Guidance for Industry
S8 Immunotoxicity Studies for Human Pharmaceuticals
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U.S. Department of Health and Human Services
Food and Drug Administration
Center for Drug Evaluation and Research (CDER)
Center for Biologics Evaluation and Research (CBER)
April 2006
ICH
Guidance for Industry
S8 Immunotoxicity Studies for Human Pharmaceuticals
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http://www.fda.gov/cber/guidelines.htm
U.S. Department of Health and Human Services
Food and Drug Administration
Center for Drug Evaluation and Research (CDER)
Center for Biologics Evaluation and Research (CBER)
April 2006
ICH
Guidance for Industry
S8 Immunotoxicity Studies for Human
Pharmaceuticals
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 objectives of this guidance are to provide (1) recommendations
on nonclinical testing approaches to identify compounds that have
the potential to be immunotoxic and (2) guidance on a
weight-of-evidence decisionmaking approach for immunotoxicity
testing. Immunotoxicity is, for the purpose of this guidance,
defined as unintended immunosuppression or enhancement. Drug-induced
hypersensitivity and autoimmunity are excluded.
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.
Evaluation of potential adverse effects of
human pharmaceuticals on the immune system should be incorporated
into standard drug development. Toxicity to the immune system
encompasses a variety of adverse effects. These include suppression
or enhancement of the immune response. Suppression of the immune
response can lead to decreased host resistance to infectious agents
or tumor cells. Enhancing the immune response can exaggerate
autoimmune diseases or hypersensitivity. Drug or drug-protein
adducts might also be recognized as foreign and stimulate an
antidrug response. Subsequent exposures to the drug can lead to
hypersensitivity (allergic) reactions. Much of the science and
method development and validation efforts in the past have been
focused on evaluating drug development candidates for their
potential for either immunosuppression or contact sensitization. No
standard approaches for human pharmaceuticals are currently
available for testing for respiratory or systemic allergenicity (antigenicity)
or drug-specific autoimmunity; testing for these endpoints is not
currently required in any region. There are no regional differences
in testing approaches of skin sensitization.
Immunosuppression or enhancement can be
associated with two distinct groups:
(1)
Drugs intended to modulate immune function for therapeutic purposes
(e.g., to prevent organ transplant rejection) where adverse
immunosuppression can be considered exaggerated pharmacodynamics
(2)
Drugs not intended to affect immune function but cause
immunotoxicity due, for instance, to necrosis or apoptosis of immune
cells or interaction with cellular receptors shared by both target
tissues and nontarget immune system cells
Antiproliferative agents used to treat cancer
are an example of drugs that produce unintended immunosuppression.
In such instances, adverse findings in nonclinical studies are
predictive of human immunotoxicity in a rather straightforward
manner. That is, specific assays to determine immunotoxicity are
probably not valuable in drug risk assessment since the target
tissues are usually rapidly dividing cell types, such as bone
marrow-derived immune system progenitor cells. Hence, the adverse
effects on immune function can be predicted based on pharmacologic
activity and can usually be reliably evaluated in nonclinical
studies. For other types of compounds not intended to suppress the
immune response, distinction between exaggerated pharmacodynamics
and nontarget effects can be less obvious. As an example, some
anti-inflammatory compounds have an effect on certain innate immune
functions but do not necessarily affect the adaptive immune
response.
This guidance is focused on providing
recommendations on nonclinical testing for immunotoxicity induced by
human pharmaceuticals. It is restricted to unintended
immunosuppression and immunoenhancement, excluding allergenicity or
drug-specific autoimmunity.
This guidance applies to new pharmaceuticals
intended for use in humans, as well as to marketed pharmaceuticals
proposed for different indications or other variations on the
current product label in which the change could result in
unaddressed and relevant immunotoxicity issues. In addition, the
guidance might also apply to drugs for which clinical signs of
immunotoxicity are observed during clinical trials and following
approval to market. The guidance does not apply to
biotechnology-derived pharmaceutical products covered by ICH S6 (1)
and other biologicals.
