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Fundamentals Of Genetic Toxicology In The Pharmaceutical Industry Sept 2010

Historical and current perspectives on genetic toxicology, with commentary and slides on assay predictivity and shortcomings, regulatory guidance, and high-throughput screens to enhance preclinical drug safety.

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Fundamentals Of Genetic Toxicology In The Pharmaceutical Industry Sept 2010

  1. 1. Fundamentals of Genetic Toxicology in the Pharmaceutical Industry Prepared by: David Amacher, Ph.D, DABT www.tigertox.com
  2. 2. What is Genetic Toxicology? <ul><li>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. </li></ul><ul><li>By altering the integrity and function of DNA 1 at the gene or chromosomal level, the damage can lead to heritable mutations ultimately resulting in genetic disorders, congenital defects, or cancer. </li></ul><ul><li>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). </li></ul><ul><li>1 DNA = deoxyribonucleic acid </li></ul>
  3. 3. Genotoxic Classification Scheme <ul><li>Deletions </li></ul><ul><li>Rearrangements </li></ul><ul><li>Breaks </li></ul>Microlesions Base-pair substitution mutations Frameshift mutations Macrolesions [cytologically visible] Numerical changes in chromosomes Stuructural changes in chromosomes Mutation [Qualitative change in 1 or a few nucleotide pairs] [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. 4. Mechanisms for Genetic Damage <ul><li>Genotoxic chemicals produce genetic damage at subtoxic levels. </li></ul><ul><li>The types of DNA 1 damage produced include: </li></ul><ul><ul><li>single- & double-strand breaks, </li></ul></ul><ul><ul><li>crosslinks between DNA bases and proteins, and </li></ul></ul><ul><ul><li>chemical additions to the DNA bases (adducts). </li></ul></ul><ul><li>DNA replication itself can introduce errors via incorrect base substitution, a process that can be exacerbated by some genotoxic agents. </li></ul><ul><li>1 DNA = deoxyribonucleic acid </li></ul>
  5. 5. Types of Genetic Damage <ul><li>Base substitution : The replacement of the correct nucleotide by an incorrect one. </li></ul><ul><ul><li>A transition involves a change of a purine for a purine or a pyrimidine for a pyrimidine </li></ul></ul><ul><ul><li>A transversion involves a change of a purine for a pyrimidine or vice versa. </li></ul></ul><ul><li>Frame shift mutation : The addition or deletion of one or a few base pairs (not in multiples of 3 [codon]) in protein-coding regions. </li></ul><ul><li>Structural chromosome aberrations : For non-radiomimetic chemicals, these can arise from errors of DNA replication on a damaged template. </li></ul><ul><ul><li>Radiomimetic chemicals can directly induce strand breaks. </li></ul></ul>
  6. 6. Types of Genetic Damage <ul><li>Numerical chromosome changes: </li></ul><ul><ul><li>Numerical aberrations are those involving non-diploid variations in chromosome number in the nucleus. </li></ul></ul><ul><ul><li>Monosomies, trisomies, & other ploidy changes arise from errors in chromosome segregation. </li></ul></ul><ul><ul><li>Aneuploidy (numerical deviation of the modal chromosome number) can result from the effects of chemicals on tubulin polymerization or spindle microtubule stability. </li></ul></ul><ul><li>Sister chromatid exchanges (SCE): </li></ul><ul><ul><li>SCE can be produced during S phase as a consequence of errors in the replication process and are apparently reciprocal exchanges </li></ul></ul>
  7. 7. DNA Repair <ul><li>DNA enzymatic repair mechanisms developed to maintain fidelity and integrity of genetic information </li></ul><ul><li>Enzymes are able to remove and replace damaged segments of DNA </li></ul><ul><li>Particularly useful during low-level exposure where excision repair enzymes are not fully saturated by excessive DNA damage </li></ul><ul><li>Stimulation of repair activity following treatment at sublethal concentrations can indicate presence of DNA-directed toxicity </li></ul><ul><li>Cells in S phase * (DNA synthesis) are most susceptible to genetic injury because they have less time to repair the damage prior to mitosis. </li></ul><ul><ul><li>* See supplemental slides at end of slide deck. </li></ul></ul>
