential 
confounders for evaluating safety/tolerability, such as STIs or baseline cervicovaginal 
abnormalities.  Safety evaluations should focus on local toxicities of the cervicovaginal area and 
the female reproductive tract, as well as systemic toxicities.  Initial studies can obtain data 
reflecting regular drug product use for at least one complete ovulatory/menstrual cycle.  Studies 
                                                 
6 See the guidance for industry Acceptance of Foreign Clinical Studies. 
8 
 Contains Nonbinding Recommendations 
of at least 3 to 6 months dosing duration are needed to identify toxicities from cumulative drug 
product use. Generally, data from at least 100 to 200 subjects are needed to characterize the 
preliminary safety profile.  
 
Systemic absorption should be determined early in drug development because of important 
safety and drug development implications.  For systemically absorbed drug products, the 
potential for systemic adverse events should be monitored from early trials onward.  Knowledge 
about systemic absorption influences the type of clinical pharmacology studies needed for drug 
product development.  For example, typical clinical pharmacology studies (e.g., drug interaction, 
hepatic impairment, renal impairment) may not be needed if systemic exposure is low or 
undetectable. For drug products containing either a drug originally approved for HIV treatment 
or a new molecular entity in the same class as an approved drug for HIV treatment, the presence 
of sub-inhibitory but detectable systemic drug concentrations can potentially result in resistance  
or cross-resistance, respectively, in the event of HIV seroconversion.  
 
Local pharmacokinetic (PK) evaluations should include serial assessment of cervicovaginal fluid 
concentrations at different time points after dosing to estimate the level of local exposure as well 
as to determine elimination of the drug locally.  Local drug distribution can be quantified by 
collection of cervicovaginal fluid samples from multiple regions of the vaginal lumen.  
Researchers have used magnetic resonance imaging and other imaging techniques as another 
mechanism to determine local and regional drug distribution.  Hypothetically, to be optimally 
effective, a vaginal microbicide should uniformly coat the entire vaginal lumen and cervix.  
However, there is presently no clear evidence linking extent of local coverage or tissue 
distribution or local drug exposure to clinical outcome for vaginal microbicides.  Tissue samples 
obtained by biopsy can be used to quantify the extent to which the drug is taken up by 
cervicovaginal epithelium. In addition, local PK data collected in early studies should include an 
assessment of microbicide drug concentrations during menses, both with and without tampon 
use. For vaginal ring microbicides, sponsors should determine local tissue drug concentrations at 
different time points after ring removal to provide an estimate of the elimination of the drug 
locally following ring expulsion or loss.  
 
b.  Drug product characteristics that affect end-user acceptability  
 
Trials should assess drug product characteristics that are expected to affect user acceptability 
early in drug development.  Although phase 1 acceptability data may not reflect phase 3 or 
postmarketing acceptability, early data can guide drug product reformulation needed to optimize 
acceptability before embarking on large scale trials.  Poor or low acceptability may be secondary 
to unfavorable physical attributes (e.g., viscosity, odor, color, taste).  For semisolid gel 
formulations, vaginal retention should be assessed because excessive vaginal leakage after 
application may be undesirable and also lead to poor distribution and possibly compromised drug 
effect. 
 
c.  Dose selection 
 
Dose selection poses a unique challenge to microbicide development.  The ability to use 
PK/pharmacodynamic (PD) models for dose selection is contingent upon establishing 
9 
 Contains Nonbinding Recommendations 
measurable and valid PD endpoints.  Therefore, typical dose-response or exposure-response 
analyses are unlikely to be helpful for microbicides unless validated surrogate markers of 
protection are identified. In vitro antiviral activity data combined with local exposure data (e.g., 
vaginal and cervical concentrations) can be used to predict a minimally efficacious dose.  
Protein-binding of the drug product in vaginal fluid also should be taken into consideration for 
highly protein-bound drug products when selecting doses.  Additionally, animal toxicity findings 
and safety findings in phase 1 clinical trials can provide further information guiding human dose 
selection. Another consideration is systemic absorption; in some instances higher doses may not 
be preferred because of increased systemic absorption and a greater likelihood of systemic 
adverse events. 
 
4.  Efficacy Considerations  
 
Despite advances in the HIV and microbicide field, there are several challenges in the clinical 
development of vaginal microbicides.  Some  challenges in evaluating clinical efficacy are 
addressed below. 
 
In addition to biological efficacy, variables such as drug product adherence, concurrent use of 
other prevention methods (e.g., condoms, oral PrEP), and frequency of high-risk sexual behavior 
are closely linked to the overall effectiveness of a vaginal microbicide.  Because usage rates of 
microbicide and/or other prevention methods as well as sexual behavior patterns are expected to 
fluctuate over time, and long-term safety events (e.g., epithelial disruptions) may potentially 
reduce efficacy over time, longer duration efficacy trials are preferred because they can capture 
the effect of these variables and are more likely to reflect the real-world effects (Lagakos and 
Gale 2008). 
 
Efficacy trials should measure the rate of new HIV infections.  Given the relatively low 
incidence of seroconversion even in high-prevalence populations, a large sample size usually is 
necessary to provide adequate power to detect a statistically significant effect on HIV 
seroincidence. As with other drug trials, sample size calculations should account for the 
anticipated effect size of the drug product, as well as loss to follow-up and dropouts caused by 
adverse events. An additional consideration is the anticipated pregnancy rate, because pregnant  
women in microbicide trials should be taken off the drug product unless specific criteria are met 
(see section III.A.5.c., Safety in specific populations (Pregnant women)). 
 
Risk-reduction counseling and promoting condom use are ethical imperatives during the conduct 
of microbicide trials.  Additionally, an approved oral PrEP agent can be offered in the trial as  
part of the background prevention package.  Offering oral PrEP depends on several 
considerations including acceptability as standard HIV prevention locally and implementation in 
regions where trials are conducted.  Alternatively, trials can be designed to enroll subjects who 
refuse oral PrEP as a result of intolerance, side effects, or personal preference.  Importantly, 
condom promotion and other prevention interventions are likely to reduce the infection rate in 
the trial and further increase the trial sample size.    
 
Accelerated approval (21 CFR part 314, subpart H), based on a surrogate endpoint considered 
reasonably likely to predict clinical benefit for a serious or life-threatening disease, is not 
10 
  Contains Nonbinding Recommendations 
applicable for an HIV prevention indication. The endpoint of HIV infection, although a 
laboratory measurement, is considered to be reliably predictive of progressive clinical disease.  
At this time, other surrogates that are predictive of laboratory infection of HIV have not been 
defined. The assay used to diagnose HIV infection should be the most reliable assay available at 
the time of the trial.  
 
