**Guidance for Industry**

**Antiviral Drug Development --- Conducting Virology Studies and
Submitting the Data to the Agency**

***DRAFT GUIDANCE***

**This guidance document is being distributed for comment purposes
only.**

Comments and suggestions regarding this draft document should be
submitted within 60 days of publication in the *Federal Register* of the
notice announcing the availability of the draft guidance. Submit
comments to the Division of Dockets Management (HFA-305), Food and Drug
Administration, 5630 Fishers Lane, rm. 1061, Rockville, MD 20852. All
comments should be identified with the docket number listed in the
notice of availability that publishes in the *Federal Register*.

For questions regarding this draft document contact Lisa Naeger, Ph.D.,
or Julian J. O'Rear, Ph.D., at 301-827-2330.

**U.S. Department of Health and Human Services**

**Food and Drug Administration**

**Center for Drug Evaluation and Research (CDER)**

**May 2005**

**Clinical Antimicrobial**

**Guidance for Industry**

**Antiviral Drug Development --- Conducting Virology Studies and
Submitting the Data to the Agency**

*Additional copies are available from:*

*Office of Training and Communications*

*Division of Drug Information, HFD-240*

*Center for Drug Evaluation and Research*

*Food and Drug Administration*

*5600 Fishers Lane*

*Rockville, MD 20857*

*(Tel) 301-827-4573*

*http://www.fda.gov/cder/guidance/index.htm*

**U.S. Department of Health and Human Services**

**Food and Drug Administration**

**Center for Drug Evaluation and Research (CDER)**

**May 2005**

**Clinical Antimicrobial**

**TABLE OF CONTENTS**

#  {#section .TOC-1 .unnumbered}

# I. INTRODUCTION [1](#i.-introduction)II. background [1](#ii.-background) {#i.-introduction-1ii.-background-1 .TOC-1}

# III. nonclinical virology reports [2](#iii.-nonclinical-virology-reports) {#iii.-nonclinical-virology-reports-2 .TOC-1}

## A. Overview [2](#a.-overview) {#a.-overview-2 .TOC-2}

## B. Recommended Components of Nonclinical Virology Reports [3](#b.-recommended-components-of-nonclinical-virology-reports) {#b.-recommended-components-of-nonclinical-virology-reports-3 .TOC-2}

### 1. Mechanism of Action [3](#mechanism-of-action) {#mechanism-of-action-3 .TOC-3}

### 2. Antiviral Activity [4](#antiviral-activity) {#antiviral-activity-4 .TOC-3}

#### a. Antiviral activity in vitro [4](#a.-antiviral-activity-in-vitro) {#a.-antiviral-activity-in-vitro-4 .TOC-4}

#### b. Antiviral activity in vitro in the presence of serum proteins [5](#b.-antiviral-activity-in-vitro-in-the-presence-of-serum-proteins) {#b.-antiviral-activity-in-vitro-in-the-presence-of-serum-proteins-5 .TOC-4}

#### c. Inhibitory quotient [6](#c.-inhibitory-quotient) {#c.-inhibitory-quotient-6 .TOC-4}

#### d. Antiviral activity in vivo [6](#d.-antiviral-activity-in-vivo) {#d.-antiviral-activity-in-vivo-6 .TOC-4}

### 3. Cytotoxicity/Therapeutic Index [6](#cytotoxicitytherapeutic-index) {#cytotoxicitytherapeutic-index-6 .TOC-3}

### 4. In Vitro Combination Activity Analysis [7](#in-vitro-combination-activity-analysis) {#in-vitro-combination-activity-analysis-7 .TOC-3}

### 5. Resistance [7](#resistance) {#resistance-7 .TOC-3}

#### a. Selection of resistant virus in vitro [7](#a.-selection-of-resistant-virus-in-vitro) {#a.-selection-of-resistant-virus-in-vitro-7 .TOC-4}

#### b. Genotypic analysis [8](#b.-genotypic-analysis) {#b.-genotypic-analysis-8 .TOC-4}

#### c. Phenotypic analysis [9](#c.-phenotypic-analysis) {#c.-phenotypic-analysis-9 .TOC-4}

#### d. Cross-resistance [10](#d.-cross-resistance) {#d.-cross-resistance-10 .TOC-4}

# Iv. Proposal for Monitoring Resistance Development [10](#iv.-proposal-for-monitoring-resistance-development) {#iv.-proposal-for-monitoring-resistance-development-10 .TOC-1}

# V. Virology Study Reports [12](#v.-virology-study-reports) {#v.-virology-study-reports-12 .TOC-1}

# VI. SUMMARY [12](#vi.-summary) {#vi.-summary-12 .TOC-1}

# APPENDIX 1: Template for Submitting HIV Resistance Data [13](#template-for-submitting-hiv-resistance-data) {#appendix-1-template-for-submitting-hiv-resistance-data-13 .TOC-1}

# APPENDIX 2: Template for Submitting HBV Resistance Data [18](#template-for-submitting-hbv-resistance-data) {#appendix-2-template-for-submitting-hbv-resistance-data-18 .TOC-1}

# APPENDIX 3: Template for Submitting HCV Resistance Data [22](#template-for-submitting-hcv-resistance-data) {#appendix-3-template-for-submitting-hcv-resistance-data-22 .TOC-1}

**Guidance for Industry**[^1]

**Antiviral Drug Development --- Conducting**

**Virology Studies and Submitting the Data to the Agency**

This draft guidance, when finalized, will represent the Food and Drug
Administration's (FDA's) 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

The purpose of this guidance is to assist sponsors in the development of
antiviral drug products and to serve as a starting point for
understanding the nonclinical and clinical virology data important to
support clinical trials of antiviral agents. This guidance focuses on
nonclinical and clinical virology reports, which are essential
components in the review of investigational antiviral drugs. Topics in
this guidance include studies defining the mechanism of action,
establishing specific antiviral activity of the investigative drug,
providing data on the development of viral resistance to the
investigational drug, and providing data identifying cross-resistance to
approved drugs having the same target.

FDA's guidance documents, including this guidance, do not establish
legally enforceable responsibilities. Instead, guidances describe the
Agency's 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 recommendations in this guidance are based on the review experience
with antiviral drugs of the Division of Antiviral Drug Products (DAVDP)
as well as input from pharmaceutical sponsors and the scientific
community. Because of the experience, history, and lessons learned with
HIV-1 studies, this guidance focuses on studies commonly used to
evaluate HIV-1 drugs and uses them as a paradigm for future studies of
drugs to treat other viruses. Since the field of virology is dynamic and
continually evolving, this guidance will be revised as new information
accumulates and as circumstances warrant.

Guidances on the overall organization of investigational new drug (IND)
applications and new drug applications (NDAs) can be found at
www.fda.gov/cder/regulatory/applications/default.htm. Sponsors are
encouraged to contact the Agency early in the development of an
investigational antiviral drug to facilitate the review and approval
process. To assist prospective sponsors, the Agency accepts informal
submissions in an abbreviated IND format (i.e., pre-INDs) for review and
comment. Pre-INDs are especially useful in instances where applicants
are unfamiliar with the process for evaluating investigational drug
products in humans. Information about submitting pre-INDs can be found
at www.fda.gov/cder/ode4/preind/getting.htm. The Agency accepts
electronic submissions to expedite the review process. FDA Web sites can
be consulted for information on electronic application submission.

# III. nonclinical virology reports

## A. Overview

Nonclinical virology reports aid in evaluating the safety and efficacy
of an investigational drug before it is tested in humans. The reports
can include studies identifying the mechanism of action, establishing
specific antiviral activity of the compound in a model system, and
providing data on the development of viral resistance to the
investigational drug. Additionally, co-administration of an
investigational drug with other drugs approved for the same indication
is often necessary to treat virus infections in clinical settings. In
these cases, it is desirable to have in vitro combination activity
studies designed to identify possible negative interactions on antiviral
activity (i.e., antagonism) of the investigational drug with other
antiviral drugs.

