U.S. Department of Health and Human Services
Food and Drug Administration
Center for Drug Evaluation and Research (CDER)
June 2006
Clinical Antimicrobial
Guidance for Industry
Antiviral Product Development — Conducting and Submitting
Virology Studies 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)
June 2006
Clinical Antimicrobial
Guidance for Industry
Antiviral Product Development — Conducting and Submitting Virology
Studies to the Agency
This
guidance represents 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.
The purpose of this guidance is to assist
sponsors in the development of antiviral drugs and biological
products (i.e., therapeutic proteins and monoclonal antibodies)
from the initial pre-IND through the new drug application (NDA)
and postmarketing stages. This guidance should serve as a
starting point for understanding what nonclinical and clinical
virology data are important to support the submission of an
investigational new drug application (IND), NDA, or biologics
license application (BLA) for approval of an antiviral product.
This guidance focuses on nonclinical and clinical virology study
reports and makes recommendations for collecting and submitting
resistance data to the Food and Drug Administration (FDA). Nonclinical
and clinical virology study reports, based on collected data, are
essential for the FDA’s review of antiviral drug investigational
and marketing applications. Specific topics discussed in this
guidance include:
- Defining the mechanism of action
- Establishing specific antiviral activity
of the investigational product
- Assessing the potential for antagonism of
other antiviral products that might be used in combination with
the investigational product
- Providing data on the development of viral
resistance to the investigational product
- Providing data that identify
cross-resistance to approved antiviral products having the same
target
FDA’s guidance documents, including this
guidance, do not establish legally enforceable responsibilities.
Instead, guidances describe the FDA’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 FDA guidances means that something is
suggested or recommended, but not required.
The recommendations in this guidance are
based on the antiviral product review experience of the Division
of Antiviral Products and input from pharmaceutical sponsors and
the scientific community. Because of the experience, history, and
lessons learned with HIV-1 studies, this guidance employs studies
commonly used to evaluate HIV-1 products as a paradigm for studies
of products to treat other viruses. Although assays and model
systems vary with different viruses, many of the principles in
this guidance can be applied to antiviral products in development
for the treatment of other viral infections (e.g., hepatitis B
virus, hepatitis C virus, herpes simplex virus, varicella zoster
virus, influenza virus,
rhinovirus, cytomegalovirus, and human papillomavirus). Since the
field of virology is dynamic and continually evolving, this
guidance will be revised as new information accumulates and as
circumstances warrant.
Sample formats have been developed to assist
sponsors in providing resistance data to the Agency (see the
stand-alone documents accompanying this guidance). The sample
formats are being provided as stand-alone documents to help
sponsors provide the appropriate information to the Agency. These
sample formats will be updated as needed, and additional formats
for other viruses may be provided.
Guidances on the overall organization of INDs
and NDAs can be found at
www.fda.gov/cder/regulatory/applications/default.htm
Sponsors are encouraged to contact the division early in the
development of an investigational antiviral product to facilitate
the review and approval process. To assist prospective sponsors,
the FDA accepts submissions in an abbreviated IND format (i.e.,
pre-INDs) for review and comment. Pre-INDs are especially useful
in instances when sponsors are unfamiliar with the process for
evaluating investigational products in humans. Information about
submitting pre-INDs to the division (“Getting Started with the
Pre-IND Process”) can be found at
www.fda.gov/cder/ode4/preind/getting.htm The FDA accepts
electronic submissions to expedite the review process. FDA Web
sites can be consulted for information on electronic application
submission.
Sponsors are advised to consult the division
for additional guidance on the development of investigational
products against orthopoxviruses, influenza virus, severe acute
respiratory syndrome (SARS) virus, or other emerging infections.
For antiviral products that treat smallpox and other
orthopoxviruses, related information can be found in the draft
guidance for industry Vaccinia Virus — Developing Drugs to
Mitigate Complications from Smallpox Vaccination.
Sponsors should give careful attention to observing all
provisions of the Select Agent rule
and other applicable governmental and institutional biosafety and
biosecurity provisions.
Nonclinical
virology studies aid in the evaluation of the safety and efficacy
of an investigational product before it is tested in humans.
Recommended studies can identify the mechanism of action, help
establish specific antiviral activity of the product in a model
system, and provide data on the development of viral resistance to
the investigational product. Additionally, co-administration of
an investigational product with other products approved for the
same indication is often necessary to treat viral 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 product with other antiviral products.
