Toxicology for Industrial and Regulatory Scientists Genetic Toxicology Mark W. Powley, Ph.D. Pharmacology/Toxicology Reviewer Division of Antiviral Products FDA/CDER/OND April 29, 2015 1
Outline Background Assays Drug Development and Regulations Results and Decision Making Drug Impurities FDA Case Studies The Future Disclaimer: The views expressed in this presentation are those of the speaker and not, necessarily, of the FDA. 2
Genetic Toxicology: Background 3
Background What is genetic toxicology? sub-discipline of toxicology focused on the ability of an agent to cause genetic damage drugs consumer products chemicals radiation viruses 4
Background Why is genetic toxicology important? t? genotoxic and carcinogenic agents are ubiquitous genetic e damage is constantly occurring Cosmic Radiation Brussel Sprouts - allyl isothiocyanate Coffee - benzo(a)pyrene - benzaldehyde -benzene - benzofuran - caffeic acid - catechol - 1,2,5,6-dibenz(a)anthracene - ethyl benzene -furan -furfural -hydrogen peroxide - hydroquinone - d-limonene - 4-methylcatechol 5
Background Why is genetic toxicology important? genetic damage is tied to multistep process of carcinogenesis (primary concern) carcinogenic effects will not be evident for many years, ultimately defeating the purpose of clinical monitoring 6 Klaassen, Curtis D. (2013) Casarett and Doull's Toxicology - The Basic Science of Poisons (8th Edition). McGraw-Hill.
Background DNA genetic material; macromolecule essential for life organization gene functional unit of DNA containing information necessary for synthesizing proteins chromosome structural t unit of DNA 7
Background DNA damage vs. mutation damage DNA strand breaks, adducts, etc. may be repaired or result in cell death mutation genetic damage that becomes fixed and passed along to daughter cells cannot be repaired note: not all damage will result in a mutation and not all mutations will result in cancer 8
Background Effects on Concern gene mutations base-pair substitution, frameshift mutations, additions, i and deletions may result in loss of expression expression of non-functional or poorly functional protein 9
Examples of Gene Mutations resulting from base-pair substitution 10
Background Effects of Concern chromosomal damage structural chromosomal breakage resulting in genetic information being deleted, added, or rearranged may result in loss/gain of gene expression expression in unintended cells expression at unintended time 11
Examples of Structural Chromosome Damage 12
Background Effects of Concern chromosomal damage numerical chromosomal damage resulting in change in chromosome number two forms polyploidy change in whole set of chromosomes aneuploidy change in part of chromosome set 13
Background Effects of Concern aneuploidy noted in large % of tumor cells it s debated whether aneuploidy causes tumorigenicity i it or is a byproduct of the process may result in loss or gain of expression Torres E.M., et al. (2008) Genetics 179:737-746 14
Background Additional Considerations location of damage although preferential localization is possible, damage generally occurs randomly bad activation of proto-oncogene oncogene resulting in altered regulation of growth and differentiation (e.g., Ras) inactivation of tumor suppressor gene resulting in impaired response to genetic damage (e.g., p53) not so bad damage to less critical region of genome 15
Background Additional Considerations extent of damage damage must be heritable (passed on to daughter cells) too much damage and certain types of damage may result in cell death repair capacity is critical element 16
Background Additional Considerations repair of damage multiple pathways for repairing genetic damage base excision repair nucleotide excision repair homologous recombination mismatch repair some pathways are more accurate than others e.g., polymerases involved in translesion synthesis Pol η (error-free) vs. Pol ι (error-prone) 17
Background Additional Considerations Threshold is there a dose below which no effects are observed? threshold no threshold 18
Genetic Toxicology: Assays 19
Assays Bacterial Reverse-Mutation Assay principle p histidine or tryptophan dependent-bacteria are treated with test article with and without metabolic activation reverse mutation restores functional capacity of gene responsible for synthesizing histidine or tryptophan revertant colonies (bacteria that grow in absence of fhistidine idi or tryptophan) are counted using automated system 20
