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Fundamentals of Genetic Toxicology in the Pharmaceutical Industry

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Title: Fundamentals of Genetic Toxicology in the Pharmaceutical Industry Author: David Last modified by: Cynthia Davenport Created Date: 6/15/2009 1:00:52 PM – PowerPoint PPT presentation

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Title: Fundamentals of Genetic Toxicology in the Pharmaceutical Industry


1
Fundamentals of Genetic Toxicology in the
Pharmaceutical Industry
  • Prepared by
  • David Amacher, Ph.D, DABT
  • www.tigertox.com

2
What is Genetic Toxicology?
  • Genetic Toxicology refers to the assessment of
    the deleterious effects of chemicals or physical
    agents on the hereditary material and related
    genetic processes of living cells.
  • By altering the integrity and function of DNA1 at
    the gene or chromosomal level, the damage can
    lead to heritable mutations ultimately resulting
    in genetic disorders, congenital defects, or
    cancer.
  • Targets of DNA damage include somatic cells
    (detrimental to the exposed individual), germinal
    cells (potentially heritable effects), and
    mitochondria (detrimental to the exposed
    individual progeny via maternal inheritance).
  • 1 DNA deoxyribonucleic acid

3
Genotoxic Classification Scheme
Mutation
Macrolesions cytologically visible
Microlesions
Base-pair substitution mutations
Frameshift mutations
Numerical changes in chromosomes
Stuructural changes in chromosomes
Qualitative change in 1 or a few nucleotide
pairs
  • Deletions
  • Rearrangements
  • Breaks

Quantitative change in 1 or a few nucleotide
pairs
Diagram from Bruswick, D.J. Alterations of germ
cells leading to mutagenesis and their detection.
Environ. Health Perspect. 24(1978)105-112.
4
Mechanisms for Genetic Damage
  • Genotoxic chemicals produce genetic damage at
    subtoxic levels.
  • The types of DNA1 damage produced include
  • single- double-strand breaks,
  • crosslinks between DNA bases and proteins, and
  • chemical additions to the DNA bases (adducts).
  • DNA replication itself can introduce errors via
    incorrect base substitution, a process that can
    be exacerbated by some genotoxic agents.
  • 1 DNA deoxyribonucleic acid

5
Types of Genetic Damage
  • Base substitution The replacement of the correct
    nucleotide by an incorrect one.
  • A transition involves a change of a purine for a
    purine or a pyrimidine for a pyrimidine
  • A transversion involves a change of a purine for
    a pyrimidine or vice versa.
  • Frame shift mutation The addition or deletion
    of one or a few base pairs (not in multiples of 3
    codon) in protein-coding regions.
  • Structural chromosome aberrations For
    non-radiomimetic chemicals, these can arise from
    errors of DNA replication on a damaged template.
  • Radiomimetic chemicals can directly induce strand
    breaks.

6
Types of Genetic Damage
  • Numerical chromosome changes
  • Numerical aberrations are those involving
    non-diploid variations in chromosome number in
    the nucleus.
  • Monosomies, trisomies, other ploidy changes
    arise from errors in chromosome segregation.
  • Aneuploidy (numerical deviation of the modal
    chromosome number) can result from the effects of
    chemicals on tubulin polymerization or spindle
    microtubule stability.
  • Sister chromatid exchanges (SCE)
  • SCE can be produced during S phase as a
    consequence of errors in the replication process
    and are apparently reciprocal exchanges

7
DNA Repair
  • DNA enzymatic repair mechanisms developed to
    maintain fidelity and integrity of genetic
    information
  • Enzymes are able to remove and replace damaged
    segments of DNA
  • Particularly useful during low-level exposure
    where excision repair enzymes are not fully
    saturated by excessive DNA damage
  • Stimulation of repair activity following
    treatment at sublethal concentrations can
    indicate presence of DNA-directed toxicity
  • Cells in S phase (DNA synthesis) are most
    susceptible to genetic injury because they have
    less time to repair the damage prior to mitosis.
  • See supplemental slides at end of slide deck.