As more antiviral products are developed to
treat particular viral diseases, cross-resistance (i.e., viral
resistance to one product causing resistance to more than one
product 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
products:
- Determining the antiviral activity of an
investigational product against relevant viruses resistant to
other approved products with the same target molecule or complex
- Determining the antiviral activity of
approved products against viruses resistant to an
investigational product with the same target molecule or complex
We recommend conducting nonclinical studies
(i.e., mechanism of action, antiviral activity in vitro,
cytotoxicity and therapeutic indexes, and effects of serum protein
binding on antiviral activity) before the initiation of phase 1
clinical studies. When developing products for viruses where in
vitro infection systems exist, sponsors should complete in vitro
drug combination activity studies of the investigational product
with other approved products against the same virus (i.e., all
approved and available investigational products that target the
same protein and at least one representative product from each of
the other existent drug classes) before the initiation of clinical
trials that will examine the efficacy of the investigational
product in combination with other antiviral products.
Furthermore, we recommend examining the in vitro selection of
resistant viruses to the investigational product, the phenotypic
and genotypic characterization of resistant viruses, and
cross-resistance before initiation of clinical studies in patients
infected with a particular virus. Complete study reports on
nonclinical and clinical virology studies can be submitted to the
FDA upon their completion and need not be held until submission of
the NDA. Specific details of each of the nonclinical studies
recommended by the division are discussed in the following
sections.
Mechanism of action studies should be
conducted before the initiation of phase 1 clinical studies.
There are many steps in a virus life cycle that can be targeted by
potential antiviral product candidates. Products 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 product’s ability to
specifically inhibit viral replication or a virus-specific
function
·
Establish the site of the product’s action (e.g.,
viral replicase, 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 product 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 product 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
product should be demonstrated for the viral target over cellular
or host proteins, especially in those cases in which a viral
enzyme has a cellular counterpart. For example, if the
investigational product targets a viral polymerase, we recommend
showing the activity of the product against the viral polymerase
in comparison with its activity against host DNA polymerases such
as DNA polymerase a,
b, and
g.
Immunomodulatory products raise additional
issues. These products can have the potential for unintended
effects on viral replication and other adverse effects resulting
from actions on the immune system. For investigational products
that act through a general immune stimulatory mechanism, we
recommend that sponsors show a reduction in antiviral activity and
identify the specific immune system components that are involved.
Sponsors should consult the division for specific advice regarding
the development of immunomodulatory products for the treatment of
viral diseases as well as products with other nonviral host
targets.
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 product 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.C., Cytotoxicity and
Therapeutic Indexes) using relevant cell types and virus isolates
can be used to guide the selection of appropriate dose ranges in
early clinical trials. Sponsors are encouraged to obtain
antiviral activity data using primary human target cells, if
possible. Because of viral genetic variation, the antiviral
activity of the investigational product 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 product include:
- Assessing specific antiviral activity of
the investigational product against a broad range of clinical
and laboratory viral isolates including different clades,
subtypes, or genotypes
- Evaluating the antiviral activity of the
investigational product against mutant viruses that are
resistant to products with the same target molecule or complex
as the investigational product as well as a representative
sample of viruses resistant to other approved products 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 product
compared to replication in the absence of the product. The
effective concentration is the concentration of product at which
virus replication is inhibited by 50 percent (e.g., EC50
for cell-based assays; IC50 for biochemical or
subcellular assays). 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. We suggest that host
cell lines be low in passage number for reproducible results.
It is important that the effective
concentration be consistent with data supporting the mechanism of
action. An investigational product 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. Resistance analyses can provide in
vivo validation of the proposed mechanism of action when
biochemical data are not consistent with antiviral activity data.
For nucleoside or nucleotide analogs, we recommend that sponsors
determine the intracellular half-life (t1/2) 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 the active moiety of an antiviral
product enters cells, if it has a proposed intracellular site of
action, and if the intracellular concentration correlates with
biochemical studies. Cell-based assays and host cell lines for
studying viruses such as hepatitis B virus (HBV) and hepatitis C
virus (HCV) replication have advanced, but at the present time are
limited. 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
For analyses of HCV replication, replicon
systems have been developed that permit studies of viral
replication and can be used to assess antiviral activity of some
anti-HCV products.,
In addition, recent studies indicate that cell-based systems for
the replication of HCV in vitro are in development and might be
available to assess antiviral activity.