Assays Bacterial Reverse-Mutation Assay detects gene mutations base-pair substitutions frameshift mutations multiple bacterial strains used each has different combination of modifications such as cell wall permeability repair capacity 21
Bacterial Reverse-Mutation Assay Mortelmans K., Zeiger E. (2000) Mut. Res. 455:29-60 22
Assays In Vitro Chromosomal Aberration Assay in vitro cytogenetics assay principle cells are exposed to test article with and without metabolic activation cells are arrested in metaphase, collected, and stained chromosomes are analyzed for aberrations by light microscopy 23
Assays In Vitro Chromosomal Aberration Assay detects structural damage to chromosomes/chromatids numerical changes conducted in different cell types, most commonly Chinese Hamster Ovary cells human peripheral blood lymphocytes well described karyotypes with big chromosomes 24
In Vitro Chromosomal Aberration Assay normal chromosomes tb dc tb = chromatid break dc = dicentric chromosome images courtesy of Dr. Leon Stankowski BioReliance Corporation 25
Assays Mouse Lymphoma Assay thymidine kinase gene mutation assay (Tk +/- assay) principle cells are treated with test article with and without metabolic activation then challenged with toxic thymidine analog (trifluorothymidine; TFT) forward mutation decreases functional capacity of gene responsible for thymidine phosphorylation so TFT will not block DNA synthesis mutant colonies (cells that grow in presence of TFT) are sized and counted using automated system 26
Assays Mouse Lymphoma Assay detects point mutations chromosomal damage deletions rearrangements mitotic recombination/gene conversion aneuploidy 27
Assays Mouse Lymphoma Assay sizing mutant colonies may help establish mechanism large colonies (normal growth rate) suggest gene mutation small colonies (slow growth rate) suggest chromosome level effect 28
Mouse Lymphoma Assay negative control positive control images courtesy of Dr. Leon Stankowski BioReliance Corporation 29
Assays In Vivo Micronucleus Assay in vivo cytogenetics assay in rodents micronuclei cytoplasmic chromatin-containing bodies that are not incorporated into daughter cells can be whole chromosomes or fragments principle bone marrow or peripheral p blood is collected from rodents treated with test article, smears made, and stained micronucleated polychromatic erythrocytes t are counted using light microscopy or flow cytometry 30
Assays In Vivo Micronucleus Assay damage detected structural chromosomal damage mitotic ti spindle apparatus damage other accounts for in vivo conditions route of administration ADME 31
Assays In Vivo Micronucleus Assay mechanistic information special staining procedures allow differentiation between clastogenicity and aneugenicity CREST staining uses antibodies to detect kinetochores in micronuclei kinetochore positive indicates presence of whole chromosome (aneugenic mechanism) kinetochore negative indicates presence of chromosome fragment (clastogenic mechanism) 32
In Vivo Micronucleus Assay Giemsa stained bone marrow Acridine orange stained bone marrow images courtesy of Dr. Leon Stankowski BioReliance Corporation 33
Assays In Vivo Comet Assay single cell gel electrophoresis assay comet fragmented DNA that migrates from nucleus to form a tail principle rodents are treated with test article tissues are collected, cells isolated, and subjected to gel electrophoresis comet tail is measured by image analysis 34
Assays In Vivo Comet Assay detects strand breaks alkali-labile sites DNA-DNA or DNA-protein crosslinks able to evaluate cells from multiple tissues select tissues based on toxicity, exposure, etc. 35
In Vivo Comet Assay images courtesy of Dr. Leon Stankowski BioReliance Corporation 36
Assays Other Assays Gene Mutations in vivo gene mutation assay in transgenic animals (e.g., lacz, laci, gpt, etc.) detects gene mutations in any tissue of interest in vivo Pig-a gene mutation tti assay dt detects t gene mutations in bone marrow cells in vitro gene mutation assay in mammalian cells (e.g., hprt) 37
Assays Other Assays Chromosomal Damage in vitro MN similar to in vivo assay in vivo CA similar to in vitro assay Primary Damage in vivo and in vitro unscheduled DNA synthesis (UDS) detects DNA repair DNA adducts detects covalent binding 38
Assays Other Considerations metabolic activation CYP450 O may use tissue fractions from animals treated with known enzyme inducers to maximize metabolic activity typically use rat liver S9 plus necessary cofactors, etc., but alternative tissues sources may be acceptable e.g., human liver S9 appropriate when unique human metabolite is a concern S9 is a subcellular fraction containing both microsomes (e.g., includes CYP450s) and cytosol 39