As more antiviral drugs are developed to treat particular viral
diseases, cross-resistance (i.e., viral resistance to one drug causing
resistance to more than one agent within that drug class) can become a
major issue in clinical settings. Therefore, from a scientific
perspective, the following information is critical in the development of
antiviral drug products:

-   Determining the antiviral activity of an investigational drug
    against viruses resistant to other drugs with the same molecular
    target

-   Determining the antiviral activity of approved drugs against viruses
    resistant to the investigational drug with the same molecular target

Specific details of each of the nonclinical studies recommended by the
FDA can be found in Section III.B., Recommended Components of
Nonclinical Virology Reports. We recommend conducting nonclinical
studies (i.e., mechanism of action, antiviral activity in vitro,
cytotoxicity/therapeutic index, and effects of serum protein binding on
antiviral activity) before the initiation of phase 1 clinical studies.
We suggest that sponsors complete in vitro drug combination activity
studies of the investigational drug with the approved drugs before the
initiation of clinical trials that will examine the efficacy of the
investigational drug in combination with approved drugs. Furthermore, we
recommend examining the in vitro selection of resistant viruses to the
investigational drug, the phenotypic and genotypic characterization of
the resistant viruses, and cross-resistance analyses before initiation
of clinical studies in patients infected with the particular virus.
Complete virology study reports on nonclinical and clinical studies can
be submitted upon their completion and need not be held until submission
of the NDA.

## B. Recommended Components of Nonclinical Virology Reports 

### 1. Mechanism of Action

We prefer that mechanism of action studies be conducted before the
initiation of phase 1 clinical studies. Nonclinical virology reports on
the mechanism of action should include background information and data
identifying the mechanism of action of the investigational drug and its
metabolites. There are many steps in a virus life cycle that can be
targeted by potential drug candidates. Compounds can act directly to
inhibit a virus by targeting a specific viral-encoded function (e.g., an
enzyme inhibitor) or act indirectly (e.g., interferon induction of host
cell response). We recommend that mechanism of action studies:

-   Demonstrate the investigational drug's ability to specifically
    inhibit viral replication or a virus-specific function

-   Establish the site of the drug's action (e.g., viral replicase,
    integrase, protease)

Biochemical, structural, cellular, or genetic data can be presented to
support the proposed mechanism of action. Data that demonstrate the
mechanism of action include, but are not limited to, receptor binding,
inhibition of enzymatic activity, X-ray crystallographic structure
determination of bound inhibitor complex, and characterization of
resistance mutations in the gene encoding the target.

A well-characterized mechanism of action is useful in predicting
toxicities and in designing studies to assess the development of
resistance. A clear understanding of the mechanism of action for an
investigational antiviral drug can provide insight into the regions of
the viral genome where resistance mutations could develop. These regions
are not limited to the site of action (viral-encoded target) of the
investigational drug but can include the enzyme substrate or another
viral-encoded protein existing in a complex with the target protein.
Characterization of the resistance mutations can provide in vivo
validation of the mechanism of action studies.

The specificity of the investigational drug should be demonstrated for
the viral target over cellular/host proteins, especially in those cases
in which a viral enzyme has a cellular counterpart. For example, if the
investigational drug targets a viral polymerase, we recommend showing
the activity of the drug against the viral polymerase in comparison with
its activity against host DNA polymerases such as DNA Polymerase α, β,
and γ.

Immunomodulatory drugs raise additional issues. These drugs can have the
potential for unintended adverse effects resulting from the drug's
actions on the immune system in addition to effects on viral
replication. We recommend that an indirect antiviral effect and the
specific immune system components that are involved in the drug's action
be identified for investigational drugs that act through a general
immune stimulatory mechanism. We recommend consulting the DAVDP for
specific advice regarding the development of immunomodulatory drugs for
the treatment of viral diseases.

### 2. Antiviral Activity 

#### a. Antiviral activity in vitro

For many human viruses, there are cell culture systems or animal hosts
in which the infectious agent can undergo a complete virus life cycle.
In these cases, we recommend that the sponsor document that the
investigational drug and/or its metabolites show specific, quantifiable
antiviral activity in vitro before initiating tests in humans (i.e.,
before initiation of phase 1 studies). It is important that these data
support clinical testing in humans by providing clear evidence of
antiviral effects at drug concentrations that can be achieved in vivo
with acceptable risk-benefit. Additionally, in vitro antiviral activity
and cytotoxicity assessments (see Section III.B.3.,
Cytotoxicity/Therapeutic Index) can be used to guide the selection of
appropriate dose ranges in early clinical trials by establishing a
dose/response relationship using a broad range of relevant cell types
and virus isolates. If possible, sponsors are encouraged to obtain
antiviral activity data using primary human target cells. Because of
viral genetic variation, the antiviral activity of the investigational
drug should be examined for multiple clinical isolates and viral
isolates representative of the virus population in clinical trials.
Antiviral activity evaluations that are recommended to support the
development of the investigational drug include:

-   Assessing specific antiviral activity of the investigational drug
    against a broad range of clinical and laboratory viral isolates
    including different clades, subtypes, or genotypes

-   Evaluating the antiviral activity of the investigational drug
    against mutant viruses that are resistant to drugs with the same
    molecular target as the investigational drug as well as viruses
    resistant to other drugs for the same indication

We recommend determining specific antiviral activity using a
quantitative assay to measure virus replication in the presence of
increasing concentrations of the drug compared to replication in the
absence of drug. The inhibitory concentration (IC~50~) is the
concentration of the drug at which virus replication is inhibited by 50
percent. Assays that evaluate antiviral activity and cytotoxicity
include but are not limited to virus inactivation assays, plaque
reduction assays, cytopathic effect inhibition assays, peripheral blood
mononuclear cell (PBMC) assays, and binding and fusion assays. Other
factors that can be assessed in these assays include the effect of an
increasing multiplicity of infection and the effect of pretreatment of
virus or cells before infection versus treatment post-infection.

It is important that the inhibitory concentration be consistent with
data supporting the mechanism of action. An investigational drug that
inhibits virus replication at concentrations lower than biochemical data
for the proposed mechanism indicates that another target or mechanism of
inhibition may be affected. For nucleoside or nucleotide analogs, we
recommend that sponsors determine the intracellular half-life (t~1/2~)
of the triphosphate form of the active drug moiety in stationary and
dividing cells from the target tissue.

For some human viruses (e.g., Hepatitis B and Hepatitis C viruses), no
satisfactory cell culture or animal model exists, and in these cases,
inhibition of an essential viral function or activity against related
viruses can be used to indicate potential activity. When no satisfactory
cell culture or animal model exists for the target human virus, it is
particularly important to know whether or not a drug's active moiety
enters cells, if it has a proposed intracellular site of action, and if
the intracellular concentration is consistent with biochemical studies
identifying an inhibitory concentration. Cell-based assays and host cell
lines for studying viruses such as Hepatitis B Virus (HBV) and Hepatitis
C Virus (HCV) replication may advance and improve, but at the present
time are limited. For analysis of HCV replication, a replicon system has
been developed that permits studies of viral replication and can be used
to assess antiviral activity of some anti-HCV drugs. Currently, assays
that examine HBV replication include, but are not limited to:

-   Measuring HBV DNA polymerase activity in biochemical assays

-   Cell culture assays with baculovirus-mediated cell transfer or
    transfection of HBV genomes into human hepatoma cell lines followed
    by quantitative Southern blot analysis with HBV DNA probes

-   Cell culture assays with stably-transfected cell lines containing
    the HBV genome

-   Quantitative PCR of extracellular HBV DNA

#### b. Antiviral activity in vitro in the presence of serum proteins

Serum proteins can bind to and sequester many drugs and may interfere
with a drug's antiviral activity. We recommend that sponsors ascertain
if the investigational drug is significantly bound by serum proteins.
Common methods for determining protein binding include equilibrium
dialysis, ultrafiltration methods, and fluorescence-based high
throughput human serum albumin and α-acidic glycoprotein protein
binding. If the investigational drug is highly protein bound, sponsors
are encouraged to examine the in vitro antiviral activity of the
investigational drug in the presence of increasing concentrations of
human serum up to 40 to 50 percent. An IC~50~ for 100-percent human
serum can be extrapolated from this data and the serum adjusted IC~50~
values reported. Sponsors are also encouraged to determine IC~50~ values
in the presence of 2 mg/mL\
α-acidic glycoprotein.