8
DNA Repair Mechanisms
  • Base excision repair To repair DNA1 base
    damages
  • A glycosylase removes the damaged base producing
    an apurinic or apyrimidinic site,
  • A DNA polymerase fills the gap with the
    appropriate base
  • A ligase seals the repaired patch.
  • Nucleotide excision repair To remove bulky
    lesions from DNA, a process involving as many as
    30 proteins to remove damaged oligonucleotides
    from DNA in steps involving damage recognition,
    incision, excision, repair synthesis, and
    ligation.
  • 1 DNA deoxyribonucleic acid

9
DNA Repair Mechanisms
  • Double-strand break repair These are homologous
    recombination or nonhomologous end-joining
    processes which repair broken chromosomes.
  • Homologous recombination steps include
  • Exonuclease or helicase activity produces a
    3-ended single-stranded tail.
  • Holliday junction DNA complex is formed via
    strand invasion. This junction is cleaved to
    produce two DNA molecules, neither containing a
    strand break.
  • A second mechanism, the nonhomologous end-joining
    processes, involves a DNA-dependent protein
    kinase.
  • Unrepaired breaks result in checked cell cycle
    progression or the induction of apoptosis.

10
DNA Repair Mechanisms
  • Mismatch repair Mismatched bases can be formed
    during DNA replication, genetic recombination, or
    chemically-induced DNA damage. A specific
    protein recognizes binds to the mismatch and
    additional proteins stabilize it. This is
    followed by excision, resynthesis, and ligation.
  • O6-methylguanine-DNA methyltransferase repair By
    transferring methyl groups from O6-methylguanine
    in affected DNA, this repair mechanism protects
    against simple aklylating agents.

11
Testing Requirements
  • The fundamental purpose of genetic toxicology
    testing is to safeguard the human gene pool from
    chemical damage. There are two basic types of
    screening assays (1) tests for gene mutations,
    (2) tests for chromosomal aberrations.
  • A gene mutation assay is generally considered
    sufficient to support all single-dose clinical
    trials1. In support of multiple-dose clinical
    trials, 2 batteries of tests, Option 1 and Option
    2, are described2. Option 2, if selected, should
    be completed prior to first human use in
    multiple-dose studies1. The in vitro components
    of Option 1, if selected, should be completed
    prior to first multiple-dose human studies1. The
    in vivo component of Option 1 should be completed
    prior to Phase 21.
  • If an equivocal or positive finding occurs,
    additional tests should be performed as described
    in S2(R1)2,3 by the FDA4.
  • The standard battery of tests S2(R1)2 should be
    completed prior to the initiation of Phase II
    studies. Testing must be GLP-compliant (21 CFR
    part 55).
  • 1 ICH Harmonized Tripartite Guideline M3(R2)
    Nonclinical Safety Studies for the Conduct of
    Human Clinical Trials and Marketing Authorization
    for Pharmaceuticals. (Final, January 2010).
  • (http//www.fda.gov/downloads/Drugs/GuidanceCompli
    anceRegulatoryInformation/Guidances/ucm073246.pdf)
  • 2 ICH Harmonized Tripartite Guideline. Guideline
    for Industry S2(R1) Genotoxicity Testing and
    Data Interpretation for Pharmaceuticals Intended
    for Human Use (Step 3, March 2008).
    (http//www.fda.gov/cder/guidance/index.htm)
  • 3 See also supplemental slides.
  • 4 FDA Guidance for Industry and Review Staff
    Recommended Approaches to Integration of Genetic
    Toxicology Study Results (January, 2006).
    (http//www.fda.gov/cder/guidance/6848fnl.pdf).