Sponsors can request that the FDA expedite drug product development through mechanisms such 
as priority review and fast track designation.7  The fast track designation allows for frequent 
interactions with the FDA and permits rolling review when an NDA is submitted.  Proposals for 
fast track designation can be considered at any time during development depending on 
appropriate fulfillment of the designated criteria.8    
 
5.  Safety Considerations 
 
a.  Adequacy of the safety database 
 
Microbicide efficacy trials will have a large sample size; therefore, it is anticipated the safety 
database will consist of several thousand subjects exposed to the proposed to-be-marketed dose 
for at least 12 months.  A proportion of phase 3 subjects should be followed for a longer duration 
as outlined in section III.B.5., Trial Duration.  Data from more subjects may be needed if safety 
concerns are identified during development.  Alternatively, a smaller safety database may be 
considered adequate if the drug product is already approved for use by another route/formulation 
or is unapproved but has been evaluated substantially using other routes of administration.  
Sponsors are strongly encouraged to discuss their proposed safety database with the FDA at an 
end-of-phase 2 meeting or earlier.  
 
b.  General safety considerations 
 
Local vaginal and cervical safety is a critical consideration for a vaginal microbicide.  Sponsors 
should assess local toxicity through symptoms  and signs for genitourinary and reproductive 
adverse events and by pelvic examination.  Evaluations should focus on symptoms and signs 
representing genital irritation, inflammation, or mucosal breakdown.  Pelvic examination should 
include visual inspection and speculum examination.  Comparative safety data from randomized, 
double-blind trials using an appropriate placebo control are preferred to allow clear interpretation 
of safety findings.  Severity grading of cervicovaginal abnormalities should be based on accepted  
grading criteria for genital toxicity such as the National Institutes of Health DAIDS genital 
toxicity table.9   
 
                                                 
7 See 
http://www.fda.gov/ForConsumers/ByAudience/ForPatientAdvocates/SpeedingAccesstoImportantNewTherapies. 
 
8 See the guidance for industry Expedited Programs for Serious Conditions  Drugs and Biologics. 
 
9 See http://rsc.tech-res.com/safetyandpharmacovigilance/gradingtables.aspx. 
11 
  Contains Nonbinding Recommendations 
Colposcopy should be performed in at least one phase 1 trial following multiple drug exposures 
in sexually active women conducted early in drug product development.  This technique was 
originally developed to detect local malignancy; specifically for microbicide trials, the  
examination should focus on findings reflecting potential drug toxicities including epithelial 
disruption. Sponsors should refer to standard criteria for colposcopy technique and training for 
vaginal microbicides.10  Depending on the safety profile and colposcopic findings in phase 1, we 
will determine the need for colposcopy in subsequent phase trials.  We do not consider vaginal 
biopsies necessary unless indicated by local toxicity findings.  Trials should also monitor for 
adverse events reflecting drug effects on the uterus, fallopian tubes, and ovaries that may arise 
from regional drug distribution. 
 
Sponsors should perform assessments for microbicide effects on vaginal pH, balance of vaginal 
microflora, and the frequency of other STIs.  Significant shifts in local microflora may have 
clinical implications because the normal vaginal microflora is thought to play a role in 
preventing HIV-1 infection and other STIs (Myer, Kuhn, et al. 2005).  Certain types of 
microflora imbalance or decreases in particular flora species can also increase the likelihood of 
bacterial vaginosis, urinary tract infections including urosepsis, and pelvic infections.  
 
Systemic adverse reactions may arise if a drug product is systemically absorbed following 
vaginal administration. At a minimum, sponsors should evaluate systemic safety through 
adverse event assessment and routine laboratory tests such as hematology and chemistry 
parameters.  The need for additional evaluation depends on the level of systemic absorption and 
expected or known risks of the drug product as indicated by nonclinical findings or toxicities 
observed in earlier human trials.  For a microbicide originally approved as an oral formulation, 
sponsors should consider the established safety profile when forming a targeted safety 
assessment plan for trial protocols.  For example, if thyroid function abnormality is a recognized 
toxicity with the oral formulation, appropriate laboratory testing of thyroid function should be 
included if significant systemic absorption is expected.  Grading of nongenital adverse events 
and laboratory abnormalities should follow commonly used and accepted toxicity grading 
schemes.  
 
For drug products intended for vaginal retention for a specified time period (e.g., a 28-day 
intravaginal ring), sponsors should collect safety data reflecting greater than the prescribed 
duration of exposure to characterize safety in case of drug product overuse.  
 
Safety evaluations in phase 1 trials generally can be performed at weekly intervals or less 
frequently. In late-stage trials, sponsors should perform evaluations within the first month of 
starting the drug product and then at least once every 1 to 3 months.  The protocol should have 
provisions to contact subjects or allow unscheduled visits for managing adverse events as 
needed. All visits should include safer sex and HIV risk reduction counseling and provision of 
male condoms to all subjects.  
 
                                                 
10 See the CONRAD/World Health Organization Manual for the Standardization of Colposcopy for the Evaluation 
of Vaginal  Products at http://www.who.int/reproductivehealth/publications/rtis/RHR_04.2/en/index.html.  
12 
  Contains Nonbinding Recommendations 
In addition to evaluating genital toxicity in women, sponsors should conduct male tolerance 
studies to identify penile toxicity.  Ideally, penile safety data should be obtained before dosing 
sexually active women in phase 1 trials.   
 
c.  Safety in specific populations 
 
In addition to the general safety considerations discussed previously, this section outlines safety 
perspectives for specific populations, namely pregnant women and adolescents.   
 
Pregnant women 
 
To protect the fetus from research-related risks, microbicide trials have historically excluded 
pregnant women from enrollment and disallowed women who became pregnant to continue to 
use the investigational drug product.  In the real-world setting, however, an approved 
microbicide may be used by pregnant women despite lack of data in this special population.  We  
recognize that complete lack of safety data in pregnant women at time of NDA submission is not 
optimal.  Ideally, microbicide safety data in pregnant women should be obtained methodically in 
a prospective controlled trial setting with careful monitoring of subjects.  Because of these 
considerations, the FDAs thinking regarding the evaluation of women who become pregnant 
while participating in microbicide trials is evolving.  In addition, both the woman and the fetus 
may stand to benefit from a microbicide that could potentially prevent HIV infection.    
 
In some instances, use of an investigational microbicide may be allowed in women who become  
pregnant while participating in microbicide clinical trials.  The decision to allow use of the drug 
product in women who become pregnant while participating in microbicide clinical trials 
depends on the safety and PK profile of the drug product in nonclinical studies and early clinical 
trials. To assess whether pregnant women should continue drug product use, the FDA will 
consider the following information in the decision-making process: 
 
 	  Completed reproductive toxicology studies including data from fertility and early 
embryonic development study, the embryo-fetal development study, and the pre- and 
postnatal development study.  
 