#### c. Inhibitory quotient

Information on plasma and intracellular drug concentrations is important
in assessing the dose/response of antiviral therapy and evaluating the
potential for resistance development; therefore, it is useful to
determine an inhibitory quotient (IQ).[^2] (For more information on
determining IC~50~ values, see Section III.B.2.a., Antiviral activity in
vitro.) We view IQ ratios as a useful tool integrating in vivo drug
concentrations and antiviral activity. It is a measure that
characterizes the relationship between drug exposure and the
susceptibility of a virus to a drug. A high IQ indicates an effective
drug concentration can be achieved in a patient to inhibit the virus and
minimize the development of drug resistance. Since one dose may not be
adequate for all patient populations, IQ ratios can be used to aid in
the selection of a dose or doses to further evaluate in phase 3 clinical
studies.

#### d. Antiviral activity in vivo

In cases where no cell culture or replicon system has been shown to be
predictive of antiviral activity in humans, measurement of viral titers
after drug treatment in animal model systems can be used to assess
antiviral activity of the investigational drug. Analyses from animal
models include the morbidity and mortality of animals following
documented infection, histologic examination of tissues, quantification
of viral titers over time, isolation and characterization of resistant
isolates in animals that experience viral rebound, quantification of
viral antigens and antibodies, pharmacokinetics of the investigational
drug, and a description of symptoms (e.g., neurologic, weight loss).
Antiviral drugs for smallpox and other orthopoxviruses require special
considerations. Consult the DAVDP for guidance on the development of
investigational drugs against orthopoxviruses.

### 3. Cytotoxicity/Therapeutic Index

> It is important to establish that an investigational drug has
> antiviral activity at concentrations that can be achieved in vivo
> without inducing toxic effects to cells. Furthermore, in a cell
> culture model, the apparent antiviral activity of an investigational
> drug can result from host cell death after drug exposure. Cytotoxicity
> tests use a series of increasing concentrations of the antiviral drug
> to determine what concentration results in the death of 50 percent of
> the host cells. (See also Section III.B.2.a., Antiviral activity in
> vitro.) This value is referred to as the median cellular cytotoxicity
> concentration and is identified by CC~50~ or CCIC~50~. The relative
> effectiveness of the investigational drug in inhibiting viral
> replication compared to inducing cell death is defined as the
> therapeutic or selectivity index (i.e., CC~50~ value/IC~50~ value). It
> is desirable to have a high therapeutic index giving maximum antiviral
> activity with minimal cell toxicity. We recommend determining CC~50~
> values in both stationary and dividing cells from multiple human cell
> types and tissues for potential cell-cycle, species, or
> tissue-specific toxicities. We prefer that studies determining
> cytotoxicity/therapeutic index be conducted before the initiation of
> phase 1 clinical studies.

Because of the myelosuppressive effects of certain antiviral drugs, we
recommend assessing the potential effects of certain candidate drugs on
the growth of human bone marrow progenitor cells in colony formation
assays. Some investigational antiviral drugs are potential inhibitors of
DNA polymerases. The cellular DNA polymerases are responsible for normal
nuclear and mitochondrial DNA synthesis and repair. The inhibition of
human pol γ, the enzyme responsible for mitochondrial DNA synthesis, has
been linked to defects in mitochondrial function. Therefore, it is
important to monitor the effects of certain investigational drugs (e.g.,
nucleoside analogs) on mitochondrial toxicity by examining mitochondrial
morphology, glucose utilization, lactic acid production, and
mitochondrial DNA content.

### 4. In Vitro Combination Activity Analysis 

Within an infected individual, viruses can exist as a heterogeneous
population of variant viruses, some of which may show reduced
susceptibility to one or more antiviral drugs. Therefore, for some
viruses, administration of multiple antiviral drugs (e.g., three-drug
combination antiretroviral therapy against HIV-1) can be more effective
than a single drug in establishing and maintaining inhibition of virus
replication. However, the interactions of drugs are complex and can
result in antagonistic, additive, or synergistic effects with respect to
antiviral activity. For this reason, we recommend that sponsors evaluate
the in vitro antiviral activity of investigational drugs in two- or
three-drug combinations with other drugs approved for the same
indication. We also recommend completing the in vitro drug combination
activity studies of approved drugs with the investigational drug prior
to initiation of the clinical trials that will evaluate the efficacy of
the combination of the investigational drug with approved drugs. Often
subjects are infected with two or more viral diseases (e.g., HIV and HBV
or HCV); therefore, we recommend that the in vitro antiviral activity of
antiviral drugs used in co-infected patients for different indications
be assessed in in vitro combination activity studies.

### 5. Resistance

#### a. Selection of resistant virus in vitro

This guidance focuses on resistance to antiretroviral agents caused by
mutations in the viral genome that result in reduced phenotypic
susceptibility to a given drug product. We recommend that the in vitro
selection of resistant viruses to the investigational drug, the
phenotypic and genotypic characterization of resistant viruses, and
cross-resistance analyses be examined before initiation of clinical
studies in patients infected with the particular virus. The completion
of in vitro resistance selection studies is recommended to assess the
potential of a target virus to mutate and develop reduced susceptibility
(i.e., resistance) to the investigational drug. Resistance, as it is
used here, is not an absolute term but relative.[^3]

Selection in cell culture of virus resistant to the investigational drug
can provide insight into whether the genetic threshold for resistance
development is high or low. Several factors specific to the
investigational drug and the target virus affect the development of
resistance (e.g., drug concentration). For some drugs, the development
of resistance in the presence of high concentrations of the
investigational drug occurs as a result of a single viral mutation
whereas others require multiple mutations in the virus. A product with a
low genetic threshold may select for resistance with only one or two
mutations. In contrast, a product with a high genetic threshold may
require several mutations to result in viral strains with reduced
susceptibility. The rate of appearance of mutant viruses is dependent
upon the rate of replication of the virus, the number of virus genomes
produced, the fidelity of the replicative machinery, and host factors.
Consideration of these factors can help in designing tests to detect the
appearance in vitro of virus resistant to high concentrations of the
drug. For example, many cell culture systems do not produce sufficient
virus titers in those instances where multiple mutations are required to
develop resistance to high concentrations of the investigational drug.
In these instances, serial passage of the virus in cell culture under
conditions of increasing concentrations of the investigational drug can
lead to the isolation of resistant virus. Sponsors are encouraged to
assess the development of resistance in vitro over the concentration
range spanning the anticipated in vivo concentration and to determine if
the same or different patterns of resistance mutations develop by
repeating the selection of variants resistant to the investigational
drug several times.

If the targeted virus replicates in a cell culture system, two basic
methods can be employed to isolate viruses that have reduced
susceptibility to the investigational drug.