Currently, assays that examine HCV replication using the HCV
replicon system include, but are not limited to:
- Measuring the level of HCV RNA by RT-PCR
in HCV replicon cells
- Measuring HCV RNA polymerase, HCV serine
protease, or reporter enzyme (e.g., luciferase) activity in
biochemical assays
Serum proteins
can bind to and sequester many products and interfere with a
product’s antiviral activity. We recommend that sponsors
ascertain if the investigational product 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
a-acidic glycoprotein
protein binding. If the investigational product is highly protein
bound, sponsors are encouraged to examine the in vitro antiviral
activity of the investigational product in the presence of a
series of dilutions of human serum up to 40 percent (e.g., 5
percent, 10 percent, 20 percent, 40 percent). An EC50
value for 100 percent human serum can be extrapolated from these
data and the serum-adjusted EC50 values reported. In
addition, sponsors are encouraged to determine EC50
values in the presence of physiological concentrations of
a-acidic glycoprotein and
human serum albumin.
Information on
plasma and intracellular product 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). An IQ is the Cmin/serum-adjusted
EC50 value. (For more information on determining EC50
values, see Section III.B.1., Antiviral Activity in Vitro.) We
view IQ values as a useful tool integrating in vivo product
concentrations and antiviral activity for an individual product.
It is a measure that characterizes the relationship between
product exposure and the susceptibility of a virus to a product.
A high IQ indicates that an effective product concentration can be
achieved in a patient to inhibit the virus and minimize the
development of resistance. Since one dose may not be adequate for
all patient populations, IQ values can be used to aid in the
selection of doses to further evaluate in phase 3 and phase 4
clinical studies.
In cases where no in vitro cell culture or
replicon system has been shown to be predictive of antiviral
activity in humans, measurement of viral titers after treatment in
animal model systems can be used to assess antiviral activity of
the investigational product. Analyses from animal models include
the morbidity and mortality of animals following documented
infection, histological 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 product, and a description of symptoms
(e.g., neurological, weight loss).
It is important to establish that an
investigational product has antiviral activity at concentrations
that can be achieved in vivo without inducing toxic effects to
cells. Furthermore, in a cell culture model, apparent antiviral
activity of an investigational product can be the result of host
cell death after exposure to the product. Cytotoxicity tests use
a series of increasing concentrations of the antiviral product to
determine what concentration results in the death of 50 percent of
the host cells (see also Section III.B.1., Antiviral Activity in
Vitro). This value is referred to as the median cellular
cytotoxicity concentration and is identified by CC50 or
CCIC50. The relative effectiveness of the
investigational product in inhibiting viral replication compared
to inducing cell death is defined as the therapeutic or
selectivity index (i.e., CC50 value/EC50
value). It is desirable to have a high therapeutic index giving
maximum antiviral activity with minimal cell toxicity. We
recommend determining CC50 values in both stationary
and dividing cells from multiple relevant human cell types and
tissues to ascertain the potential for cell-cycle, species, or
tissue-specific toxicities. Studies determining cytotoxicity and
therapeutic indexes should be conducted before the initiation of
phase 1 clinical studies.
Because of the myelosuppressive effects of
some antiviral products, we recommend assessing the potential
effects of certain investigational products (e.g., nucleoside
analogs) on the growth of human bone marrow progenitor cells in
colony formation assays. Additionally, some investigational
antiviral products are potential inhibitors of cellular DNA
polymerases, which are responsible for normal nuclear and
mitochondrial DNA synthesis and repair. Sponsors should determine
IC50 values for investigational antiviral products
against cellular polymerases and show specificity for the viral
target over cellular polymerases. The inhibition of human pol
g, the enzyme responsible
for mitochondrial DNA synthesis, has been linked to defects in
mitochondrial function that can lead to adverse events in humans.
Therefore, it is important to examine the effects of certain
investigational products (e.g., nucleoside analogs) on human pol
g activity and on
mitochondrial toxicity (e.g., lactic acid production,
mitochondrial DNA content, mitochondrial morphology, glucose
utilization).
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
products. Therefore, for some viruses, administration of multiple
antiviral products (e.g., three-drug combination antiretroviral
therapy against HIV-1) can be more effective than a single product
in establishing and maintaining inhibition of virus replication.
However, the interactions of products are complex and can result
in antagonistic, additive, or synergistic effects with respect to
antiviral activity. For this reason, sponsors should evaluate the
in vitro antiviral activity of investigational products in
two-drug combinations with other products approved for the same
indication. Specifically, combinations that should be tested
include the investigational product with all approved products
that target the same protein and at least two appropriate products
from each class of products approved for the same indication. We
recommend completing the in vitro drug combination activity
studies of the investigational product with approved products
before initiation of clinical trials that will evaluate the
efficacy of the investigational product in combination with other
antiviral products. Often patients are infected with two or more
viral diseases (e.g., HIV and HBV or HCV); therefore, we also
recommend that the in vitro antiviral activity of antiviral
products used in co-infected patients for different indications be
assessed in in vitro combination activity studies.