Assays Other Considerations positive and negative controls standardized for the various assays assure that assay is working as expected help with interpreting assay results compare with both concurrent and historical control values 40
Assays Other Considerations top dose bacterial reverse-mutation assay standard top dose is 5000 μg/plate may be limited by toxicity (e.g., reduction in background lawn and/or revertants) or test article solubility 41
Assays Other Considerations top dose - in vitro mammalian cell assays standard top dose is lower of 1 mm or 0.5 mg/ml consider cytotoxicity, too much will confound interpretation in vitro chromosomal aberrations and micronucleus: ~ 50% cytotoxicity (e.g., reduction in cell growth) mouse lymphoma assay: 80-90% cytotoxicity (e.g., reduction in relative total growth) 42
Assays Other Considerations top dose - in vivo assays short term study (1 to 3 doses) 2000 mg/kg maximum tolerated dose (MTD) based on expected lethality note need to demonstrate adequate exposure through toxicity or toxicokinetic data 43
Assays Other Considerations top dose - in vivo assays multiple-dose study 2000 mg/kg up to 14 days, 1000 mg/kg for >14 days maximum feasible dose (MFD) formulation issue MTD based on expected lethality systemic exposure saturation toxic effects on blood and bone marrow are critical considerations for micronucleus assay dose selection criteria is different if integrated into repeat-dose general toxicology study 44
How good are the assays at detecting various effects? Dearfield K.L., et al. (2011) Environ. Mol. Mut. 52:177-204 45
Assays How good are these assays at predicting rodent carcinogenicity? Genetic toxicology assays are designed to identify carcinogens acting via direct genetic damage. However, many rodent carcinogens act via other mechanisms (e.g., enzyme induction, hormonal effects, immunosuppressive effects, etc.). 46
Genetic Toxicology: Drug Development and Regulations 47
Drug Development and Regulations Drug Development involves a series of risk:benefit analyses besides empirical data there are other important considerations: clinical indication: life-threatening vs. less serious duration of treatment: acute vs. chronic patient population: healthy volunteers vs. patients, adults vs. pediatric other available drugs 48
Drug Development and Regulations How is genetic toxicology data used to support drug development? screening screening assays help identify and eliminate genotoxic molecules during candidate selection may also help guide assay selection for GLP battery mechanistic understanding experiments conducted to further explain results from primary assays typically use second tier assays 49
Drug Development and Regulations How is genetic toxicology data used to support drug development? hazard identification major focus of genetic toxicology in drug development fulfill regulatory requirements data used as part of risk:benefit analysis determine mutagenic/carcinogenic potential mutagenicity is integral to carcinogenicity in most cases rodent carcinogenicity data will eventually be available due to duration and cost, carcinogenicity assays are not performed until much later in development 50
Drug Development and Regulations Testing Paradigm genetic toxicology testing to support drug development employs a battery approach designed to detect genotoxicity arising through multiple mechanisms (e.g., fishing expedition) gene mutations mutagenicity chromosomal damage (in vitro and/or in vivo) clastogenicity aneugenicity 51
Drug Development and Regulations Testing Paradigm predictive of rodent carcinogenicity correlation between genotoxic agents and rodent carcinogens is not perfect rodent carcinogens sometimes work through non-genotoxic mechanisms step-wise approach in regards to required timing serves as a place-holder until rodent carcinogenicity is available more data required as development proceeds 52
Drug Development and Regulations ICH S2(R1) Battery Option 1 i. A test for gene mutation in bacteria; ii. A cytogenetic test for chromosomal damage (the in vitro metaphase chromosome aberration test or in vitro micronucleus test), or an in vitro mouse lymphoma tk gene mutation assay; iii. An in vivo test for genotoxicity, generally a test for chromosomal damage using rodent hematopoietic cells, either for micronuclei or for chromosomal aberrations in metaphase cells. 53