  8. 8. DNA Repair Mechanisms <ul><li>Base excision repair: To repair DNA 1 base damages: </li></ul><ul><ul><li>A glycosylase removes the damaged base producing an apurinic or apyrimidinic site, </li></ul></ul><ul><ul><li>A DNA polymerase fills the gap with the appropriate base </li></ul></ul><ul><ul><li>A ligase seals the repaired patch. </li></ul></ul><ul><li>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. </li></ul><ul><li>1 DNA = deoxyribonucleic acid </li></ul>
  9. 9. DNA Repair Mechanisms <ul><li>Double-strand break repair : These are homologous recombination or nonhomologous end-joining processes which repair broken chromosomes. </li></ul><ul><ul><li>Homologous recombination steps include: </li></ul></ul><ul><ul><ul><li>Exonuclease or helicase activity produces a 3’-ended single-stranded tail. </li></ul></ul></ul><ul><ul><ul><li>“ Holliday junction DNA complex” is formed via strand invasion. This junction is cleaved to produce two DNA molecules, neither containing a strand break. </li></ul></ul></ul><ul><ul><li>A second mechanism, the nonhomologous end-joining processes, involves a DNA-dependent protein kinase. </li></ul></ul><ul><li>Unrepaired breaks result in checked cell cycle progression or the induction of apoptosis. </li></ul>
  10. 10. DNA Repair Mechanisms <ul><li>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. </li></ul><ul><li>O 6 -methylguanine-DNA methyltransferase repair : By transferring methyl groups from O 6 -methylguanine in affected DNA, this repair mechanism protects against simple aklylating agents. </li></ul>
  11. 11. Testing Requirements <ul><li>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. </li></ul><ul><li>A gene mutation assay is generally considered sufficient to support all single-dose clinical trials 1 . In support of multiple-dose clinical trials, 2 batteries of tests, Option 1 and Option 2, are described 2 . Option 2, if selected, should be completed prior to first human use in multiple-dose studies 1 . The in vitro components of Option 1, if selected, should be completed prior to first multiple-dose human studies 1 . The in vivo component of Option 1 should be completed prior to Phase 2 1 . </li></ul><ul><li>If an equivocal or positive finding occurs, additional tests should be performed as described in S2(R1) 2,3 & by the FDA 4 . </li></ul><ul><li>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). </li></ul><ul><li>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). </li></ul><ul><li>(http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm073246.pdf ) </li></ul><ul><li>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 ) </li></ul><ul><li>3 See also supplemental slides . </li></ul><ul><li>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 ). </li></ul>
  12. 12. Special Requirements for Testing during Preclinical Drug Development <ul><li>If positive or equivocal findings are found in vitro , then in vivo genetic tox tests are required prior to Phase I. </li></ul><ul><li>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. </li></ul><ul><li>FDA & EMA require phototoxicity testing by systemic or cutaneous applications of drugs that absorb light & penetrate into skin in relevant concentrations. </li></ul><ul><li>FDA = US Food and Drug Administration; EMA = European Medicines Agency </li></ul>
  13. 13. Examples of Genetic Toxicology Assays 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. 44:83-96, 2006) † Poor correlation with carcinogenicity (see: Regul. Toxicol. Pharmacol. 44:83-96, 2006) 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
  14. 14. The standard test battery <ul><li>Assessment in a bacterial reverse mutation assay. These assays detect the majority of genotoxic rodent and human carcinogens. </li></ul><ul><ul><li>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: </li></ul></ul><ul><ul><ul><li>TA98; TA100; TA1535; TA1537 or TA97 or TA97a; TA102 or E. coli WP2 uvr A or E . coli WP2 uvr A (pKM101). </li></ul></ul></ul>
  15. 15. The standard test battery <ul><li>Evaluation in mammalian cells in vitro and/or in vivo </li></ul><ul><ul><li>In vitro mammalian cell systems include: Mouse lymphoma L5178Y cells, Chinese hamster cells, primary rat hepatocytes, & human peripheral lymphocytes. </li></ul></ul><ul><ul><li>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. </li></ul></ul><ul><ul><li>ADME = absorption, distribution, metabolism, and excretion </li></ul></ul>ADME = absorption, distribution, metabolism, and excretion.