This guidance focuses on resistance to
antiviral products caused by mutations in viral genomes that
result in reduced phenotypic susceptibility to a given antiviral
product. Resistance, as it is used here, is not an absolute term,
but relative. We recommend that the in vitro selection of
resistant viruses to the investigational product, 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.
We understand that the resistance data generated in vitro are not
necessarily predictive of clinical resistance. However, in vitro
resistance selection studies are recommended to assess the
potential barrier of a target virus to develop reduced
susceptibility (i.e., resistance) to the investigational product
and to aid in designing clinical studies.
Selection in
cell culture of virus resistant to the investigational product can
provide insight into whether the genetic threshold for resistance
development is high or low. 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 multiple mutations to select for resistance. Several
factors specific to the investigational product and the target
virus affect the development of resistance (e.g., product
concentration). The rate of appearance of mutant viruses depends
on 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 resistant virus in vitro. For example,
when multiple mutations are required to develop resistance to high
concentrations of the investigational product, many cell culture
systems do not produce sufficient virus titers to select resistant
virus. In these instances, serial passage of the virus in cell
culture under conditions of increasing concentrations of the
investigational product 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. Selection of variants
resistant to the investigational product should be repeated more
than once (e.g., with different strains of wild-type, with
resistant strains, under high and low selective pressure) to
determine if the same or different patterns of resistance
mutations develop, and to assess the relationship of product
concentration to the genetic barrier to resistance.
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
product:
·
A high initial virus inoculum is propagated for
several passages at a fixed product concentration, using multiple
cultures to test different concentrations.
·
A low initial virus inoculum is passaged in the
presence of increasing product concentrations starting near the EC50
value for the parental virus.
Virus production is monitored by determining
the genotype and phenotype of isolates throughout the selection
process to detect the outgrowth of resistant viruses.
HCV resistance to investigational products
can be examined using HCV replicon systems. Methods that select
HCV resistance using HCV replicon cells include the following:
- HCV replicon cells are cultured at low
density in the presence of neomycin and a fixed concentration of
the investigational product using multiple cultures to test
different concentrations. The cells containing resistant
replicons will form colonies that are then expanded for
genotypic and phenotypic characterization.
- HCV replicon cells are passaged in the
presence of a fixed product concentration, but in the absence of
neomycin, using multiple cultures to test different
concentrations. The HCV replicon cells from each passage are
harvested and stored for phenotypic and genotypic
characterization.
Genotypic analysis of resistant viruses
selected in vitro determines the mutations that might contribute
to reduced susceptibility to the investigational product.
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 product. Sponsors should determine the
entire coding sequence of the gene for the target protein and
compare the pattern of mutations leading to resistance of the
investigational product with the pattern of mutations of other
products in the same class. For larger viruses (e.g.,
herpesviruses, poxviruses), the relevant portions of the viral
gene targeted by the investigational product should be sequenced
and analyzed for mutations that could contribute to product
resistance (e.g., marker rescue). We recommend that resistance
pathways be characterized in several genetic backgrounds (i.e.,
strains, subtypes, genotypes) and that isolates be obtained
throughout the selection process to identify the order in which
multiple mutations appear.
For 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.
Phenotypic analysis determines if mutant
viruses have reduced susceptibility to the investigational
product. When mutations that may be associated with resistance
are identified by genotypic analysis, the ability of each of these
mutations to confer phenotypic resistance should be evaluated in a
recombinant virus system if possible (e.g., by using site-directed
mutagenesis, PCR amplification of relevant portions of virus
genome to introduce these mutations into a standard laboratory
genetic background, or other suitable system). Recombinant
viruses can then be tested in vitro for susceptibility to the
product to determine an EC50 value. The fold resistant
change should be calculated as the EC50 value of the
isolate/EC50 value of the reference or parental
strain. Phenotypic results can be determined with any standard
virus assay (e.g., protein assay, viral RNA assay, polymerase
assay, MTT cytotoxic assay, reporter gene expression). The shift
in susceptibility (or fold resistant change) for a viral isolate
should be measured by determining the EC50 values for
the isolate and comparing it to the EC50 value of a
reference (well-characterized wild-type laboratory strain) or
parental virus done under the same conditions and at the same
time. The use of the EC50 value for determining shifts
in susceptibility is preferred because it can be determined with
greater precision than an EC90 or EC95
value. The utility of a phenotypic assay depends on its
sensitivity (i.e., its ability to measure shifts in susceptibility
(fold resistance change) in comparison to reference, parental
strains, or baseline clinical isolates). Calculating the fold
resistant change (EC50 value of isolate/EC50
value of reference strain) allows for comparisons among phenotypic
assays.