12
Special Requirements for Testing during
Preclinical Drug Development
  • If positive or equivocal findings are found in
    vitro, then in vivo genetic tox tests are
    required prior to Phase I.
  • For women of child bearing potential, pregnant
    women, children, and for compounds bearing
    structural alerts, both in vitro and in vivo
    genetic tox assays must be completed prior to any
    clinical trials.
  • FDA EMA require phototoxicity testing by
    systemic or cutaneous applications of drugs that
    absorb light penetrate into skin in relevant
    concentrations.
  • FDA US Food and Drug Administration EMA
    European Medicines Agency

12
13
Examples of Genetic Toxicology Assays
Gene Mutations Chromosome Damage Primary DNA Damage
Salmonella Mammalian Microsome (Ames) Assay In vitro metaphase chromosomal aberrations Transformation Assays (BALB/c or SHE)
Mouse Lymphoma TK Assay (MLA) In vitro micronucleus test Comet Assay for DNA strand breakage (in vitro in vivo)
CHO/HGPRT Assay In vivo metaphase chromosomal aberrations Alkaline Elution Assay for DNA strand breakage
Transgenic Assays (e.g. Big Blue mouse rat, Muta Mouse, lacZ plasmid mouse) In vivo micronucleus test Unscheduled DNA synthesis (UDS) (in vitro in vivo).
DNA covalent binding assay
  • CHO Chinese Hamster Ovary, HGPRT
    hypoxanthine-guanine phosphoribosyl transferase
    gene in CHO cells, SHE Syrian Hamster Embryo,
    BALB BALB/3T3 cell transformation assay, Comet
    also called single cell gel electrophoresis
    (SCGE)., DNA deoxyribonucleic acid.
  • ICH S2B assay
  • Good correlation with carcinogenicity (see
    Regul. Toxicol. Pharmacol. 4483-96, 2006)
  • Poor correlation with carcinogenicity (see
    Regul. Toxicol. Pharmacol. 4483-96, 2006)

14
The standard test battery
  • Assessment in a bacterial reverse mutation assay.
    These assays detect the majority of genotoxic
    rodent and human carcinogens.
  • Bacterial mutagens are detected by selecting
    tester strains that detect base substitution
    frameshift point mutations. Bacterial mutation
    assays for base pair substation and frameshift
    point mutations include the following base set of
    strains
  • TA98 TA100 TA1535 TA1537 or TA97 or TA97a
    TA102 or E. coli WP2 uvrA or E. coli WP2 uvrA
    (pKM101).

15
The standard test battery
  • Evaluation in mammalian cells in vitro and/or in
    vivo
  • In vitro mammalian cell systems include Mouse
    lymphoma L5178Y cells, Chinese hamster cells,
    primary rat hepatocytes, human peripheral
    lymphocytes.
  • In vivo tests provide additional relevant ADME
    factors. Commonly used in vivo systems include
    Rodent bone marrow or lymphocytes following in
    vivo exposure, rat liver or other target organs
    following in vivo exposure, transgenic mice.
  • ADME absorption, distribution, metabolism, and
    excretion

ADME absorption, distribution, metabolism, and
excretion.
16
Assays for Detecting DNA Damage Repair
  • Chromososmal aberrations are assayed in vitro by
    metaphase analysis in cultured cells and in vivo
    by metaphase analysis of rodent bone marrow or
    lymphocytes. Chromosomal structural or numerical
    changes are detected in vitro via
    cytokinesis-blocked micronucleus assay in human
    lymphocytes or mammalian cell lines and in vivo
    by the micronucleus assay in rodent bone marrow
    or blood.
  • In mammalian cells, DNA1 repair is commonly
    assayed by measuring unscheduled DNA synthesis
    (UDS). Also, comparisons of a test chemical in
    DNA repair-deficient vs. DNA repair-proficient
    bacteria strains (e.g., E. coli polA- polA or
    Bacillis subtilis rec- rec) is another
    indirect use of DNA repair for the detection of
    DNA damage.
  • 1 DNA deoxyribonucleic acid.