	   Completed genotoxicity studies 
 
	   Chronic toxicity studies in two species to support the duration of exposure in human trials 
 
	   Data on systemic absorption of the microbicide in nonpregnant female subjects 
 
Additionally, the microbicide trial design should have the following provisions for women who 
become pregnant: 
 
	   Women who become pregnant during the trial should be re-consented.  Information about 
potential risk to the fetus following microbicide exposure should be included in the 
original informed consent and discussed again with the pregnant subject.  Data collected 
should include but not be limited to timing of pregnancy relative to length of time on the 
13 
  Contains Nonbinding Recommendations 
investigational drug product, duration of fetal drug exposure, and pregnancy outcome.  
Women who choose to discontinue the investigational drug product but remain in the trial 
should be followed for pregnancy and fetal outcome data. 
 
 	  Pregnant women who choose to continue receiving the investigational drug product in the 
trial should undergo increased safety monitoring including more frequent visits, 
laboratory testing, and fetal monitoring.  In addition, the protocol should include a 
toxicity monitoring and safety management plan for pregnant women and their fetuses.  
Protocol safety monitoring should take into account current standards of antepartum  care 
in the trial countries. 
 
 	  PK data should be collected in a subset of pregnant women who re-consent and who 
choose to continue the investigational drug product.  Assessments should be performed 
during pregnancy (at least at one time point in each trimester), in the postpartum period, 
and during lactation to characterize changes in systemic exposure related to any alteration 
in local absorption.11  Data should be obtained at time points in each pregnancy trimester, 
at a minimum.  Protocols should also include, when possible, collection of cord blood for 
the evaluation of systemic exposure to the fetus at the time of delivery.   
 
 	  The exposed infant should be followed from  birth up to 1 year for collection of data 
during this time frame.  Sponsors should obtain FDA concurrence for the type of data to 
be collected.  
 
 	  Women who become pregnant during the trial should be followed in a pregnancy 

exposure registry such as the Microbicide Trials Network Registry MTN-016. 

 
The FDAs decision on a sponsors proposal to dose pregnant women will be made on a case-by-
case basis.  
 
Adolescents  
 
In the United States, high-risk heterosexual contact is estimated to account for approximately 90 
percent of transmission in adolescent females aged 13 to 19 years (CDC 2010).  In certain 
countries in Sub-Saharan Africa, HIV prevalence is estimated as high as 20 percent among 
females aged 15 to 24 years (UNAIDS 2011).  Local sociocultural norms and biological factors 
may contribute to the high infection rate in this age group.  Younger women with older sexual 
partners may be less successful in negotiating male condom use.  Cervicovaginal differences 
between adolescence and adulthood, such as the extent of cervical ectopy, may also play a role.  
In adolescence, a larger zone of ectopy associated with highly vascularized epithelial lining and 
greater mucosal fragility may increase HIV susceptibility (Moss, Celemetson, et al. 1991).   
 
Conducting clinical trials in adolescents involves important ethical, regulatory, and parental and 
adolescent consent considerations (Nelson, Lewis, et al. 2010).  Generally for drug products 
                                                 
11 See the draft guidance for industry Pharmacokinetics in Pregnancy  Study Design, Data Analysis, and Impact 
on Dosing and  Labeling. When  final, this guidance will represent the FDAs current thinking on this topic.  
14 
  Contains Nonbinding Recommendations 
approved for adult use, separate clinical studies are needed to establish safety in the pediatric age 
group, if appropriate.12  A central consideration in pediatric research is whether the intervention 
offers the prospect of direct benefit to the enrolled subject.  In addition, sponsors should consider 
the level of risk associated with the intervention (e.g., an intervention that involves greater than 
minimal risk must offer the prospect of direct benefit to the individual subject and meet certain 
conditions to involve children as subjects (21 CFR 50.52; see also 21 CFR part 50, subpart D)).  
Other influential factors include justification of the intervention in the context of disease 
severity, comparability of the intervention to available alternatives, and past experience for a 
drug product or the class. Specifically for vaginal microbicides, key considerations in the 
decision-making process include the serious and life-threatening nature of HIV disease, 
relatively higher HIV prevalence among adolescents, the likelihood of drug product usage after 
approval, and the safety profile and efficacy of the drug product.  
 
After approval for adult use, a vaginal microbicide may be used by individuals younger than 18 
years, even in the absence of safety data in adolescents.  Adolescent safety data should therefore 
be collected before microbicide approval.  Sponsors should consider a two-stage approach 
whereby initial safety data are collected from older subjects 16 to 18 years of age, followed by 
recruitment of adolescents younger than 16 years, depending on the clinical needs and pediatric 
research requirements of the participating trial sites.  Sponsors should make every effort to 
submit safety data from older adolescents with the NDA submission.  
 
Retention in trials and adherence to the drug product are challenges inherent to this age group 
that should be taken into consideration.  Sexual practices vary by region; therefore, sponsors are 
strongly encouraged to obtain some of their data from adolescents in the United States. 
 
In addition, adolescents are also at risk for pregnancy.  Therefore, the considerations discussed in 
the previous section regarding pregnant women apply to adolescent subjects who become  
pregnant as well. 
 
d.  Other safety considerations 
 
This section focuses on additional safety considerations such as assessing safety following rectal 
application of semisolid microbicide preparations, and safety in the postmenopausal age group.  
 
Rectal safety  
 
Vaginal and rectal compartments differ in several respects including structural anatomy, local 
epithelial lining, and the population of target immune cells (Poles, Elliott, et al. 2001).  As a 
result, a microbicide shown to be safe and effective for vaginal use should not be assumed to be 
safe or effective with rectal administration.  Use of an unsafe drug product may be deleterious if 
it increases HIV susceptibility through irritation or breakdown of the more fragile rectal mucosa.  
After approval for vaginal use, microbicides may be used by women and men off-label rectally 
with the intention of preventing infection; therefore, rectal safety data for semisolid microbicide 
                                                 
12 For the purposes of this guidance, reference to  pediatric subjects includes only the adolescent age group. 
15 
  Contains Nonbinding Recommendations 
formulations should be available at the time of drug product registration to provide information 
on any potential significant safety concerns.  
 