  16. 16. Assays for Detecting DNA Damage & Repair <ul><li>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. </li></ul><ul><li>In mammalian cells, DNA 1 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 pol A- & pol A+ or Bacillis subtilis rec- & rec+ ) is another indirect use of DNA repair for the detection of DNA damage. </li></ul><ul><li>1 DNA = deoxyribonucleic acid. </li></ul>
  17. 17. Assays for Detecting DNA Damage & Repair <ul><li>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. </li></ul><ul><ul><li>Direct measures of DNA damage involve the detection of chemical adducts or DNA strand breaks. </li></ul></ul><ul><ul><li>Indirect assays measure DNA repair processes. </li></ul></ul><ul><li>Assays for bulky DNA adducts include : </li></ul><ul><ul><li>the 32 P-postlabeling assay. </li></ul></ul><ul><ul><li>DNA strand-breakage assays include alkaline elution assay and electrophoretic methods. </li></ul></ul><ul><ul><li>Single-cell gel electrophoresis (the Comet assay) is now widely used to measure DNA damage. </li></ul></ul>
  18. 18. Options for the standard battery <ul><li>Option 1 </li></ul><ul><li>A test for gene mutation in bacteria. </li></ul><ul><li>A cytogenetic test for chromosomal damage (choice of three) </li></ul><ul><li>An in vivo test for chromosome damage using rodent hematopoietic cells (either micronuclei or chromosomal aberrations in metaphase cells). </li></ul>
  19. 19. Options for the standard battery <ul><li>Option 2 </li></ul><ul><li>A test for gene mutation in bacteria. </li></ul><ul><li>An in vivo assessment with two tissues (e.g., micronuclei using rodent hematopoietic cells + a second in vivo assay (e.g., liver UDS 1 assay, transgenic mouse assay, Comet assay, etc.) </li></ul><ul><li>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 . </li></ul><ul><li>1 </li></ul><ul><li>UDS = unscheduled DNA synthesis. </li></ul>
  20. 20. Outcomes & Follow-up <ul><li>If results from any of the 3 assays in the ICH 1 genotoxicty standard battery are positive, complete a 4th test from the ICH battery to confirm. </li></ul><ul><li>Equivocal studies should be repeated to determine reproducibility. </li></ul><ul><li>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. </li></ul><ul><li>1 ICH = International Conference for Harmonization </li></ul>
  21. 21. Confounding Factors <ul><li>Examples of some experimental factors that may produce study artifacts: </li></ul><ul><ul><li>Accelerated erythropoiesis (micronucleus assay) </li></ul></ul><ul><ul><li>Non-physiological conditions (mammalian cells) </li></ul></ul><ul><ul><li>Positive only @ highly cytotoxic concentrations ( in vitro assays) </li></ul></ul><ul><ul><li>Presence of mutagenic impurities or precursors </li></ul></ul><ul><ul><li>Pharmacologically related indirect threshold mechanisms </li></ul></ul><ul><ul><li>Metabolic differences between “induced” rat S9 (overrepresentation of CYP1A & 2B enzymes) vs. human cells </li></ul></ul><ul><ul><li>Non-DNA thresholds </li></ul></ul><ul><ul><li>S9 = metabolic activation, CYP = cytochrome P-450 enzymes, DNA = deoxyribonucleic acid. </li></ul></ul>
  22. 22. Rationale for Early Genetic Toxicology Evaluation (Screening Assays) <ul><li>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. </li></ul><ul><li>Mutagenicity pre-screening in drug discovery/lead optimization can identify potential mutagens & remove them from development at an early stage. </li></ul><ul><li>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. </li></ul>