Existing guidance documents on sensitization or
hypersensitivity remain in force and are not affected by this
document. It is beyond the scope of this guidance to provide
specific guidance on how each immunotoxicity study should be
performed. General methodology guidance is provided in the Appendix.
The general principles that apply to this
guidance are:
(1)
All new human pharmaceuticals should be evaluated for the potential
to produce immunotoxicity.
(2)
Methods include standard toxicity studies (STS) and additional
immunotoxicity studies conducted as appropriate. Whether additional
immunotoxicity studies are appropriate should be determined by a
weight-of-evidence review of factor(s) in section II.A (2.1).
The description of the guidance below will
follow the recommended decision process in immunotoxicity evaluation
as shown in the flow diagram (Figure 1). More detailed descriptions
of the testing methods are in the Appendix.
II.
GUIDANCE (2)
Factors to consider that might prompt
additional immunotoxicity studies can be identified in the following
areas: (1) findings from STS, (2) the pharmacological properties of
the drug, (3) the intended patient population, (4) structural
similarities to known immunomodulators, (5) the disposition of the
drug, and (6) clinical information.
The initial screen for potential immunotoxicity
involves standard toxicity studies. Data from rodent and nonrodent
studies from early short term to more chronic repeat-dose studies
should be taken into consideration. Additional details on the
parameters that should be evaluated and the reporting of
histopathology findings are provided in the appendix.
Data from STS should be evaluated for signs of
immunotoxic potential. Signs that should be taken into consideration
are the following:
(1)
Hematological changes such as leukocytopenia/leukocytosis,
granulocytopenia/ granulocytosis, or lymphopenia/ lymphocytosis
(2)
Alterations in immune system organ weights and/or histology (e.g.,
changes in thymus, spleen, lymph nodes, and/or bone marrow)
(3)
Changes in serum globulins that occur without a plausible
explanation, such as effects on the liver or kidney, can be an
indication that there are changes in serum immunoglobulins
(4)
Increased incidence of infections
(5)
Increased occurrence of tumors can be viewed as a sign of
immunosuppression in the absence of other plausible causes such as
genotoxicity, hormonal effects, or liver enzyme induction.
Changes in these parameters could reflect
immunosuppression or enhanced activation of the immune system.
Immunosuppression is usually reflected by reduced values of immune
parameters, whereas immunoenhancement is usually reflected by
increased values. However, these relationships are not absolute and
can be inverted in some cases.
Similar to the
assessment of risk with toxicities in other organ systems, the
assessment of immunotoxicity should include the following:
·
Statistical and biological significance of the changes
·
Severity of the effects
·
Dose/exposure relationship
·
Safety factor above the expected clinical dose
·
Treatment duration
·
Number of species and endpoints affected
·
Changes that may occur secondarily to other factors
(e.g., stress, see the appendix, section A.4 (1.4)),
·
Possible cellular targets and/or mechanism of action
·
Doses that produce these changes in relation to doses
that produce other toxicities
·
Reversibility of effect(s)
If the pharmacological properties of a test
compound indicate it has the potential to affect immune function
(e.g., anti-inflammatory drugs), additional immunotoxicity testing
should be considered. Information obtained from the nonclinical
pharmacology studies on the ability of the compound to affect the
immune system could be used in a weight-of-evidence approach to
decide if additional immunotoxicity studies are needed.
Additional immunotoxicity studies might be warranted if the majority
of the patient population for whom the drug is intended is
immunocompromised by a disease state or concurrent therapy.
Compounds
structurally similar to compounds with known immunosuppressive
properties should also be considered for additional immunotoxicity
testing.
If the compound
and/or its metabolites are retained at high concentrations in cells
of the immune system, additional immunotoxicity testing should be
considered.
Clinical findings
suggestive of immunotoxicity in patients exposed to the drug could
call for additional nonclinical immunotoxicity testing.