Gastrointestinal (GI) toxicity or anorectal toxicity may be caused by the active ingredient, 
excipient, or physical attributes of the drug product such as osmolarity.  Sponsors should conduct 
rectal safety trials in healthy adult men and women.  Subjects with underlying anorectal or GI 
conditions including evidence of inflammation at screening anoscopy, history of gastrointestinal 
bleeding, or untreated rectal STIs should be excluded.  Trials should initially evaluate single or 
multiple rectal doses in subjects who are not planning to engage in anal intercourse for the trial 
duration.  Initial trials should include a minimum dosing duration of approximately 7 to 10 days.  
Rectal application of lubricants and suppositories or rectal douching should not be allowed.  
Subjects should be monitored for symptoms and signs of anorectal, GI, and systemic adverse 
events. Safety laboratory testing should include routine safety parameters such as complete 
blood count, liver, and renal markers.  Lower GI endoscopy should be conducted for visual 
inspection of mucosal toxicity.  
 
Sponsors should consider drug product discontinuation in subjects developing GI or abdominal 
adverse events, and evaluations exploring potential drug toxicity should be undertaken in 
consultation with a gastroenterology expert. The protocol should include an adequate safety 
monitoring plan as well as individual and trial stopping criteria.  Nonclinical safety findings and 
the overall clinical safety profile should be used as guides in making the decision for longer 
duration safety trials. Plasma drug levels should be determined because systemic exposure with 
rectal administration may not mimic levels attained with vaginal use. 
 
Randomized, double-blind, and placebo-controlled trials are preferred.  Until data supporting 
placebo effects of hydroxyethyl cellulose (HEC) gel in the rectal compartment are available, 
safety comparisons with HEC gel should be interpreted with caution.  Variations in osmolarity 
have been shown to affect rectal mucosal integrity (Fuchs, Lee, et al. 2007).  Physical attributes 
such as osmolarity and pH of the placebo gel should match the microbicide gel being evaluated.   
 
Safety in postmenopausal women 
 
Microbicide clinical trials typically enroll sexually active adult women up to the age of 40 or 45 
years. However, in the United States, the incidence of new HIV infections has risen in women 
over the age of 50. Postmenopausal women experience thinning of vaginal mucosa and 
increased expression of cervical CCR5 receptors (Meditz, Moreau, et al. CROI) that may 
enhance susceptibility to HIV infection.  An approved vaginal microbicide likely would be used 
in women of all ages; therefore, sponsors are strongly encouraged to collect safety data in this 
population. Safety, tolerability, and PK data can be collected in a separate trial or by allowing 
limited enrollment of older women, including postmenopausal women, in phase 3 trials or in a 
separate trial. 
 
16 
  Contains Nonbinding Recommendations 
B.  Specific Considerations for Efficacy Trials  
 
1.  Trial Design, Randomization, and Endpoints 
 
Microbicide efficacy trials should be randomized, double-blinded, and conducted across multiple 
sites. HIV seroconversion should be the primary endpoint.  Demonstrating reduction in HIV 
seroconversion requires phase 3 trials with large sample sizes.  Phase 2 trials, which do not have 
sufficient power to show differences in seroconversion, can be conducted to obtain safety data 
before initiation of large phase 3 trials.  For logistical reasons, such as maintaining trial sites and 
maintaining continued enrollment, phase 2 and phase 3 trials can be combined, with a phase 2b 
lead-in as an initial part of a phase 3 trial. New infections and person-years of exposure obtained 
in the phase 2 lead-in portion can contribute to the safety and efficacy of the phase 3 portion.  
Safety and tolerability from the first several hundred women enrolled in the phase 2b lead-in 
should be evaluated before accrual in the phase 3 portion is initiated.  If seroconversion 
endpoints from the phase 2 lead-in portion are unblinded and evaluated before starting the phase 
3 portion, then statistically appropriate methods should be used for combining the results of the 
phase 2 and phase 3 data. This design offers the advantages of enrolling fewer new subjects than 
if the phase 2 and phase 3 trials were conducted separately, and of allowing more safety 
evaluations before expanding the trial enrollment.   
 
2.  Choice of Comparator  
 
We recommend a vaginal microbicide placebo as the comparator in efficacy trials when 
investigational drug products are added to a background of other HIV prevention modalities such 
as condoms, counseling, and even oral PrEP (as deemed appropriate and acceptable by local 
jurisdictions; see section III.A.4., Efficacy Considerations).  The vehicle (or excipient) 
component may not serve as an acceptable placebo unless it is known that there are no beneficial 
or harmful effects resulting from the vehicle.  Clinical data support HEC gel as an acceptable 
placebo because outcomes in the HEC gel arm were shown to be no more than 2.4 percent worse 
than the condom-only arm (or no-treatment arm) in HIV prevention trial HPTN 035 (Karim, 
Richardson, et al. 2011). 
 
Following approval of a vaginal microbicide, an active-controlled noninferiority trial design 
comparing the candidate microbicide to the approved microbicide could be considered 
appropriate. A noninferiority design depends on the ability to define the magnitude of the 
contribution of the new active control such that a reliable noninferiority margin can be 
calculated.13   
 
Designing a trial using oral emtricitabine/tenofovir as a comparator will be challenging.  A 
superiority trial will likely need a large number of subjects to demonstrate a treatment effect in 
the setting of even moderately good adherence.  A noninferiority comparison to oral 
emtricitabine/tenofovir presents methodological challenges because of uncertainty of the assay 
sensitivity of oral emtricitabine/tenofovir as an active control.  In previous trials of oral  
                                                 
13 See the draft guidance for industry Non-Inferiority Clinical Trials. When  final, this guidance will represent the 
FDAs current  thinking on this topic.  
17 
  Contains Nonbinding Recommendations 
emtricitabine/tenofovir the overall preventive treatment effect ranged from 0 to 75 percent 
depending on subject adherence (Grant, Lama, et al. 2010; Baeten, Donnell, et al. 2012; Thigpen, 
Kebaabetswe, et al. 2012; Van Damme, Corneli, et al. 2012).  Given this historical data, defining 
a noninferiority margin will be problematic.  Sponsors considering an active-controlled 
noninferiority design should discuss protocol proposals with the FDA well in advance of trial 
initiation. Use of a future-approved vaginal microbicide as an active control could result in 
similar methodologic challenges as for oral emtricitabine/tenofovir depending on the 
effectiveness (and associated confidence intervals) of the approved vaginal microbicide in 
previously conducted trials. 
 
3.  Enrollment Criteria  
 
Healthy, non-HIV-infected, sexually active adult women at high risk of acquiring HIV should be 
enrolled in phase 3 trials.  Phase 2 trials also should enroll sexually active subjects.  It may be 
reasonable to enroll moderate- to high-risk subjects in some phase 2 trials to obtain safety data in 
the target population early in development.  In particular, enrolling high-risk subjects is 
appropriate with the phase 2b lead-in design for reasons discussed previously (see section 
III.B.1., Trial Design, Randomization, and Endpoints).  
 