Antiviral products targeting the same protein
(typically products of the same drug class) may develop mutations
that lead to reduced susceptibility to one antiviral product and
can result in decreased or loss of susceptibility to other
antiviral products 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. We recommend that the effectiveness of
the investigational product against viruses resistant to other
approved products in the same drug class and the effectiveness of
approved products against viruses resistant to the investigational
product be evaluated by phenotypic analyses. Additionally, we
recommend that cross-resistance be analyzed between drug classes
in instances where more than one drug class 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 suggest that multiple recombinant and clinical
isolates representative of the breadth of diverse mutations and
combinations of mutations known to confer reduced susceptibility
to products in the same drug class be tested for phenotypic
susceptibility to the investigational product. If phenotyping is
performed in cell lines with recombinant viruses, the EC50
value should be validated with clinical isolates.
It is important to disseminate information
about an antiviral product’s resistance profile to health care
professionals and patients so that they can make optimal treatment
decisions. Furthermore, crucial decisions in protocol design and
product development plans often hinge on resistance and
cross-resistance data. Therefore, it is strongly recommended that
comprehensive resistance testing be undertaken during all phases
of product development consistent with the way the product will be
used in clinical practice.
For some viruses, measurements of changes in
viral concentrations are accepted endpoints to determine clinical
effectiveness of antiviral products. In these cases, assays that
measure and monitor viral load can be used. Well-characterized
genotypic and phenotypic assays provide the basis for examination
of the emergence of resistant virus to investigational products
and attempt to show a relationship between viral resistance and
clinical virologic failure. In addition, phenotypic and genotypic
results are used to define treatment options and predict the
utility of treating an individual with an investigational
product. Genotypic analysis of viral isolates from patients
failing to respond to therapy or undergoing viral rebound can help
identify mutations that contribute to reduced susceptibility to
the investigational product. In addition, we recommend genotypic
and phenotypic analyses of baseline isolates be used to determine
response to treatment outcomes based on baseline mutations and
polymorphisms and baseline phenotypic drug susceptibilities. In
cases where measurements of viral concentrations have not been
established as principal endpoints, development of assays and
monitoring of resistance emergence will be important for
exploratory analyses of relationships between virologic
measurements and clinical outcomes and can assist in refining
designs of future studies.
We recommend that sponsors develop and submit
a plan for monitoring the development of resistant viruses in
clinical studies before the initiation of clinical studies in
virus-infected individuals. A resistance monitoring plan should
include, but not be limited to:
- A description of the assays that will be
used to monitor viral loads
- Viral load assay protocols and performance
characteristics (if required)
- The genotypic and phenotypic assays that
will be used
- Genotypic and phenotypic assay protocols
and performance characteristics (if required)
- The methods for sample collection and
storage
- The methods for sample handling and
shipping (frozen or ambient)
- A description of additional resistance
analyses
- Time points when samples for viral loads,
genotypic and phenotypic assays, and other resistance analyses
will be collected (i.e., baseline, week 24, week 48, following
regimen failure or discontinuation)
The resistance monitoring plan should be
included with the overall clinical development plan in the IND.
We also recommend developing and submitting
with the IND plans for genotypic and phenotypic baseline studies
and resistance substudies early in product 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 product
and its cross-resistance potential with other antiviral products.
For viruses such as HBV and HCV, baseline and post-treatment
genotypic analyses are key to monitoring the development of
genotypic resistance. Comparing genotypic analyses of samples
from patients exhibiting virologic breakthrough (or rebound) with
baseline samples can identify mutations associated with
resistance. The extent and type of resistance monitoring and
analysis should be discussed and agreed to with the division in
advance.