-   In the first method, a high initial virus inoculum is propagated for
    several passages at a fixed drug concentration, using multiple
    cultures to test different concentrations. This approach is useful
    in identifying drugs for which there is a low genetic barrier to
    resistance.

-   In the second method, virus is passaged in the presence of
    increasing drug concentrations starting at half the IC~50~ value for
    the parental virus. Virus production is monitored to detect the
    selection of resistant virus that is then characterized with respect
    to genotype and phenotype.

#### b. Genotypic analysis 

Genotypic analysis of resistant viruses selected in vitro determines
which mutations might contribute to reduced susceptibility to the
investigational drug. Identifying resistance mutations by DNA sequence
analysis of the relevant portions of the virus genome can be useful in
predicting clinical outcomes and supporting the proposed mechanism of
action of the investigational drug. It is highly recommended that
sponsors determine the entire coding sequence of the gene for the target
protein and compare the pattern of mutations leading to resistance of
the investigational drug with the pattern of mutations of other drugs in
the same class. In the case of larger viruses, we suggest that the
relevant portions of the viral gene targeted by the investigational
agent be sequenced and analyzed for mutations that could contribute to
drug resistance. It is preferable to characterize resistance pathways in
several genetic backgrounds (i.e., strains, subtypes, genotypes) and to
obtain isolates during the selection process to identify the order in
which multiple mutations appear.

We recommend that sponsors provide the performance characteristics and
the details of the methodology of investigational genotypic assays.
Sponsors are encouraged to identify sequencing primers, state how many
bases can be read from the primer accurately, and define the sensitivity
of the genotypic assay used for detecting minority viral subpopulations.
It is important that sponsors define what percentage of the population a
mutation has to represent to be detected in their genotypic assay.

##### 

#### c. Phenotypic analysis 

Phenotypic analysis determines if mutant viruses have reduced
susceptibility to the investigational drug. Once resistance-associated
mutations are identified, the ability of each of these mutations to
confer phenotypic resistance should be evaluated in a recombinant virus
system (e.g., by using site-directed mutagenesis or PCR amplification of
relevant portions of virus genome to introduce these mutations into a
standard laboratory genetic background). Recombinant viruses should then
be tested in vitro for drug susceptibility to determine an IC~50~ value.
Phenotypic results can be determined with any standard virus assay: p24
assay, viral RNA assay, RT assay, MTT cytotoxic assay, reporter gene
expression, and others. The shift in susceptibility (or fold resistant
change) for a viral isolate should be measured by determining the IC~50~
values for the isolate and comparing it to the IC~50~ value of a
reference virus done under the same conditions and at the same time. The
fold resistant change should be calculated as the IC~50~ value of the
isolate/IC~50~ value of the reference strain. We recommend that a
well-characterized wild-type laboratory strain grown in cell culture
serve as a reference standard.

The utility of a phenotypic assay depends upon its sensitivity (i.e.,
its ability to measure shifts in susceptibility (fold resistance change)
in comparison to reference strains or baseline clinical isolates).
Calculating the fold resistant change (IC~50~ value of isolate/IC~50~
value of reference strain) allows for comparisons between phenotypic
assays.

Well-characterized genotypic and phenotypic assays should be developed
to detect the emergence of resistant virus during the development of
candidate drugs. Sponsors can choose to conduct phenotypic and genotypic
analyses themselves or send samples to a third party certified by
Clinical Laboratory Improvement Amendments (CLIA). Proper handling
procedures should be followed for laboratory samples. If the assay is
investigational, we recommend that sponsors provide the performance
characteristics (e.g., accuracy, precision, limits of detection and
quantification, specificity, linearity, range, robustness, stability) of
an investigational phenotypic assay and genotypic assay, as well as
sources of viruses (e.g., blood, plasma), their storage and stability,
and cell culture procedures. For definitions on assay validation, refer
to the CDER guidance for industry *Bioanalytical Method Validation* and
the ICH guideline *Q2A Text on Validation of Analytical Procedures*.
Sponsors are encouraged to use approved assays that are characterized
and validated. (If an investigational assay is not performed according
to the manufacturer's specifications, the assay results might not be
accepted.) We recommend that sponsors be consistent in the genotypic and
phenotypic assays used in studies and that the same assay be used for a
particular patient throughout the study.

#### d. Cross-resistance 

Antiviral drugs targeting the same protein (typically agents of the same
drug class) may develop mutations that lead to reduced susceptibility to
one antiviral drug, and can result in decreased or loss of
susceptibility to other antiviral drugs in the same drug class. This
observation is referred to as cross-resistance. Cross-resistance is not
necessarily reciprocal, so it is important to evaluate both
possibilities. For example, if virus X is resistant to drug A and drug
B, and virus Y is also resistant to drug A, virus Y may still be
sensitive to drug B. Cross-resistance analyses are important in the
development of treatment strategies (i.e., establishing the order in
which drugs are given). We recommend that the effectiveness of the
investigational drug against viruses resistant to other approved drugs
in the same class and the effectiveness of approved drugs against
viruses resistant to the investigational drug be evaluated by phenotypic
analyses. Additionally, we recommend that cross-resistance be analyzed
between classes in instances where more than one class of drugs targets
a single protein or protein complex (e.g., nucleoside reverse
transcriptase inhibitors (NRTIs) and non-nucleoside reverse
transcriptase inhibitors (NNRTIs), which both target the HIV-encoded
reverse transcriptase). We recommend that multiple clinical isolates be
examined by phenotypic assays with the investigational drug and clinical
isolates representative of the breadth of diverse mutations and
combinations known (if known) to confer reduced susceptibility. If
phenotyping is performed in cell lines, the IC~50~ value obtained in the
cell line should be validated with clinical isolates.

# Iv. Proposal for Monitoring Resistance Development

Given the importance of drug resistance in treatment decisions and the
need to disseminate to health care providers information about an
antiviral drug's resistance profile, it is strongly recommended that
comprehensive resistance testing be undertaken in all phases of drug
development. Crucial decisions in protocol design and drug development
plans often hinge on resistance and cross-resistance data.

For some viruses, measurements of changes in viral concentrations are
accepted endpoints to determine clinical effectiveness of antiviral
drugs. Sponsors can choose to quantify viral loads themselves or send
samples to companies that have previously been certified by CLIA. In the
latter case, proper handling procedures should be followed for
laboratory samples. If the antiviral assay has not been previously
reviewed and found acceptable by the FDA, we recommend that the sponsor
provide a complete description of the methodology and the quantitative
assay performance characteristics including upper and lower limits of
detection, linearity, and variability and the sources of viruses (e.g.,
blood, plasma), their storage and stability, and cell culture
procedures. Assay results from an assay that is not performed to the
manufacturer's specifications might not be accepted. We recommend that
sponsors consistently use the same assay for any particular analysis or
measurement in studies and that the same assay be used for a particular
patient throughout the study.

Prior to the initiation of clinical efficacy studies, we recommend that
sponsors submit a plan to monitor the development of resistant viruses
in the clinical studies. We recommend including these plans in the
nonclinical reports and the overall clinical development plan in the
IND. The resistance monitoring plan should include:

-   A description of the assays that will be used to monitor viral loads

-   How the assays will be performed

-   The methods for sample collection and storage

-   The methods for sample handling (frozen or ambient)

-   The genotypic and phenotypic assays that will be used

-   Genotypic and phenotypic assay protocols

-   Time points that samples for viral loads, genotypic and phenotypic
    assays, and other resistance analyses will be collected (i.e.,
    baseline, week 24, week 48, discontinuations)

Phenotypic and genotypic results are used to define treatment options
and to predict the utility of treating an individual with an
investigational drug. Genotypic analysis of virus isolates from patients
failing to respond to therapy or undergoing viral rebound can help
identify mutations that contribute to reduced susceptibility to the
investigational drug. In addition, genotypic and phenotypic analyses of
baseline isolates are encouraged to determine response to therapy
outcomes based on baseline mutations/polymorphisms and baseline
phenotypic drug susceptibilities. We recommend submitting plans for
genotypic and phenotypic baseline studies and resistance substudies
early in drug development.