A
weight-of-evidence review should be performed on information from
all the factors outlined above to determine whether a cause for
concern exists. A finding of sufficient magnitude in a single area
should trigger additional immunotoxicity studies. Findings from two
or more factors, each one of which would not be sufficient on its
own, could trigger additional studies. If additional immunotoxicity
studies are not performed, the sponsor should provide justification.
If a cause for concern is identified,
additional immunotoxicity studies should be performed to verify the
immunotoxic potential of the compound. These studies can also help
determine the cell type affected, reversibility, and the mechanism
of action. This type of information can also provide more insight
into potential risk and possibly lead to biomarker selection for
clinical studies.
If the weight-of-evidence review indicates that
additional immunotoxicity studies are called for, there are a number
of assays that can be used. If there are changes in standard
toxicity testing data suggesting immunotoxicity, the type of
additional immunotoxicity testing that is considered appropriate
will depend on the nature of the immunological changes observed and
the concerns raised by the class of compound. It is recommended that
an immune function study be conducted, such as a T-cell dependent
antibody response (TDAR). If specific cell types that are affected
in STS are not known to participate in a TDAR, assays that measure
function of that specific affected cell type might be conducted (see
the appendix). Where a specific target is not identified, an immune
function study such as the TDAR is recommended.
In addition,
immunophenotyping of leukocyte populations, a nonfunctional assay,
can be conducted to identify the specific cell populations affected
and might provide useful clinical biomarkers.
To assess drug-induced immunotoxicity, a
generally accepted study design in rodents is a 28-day study with
consecutive daily dosing. Adaptations of immunotoxicity assays have
been described using nonrodent species. The species, strain, dose,
duration, and route of administration used in additional
immunotoxicity studies should be consistent, where possible, with
the standard toxicity study in which an adverse immune effect was
observed. Usually both sexes should be used in these studies,
excluding nonhuman primates. Rationale should be given when one sex
is used in other species. The high dose should be above the no
observed adverse effect level (NOAEL) but below a level inducing
changes secondary to stress (see appendix, section A.4 (1.4)).
Multiple dose levels are recommended in order to determine
dose-response relationships and the dose at which no immunotoxicity
is observed.
Results from
additional immunotoxicity studies should be evaluated as to whether
sufficient data are available to reasonably determine the risk of
immunotoxicity.
1.
Additional studies might show that no risk of immunotoxicity
can be detected and no further testing is called for.
2.
Additional studies might demonstrate a risk of immunotoxicity
but fail to provide sufficient data to make a reasonable
risk-benefit decision. In this case, further testing might help
provide sufficient information for the risk-benefit decision.
3.
If the overall risk-benefit analysis suggests that the risk
of immunotoxicity is considered acceptable and/or can be addressed
in a risk management plan (see ICH E2E (2)), then no further testing
in animals might be called for.
IV. TIMING OF IMMUNOTOXICITY TESTING
IN RELATION TO CLINICAL STUDIES (4)
If the weight-of-evidence review indicates that
additional immunotoxicity studies are appropriate, these should be
completed before exposure of a large population of patients, usually
phase 3. This will allow for the incorporation of monitoring immune
system parameters in the clinical studies if appropriate. The
timing of the additional immunotoxicity testing might be determined
by the nature of the effect by the test compound and the type of
clinical testing that would be called for if a positive finding is
observed with the additional immunotoxicity testing. If the target
patient population is immunocompromised, immunotoxicity testing can
be initiated at an earlier time point in the development of the
drug.
1. ICH S6 Preclinical Safety Evaluation of
Biotechnology-derived Pharmaceuticals (FDA,
1997).
2. ICH E2E Pharmacovigilance Planning
(FDA, 2005).
APPENDIX: Methods to Evaluate
Immunotoxicity
A. Standard Toxicity Studies (1)
The following table lists the parameters that
should be evaluated in standard toxicity studies for signs of
immunotoxicity. These parameters (excluding hematology and clinical
chemistry) and methods for obtaining samples and evaluating tissue
sections are described in more detail in documents from professional
toxicological pathology societies.