Drug Development and Regulations ICH S2(R1) Battery Option 2 i. A test for gene mutation in bacteria; ii. An in vivo assessment of genotoxicity with two tissues, usually an assay for micronuclei using rodent hematopoietic cells and a second in vivo assay. 54
Drug Development and Regulations Option 1 vs 2 Option 1 similar to previous battery substantial regulatory experience preferred when Ames may not be appropriate (e.g., for antibacterials) nonclinical systemic exposure clinical 55
Drug Development and Regulations Option 1 vs 2 Option 2 currently there is little experience as a standalone battery similar strategy historically used to follow-up to positive in vitro mammalian cell results preferred when short-lived reactive metabolites are expected to be formed in liver 56
Drug Development and Regulations Key Regulatory Drivers ICH M3(R2) Guidance on Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals (2009) provides recommendations for timing of genetic toxicology testing testing to support exploratory clinical trials 57
Drug Development and Regulations Additional Regulatory Guidance/Guidelines FDA Recommended Approaches to Integration of Genetic Toxicology Study Results provides recommendations for: dealing with positive results by considering weight of evidence, mechanism of action, and follow-up testing limiting risk in clinical trials 58
Drug Development and Regulations Other Resources FDA Gid Guidance for Id Industry and doh Other Stakeholders, khld Toxicological Principles for the Safety Assessment of Food Ingredients (aka Redbook 2000) OECD Guidelines guidelines describe approved protocols for the assays Other publications, etc. 59
Genetic Toxicology: Results and Decision Making 60
Results and Decision Making Positive Results Does a positive genetox finding mean the end of development? not necessarily depends on many factors severity of the clinical indication healthy subjects vs. patient population single vs. multiple-dose clinical trial others 61
Results and Decision Making Positive Results many marketed drugs are genotoxic Snyder R.D. (2009) Environ. Mol. Mutagen. 50:435-450 62
Results and Decision Making Positive Results Step 1. Evaluate weight-of-evidence evidence What assay(s) was positive? a weak positive (e.g., increase at high dose slightly outside historical control range) in an in vitro mammalian cell assay vs. Ames Was there a positive result in a single assay or multiple assays? multiple assays more of a concern 63
Results and Decision Making Positive Results Step 1. Evaluate weight-of-evidence evidence Is the positive biologically relevant? Ames positive due to bacterial specific metabolite, impurity, etc. in vitro chromosomal aberrations positive due to non-physiologic py conditions (e.g., g,p ph, etc.) or extreme cytotoxicity in vivo micronuclei due to effects on erythropoiesis or body temperature 64
Results and Decision Making Positive Results Step 2. Evaluate mechanism of action Can you identify a mechanism? may help with Step 3 Does the mechanism have a threshold? nucleotide imbalance, damage to spindle proteins, inhibition of DNA synthesis, inhibition of topoisomerase, etc. if yes, it may be possible to determine an appropriate margin of safety based on comparison of non-clinical and clinical systemic exposure 65
Results and Decision Making Positive Results Step 3. Experimental follow-up Positive results may lead to additional studies but notice this is not the 1 st step! You must attempt to understand biological relevance and mechanism before trying to determine best route for defining genotoxic risk. ICH S2(R1) provides some recommendations but exact remedies are not described appropriate follow-up will be based on best science 66
Experimental Follow-Up Dearfield K.L., et al. (2011) Environ. Mol. Mut. 52:177-204 67
Results and Decision Making Positive Results impact on clinical trials Ames positive rarely remain in development pipeline probably OK for single dose trial in healthy volunteers and patients questionable for multiple dose trial in healthy volunteers possibly OK in multiple dose trials for certain patient populations, duration is also important consider all factors in risk:benefit analysis 68
Results and Decision Making Positive Results impact on clinical trials in vitro chromosomal aberration positive more common finding probably OK for single dose and multiple dose trials in healthy volunteers and patients, duration is important consider all factors in risk:benefit analysis 69