We suggest that genotypic and phenotypic analyses of baseline and
post-treatment isolates be completed in a timely manner to characterize
the resistance profile of the investigational drug and its
cross-resistance potential with other antiviral drugs. Sponsors are
strongly encouraged to collect (at a minimum) phenotype and genotype
data for baseline isolates from all patients and endpoint isolates from
all virologic failures and discontinuations (not suppressed). *Virologic
failures*[^4] are protocol defined but for HIV-1 studies often
include: 1) Rebound: confirmed (two consecutive) plasma viral load
values greater than Lower Limit of Quantification (LLOQ) after achieving
confirmed level below the LLOQ during the treatment phase; 2) Never
Suppressed: viral load levels never achieve confirmed suppression with
at least 48 weeks of randomized treatment; or 3) Insufficient Viral
Response: viral load levels never achieve confirmed suppression and the
investigator identifies insufficient viral load response as the reason
for treatment discontinuation prior to week 48. Sponsors with
investigational drugs for other viral diseases can solicit advice from
the FDA early in drug development on definitions of clinical
response/failure and plans to monitor resistance in clinical studies. We
recommend that sponsors consult with the DAVDP for detailed descriptions
and examples of how to submit clinical viral resistance data. The DAVDP
has templates for submitting resistance data for HIV, HBV, and HCV. (See
Appendixes 1-3.)

# V. Virology Study Reports

Complete virology study reports are extensive and should include the raw
and analyzed data as well as all the information necessary to evaluate
the procedures used to obtain those data. Virology study reports should
convey information on nonclinical studies, clinical antiviral activity
of the investigational drug, resistance development to the
investigational drug in treated patients, and cross-resistance with
other drugs in the same drug class. The format of a virology study
report should be similar to that of a scientific publication and
typically should include the following sections: summary, introduction,
materials and methods, results, and discussion. The methods section
should describe all the protocols employed and include a description of
the statistical analyses used. Complete virology study reports on
nonclinical and clinical studies can be submitted upon their completion
and need not be held until submission of the NDA.

# VI. SUMMARY

The nonclinical virology section of this guidance identifies studies
relevant to development of antiviral drugs for the treatment of viral
infections. The goal of this guidance is to stimulate the generation of
more complete analyses for antiviral drug products. These analyses
provide data that support the introduction of an investigational drug
into humans, and provide data necessary for determining dose/response
relationships, designing clinical trials, and selecting appropriate
patient populations. Thus, these studies have an effect on the
therapeutic success of a given product. Because the nonclinical in vitro
virology studies provide useful information for the design of in vivo
studies and can be predictive of the development of resistant viruses in
vivo, we recommend conducting nonclinical studies before the initiation
of phase 1 clinical studies and examining the analysis of in vitro
selected resistant viruses before initiation of clinical studies in
patients infected with the particular virus. The preceding sections of
this guidance include recommendations for how and when to perform
nonclinical virology studies. Such information could potentially be
included in drug labeling to facilitate appropriate prescribing of
products and to maximize the chance for therapeutic success.

# APPENDIX 1:

# Template for Submitting HIV Resistance Data

One dataset combines patient data, endpoint data, genotypic data, and
phenotypic data. There are a number of ways datasets can be subdivided
(i.e., by clinical study, baseline isolates, or virologic failure
isolates) and it is recommended that this be discussed with the DAVDP
before submission.

For each study, we recommend constructing datasets as SAS transport
files containing the following information:

-   One record (row) per patient per isolate (e.g., baseline, failure,
    and other time points).

```{=html}
<!-- -->
```
-   Data in columns (with suggested column headings shown below) on all
    isolates.

```{=html}
<!-- -->
```
-   Genotypic data for (at a minimum) baseline isolates of all patients
    and the endpoint isolates of virologic failures and discontinuations
    --- on the corresponding record for each patient isolate.

-   Phenotypic data for (at a minimum) baseline isolates and the
    endpoint isolates of virologic failures and discontinuations --- on
    the corresponding record for each patient isolate.

*Virologic failures* (other definitions can be used and discussed with
the DAVDP)[^5] --- 1) Rebound: confirmed (two consecutive) plasma HBV
DNA values greater than or equal to 1 log~10~ copies/mL increase in
serum HBV DNA from the on treatment nadir; 2) Never Suppressed: plasma
HBV DNA levels never achieve confirmed suppression with at least 48
weeks of randomized treatment; or 3) Insufficient Viral Response: plasma
HBV DNA levels never achieve confirmed suppression and investigator
identifies the reason for treatment discontinuation prior to week 48 due
to insufficient viral load response: nonresponders and rebounders.

**Information to Include with Suggested Column Headings**[^6]

**I. Patient Data**

-   Patient identification number

```{=html}
<!-- -->
```
-   Isolate (e.g., baseline, week 24, week 48, discontinuation. Multiple
    isolates numbered (e.g., failure 1, failure 2))

```{=html}
<!-- -->
```
-   Date of isolate

-   Previous therapeutic agents where available

-   Treatment group

**II. Endpoint Data**

-   HIV RNA (copies/mL) at baseline

```{=html}
<!-- -->
```
-   HIV RNA (copies/mL) at predefined time points (e.g., week 24 and
    week 48)

```{=html}
<!-- -->
```
-   HIV RNA (copies/mL) at time of loss of virologic response or
    discontinuation due to adverse event

```{=html}
<!-- -->
```
-   Endpoint assessment (e.g., mean log change in viral load from
    baseline)

-   Other endpoint assessments (e.g., DAVG, mean log change in viral
    load from baseline)

```{=html}
<!-- -->
```
-   Indication of data censored for reasons other than virologic failure
    (e.g., discontinuation due to adverse event)

-   Outcome (i.e., responder, virologic failure, discontinuation while
    suppressed, discontinuation before achieving viral suppression)

-   Reason for discontinuation (i.e., adverse event, pregnancy) or
    failure (i.e., never suppressed, rebound)

-   HIV RNA (copies/ml) from additional time points can be included.

**III. Genotypic Data** (for baseline isolates of all patients and
endpoint isolates from virologic failures and discontinuations)

-   Clade

-   Genotype for the RT, protease and gp160 (for agents targeting entry
    only), one amino acid per column with the wild-type (WT) amino acid
    as column heading. Changes from WT standard sequence indicated
    (i.e., blanks indicate no change).

**Example** (This example highlights how genotype information can be
displayed but does not include all column headings as previously
suggested.)

  --------- --------- ----- ----- ----- ------ ------ ------ ------ ------ ------
  Patient   Isolate   V82   N83   I84   I85    G86    R87    N88    L89    L90
  \#                                                                       

  001       BL                                               S             M/L

  001       WK48                  V                          S             M

  002       BL        A/T         V                          D             M

  002       WK48      T           V                                        M

  003       BL        T           V                                        

  004       BL                    V                                        M
  --------- --------- ----- ----- ----- ------ ------ ------ ------ ------ ------

BL = baseline

WK48 = week 48

**IV. Protease Cleavage Sites** (for protease inhibitors only)

-   p2/NC protease cleavage site: show amino acid and position of
    cleavage site of WT in column headings (as above for genotype) and
    indicate amino acid change if mutant

```{=html}
<!-- -->
```
-   NC/p1 Gag cleavage sites: show amino acid and position of cleavage
    site of WT in column headings (as above for genotype) and indicate
    amino acid change if mutant

-   p1/p6 Gag cleavage sites: show amino acid and position of cleavage
    site of WT in column headings (as above for genotype) and indicate
    amino acid change if mutant

**V. Phenotypic Data** (minimally for baseline isolates and endpoint
isolates from virologic failures and discontinuations)

-   Candidate drug

    -   Baseline IC~50~ value for candidate drug

    -   IC~50~ value of reference strain for candidate drug

    -   Fold change of baseline IC~50~ value compared to IC~50~ value of
        reference strain of candidate drug

    -   IC~50~ value at time of endpoint assessment or failure for
        candidate drug

    -   Fold change values in IC~50~ value at time of endpoint
        assessment or failure compared to reference strain for candidate
        drug

    -   Fold change in IC~50~ value at time of endpoint assessment or
        failure compared to baseline for candidate drug

-   Approved/investigational anti-HIV agents (List first agents in the
    same class in alphabetical order followed by agents with the same
    target protein in alphabetical order. End with agents outside drug
    class in alphabetical order.)