Parameter |
Specific Component |
Hematology |
Total and
absolute differential leukocyte counts |
Clinical
Chemistry |
Globulin levels1
and A/G ratios |
Gross pathology |
Lymphoid
organs/tissues |
Organ weights |
Thymus, spleen
(optional: lymph nodes) |
Histology |
Thymus, spleen, draining lymph node and at
least one additional lymph node, bone marrow2,
Peyer’s patch,3 BALT,4 NALT4 |
1Unexplained
alterations in globulin levels could call for measurement of
immunoglobulins.
2Unexplained
alterations in peripheral blood cell lines or histopathologic findings
might suggest that cytologic evaluation of the bone marrow would be
appropriate.
3Oral
administration only.
4For
inhalation
or nasal route
only. BALT:
bronchus-associated lymphoid tissues. NALT:
nasal-associated lymphoid tissues
1.
Hematology and Clinical Chemistry (1.1)
Total leukocyte counts and absolute
differential leukocyte counts are recommended to assess
immunotoxicity. When evaluating changes in globulin levels, other
factors should be taken into account (e.g., liver toxicity,
nephrotoxicity). Changes in serum globulins can be an indication
that there are changes in serum immunoglobulins. Although serum
immunoglobulins are an insensitive indicator of immunosuppression,
changes in immunoglobulins levels can be useful in certain
situations in order to better understand target cell populations or
mechanism of action.
2.
Gross Pathology and Organ Weights (1.2)
All lymphoid tissues should be evaluated for
gross changes at necropsy. However, this can be more difficult for
the Peyer’s patches of rodents due to the small size. Spleen and
thymus weights should be recorded. To minimize variability of
spleen weights in dogs and monkeys, bleeding the animals thoroughly
at necropsy is recommended. Atrophy of the thymus with aging can
preclude obtaining accurate thymus weight.
3.
Histopathological
Examination (1.3)
Histopathological changes of the spleen and
thymus should be evaluated as an indicator of systemic
immunotoxicity. The lymphoid tissue that drains or contacts the
site of drug administration (and therefore is exposed to the highest
concentration of the drug) should be examined. These sites include
the Peyer’s patches and mesenteric lymph nodes for orally
administered drugs, bronchus-associated lymphoid tissues (BALT) for
drugs administered by the inhalation route, nasal-associated
lymphoid tissues (NALT) for drugs administered by the inhalation or
nasal route (if possible), and the most proximal regional draining
lymph nodes for drugs administered by the dermal, intramuscular,
intradermal, intrathecal, or subcutaneous routes. The specific node
selected and the additional lymph node should be at the discretion
of the sponsor based on the sponsor's experience. For intravenously
administered drugs, the spleen can be considered the draining
lymphoid tissue.
It is recommended that a “semi-quantitative”
description of changes in compartments of lymphoid tissues be used
in recording changes and reporting treatment-related changes in
lymphoid tissues.
4.
Interpretation of Stress-Related Changes (1.4)
With standard
toxicity studies, doses near or at the maximum tolerated dose can
result in changes to the immune system related to stress (e.g., by
exaggerated pharmacodynamic action). These effects on the immune
system might be mediated by increased corticosterone or cortisol
release or other mediators. Commonly observed stress-related immune
changes include increases in circulating neutrophils, decreases in
circulating lymphocytes, decreases in thymus weight, decreases in
thymic cortical cellularity and associated histopathologic changes,
and changes in spleen and lymph node cellularity. Increases in
adrenal gland weight and/or histologic evidence of adrenal cortical
hyperplasia can also be observed. Thymic weight decreases in the
presence of clinical signs, such as decreased body weight and
physical activity, are too often attributed to stress. These
findings on their own should not be considered sufficient evidence
of stress-related immunotoxicity. The evidence of stress should be
compelling in order to justify not conducting additional
immunotoxicity studies.
B. Additional Immunotoxicity Studies
(2)
1.