Results and Decision Making Positive Results impact on clinical trials in vivo micronucleus assay positive unlikely to remain in development pipeline consider all factors in risk:benefit analysis 70
Genetic Toxicology: Drug Impurities 71
Drug Impurities Why are we concerned with impurities? there will be impurities in drugs regardless of control strategy starting materials reactants synthesis intermediates degradants potential exists for genotoxicity in contrast to the API, impurities offer no direct benefit to the patient 72
Drug Impurities Are we too concerned with genotoxic impurities? iti in some cases, the answer is yes lifetime e risk of developing cancer ce in the US is ~1 in 2 for men and ~1 in 3 for women* we are all exposed to genotoxic and carcinogenic agents (e.g., remember coffee?) Note: It is important to consider how much additional risk is posed by small amounts of genotoxic impurities in drugs. *American Cancer Society (2014) Cancer Facts and Figures 73
Drug Impurities Regulatory Guidelines ICH Q3A(R2) Impurities in New Drug Substances Q3B(R2) Impurities in New Drug Products Q3C(R5) Impurities: Guideline for Residual Solvents address genotoxicity to a limited extent provides recommendations to support marketing 74
Drug Impurities primary driver for regulatory decision making regarding mutagenic impurities applicable to both clinical development and marketing provides recommendations relevant for both safety and chemistry review safety focus: structural alerts for mutagenicity experimental data setting limits 75
Drug Impurities Structural t Alerts for Mutagenicity it structural features (e.g., functional group) known or suspected to be associated with mutagenicity premise: similar chemistry similar biologic response evaluated using (quantitative) structural activity relationship [(Q)SAR] assessment 2 complementary methodologies needed in most cases, impurities lacking a structural alert will not require further qualification impurities possessing a structural alert will require experimental data to refute the positive (Q)SAR prediction or control to appropriate level 76
Drug Impurities Experimental Data primary concern is Ames mutagenicity ICH M7 recommends an OECD compliant Ames assay; however, other formats may be justified in certain cases (e.g., limited test article available, etc.) other genotoxicity it assays can be used characterize in vivo risk associated with Ames positive impurities (next slide) impurities that are negative in Ames assay are treated as routine impurities per ICH Q3A/B 77
Experimental Data 78
Drug Impurities Mutagenic Impurity Levels control mutagenic or known mutagenic impurities to levels resulting in minimal cancer risk threshold h of ftoxicological i l concern (TTC)derived d from measure of carcinogenic potency when rodent carcinogenicity data is not available default TTC = 1.5 µg/day for chronic exposure chemical specific TTC may be calculated when rodent carcinogenicity i i data is available OK to adjust for expect less than lifetime exposure TTC should not be considered fine line between acceptable and unacceptable 79
Impurity Limits 80
Genetic Toxicology: FDA Case Studies 81
FDA Case Studies Case Study #1 background API being developed for treatment of serious disease expected clinical i l treatment t t of 2 weeks, multiple l lifetime exposures possible genetic toxicology battery results Ames negative in vitro chromosomal aberration negative in vivo rodent micronucleus (acute) negative 82
FDA Case Studies Case Study #1 conclusions API is not genotoxic no additional testing needed proceed with clinical trial This is the most common result of genetic toxicology testing! 83
FDA Case Studies Case Study #2 background API being developed for serious indication expected clinical treatment of ~ 3 months, 1x/lifetime genetic toxicology battery results Ames negative experimental conditions for metabolic activation were not per OECD protocol, study only partially valid Agency performed (Q)SAR to eliminate obvious mutagenic concerns negative in vitro CA negative in vivo rodent micronucleus (acute) negative 84
FDA Case Studies Case Study #2 conclusions while not fully established, genotoxic risk appears to be minimal proceed with initial clinical trial (single dose in healthy volunteers + 3 days of dosing in patients) Sponsor asked to repeat Ames assay with appropriate conditions prior to initiating additional clinical trials 85