    -   Fold change in IC~50~ value of baseline compared to reference
        strain for all approved/investigational anti-HIV agents

    -   Fold change in IC~50~ value at time of endpoint assessment or
        failure compared to reference strain for each of the
        approved/investigational anti-HIV agents

    -   Fold change in the IC~50~ value at time of endpoint assessment
        or failure compared to baseline for each of the
        approved/investigational anti-HIV agents

**Example** (This example highlights how phenotype information can be
displayed but does not include all column headings as previously
suggested.)

+-------+-----+------+------+------+-------+--------+-------+-------+
|       | D   |      |      |      | Other |        | Other |       |
|       | rug |      |      |      | Drugs |        | Drugs |       |
|       | X   |      |      |      | in    |        | Ou    |       |
|       |     |      |      |      | Same  |        | tside |       |
|       |     |      |      |      | Drug  |        | Drug  |       |
|       |     |      |      |      | Cl    |        | Cl    |       |
|       |     |      |      |      | ass\* |        | ass\* |       |
+-------+-----+------+------+------+-------+--------+-------+-------+
| S     | IC~ | Ref  | ∆   | ∆   | ∆     | ∆     | ∆    | ∆    |
| ample | 50~ | st   | r    | r    | resis | resis  | resis | resis |
|       | va  | rain | esis | esis |       |        |       |       |
|       | lue |      |      | from | from  | from   | from  | from  |
|       | D   | IC   | from | BL   | ref   | BL     | ref   | BL    |
|       | rug | ~50~ | ref  |      |       |        |       |       |
|       | X   | v    |      | Drug | Drug  | Drug Y | Drug  | Drug  |
|       |     | alue | Drug | X    | Y     |        | A     | A     |
|       |     |      | X    |      |       |        |       |       |
|       |     | Drug |      |      |       |        |       |       |
|       |     | X    |      |      |       |        |       |       |
+-------+-----+------+------+------+-------+--------+-------+-------+
| Bas   |     |      |      |      |       |        |       |       |
| eline |     |      |      |      |       |        |       |       |
+-------+-----+------+------+------+-------+--------+-------+-------+
| End   |     |      |      |      |       |        |       |       |
| point |     |      |      |      |       |        |       |       |
+-------+-----+------+------+------+-------+--------+-------+-------+

Drug X = candidate drug

∆ resis = fold resistance change, e.g.: [IC~50~ value of baseline sample
with Drug X]{.underline}

> IC~50~ value of reference strain with Drug X

Ref strain = reference strain (or WT)

Endpoint = predefined time point for endpoint assessment (e.g., week 24,
week 48, failure or discontinuation)

\*Note: Include the ∆ resis from ref and ∆ resis from BL for all
approved anti-HIV drugs

**VI. Column with Total Number of PI Mutations in Patient Isolate** (for
baseline and endpoint isolates)

PI mutations to be counted include changes at amino acid D30, V32, M361,
M46, G48, I50, I54, G73, V82, I84, N88, L90, and any additional amino
acid changes in PR that are important/relevant to the study drug as
shown in vitro or in clinical trials. Discuss with the DAVDP in advance
for agreement on the total number of mutations.

**VII. Column with Total Number of NRTI Mutations in Patient Isolate**
(for baseline and endpoint isolates)

RT mutations to be counted include changes at amino acid M41, E44, K65,
D67, T69, K70, L74, Y115, V118, M184, L210, T215, and any additional
amino acid changes in RT that are important/relevant to the study drug
as shown in vitro or in clinical trials. Discuss with the DAVDP in
advance for agreement on the total number of mutations.

**VIII. Column with Total Number of NNRTI Mutations in Patient Isolate**
(for baseline and endpoint isolates)

RT mutations to be counted include changes at amino acid A98, L100,
K103, V106, V108, Y181, Y188, G190, P225, M230, P236 and any additional
amino acid changes in RT that are important/relevant to the study drug
as shown in vitro or in clinical trials. Discuss with the DAVDP in
advance for agreement on the total number of mutations.

**IX. Co-Receptor Usage** (for all agents targeting entry)

-   Co-receptor usage of baseline isolates. Indicate R5, X4, D for
    dual-tropic, M for mixed-tropic, or D/M if the assay cannot
    distinguish between dual or mixed, in a column.

```{=html}
<!-- -->
```
-   Baseline R5 assay value

-   Baseline X4 assay value

```{=html}
<!-- -->
```
-   Co-receptor usage of virologic failures and end-of-study isolates
    (on therapy). Indicate R5, X4, D for dual-tropic, M for
    mixed-tropic, or D/M if the assay cannot distinguish between dual or
    mixed, in a column.

```{=html}
<!-- -->
```
-   R5 assay value at failure/end of study

-   X4 assay value at failure/end of study

# APPENDIX 2:

# Template for Submitting HBV Resistance Data

One dataset combines patient data, endpoint data, genotypic data, and
phenotypic data. There are a number of ways datasets can be subdivided
(i.e., by clinical study, baseline isolates, or virologic failure
isolates) and it is recommended that this be discussed with the DAVDP
before submission.

For each study, we recommend constructing datasets as SAS transport
files containing the following information:

-   One record (row) per patient per isolate (e.g., baseline, failure,
    and other time points).

-   Data in columns (with suggested column headings shown below) on all
    isolates.

```{=html}
<!-- -->
```
-   Genotypic data for (at a minimum) baseline isolates of all patients
    and the endpoint isolates of virologic failures and discontinuations
    --- on the corresponding record for each patient isolate.

-   Phenotypic data for (at a minimum) baseline isolates and the
    endpoint isolates of virologic failures and discontinuations --- on
    the corresponding record for each patient isolate. We recognize the
    difficulty of phenotypic analysis of HBV. Sponsors are strongly
    encouraged to collect and store appropriately samples for later
    analysis if warranted. If the pathway to resistance as defined by
    genotypic analysis is straightforward (e.g., lamivudine), phenotypic
    analysis may not be necessary. We recommend that sponsors consult
    with the DAVDP to determine whether phenotypic analysis should be
    conducted. Samples should be collected from rebound and failure
    isolates for phenotypic analysis when the subject is on study drug.

*Virologic failures* (other definitions can be used and discussed with
the DAVDP)[^7] --- 1) Rebound: confirmed (two consecutive) plasma HBV
DNA values greater than or equal to 1 log~10~ copies/mL increase in
serum HBV DNA from the on treatment nadir; 2) Never Suppressed: plasma
HBV DNA levels never achieve confirmed suppression with at least 48
weeks of randomized treatment; or 3) Insufficient Viral Response: plasma
HBV DNA levels never achieve confirmed suppression, and investigator
identifies the reason for treatment discontinuation prior to week 48 due
to insufficient viral load response: nonresponders and rebounders.

