Fundamentals of Genetic Toxicology in the Pharmaceutical Industry

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Fundamentals of Genetic Toxicology in the
Pharmaceutical Industry

• Prepared by
• David Amacher, Ph.D, DABT
• www.tigertox.com

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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

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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.
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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

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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.

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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

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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.

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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

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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.

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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.

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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).

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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

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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)

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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).

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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.
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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.

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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.

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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).

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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.

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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

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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.

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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.

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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

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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.

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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.

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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).

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• Supplemental
• Slides

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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.
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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.