Assay Characterization and Validation (2.1)
In general, the immunotoxicity test selected
should be widely used and have been demonstrated to be adequately
sensitive and specific for known immunosuppressive agents. However,
in certain situations, extensive validation might have not been
completed and/or the assay might not be widely used. In these
situations, a scientific/mechanistic basis for use of the assay is
called for and, if feasible, appropriate positive controls should be
incorporated.
There can be variations of response for each
type of immunotoxicity test used by different labs. In most
situations, these changes do not affect the ability of the assay to
assess immunotoxicity. However, to ensure proper assay performance
and lab proficiency, several standard technical validation
parameters should be observed. These parameters can include
determining intra- and inter-assay precision,
technician-to-technician precision, limit of quantitation, linear
region of quantitation and test sample stability. In addition, assay
sensitivity to known immunosuppressive agents should be established.
It is recommended that each laboratory test a positive control
concomitantly with an investigational compound or periodically in
order to demonstrate proficiency of performance, except for studies
with nonhuman primates. For immunophenotyping, if properly validated
technically, the addition of positive controls for each study might
not be needed.
Immunotoxicity studies are expected to be
performed in compliance with good laboratory practice (GLP). It is
recognized that some specialized assays, such as those described
below, might not comply fully with GLP.
2.
T-cell Dependent Antibody Response (TDAR) (2.2)
The TDAR should be performed using a recognized
T-cell dependent antigen (e.g., sheep red blood cells (SRBC) or
keyhole limpet hemocyanin (KLH)) that results in a robust antibody
response. The endpoint selected should be justified as the most
appropriate for the chosen assay and the selected species.
Antigens for immunization should not be used
with adjuvants without justification. Alum might be considered
acceptable for use only in nonhuman primate studies. The relative
TDAR response can be strain-dependent, especially in mice. With
outbred rats, there can be significant variability among rats within
the same group. Inbred rat strains could be used with provision of
sufficient exposure data to bridge to the strain used in the STS.
Antibody can be measured by using an ELISA or
other immunoassay methods. One advantage of this method over the
antibody forming cell response is that samples can be collected
serially during the study. In monkeys, serial blood collection can
be important due to the high inter-animal variability in the
kinetics of the response. For these studies, data can be expressed
as the sum of the antibody response over several collection dates
(e.g., area under the curve).
When SRBC antigens are used for an ELISA, the
preparation of the capture antigen that is coated on the plates is
considered critical. Whole fixed erythrocytes or membrane
preparations can be used as the SRBC capture antigen. ELISA results
should be expressed either as concentration or as titer, but
expression as optical densities is not recommended.
3.
Immunophenotyping (2.3)
Immunophenotyping is the identification and/or
enumeration of leukocyte subsets using antibodies.
Immunophenotyping is usually conducted by flow cytometric analysis
or by immunohistochemistry.
Flow cytometry, when employed to enumerate
specific cell populations, is not a functional assay. However, flow
cytometry can be used to measure antigen-specific immune responses
of lymphocytes. Data obtained from peripheral blood can be useful
as a bridge for clinical studies in which peripheral blood
leukocytes are also evaluated. It is recommended that absolute
numbers of lymphocyte subsets as well as percentages be used in
evaluating treatment-related changes.
One of the advantages of immunohistochemistry
over flow cytometry is that tissues from standard toxicity studies
can be analyzed retrospectively if signs of immunotoxicity are
observed. In addition, changes in cell types within a specific
compartment within the lymphoid tissue can be observed. Some of the
lymphocyte markers for certain species are sensitive to formalin
fixation and can only be localized in tissue that are either fixed
with certain fixatives or flash frozen. Quantitation of leukocytes
and intensity of staining is much more difficult with
immunohistochemistry.
When immunophenotyping studies are used to
characterize or identify alterations in specific leukocyte
populations, the choice of the lymphoid organs and/or peripheral
blood to be evaluated should be based on changes observed.
Immunophenotyping can be easily added to standard repeat dose
toxicity studies, and changes can be followed during the dosing
phase and periods without drug exposure (reversal period).