Generally, in studies of
treatment-experienced patients, sponsors are strongly encouraged
to collect phenotypic and genotypic data for baseline isolates
from all patients and endpoint isolates from all virologic
failures and discontinuations (not suppressed). Virologic failure
and discontinuation samples should be collected when the patient
is still on the study product. In studies of treatment-naïve
patients, phenotypic and genotypic data for baseline and endpoint
isolates from all virologic failures and discontinuations (not
suppressed) should be obtained. Therefore, in treatment-naïve
studies, a baseline sample should be collected and stored from all
patients for potential phenotypic and genotypic analysis of
virologic failures. Additional genotypic and phenotypic
assessments and subset analyses may be appropriate depending on
the clinical study protocol or population, thus re-emphasizing the
need to collect and store baseline samples and treatment samples
throughout studies. In select cases, sponsors may propose to
collect samples at baseline and at the time of failure on a subset
of treatment-naïve patients when in vitro data indicate that
acquisition of a single mutation results in a high degree of
phenotypic resistance. However, any such proposal should be
discussed with the division in advance.
Virologic failures and discontinuation
definitions are protocol defined. Sponsors with
investigational products can solicit advice from the division
early in product development on definitions of clinical
response-failure and plans to monitor resistance in clinical
studies. We recommend that sponsors consult with the division for
detailed descriptions and examples of how to submit clinical viral
resistance data. In conjunction with this guidance, we are
providing stand-alone documents to aid sponsors in submitting
resistance data for HIV, HBV, HCV, and influenza studies. These
sample formats may be updated periodically and additional formats
for other viruses added as needed.
Sponsors can choose to quantify viral loads
and conduct phenotypic and genotypic analyses themselves or send
samples to companies that have been certified by Clinical
Laboratory Improvement Amendments (CLIA). Proper handling
procedures should be followed for laboratory samples. If an assay
is not performed according to the manufacturer’s specifications,
the assay results might not be acceptable. Sponsors are
encouraged to use approved assays (if possible) that are
characterized and validated. If the assay is investigational, we
recommend that sponsors provide the performance characteristics of
the assay (e.g., accuracy, precision, limits of detection and
quantification, specificity, linearity, range, robustness,
stability), 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 guidance for
industry Bioanalytical Method Validation, the guidance for
industry Antiretroviral Drugs Using Plasma HIV RNA Measurements
— Clinical Considerations for Accelerated and Traditional Approval,
and the ICH guideline for industry Q2A Text on Validation of
Analytical Procedures. Assays that will be used in clinical
practice should have more extensive validation than exploratory
assays that are used for the characterization of antiviral
activity and resistance of investigational products. Commercially
available assays that are routinely used should be identified, but
it may not be necessary to provide the performance
characteristics. The amount and nature of validation necessary
for an assay and mechanisms of submitting assay performance
characteristics (e.g., Data Master File) should be discussed with
the division. We recommend that sponsors consistently use the
same assay for any particular analysis or measurement in phase 3
studies and that the same assay be used for a particular patient
throughout the study. Sponsors should provide data supporting the
implementation and use of any new assays that become available
during product development.
Complete virology study reports can be
extensive and should include the primary data and derived data,
the procedures used to obtain the data, and information necessary
to evaluate the data. Virology study reports should contain
information on nonclinical studies, clinical antiviral activity of
the investigational product, resistance development to the
investigational product in treated patients, cross-resistance with
other products in the same drug class, and baseline genotypic and
phenotypic virologic response analyses (if applicable). The
formats of virology study reports 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. Virology study reports on nonclinical and clinical
studies can be submitted upon their completion and need not be
held until submission of the NDA. If the NDA is submitted in
the common
technical document (CTD) format, sponsors should submit
virology study reports and datasets in Module 5, Section 5.3.5.4,
Other Studies, under the specific heading, Antiviral Reports.
This guidance identifies virology studies
relevant to the development and application review of antiviral
products for the treatment of viral infections. The goal of this
guidance is to stimulate the generation of more complete analyses
for antiviral products. Such analyses help provide data that
support the introduction of an investigational product into humans
and provide the data necessary for determining dose-response
relationships, designing clinical trials, and selecting
appropriate patient populations. Thus, the data collected during
these studies can affect the therapeutic success of a given
product.
Because nonclinical in vitro virology studies
can provide useful information for the design of in vivo studies
and can help predict the development of resistant viruses in vivo,
we recommend conducting nonclinical studies before the initiation
of phase 1 clinical studies. In vitro selected resistant viruses
should be analyzed carefully before initiation of studies in
patients infected with a particular virus. This guidance includes
recommendations for how and when to perform virology studies.
Such information could be included in product labeling to
facilitate appropriate prescribing of antiviral products and
maximize the chance for therapeutic success. To assist sponsors
in providing data from clinical resistance studies, we have
developed stand-alone documents that accompany this guidance and
provide a format for submitting resistance data to the Agency.