Screening evaluation should include medical history, physical examination including pelvic 
examination, and the following laboratory tests: 
 
   HIV serology 
   Serum hematology and chemistry profile 
   Urine or serum beta human chorionic gonadotropin 
   Testing for STIs, bacterial vaginosis, and vaginal candidiasis  
   Papanicolaou smear  
 
At least two negative HIV serology test results are necessary to confirm lack of seroconversion 
before trial entry.  Samples for reverse transcription (or transcriptase) polymerase chain reaction 
(RT-PCR) testing should be obtained and stored at screening and baseline visits (see section 
III.C.1., Clinical Virology).  
 
Subjects diagnosed with treatable STIs should receive appropriate treatment and these subjects 
can be considered for enrollment following resolution of infection.  Subjects should not be 
pregnant and should be willing to prevent pregnancy for the trial duration.  Tampon use should 
not be considered an exclusion criterion. 
 
4.  Trial Procedures 
 
HIV seroconversion data, as measured by an approved HIV antibody assay, should be obtained 
through monthly testing.  Sponsors should conduct safety evaluations at scheduled intervals as 
discussed previously. Data pertaining to type and duration of hormonal contraception used 
should be obtained as part of concomitant medication assessment to allow analysis (subgroup or 
sensitivity analyses) for any effects of hormonal contraception on microbicide safety or efficacy.  
Data related to sexual behavior, coital frequency, condom use, microbicide adherence, and 
18 
  Contains Nonbinding Recommendations 
tampon use should be obtained through interviews, subject diary cards, or other methods for 
obtaining data for self-reported behaviors.  Subjects who seroconvert during the course of the 
trial should be referred for HIV treatment according to the local standard of care.  
 
5.  Trial Duration 
 
All enrolled subjects should be followed for a minimum period of 12 months and should be 
followed until the last enrolled subject completes the trial and at least 50 percent of the subjects 
have received 24 months follow-up.  Data reflecting safety over a dosing duration of at least 12 
months is important because an approved microbicide may be used indefinitely and new safety 
concerns from chronic exposure may arise.  Longer duration data also captures drug product 
adherence, which may reduce over time with diminishing effect as observed in some biomedical 
prevention trials (Karim, Karim, et al. 2010; Grant, Lama, et al. 2010).  We do not recommend 
large trials of short duration because they are not as informative with respect to longer term use.  
 
If interim analyses suggest the targeted number of HIV seroconversions may be reached in 
advance of the recommended duration of follow-up, sponsors are advised to engage in 
discussions with the FDA before making decisions about trial termination.  Before trial 
termination, sponsors should provide the FDA with estimates of the exposure including the 
number of subjects with 12, 18, and 24 months follow-up at the time of intended trial 
termination.  Estimations should account for expected lost-to-follow-up and other anticipated 
reasons for subject discontinuation. 
 
6.  Statistical Considerations 
 
a.  Endpoint analysis 
 
The primary endpoint should be HIV seroconversion rate per person-year of drug product use.  
Drug product use should be calculated as the time from provision to the subject of the 
investigational drug product or placebo until the time when the subject has completed or been 
discontinued from the trial.  Drug product use should not be adjusted for actual use or 
compliance.  Calculations based on person-years of drug product use are preferred to the 
absolute seroconversion rate because the former accounts for differential dropout rates between 
arms.  In the event of markedly different dropout rates between arms, sensitivity analyses should 
be provided. Cox proportional hazards regression or Poisson model analysis generally are 
accepted methods of analysis.  Cox proportional hazards regression also can be used with 
adjustment for protocol-specified baseline covariates.  Survival curves and hazard curves should 
be plotted to ensure absence of convergence or crossover in hazard rates.  
 
b.  Strength of evidence 
 
Sponsors should provide evidence from at least two independent clinical trials, each convincing 
on its own, to support drug product approval.14  A statistically significant treatment effect 
                                                 
14 See the guidance for industry Providing Clinical Evidence of Effectiveness for Human  Drug and Biological 
Products. 
19 
  Contains Nonbinding Recommendations 
compared to control for a superiority trial is a two-sided p-value less than 0.05.  Trials with 
strong internal consistency increase confidence whereas lack of consistency reduces confidence 
in the result. Conducting two independent trials simultaneously (or nearly so) may be feasible 
and could avoid potential ethical difficulties if one trial finishes first and shows a statistical  
significant treatment effect.  
 
Data from a single large phase 3 trial also may be acceptable.  Formally, two independent trials 
each statistically significant at the two-sided 0.05 level provide a strength of evidence equivalent  
to a single trial statistically significant at the two-sided 0.001 level.  However, accepting data 
from a single large trial is contingent on several variables, including the generalizability of 
results to a broader population and internal consistency across subgroups and sites within the 
trial. Sponsors are advised to refer to FDA guidance for details.15   
 
Large sample sizes and other challenges with conducting HIV prevention trials may provide an 
incentive for microbicide sponsors to combine efforts toward collaborative trials.  A 
collaborative trial design evaluating  multiple investigational drug products offers the advantage 
of a single control arm serving as comparator for each drug product.  Increasing the control arm  
size increases the power to show effect for the individual investigational drug product.  This 
design also permits the evaluation of combination products.  Safety and efficacy comparisons 
between microbicides can provide additional useful information.  
 
c.  Adherence 
 
Male condoms (or other proven prevention interventions) are expected to reduce HIV 
transmission; therefore, HIV infection is likely to occur more frequently in subjects not using 
condoms (or another intervention) irrespective of the trial arm.  A larger treatment difference is 
expected among subjects compliant with the microbicide but not compliant with condoms in case 
of an efficacious drug product. Secondary analyses in this subgroup, defined based on post-
randomization assessments of drug product adherence, may provide supportive evidence of 
efficacy. A compliance-based secondary analysis demonstrating efficacy would not be 
considered as a refutation of primary intent-to-treat analysis that failed to demonstrate efficacy.   
 
Self-reports of drug product use alone are not considered to be a reliable measure of adherence.  
Trials should incorporate objective methods such as plasma or tissue drug levels in the case of 
systemically absorbed agents or residual drug levels in drug-device combinations to provide 
estimates of drug product use over time.  Data for adherence in the placebo arm, for example by 
measuring levels of an inert taggant, can allow for sensitivity analyses.  
 
We do not recommend drug product adherence incentives, other than coaching and counseling, 
because incentives will not be available after the drug product is on the market.  A trial using 
them could exaggerate potential benefit.  Incentives that encourage subjects to return for trial 
visits or maintain contact with site staff are acceptable because these ensure data collection in 
subjects both on and off their assigned treatments. 
 