4.
Natural Killer Cell Activity Assays (2.4)
Natural killer (NK) cell activity assays can be
conducted if immunophenotyping studies demonstrate a change in
number, or if STS demonstrate increased viral infection rates, or in
response to other factors. In general, all NK cell assays are ex
vivo assays in which tissues (e.g., spleen) or blood are obtained
from animals that have been treated with the test compound. Cell
preparations are co-incubated with target cells that have been
labeled with 51Cr. New methods that involve
nonradioactive labels can be used if adequately validated.
Different effector to target cell ratios should be evaluated for
each assay to obtain a sufficient level of cytotoxicity and generate
a curve.
5. Host
Resistance Studies (2.5)
Host resistance studies involve challenging
groups of mice or rats treated with the different doses of test
compound with varying concentrations of a pathogen (bacteria,
fungal, viral, parasitic) or tumor cells. Infectivity of the
pathogens or tumor burden observed in vehicle versus test compound
treated animals is used to determine if the test compound is able to
alter host resistance. Models have been developed to evaluate a wide
range of pathogens such as Listeria monocytogenes,
Streptococcus pneumoniae, Candida albicans, influenza
virus, cytomegalovirus, Plasmodium yoelii, and Trichinella
spiralis. Tumor host resistance models in mice have used the
B16F10 melanoma and PYB6 sarcoma tumor cell lines.
Host resistance
assays can provide information on the susceptibility to particular
classes of infectious agents or tumor cells and can have an impact
on the risk management plan. In addition, they can have an important
role in identifying or confirming the cell type affected by a test
compound. Moreover, host resistance assays involve innate immune
mechanisms for which specific immune function assays have not been
developed. In conducting host resistance studies, the investigator
should carefully consider the direct or indirect (nonimmune
mediated) effects of the test compound on the growth and
pathogenicity of the organism or tumor cell. For instance,
compounds that inhibit the proliferation of certain tumor cells can
seem to increase host resistance. An in vitro assay to test direct
effects on the organism is recommended.
6.
Macrophage/Neutrophil Function (2.6)
In vitro macrophage and neutrophil function
assays (phagocytosis, oxidative burst, chemotaxis, and cytolytic
activity) have been published for several species. These assays
assess macrophage/neutrophil function of cells exposed to the test
compound in vitro or obtained from animals treated with the test
compound (ex vivo assay). In vitro exposure to test compound can
also be investigated. An in vivo assay can also be used to assess
the effects on the reticuloendothelial cell to phagocytize
radioactively or fluorescently labeled targets.
7.
Assays to Measure Cell-Mediated Immunity (2.7)
Assays to measure cell-mediated immunity have
not been as well established as those used for the antibody
response. These are in vivo assays where antigens are used for
sensitization. The endpoint is the ability of drugs to modulate the
response to challenge. Delayed-type hypersensitivity (DTH) reactions
with protein immunization and challenge have been reported for mice
and rats. Models in which contact sensitizers are used have been
explored in mice but have not been well validated or extensively
used. Cytotoxic T-cell response can be generated in mice using a
virus, tumor cell line, or allograft as the antigenic challenge.
Monkey DTH reactions have also been reported. However, these
reactions in monkeys are very difficult to consistently reproduce.
In addition, one should make sure that the DTH response is not
mistaken for an antibody and complement mediated Arthus reaction.
This guidance was developed
within the Expert Working Group (Safety) of the International
Conference on Harmonisation of Technical Requirements for
Registration of Pharmaceuticals for Human Use (ICH) and has been
subject to consultation by the regulatory parties, in accordance
with the ICH process. This document has been endorsed by the
ICH Steering Committee at Step 4 of the ICH process,
August 2005. At Step 4 of the process, the final draft
is recommended for adoption to the regulatory bodies of the
European Union, Japan, and the United States.
Arabic numbers reflect the organizational breakdown in the document
endorsed by the ICH Steering Committee at Step 4 of the ICH
process, August 2005.
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