  23. 23. Discovery/Lead Optimization Screening Assays <ul><li>Non-GLP Assays can be used at early stages in drug discovery to select chemical candidates for further development. </li></ul><ul><li>Early screening assay advantages include: </li></ul><ul><ul><li>Low cost </li></ul></ul><ul><ul><li>Rapid turn-around time </li></ul></ul><ul><ul><li>Require minimal amounts of test articles </li></ul></ul><ul><ul><li>Can be highly predictive </li></ul></ul>
  24. 24. Rapid Pre-screening Methods <ul><li>Examples of Modified or High-throughput methods for early screening include: </li></ul><ul><ul><li>Computer-assisted ( in silico) SAR methods for predictive toxicity screening 1 </li></ul></ul><ul><ul><li>Modified assays such as the in vitro assessment of micronucleus induction in CHO cells 2 , the Ames II assay (TA98 & TAMix), the in vitro Comet assay 3 , or well-based (e.g. ,96- or 384-well format) modifications of the yeast DEL assay 4 </li></ul></ul><ul><ul><li>Proprietary assays such as Vitotox ™ (mutagenicity), RadarScreen ® (clastogenicity), & GreenScreen ® GC 5 </li></ul></ul><ul><ul><li>1 Mohan et al. Mini Rev. Med. Chem. 7(5): 499-507, 2007. </li></ul></ul><ul><ul><li>2 Jacobson-Kram & Contrera Toxicol. Sci. 96(1): 16-20, 2007. </li></ul></ul><ul><ul><li>3 Witte et al. Toxicol. Sci. 97: 21-26, 2007 </li></ul></ul><ul><ul><li>4 Schoonen et al. EXS 99: 401-452, 2009. </li></ul></ul><ul><ul><li>5 Hontzeas et al. Mutat. Res. 634(1-2): 228-234, 2007. </li></ul></ul><ul><li>SAR = structure-activity relationship; CHO = Chinese Hamster Ovary. </li></ul>
  25. 25. Suggested sources for further reading <ul><li>Genetic Toxicology and Cancer Risk Assessment by Wai Nang Choy. 1st edition, 2001, Informa Healthcare. </li></ul><ul><li>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. </li></ul><ul><li>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. </li></ul><ul><li>Genetic Toxicology by David Brusick in: Principles & Methods of Toxicology. 5th edition, Wallace A Hayes editor, 2007, Informa Healthcare. </li></ul>
  26. 26. Further Information <ul><li>About the Author: </li></ul><ul><li>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 . </li></ul><ul><li>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 ( http://www.tigertox.com/2010/09/20/genetic-toxicology-historical-and-current-perspectives/ ). </li></ul>
  27. 27. <ul><li>Supplemental </li></ul><ul><li>Slides </li></ul>
  28. 28. Mammalian Somatic Cell Cycle <ul><li>G1 (interphase) – energy stores replenished & daughter cell growth </li></ul><ul><li>S phase (DNA synthesis ) – DNA is duplicated in process of replication. </li></ul><ul><li>G2 – energy reserves restored. </li></ul><ul><li>Mitosis - DNA is divided into two identical sets before the cell divides. </li></ul><ul><li>Cytokinesis - the division of the cytoplasm of a parent cell. It occurs at the end of Mitosis or the beginning of Interphase. </li></ul>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. 29. Genotoxicity ICH Regulatory Guidelines <ul><li>ICH Harmonized Tripartite Guideline S2(R1). Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use (Step 3, March 2008). </li></ul><ul><ul><li>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. </li></ul></ul>