                                                 
15 Ibid.  
20 
  Contains Nonbinding Recommendations 
d.  Interim analysis and data monitoring committee 
 
The plan for interim analyses to assess futility and safety should be finalized before trial 
initiation, and included in the statistical analysis plan.  Based on interim  findings, a trial may be 
terminated early for futility if the conditional power is low.  Interim findings such as rate of  
condom usage or specific local practices affecting HIV transmission rate should guide sample 
size adjustments in an ongoing trial.  Such increases in sample size also should be made in 
accordance with accepted guidelines for adaptive trial design as documented in the published 
statistical literature on sample size changes.  Safety concerns, including a greater number of HIV 
seroconversions in the investigational arm, should influence considerations for continuing 
enrollment or halting the trial.  Interim analysis results should be reviewed by an independent 
data monitoring committee to avoid an effect on trial conduct or recruitment.  A detailed charter 
with the composition of the committee members and the operational details should be provided 
for FDA review.16  Sponsors should remain blinded to individual subject and investigator data. 
 
e.  Missing data and sensitivity analysis 
 
The sponsor should minimize lost-to-follow-ups and other types of missing data with appropriate 
planning, including use of incentives that do not interfere with the clinical trial interpretations.  
 
In addition to the analysis mentioned in section III.B.6.a., Endpoint analysis, sponsors should 
perform sensitivity analysis where all lost-to-follow-ups are imputed by the placebo arm hazard. 
 
7.  Combination Products 
 
This section discusses combination products, including drug components.  Specifically, products 
including two or more microbicide drug products or microbicide-device products are discussed.  
Sponsors are encouraged to refer to other FDA guidance for developing combination products.17   
 
In general, the following information evaluating the combination microbicide is needed before 
efficacy studies of a combination product:  cell culture combination activity data, nonclinical 
toxicity for each drug, human safety data from clinical trials for each drug, information 
supporting selection of proposed doses, and drug-drug interaction data (if applicable).  If 
overlapping toxicity is observed in nonclinical studies, then nonclinical toxicity studies with the 
combination product may be necessary.  Trial designs should include provisions for 
demonstrating the contribution of each component to the desired effect.  Establishing the 
contribution of each component generally can be accomplished using factorial designs or 
modified factorial designs.  Because the development pathway depends on the specific drugs 
combined, the approach may vary for different types of combination products.  We encourage 
sponsors to discuss their specific combination product early in the development program.    
                                                 
16 See the guidance for clinical trial sponsors Establishment and Operation  of Clinical Trial Data  Monitoring  
Committees. 
 
17 See the guidances for industry Nonclinical Safety Evaluation  of Drug or Biologic  Combinations and  
Codevelopment of Two or More New Investigational Drugs for Use in Combination.  
21 
  Contains Nonbinding Recommendations 
 
If one of the components of the combination is marketed as the same vaginal formulation and 
dose, regulatory requirements may not follow the paradigm outlined above; therefore, sponsors 
are advised to seek FDA input early in the development process. 
 
Additional considerations for microbicide-device combinations and combinations for multiple 
indications are discussed below. 
 
a.  Vaginal microbicide plus device   
 
Examples of microbicide-device combination include a microbicide combined with a condom, 
cervical cap, or diaphragm.  A microbicide-device combination will be reviewed by both CDER 
and CDRH; the primary mode of action of the combination product will determine which Center 
has the lead. Both Centers work together through the review process by providing expertise for 
individual components of the combination.  The OCP has a formal product jurisdiction process 
that can be initiated by submitting a Request for Designation in cases where the lead Center is 
unclear.18  In some combinations, the sole function of the device is drug delivery.  For this type 
of product, the primary focus of review will be the active microbicide component.  
 
As previously mentioned in section III.A.1.c., Other nonclinical studies, sponsors should conduct 
studies evaluating effects of the microbicide on the integrity and function of the device.  Stability 
data for the combination product will be needed if the microbicide formulation used in the 
microbicide-device combination is different from the stand-alone microbicide.  Sponsors should 
also consider biocompatibility testing on the device with the active drug component.  
 
b.  Combination product intended for multiple indications  
 
Developing combination products where each constituent is intended for a different function or  
indication (e.g., HIV prevention plus contraception) involves complex regulatory considerations.  
A key consideration for such a multi-indication combination product is whether individual 
components are investigational or marketed for the respective indication being pursued.  If a 
component is an unmarketed investigational drug, sponsors should consider whether the drug 
will be approved as an oral formulation or as another vaginally administered formulation for the 
indication being pursued.  We anticipate that a combination product seeking multiple indications 
will need input from multiple CDER divisions, each providing regulatory expertise for the 
specific indication. 
 
The development pathway for multi-indication combination products involves unique and 
complex regulatory challenges.  Because regulatory advice depends on combination product 
characteristics and consultations within CDER, sponsors are encouraged to approach the FDA 
with questions for their specific combination product and the type of indications they plan to 
pursue. 
 
                                                 
18 See the guidance for industry How to Write a Request for Designation (RFD).  
22 
  Contains Nonbinding Recommendations 
8.  Risk-Benefit Considerations 
 
For the overall risk-benefit assessment, the totality of data should be considered.  Benefit in HIV 
prevention trials should be measured as the percent reduction in HIV transmission during the 
trial period. Microbicide efficacy trials should be powered to demonstrate at least a 33 percent 
reduction in HIV seroconversion. Lower rates of reduction in a clinical trial may not translate 
into a clinically relevant effect because lower rates of adherence or other factors with real-world 
use may further reduce the actual risk reduction.  However, we recognize that lower reductions 
may have an effect on transmission rates in high HIV prevalence areas.  The percent reduction in 
HIV and the toxicity profile of a microbicide are critical in the decision-making process.  Other 
considerations in deciding risk-benefit include the potential for behavioral disinhibition in the 
real-world setting, including condom migration (decrease in condom usage secondary to 
increased microbicide uptake), that can adversely affect transmission rates.  Behavioral data, 
rates of other STIs, and frequency of self-reported condom use collected in trials include the 
types of data important for evaluation for the final assessment.  Resistance development may be 
an additional concern, particularly for a systemically absorbed antiretroviral drug product. 
 
C.  Other Considerations 
 
1.  Clinical Virology  
 
Sensitivity and specificity of all assays used to verify HIV status should be validated for a panel 
of viral isolates representing the most common circulating strains endemic to the trial region.  
Sponsors should collect and store baseline samples for RT-PCR for all subjects.  Testing of 
stored baseline/screening samples with RT-PCR should be performed to confirm the infection 
status of subjects who seroconvert during the trial.  Verification by RT-PCR is necessary because 
the antibody assays typically used for baseline analyses (e.g., assay for anti-HIV antibodies) lack 
the sensitivity of the RT-PCR assay for detecting acute infection.  HIV-positive subjects missed 
by the screening HIV assay should not be considered new seroconversions and should be 
excluded from the primary analysis.  
 