**Information to Include with Suggested Column Headings**[^8]

**I. Patient Data**

-   Patient identification number

```{=html}
<!-- -->
```
-   Isolate (e.g., baseline, week 24, week 48, discontinuation. Multiple
    isolates numbered (e.g., failure 1, failure 2))

```{=html}
<!-- -->
```
-   Date of isolate

-   Days of study drug treatment on day isolate obtained

-   Days off study drug treatment on day isolate obtained

-   Previous therapeutic agents

-   Treatment group

**II. Endpoint Data**

-   HBV DNA (log~10~ copies/mL) at baseline

```{=html}
<!-- -->
```
-   HBV DNA (log~10~ copies/mL) at predefined time points (e.g., week 24
    and week 48)

```{=html}
<!-- -->
```
-   HBV DNA (log~10~ copies/mL) at time of loss of virologic response or
    discontinuation due to adverse event

```{=html}
<!-- -->
```
-   Endpoint assessment (e.g., log~10~ change in viral load from
    baseline)

```{=html}
<!-- -->
```
-   Indication of data censored for reasons other than virologic failure
    (e.g., discontinuation due to adverse event)

-   HBV DNA (log~10~ copies/ml) from additional time points can be
    included.

**Note:** We recommend that sponsors analyze HBV DNA with a sensitive
and specific HBV DNA assay with a lower limit of quantification less
than 1,000 copies/mL.

**III. Genotypic Data** (for baseline isolates of all patients and
endpoint isolates from virologic failures and discontinuations)

-   HBV subtype

```{=html}
<!-- -->
```
-   Genotype information for all the RT or relevant coding region
    sequenced, one amino acid per column with the wild-type (WT) amino
    acid as column heading identified using the one amino acid
    abbreviation. Changes from the WT standard sequence should be
    indicated (i.e., blanks indicate no change) and known polymorphic
    amino acid residues flagged in the column heading.

**Example** (This example highlights how genotype information can be
displayed but does not include all column headings as previously
suggested.)

  ----------- ------------- ---------- --------- ---------- -------- --------
  Patient \#  Isolate       Subtype    rtY203    rtM204     rtD205   rtD206

  001         BL            A                                        

  001         WK48          A                    V                   

  002         BL            C                                        

  002         WK48          C                    I                   

  003         BL            A                                        

  004         BL            B                    V                   
  ----------- ------------- ---------- --------- ---------- -------- --------

BL = baseline

WK48 = week 48

**IV. Phenotypic Data** (minimally for baseline isolates and endpoint
isolates from virologic failures and discontinuations)

-   Candidate drug

    -   Baseline IC~50~ value for candidate drug

    -   IC~50~ value of reference strain for candidate drug (a widely
        available standard laboratory strain is recommended as the
        reference strain)

    -   Fold change of baseline IC~50~ value compared to IC~50~ value of
        reference strain of candidate drug

    -   IC~50~ value at time of endpoint assessment or failure for
        candidate drug

    -   Fold change values in IC~50~ value at time of endpoint
        assessment or failure compared to reference strain for candidate
        drug

    -   Fold change in IC~50~ value at time of endpoint assessment or
        failure compared to baseline for candidate drug

```{=html}
<!-- -->
```
-   Approved/investigational agents in the same class (cross-resistance,
    to be determined in cases of demonstrated reduced susceptibility to
    the treatment drug)

```{=html}
<!-- -->
```
-   Fold change in IC~50~ value of baseline compared to reference strain
    for each of the approved/investigational agents in the same class

-   Fold change in IC~50~ value at time of endpoint assessment or
    failure compared to reference strain for each of the
    approved/investigational agents in the same class

-   Fold change in the IC~50~ value at time of endpoint assessment or
    failure compared to baseline for each of the
    approved/investigational agents in the same class

**Example** (The example highlights how phenotype information can be
displayed.)

+---------+------+----------+--------+---------+---------+----------+
|         | Drug |          |        |         | Other   |          |
|         | X    |          |        |         | Drugs   |          |
|         |      |          |        |         | in Same |          |
|         |      |          |        |         | Drug    |          |
|         |      |          |        |         | Class\* |          |
+---------+------+----------+--------+---------+---------+----------+
| Sample  | IC   | Ref      | ∆     | ∆      | ∆      | ∆ resis |
|         | ~50~ | strain   | resis  | resis   | resis   |          |
|         | v    |          |        | from BL |         | from BL  |
|         | alue | IC~50~   | from   |         | from    |          |
|         | Drug | value    | ref    | Drug X  | ref     | Drug Y   |
|         | X    |          |        |         |         |          |
|         |      | Drug X   | Drug X |         | Drug Y  |          |
+---------+------+----------+--------+---------+---------+----------+
| B       |      |          |        |         |         |          |
| aseline |      |          |        |         |         |          |
+---------+------+----------+--------+---------+---------+----------+
| E       |      |          |        |         |         |          |
| ndpoint |      |          |        |         |         |          |
+---------+------+----------+--------+---------+---------+----------+

Drug X = candidate drug

BL = baseline

Endpoint = predefined time point for endpoint assessment (e.g., week 24,
week 48, failure or discontinuation)

∆ resis = fold resistance change, e.g.: [IC~50~ value of baseline sample
with Drug X]{.underline}

> IC~50~ value of reference strain with Drug X

Ref strain = reference strain (or WT)

\*Note: Include the ∆ resis from ref and ∆ resis from BL for all
approved anti-HBV drugs

# APPENDIX 3:

# Template for Submitting HCV Resistance Data

One dataset combines patient data, endpoint data, genotypic data, and
phenotypic data. There are a number of ways datasets can be subdivided
(i.e., by clinical study, baseline isolates or virologic failure
isolates) and it is recommended that this be discussed with the DAVDP
before submission.

For each study, we recommend constructing datasets as SAS transport
files containing the following information:

-   One record (row) per patient per isolate (e.g., baseline, failure,
    and other time points).

-   Data in columns (with suggested column headings shown below) on all
    isolates.

```{=html}
<!-- -->
```
-   Genotypic data for (at a minimum) baseline isolates of all patients,
    the endpoint isolates of virologic failures and discontinuations,
    and follow-up for sustained virologic response --- on the
    corresponding record for each patient isolate.

-   Phenotypic data for (at a minimum) baseline isolates, the endpoint
    isolates of virologic failures and discontinuations, and follow-up
    for sustained virologic response --- on the corresponding record for
    each patient isolate. We recognize the difficulty of phenotypic
    analysis of HCV. Sponsors are strongly encouraged to collect and
    store appropriately samples for later analysis if warranted. If the
    pathway to resistance as defined by genotypic analysis is
    straightforward, phenotypic analysis may not be necessary. We
    recommend that sponsors consult with the DAVDP to determine whether
    phenotypic analysis should be conducted. Samples should be collected
    from rebound and failure isolates for phenotypic analysis when the
    subject is on study drug.

*Virologic failures* (other definitions can be used and discussed with
the DAVDP)[^9] --- 1) Rebound: confirmed (two consecutive) plasma HBV
DNA values greater than or equal to 1 log~10~ copies/mL increase in
serum HBV DNA from the on treatment nadir; 2) Never Suppressed: plasma
HBV DNA levels never achieve confirmed suppression with at least 48
weeks of randomized treatment; or 3) Insufficient Viral Response: plasma
HBV DNA levels never achieve confirmed suppression and investigator
identifies the reason for treatment discontinuation prior to week 48 due
to insufficient viral load response: nonresponders and rebounders.