FDA Case Studies Case Study #3 background API being developed for non-life threatening indication expected clinical treatment of 2 weeks, multiple lifetime exposures possible genetic toxicology battery results Ames negative in vitro chromosomal aberrations negative in vivo rodent micronucleus (acute) positive possible clastogen or aneugen 86
FDA Case Studies Case Study #3 additional genetic toxicology testing in vivo chromosomal aberrations negative unlikely to be a clastogen in vivo rodent micronucleus w/ CREST staining micronuclei were primarily CREST positive indicating aneugenic mechanism in vivo comet liver and duodenum selected for analysis because target organs of toxicity data interpretation confounded by apoptosis 87
FDA Case Studies Case Study #3 conclusions totality of information suggested API was an aneugen sufficient margins of safety were identified proceed with clinical trials 88
FDA Case Studies Case Study #4 background API being developed for non-life threatening e indication expected chronic clinical treatment genetic toxicology battery results Ames negative in vitro chromosomal aberration positive possible clastogen in vivo rodent micronucleus (acute) positive possible clastogen or aneugen effect at high dose only 89
FDA Case Studies Case Study #4 additional genetic toxicology testing mechanistic data high concentrations of drug causes nucleotide imbalance in vitro high doses of drug cause body temperature in rat in vivo UDS negative unlikely to be DNA reactive in vivo rodent micronucleus (repeat dose) negative unlikely to be clastogen or aneugen 90
FDA Case Studies Case Study #4 conclusions mechanistic data suggest positive in vitro results were not due to direct DNA damage (i.e., nucleotide imbalance) and in vivo micronuclei formation was the result of a non-genotoxic effect sufficient margins of safety were identified proceed with clinical trials 91
FDA Case Studies Case Study #5 background API being developed for non-life threatening indication expected chronic clinical treatment genetic toxicology battery results Ames negative in vitro chromosomal aberration positive possible clastogen in vivo rodent micronucleus (acute) negative 92
FDA Case Studies Case Study #5 additional genetic toxicology testing in vivo comet assay negative liver and jejunum selected for analysis because target organs of toxicity unlikely to be DNA reactive 93
FDA Case Studies Case Study #5 Conclusions negative result in 2 nd in vivo assay (i.e., comet) suggests drug has minimal genotoxic potential proceed with clinical trials 94
Genetic Toxicology: The Future 95
The Future Improvements in Regulatory Decision Making experience with S2(R1) is helping refine and clarify regulatory expectations ICH M7 has been finalized but implementation will be gradual; expect improvements in application as experience is gained by regulators and Sponsors Quantitative vs. Qualitative Use of Genetic Toxicology application of thresholds for both indirect and direct acting genotoxicants Toxicogenomics identify gene signatures associated with DNA reactive carcinogens vs. non-genotoxic carcinogens vs. non-carcinogens 96
The Future New Assays high-throughput screening variations of standard genetox assays currently screening tools used for internal decision making (e.g., lead optimization) cytotoxicity in cell lines with variety of mutated DNA repair pathways (e.g., Tox21) compare wild-type with mutated cells to action Others? identify effects and establish mechanism of 97
Additional Information 98
References Primary Regulatory Recommendations ICH S2(R1) Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use FDA Guideline for Industry and Review Staff Recommended d Approaches to the Integration ti of Genetic Toxicology Study Results ICH M3(R2) Guidance on Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals 99
References Impurities ICH M7 - Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk ICH Q3A(R2) Impurities in New Drug Substances ICH Q3B(R2) Impurities in New Drug Products 100
References Methods OECD Guidelines for the Testing of Chemicals, Section 4 Health Effects FDA Guideline for Industry and Other Stakeholders: Toxicological Principles for the Safety Assessment of Food Ingredients (Redbook 2000) 101
Websites ICH US FDA http://www.ich.org/products/guidelines.html p g http://www.fda.gov/drugs/guidancecomplianceregulatoryinformati on/guidances/ucm065014.htm OECD http://www.oecd-ilibrary.org/environment/oecdguidelines-for-the-testing-of-chemicals-section-4-healtheffects_20745788 102
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