17
Assays for Detecting DNA Damage Repair
  • Standardized assays are used to identify
    germ-cell mutagens, somatic-cell mutagens, and
    potential carcinogens through the detection of
    gene mutations, chromosomal aberrations, and/or
    aneuploidy following chemical exposure.
  • Direct measures of DNA damage involve the
    detection of chemical adducts or DNA strand
    breaks.
  • Indirect assays measure DNA repair processes.
  • Assays for bulky DNA adducts include
  • the 32P-postlabeling assay.
  • DNA strand-breakage assays include alkaline
    elution assay and electrophoretic methods.
  • Single-cell gel electrophoresis (the Comet assay)
    is now widely used to measure DNA damage.

18
Options for the standard battery
  • Option 1
  • A test for gene mutation in bacteria.
  • A cytogenetic test for chromosomal damage (choice
    of three)
  • An in vivo test for chromosome damage using
    rodent hematopoietic cells (either micronuclei or
    chromosomal aberrations in metaphase cells).

19
Options for the standard battery
  • Option 2
  • A test for gene mutation in bacteria.
  • An in vivo assessment with two tissues (e.g.,
    micronuclei using rodent hematopoietic cells a
    second in vivo assay (e.g., liver UDS1 assay,
    transgenic mouse assay, Comet assay, etc.)
  • For compounds giving negative results, the
    completion of either test battery (Options 1 or
    2), performed and evaluated as recommended, will
    usually suffice with no further testing required.
  • 1
  • UDS unscheduled DNA synthesis.

20
Outcomes Follow-up
  • If results from any of the 3 assays in the ICH1
    genotoxicty standard battery are positive,
    complete a 4th test from the ICH battery to
    confirm.
  • Equivocal studies should be repeated to determine
    reproducibility.
  • If a positive response is seen in 1 or more
    assays, the sponsor should choose from the
    following options (a) weight of evidence
    decision, (b) mechanism of action decision, or
    (c) conduct additional supporting studies.
  • 1 ICH International Conference for
    Harmonization

21
Confounding Factors
  • Examples of some experimental factors that may
    produce study artifacts
  • Accelerated erythropoiesis (micronucleus assay)
  • Non-physiological conditions (mammalian cells)
  • Positive only _at_ highly cytotoxic concentrations
    (in vitro assays)
  • Presence of mutagenic impurities or precursors
  • Pharmacologically related indirect threshold
    mechanisms
  • Metabolic differences between induced rat S9
    (overrepresentation of CYP1A 2B enzymes) vs.
    human cells
  • Non-DNA thresholds
  • S9 metabolic activation, CYP cytochrome
    P-450 enzymes, DNA deoxyribonucleic acid.

22
Rationale for Early Genetic Toxicology Evaluation
(Screening Assays)
  • Frequently, presumptive mutagens are dropped from
    development. If continued, potential clastogens
    require disclosure consent in clinical trials,
    unfavorable labeling, can result in diminished
    market potential.
  • Mutagenicity pre-screening in drug discovery/lead
    optimization can identify potential mutagens
    remove them from development at an early stage.
  • Early in vitro clastogenicity screening can
    facilitate the efficient planning of follow-up in
    vivo testing. The latter can often be integrated
    into other toxicity studies to expedite
    preclinical development and reduce cost.