**Information to Include with Suggested Column Headings**[^10]

**I. Patient Data**

-   Patient identification number

```{=html}
<!-- -->
```
-   Isolate (e.g., baseline, week 24, week 48, discontinuation. Multiple
    isolates numbered (e.g., failure 1, failure 2))

```{=html}
<!-- -->
```
-   Date of isolate

-   Days of study drug treatment on day isolate obtained

-   Days off study drug treatment on day isolate obtained

-   Previous therapeutic agents where available

-   Treatment group

**II. Endpoint Data**

-   HCV RNA (log~10~ copies/mL) at baseline

```{=html}
<!-- -->
```
-   HCV RNA (log~10~ copies/mL) at predefined time points (e.g., week 24
    and week 48)

```{=html}
<!-- -->
```
-   HCV RNA (log~10~ copies/mL) at time of loss of virologic response or
    discontinuation due to adverse event

```{=html}
<!-- -->
```
-   Endpoint assessment (e.g., log~10~ change in viral load from
    baseline)

-   Follow-up for determination of sustained virologic response

```{=html}
<!-- -->
```
-   Indication of data censored for reasons other than virologic failure
    (e.g., discontinuation due to adverse event)

-   HCV RNA (log~10~ copies/ml) from additional time points can be
    included.

**Note**: We recommend that sponsors analyze HCV RNA with a sensitive
and specific HCV RNA assay with lower limits of quantification in the
range of less than 100 copies/mL.

**III. Genotypic Data** (for baseline isolates of all patients and
endpoint isolates from virologic failures and discontinuations)

-   HCV subtype (e.g., 1a, 1b, 2)

```{=html}
<!-- -->
```
-   Genotype information for all the replicases or relevant coding
    region sequenced, one amino acid per column with the wild-type (WT)
    amino acid as column heading identified using the one amino acid
    abbreviation. Changes from WT standard sequence indicated (i.e.,
    blanks indicate no change) and known polymorphisms are indicated
    with an asterisk.

**Example** (This example highlights how genotype information can be
displayed but does not include all column headings as previously
suggested.)

  ---------- ---------- ---------- ---------- ---------- ---------- ----------
  Patient \# Isolate    Subtype    E414       F415       D416       L417

  001        BL         1                                           

  001        WK48       1                     Y                     

  001        FU         1                                           

  002        BL         1                                           

  002        WK48       1                     Y                     

  002        FU         1                     Y                     
  ---------- ---------- ---------- ---------- ---------- ---------- ----------

BL = baseline

WK48 = week 48

FU = follow-up

**IV. Phenotypic Data** (minimally for baseline isolates, endpoint, and
follow-up isolates from virologic failures and discontinuations)

-   Candidate drug

```{=html}
<!-- -->
```
-   Baseline IC~50~ value for candidate drug

-   IC~50~ value of reference strain for candidate drug (a widely
    available standard lab strain is recommended as the reference
    strain)

-   Fold change of baseline IC~50~ value compared to IC~50~ value of
    reference strain of candidate drug

-   IC~50~ value at time of endpoint assessment or failure for candidate
    drug

-   Fold change values in IC~50~ value at time of endpoint assessment or
    failure compared to reference strain for candidate drug

-   Fold change in IC~50~ value at time of endpoint assessment or
    failure compared to baseline for candidate drug

-   IC~50~ value at follow-up for candidate drug

-   Fold change values in IC~50~ value at follow-up compared to
    reference strain for candidate drug

-   Fold change in IC~50~ value at follow-up compared to baseline for
    candidate drug

    -   Approved/investigational agents in the same class

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-   Fold change in IC~50~ value of baseline compared to reference strain
    for each of the approved/investigational agents in the same class

-   Fold change in IC~50~ value at time of endpoint assessment or
    failure compared to reference strain for each of the
    approved/investigational agents in the same class

-   Fold change in the IC~50~ value at time of endpoint assessment or
    failure compared to baseline for each of the
    approved/investigational agents in the same class

**Example** (The example highlights how phenotype information can be
displayed.)

+---------+------+----------+--------+--------+----------+----------+
|         | Drug |          |        |        | Other    |          |
|         | X    |          |        |        | Drugs in |          |
|         |      |          |        |        | Same     |          |
|         |      |          |        |        | Drug     |          |
|         |      |          |        |        | Class\*  |          |
+---------+------+----------+--------+--------+----------+----------+
| Sample  | IC   | Ref      | ∆     | ∆     | ∆ resis | ∆ resis |
|         | ~50~ | strain   | resis  | resis  |          |          |
|         | v    |          |        | from   | from ref | from BL  |
|         | alue | IC~50~   | from   | BL     |          |          |
|         | Drug | value    | ref    |        | Drug Y   | Drug Y   |
|         | X    |          |        | Drug X |          |          |
|         |      | Drug X   | Drug X |        |          |          |
+---------+------+----------+--------+--------+----------+----------+
| B       |      |          |        |        |          |          |
| aseline |      |          |        |        |          |          |
+---------+------+----------+--------+--------+----------+----------+
| E       |      |          |        |        |          |          |
| ndpoint |      |          |        |        |          |          |
+---------+------+----------+--------+--------+----------+----------+

Drug X = candidate drug

BL = baseline

Endpoint = predefined time point for endpoint assessment (e.g. week 24,
week 48, failure or discontinuation)

∆ resis = fold resistance change, e.g.: [IC~50~ value of baseline sample
with Drug X]{.underline}

> IC~50~ value of reference strain with Drug X

Ref strain = reference strain (or WT)

\*Note: Include the ∆ resis from ref and ∆ resis from BL for all
approved anti-HCV drugs

[^1]: This guidance has been prepared by the Division of Antiviral Drug
    Products in the Center for Drug Evaluation and Research (CDER) at
    the Food and Drug Administration.

[^2]: The C~min~/serum adjusted IC~50~ value.

[^3]: For an in-depth review of resistance issues relating to HIV-1, see
    the draft guidance for industry *Role of HIV Drug Resistance Testing
    in Antiretroviral Drug Development*. When final, this guidance will
    represent the FDA's current thinking on this topic. For the most
    recent version of a guidance, check the CDER guidance Web page at
    http://www.fda.gov/cder/guidance/index.htm.

[^4]: This definition is based on HIV-1 clinical studies. Other
    definitions can be used and discussed with the DAVDP.

[^5]: The specific criteria for defining virologic failures should be
    discussed with the DAVDP (Division of Antiviral Drug Products). This
    definition of virologic failures is intended to be used *only* for
    the identification of patients from whom isolates should be
    collected for resistance testing and is *not* intended to be used as
    a clinical or virologic endpoint in outcome analyses. Isolates from
    patients with documented clinical progression should also be
    included.

[^6]: In the SAS transport files, column headings can be given
    abbreviated column names to fit the SAS format; however, it is
    suggested that a description of column names be provided to the
    reviewer in the submission.

[^7]: The specific criteria for defining virologic failures should be
    discussed with the DAVDP (Division of Antiviral Drug Products). This
    definition of virologic failures is intended to be used *only* for
    the identification of patients from whom isolates should be
    collected for resistance testing and is *not* intended to be used as
    a clinical or virologic endpoint in outcome analyses. Isolates from
    patients with documented clinical progression should also be
    included.

[^8]: In the SAS transport files, column headings can be given
    abbreviated column names to fit the SAS format; however, it is
    suggested that a description of column names be provided to the
    reviewer in the submission.

[^9]: The specific criteria for defining virologic failures should be
    discussed with the DAVDP (Division of Antiviral Drug Products). This
    definition of virologic failures is intended to be used *only* for
    the identification of patients from whom isolates should be
    collected for resistance testing and is *not* intended to be used as
    a clinical or virologic endpoint in outcome analyses. Isolates from
    patients with documented clinical progression should also be
    included.

[^10]: In the SAS transport files, column headings can be given
    abbreviated column names to fit the SAS format; however, it is
    suggested that a description of column names be provided to the
    reviewer in the submission.