For a microbicide containing an antiretroviral drug, viral isolates from subjects who seroconvert 
should undergo genotypic testing. Microbicide failure in these subjects may represent selection 
and enrichment of resistant HIV-1 variants. This selection could occur during local replication 
within the vaginal epithelium or during systemic replication, even if there is poor bioavailability.  
When possible, isolates from a subjects partner also should be genotypically characterized to 
assess the potential for transmission of resistant virus.  If analysis of HIV-1 isolates identifies 
novel substitutions not previously analyzed during nonclinical resistance studies, the 
substitutions should be phenotypically characterized. 
 
2.  Additional Clinical Pharmacology 
 
In vivo drug interaction studies should be considered with the candidate microbicide and 
commonly used vaginal drug products, including the contraceptive ring (e.g., Nuvaring) and 
commonly used antimicrobials (e.g., metronidazole vaginal suppository or gel), to ensure local 
release characteristics and systemic exposure, where applicable, are not adversely affected by co-
23 
  Contains Nonbinding Recommendations 
administration.  The drug interaction data collected should be applicable to the U.S. population 
and U.S. medical practice.  Preferably, all relevant drug interaction studies would be completed 
before conduct of large phase 2b or phase 3 trials to allow use of these drug products in the trials.  
 
Large efficacy trials should incorporate a PK substudy to allow characterization of local and, if 
applicable, systemic microbicide exposure in women during actual conditions of use.  In 
addition, PK samples should be obtained from all women at trial visits at which HIV testing is 
performed and the samples archived for future analysis.  Because microbicides can be developed 
with various coitally dependent and coitally independent dosing schemes, sponsors should 
discuss the sampling schedule with the FDA.  Time  of previous doses and the time of the sample 
collection should be recorded for all PK samples.  In the event of seroconversion, all samples for 
the seropositive subjects should be analyzed and compared to all samples for a matched 
seronegative cohort. Drug concentration data obtained from a large clinical trial may be useful 
to evaluate dose, for assessing adherence patterns, and for further analysis in the event of a failed 
trial. 
 
3.  Chemistry, Manufacturing, and Controls 
 
Sponsors and applicants should refer to FDA and ICH quality guidances for industry for general 
chemistry, manufacturing, and controls (CMC) recommendations for drug substance (active 
pharmaceutical ingredient) and drug product (finished dosage form).  Drug product manufacture 
must be in compliance with current good manufacturing practice requirements (21 CFR parts 
210 and 211). CMC considerations specific to vaginal microbicides are discussed below. 
 
Sponsors should conduct formulation studies to evaluate drug product attributes that affect 
microbicide quality and performance.  Attributes that may affect drug product retention and 
distribution in the vagina or have effects on vaginal epithelium should be evaluated (e.g., drug 
product solubility at different pH environments, drug product stability, rheological 
characteristics, adhesion of the vehicle). The ability of the formulation to support the growth of 
pathogens should be evaluated (e.g., Staphylococcus aureus, Pseudomonas aeruginosa,  Candida 
albicans, and Escherichia coli). Biocompatibility studies should be conducted for vaginal ring 
microbicides.  Attributes that may affect release rate of the active ingredient such as solubility, 
solid-state form, or particle size of drug substance should be evaluated.   
 
In addition, manufacturing parameters potentially affecting drug product performance should be 
identified. Drug product formulations that are not inherently antimicrobial should include 
antimicrobial preservatives to protect them from microbiological growth or from microorganisms 
that are introduced inadvertently during or subsequent to the manufacturing process (United 
States Pharmacopoeia General Chapter <51>).  Preservative effectiveness studies should be 
conducted and the drug product formulated with the minimum specified content (or less) of 
preservative. Sponsors should evaluate the safety, suitability, and performance of the proposed 
container closure system and delivery system (e.g., vaginal applicator).   
 
Drug product specification should include tests for universal attributes such as identity, strength, 
and impurities.  Depending on the specific dosage form, additional tests should be included in 
the drug product specification (e.g., viscosity, pH, particle size distribution, homogeneity 
24 
  Contains Nonbinding Recommendations 
assurance, dissolution or melting rate, in vitro release rate for controlled-release drug products, 
and mechanical integrity test including tensile strength for vaginal rings).  Testing for microbial 
limits also should be part of the drug product specification.  If the active ingredient is a polymer, 
a test should be included for molecular weight distribution (e.g., size-exclusion 
chromatography).  If the drug product contains a preservative, a test for preservative content 
should be included. 
 
In vitro release testing has been shown effective in assessing the drug product quality and 
performance over time for certain semisolid dosage forms.  However, sponsors should not use in 
vitro testing as a surrogate for in vivo bioavailability or bioequivalence, unless an in vitro/in vivo 
correlation can be developed and validated. Further, in vitro release testing generally is not 
considered an appropriate measure for drawing comparisons between different types of semisolid 
formulations (e.g., cream versus ointment from the same or different manufacturer) or in 
comparison of similar formulations across different manufacturers.  Sponsors are reminded that 
the formulation evaluated in phase 3 trials should be identical to the formulation proposed for 
marketing.   
 
The stability profile for the drug product should be established under long-term and accelerated 
storage conditions using analytical methods capable of detecting physical changes and chemical 
degradation. The microbicide should remain stable at a wide range of pH including normal 
vaginal pH. Data from initial stability studies should be provided to support the dosing duration 
of the proposed clinical trials.  Stability data supporting a proposed expiration date for the 
commercial drug product are recommended at the time of NDA submission.   
 
4.  Condom/Device Function Studies 
 
Condom function studies (clinical trials evaluating effects of a microbicide on the failure rate of 
condoms) may be needed for certain microbicides and microbicide/device combinations.  As 
mentioned previously in section III.A.1.c., Other nonclinical studies, results from nonclinical 
compatibility tests will guide the need to conduct clinical trials in this area.  Sponsors are 
strongly encouraged to seek input from the FDA regarding the need for condom function studies 
and the proposed study design. If a clinical trial is needed, sponsors are advised to plan trials 
based on appropriate methodology (Taylor 2009). 
 