22
23
Discovery/Lead Optimization Screening Assays
  • Non-GLP Assays can be used at early stages in
    drug discovery to select chemical candidates for
    further development.
  • Early screening assay advantages include
  • Low cost
  • Rapid turn-around time
  • Require minimal amounts of test articles
  • Can be highly predictive

23
24
Rapid Pre-screening Methods
  • Examples of Modified or High-throughput methods
    for early screening include
  • Computer-assisted (in silico) SAR methods for
    predictive toxicity screening1
  • Modified assays such as the in vitro assessment
    of micronucleus induction in CHO cells2 , the
    Ames II assay (TA98 TAMix), the in vitro Comet
    assay3, or well-based (e.g. ,96- or 384-well
    format) modifications of the yeast DEL assay4
  • Proprietary assays such as Vitotox
    (mutagenicity), RadarScreen (clastogenicity),
    GreenScreen GC5
  • 1 Mohan et al. Mini Rev. Med. Chem. 7(5)
    499-507, 2007.
  • 2 Jacobson-Kram Contrera Toxicol. Sci. 96(1)
    16-20, 2007.
  • 3 Witte et al. Toxicol. Sci. 97 21-26, 2007
  • 4 Schoonen et al. EXS 99 401-452, 2009.
  • 5 Hontzeas et al. Mutat. Res. 634(1-2) 228-234,
    2007.
  • SAR structure-activity relationship CHO
    Chinese Hamster Ovary.

25
Suggested sources for further reading
  • Genetic Toxicology and Cancer Risk Assessment by
    Wai Nang Choy. 1st edition, 2001, Informa
    Healthcare.
  • Genetic Toxicology by R. Julian Preston George
    R. Hoffman in Toxicology. The Basic Science of
    Poisons. 7th edition, Curtis D. Klassen editor,
    2008, McGraw-Hill.
  • Genetic Toxicology by Donald L. Putnam et al. in
    Toxicological Testing Handbook Principles,
    Applications, and Data Interpretation. 2nd
    edition, David Jacobson-Kram Kit A. Keller
    editors, 2006, Informa Healthcare.
  • Genetic Toxicology by David Brusick in
    Principles Methods of Toxicology. 5th edition,
    Wallace A Hayes editor, 2007, Informa Healthcare.

26
Further Information
  • About the Author
  • David Amacher is a senior investigative and
    biochemical toxicologist with extensive
    experience in the safety evaluation of human and
    animal health products. Dr. Amacher is a
    Diplomate of the American Board of Toxicology, a
    Fellow of the National Academy of Clinical
    Biochemistry, and serves as an Assistant Research
    Professor of Toxicology and Adjunct Professor in
    the Graduate School of the University of
    Connecticut. His professional affiliations
    include memberships in the American Society for
    Pharmacology and Experimental Therapeutics,
    Society of Toxicology, American Society of
    Biochemistry and Molecular Biology, International
    Society for the Study of Xenobiotics, American
    Association of Clinical Chemistry, and the
    American College of Toxicology.
  • An accompanying commentary on historical and
    current perspectives on genetic toxicology, assay
    predictivity and shortcomings, regulatory
    guidance, and high-throughput screens to enhance
    preclinical drug safety can be found at
    ToxInsights (www.tigertox.com).

27
  • Supplemental
  • Slides

28
Mammalian Somatic Cell Cycle
  • G1 (interphase) energy stores replenished
    daughter cell growth
  • S phase (DNA synthesis) DNA is duplicated in
    process of replication.
  • G2 energy reserves restored.
  • Mitosis - DNA is divided into two identical sets
    before the cell divides.
  • Cytokinesis - the division of the cytoplasm of a
    parent cell. It occurs at the end of Mitosis or
    the beginning of Interphase.

Referenced from http//bit.ly/byhKxT on 19 Sept
2010.
Meiosis, which occurs only in germ cells, is the
process of nuclear division that reduces the
number of chromosomes by half.
29
Genotoxicity ICH Regulatory Guidelines
  • ICH Harmonized Tripartite Guideline S2(R1).
    Genotoxicity Testing and Data Interpretation for
    Pharmaceuticals Intended for Human Use (Step 3,
    March 2008).
  • This guidance replaces and combines the ICH S2A
    S2B guidelines. The revised guidance describes
    internationally agreed upon standards for
    follow-up testing interpretation of positive
    findings in vitro in vivo in the standard
    genetic toxicology battery, including assessment
    of non-relevant findings.
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