5.  Labeling Considerations 
 
The label should emphasize that the drug product is intended for vaginal use only, and that 
efficacy with oral and rectal use are not established.  Labeling should include concerning 
findings in rectal safety trials to convey toxicity issues with potential rectal use.  For vaginal ring 
drug products, a label comprehension study may be needed to ensure instructions for intravaginal 
ring use are appropriate for the U.S. population.  For vaginal gel drug products using an 
applicator, similar studies evaluating end-user ability to correctly apply the drug product should 
be conducted before approval. Because a microbicide may not always be effective in preventing 
HIV acquisition and may not offer any protection against STIs, we anticipate that these drug 
products will be used with other prevention measures (such as condoms) as part of a 
comprehensive prevention strategy.  The product labeling should adequately convey this 
25 
  Contains Nonbinding Recommendations 
concern. In cases where the results of nonclinical condom compatibility studies, condom  
function studies, or other device compatibility studies demonstrate that the microbicide or 
microbicide/device combination has a deleterious effect on condoms or mechanical barrier 
contraceptives, the labeling should state that the microbicide or microbicide/device combination 
should not be used with such devices accordingly.  
 
 
26 
  Contains Nonbinding Recommendations 
REFERENCES 
 
 
ASTM Standard D7661, 2010, Standard Test Method for Determining Compatibility of Personal 
Lubricants with Natural Rubber Latex Condoms, ASTM International, West Conshohocken, PA, 
DOI: 10.1520/D7661-10 (www.astm.org). 
 
Aziz M and Smith KY, 2011, Challenges and Successes in Linking HIV-Infected Women to 
Care in the United States, Clin Infect Dis, 52:S231-237. 
 
Baeten JM, Donnell D, Ndase P et al., 2012, Antiretroviral Prophylaxis for HIV Prevention in 
Heterosexual Men and Women, N Engl J Med, DOI: 10.1056/NEJMoa1108524. 
 
Berges BK, Akkina SR, Folkvord JM et al., 2008, Mucosal Transmission of R5 and X4 Tropic 
HIV-1 Via Vaginal and Rectal Routes in Humanized Rag2-/- gammac -/- (RAG-hu) Mice, 
Virology, 373(2):342-51. 
 
Centers for Disease Control and Prevention (CDC), 2010, HIV/AIDS Fact Sheet for HIV in the 
United States in Women, February.  
 
Denton PW, Estes JD, Sun Z et al., 2008, Antiretroviral Pre-Exposure Prophylaxis Prevents 
Vaginal Transmission of HIV-1 in Humanized BLT Mice, PLoS Med, 5(1):e16. 
 
Fuchs EJ, Lee LA, Torbenson MS et al., 2007, Hyperosmolar Sexual Lubricant Causes Epithelial 
Damage in the Distal Colon:  Potential Implication for HIV Transmission, J Infect Dis, 
195(5):703-10. 
 
Grant RM, Lama JR, Anderson PL et al., 2010, Pre-Exposure Chemoprophylaxis for HIV 
Prevention in Men Who Had Sex With Men, N Engl J Med, 363:2587-99. 
 
Hall HI, Song R, Rhodes P et al., 2008, Estimation of HIV Incidence in United States, JAMA, 
300(5):520-529. 
 
Karim QA, Karim SA, Frohlich JA et al., 2010, Effectiveness and Safety of Tenofovir Gel, An 
Antiretroviral Microbicide, for the Prevention of HIV Infection in Women, 
Sciencexpress.org/19, July. 
 
Karim QA, Richardson BA, Ramjee G et al., 2011, Safety and Effectiveness of BufferGel and 
0.5% PRO2000 gel for the Prevention of HIV Infection in Women, AIDS, 25(7):957-66. 
 
Kulczycki A, Kim Dhong-Jin, Duerr A et al., 2004, The Acceptability of the Female and Male 
Condom:  A Randomized Crossover Trial, Perspectives on Sexual and Reproductive Health, 
36(3):114-119. 
 
Lagakos SW and Gale AR, Editors, 2008, Methodological Challenges in Biomedical HIV 
Prevention Trials, Committee on the Methodological Challenges in HIV Prevention Trials, 
Institute of Medicine of National Academies, The National Academies Press Washington D.C.  
 
27 
  Contains Nonbinding Recommendations 
Lytle CD, Rouston LB, and Cyr WH, 1992, A Simple Method to Test Condoms for Penetration 
by Viruses, Appl. Environ. Microbiol, 58:3180-3182. 
 
Meditz A, Moreau K, Gozansky et al., Healthy Post-Menopausal Women Have Higher 
Percentages of CCR5 Cervical CD4 T Cells Compared to Pre-Menopausal Women:  Implications 
for HIV Transmission, 18th Conference on Retroviruses and Opportunistic Infections Oral 
Abstract.  
 
Moss GB, Celemetson D, DCosta L et al., 1991, Association of Cervical Ectopy With 
Heterosexual Transmission of Human Immunodeficiency Virus:  Results of a Study of Couples 
in Nairobi, Kenya, Journal of Infect Dis, 164:588-91. 
 
Myer L, Kuhn L, Stein ZA et al., 2005, Intravaginal Practices, Bacterial Vaginosis, and 
Womens Susceptibility to HIV Infection:  Epidemiological Evidence and Biological 
Mechanisms, Lancet Infect Dis, 5(12):786-794. 
 
Nelson RM, Lewis LL, Struble K, and Wood SF, 2010, Ethical and Regulatory Considerations 
for the Inclusion of Adolescents in HIV Biomedical Prevention Research, J Acquir Immune 
Defic Syndr, 54:S18-21. 
 
Poles MA, Elliott J, Taing P et al., 2001, A Preponderance of CCR5(+) CXCR4(+) Mononuclear 
Cells Enhances Gastrointestinal Mucosal Susceptibility to Human Immunodeficiency Virus Type 
1 Infection, J Virol, 75:8390-9. 
 
Taylor DJ, 2009, Issues in the Design, Analysis, and Interpretation of Condom Functionality 
Studies, Contraception, 80:237-244. 
 
Thigpen MC, Kebaabetswe PM, Paxton LA, et al., 2012, Antiretroviral Preexposure Prophylaxis 
for Heterosexual HIV Transmission in Botswana, N Engl J Med, DOI: 
10.1056/NEJMoa1110711. 
 
UNAIDS, 2011, Report on the Global HIV/AIDS Epidemic.   
 
United States Pharmacopoeia General Chapter <51>:  Antimicrobial Effectiveness Testing.  
 
Van Damme L, Corneli A, Ahmed K et al., 2012, Preexposure Prophylaxis for HIV Infection 
Among African Women, N Engl J Med, DOI: 10.1056/NEJMoa1202614. 
 
Wu L and Kewalramani V, 2006, Dendritic Cell Interactions With HIV:  Infection and Viral 
Dissemination, Nat Rev Immunol, 6(11):859-868. 
 
28