ICH M7 Guidance and Tests to Investigate the In Vivo Relevance of In Vitro Mutagens Leon F. Stankowski, Jr., PhD Principal Scientist/Program Consultant PNWBIO 04 June 2014
Agenda ICH M7 guidance Assay overview Micronucleus Chromosome aberration Comet assay Transgenic rodent gene mutation assay Pig-a gene mutation assay Sample data from integrated studies 2
Guidance ICH M7: Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk Current Step 3 version (February 2013) Step 4 target t date June 2014 3
Scope new drug substances and new drug products IND or NDA existing marketed products changes in synthesis result in new impurities or increased acceptance criteria for existing impurities; changes in formulation, composition or manufacturing process result in new degradants or increased acceptance criteria for existing degradants; changes in indication or dosage that significantly affect the acceptable cancer risk level 4
Scope Exempted biological/biotechnological, peptide, oligonucleotide, radiopharmaceutical, fermentation products, herbal products, and crude products of animal or plant origin UNLESS, products such as biologicals and peptides are chemically synthesized or modified (e.g., addition of organic chemical linkers, semi-synthetic products) drug substances and drug products intended for advanced cancer (see ICH S9) drug substance known to be genotoxic at therapeutic concentrations and may be associated with an increased cancer risk 5
Impurities Two-stage process actual, observed impurities, especially when levels exceed identification thresholds (see ICH Q3A) potential impurities likely to be present in the final drug substance starting materials, reagents and intermediates identified impurities in starting materials and intermediates reasonably expected reaction by-products 6
Degradants Two-stage process actual, identified degradants (in drug substance and drug product), especially when levels exceed identification thresholds (see ICH Q3A/Q3B) potential degradants (in drug substance and drug product) likely to be present in the final drug substance starting materials, reagents and intermediates identified impurities in starting materials and intermediates reasonably expected reaction by-products 7
Classification Table 1: Impurities Classification with Respect to Mutagenic and Carcinogenic Potential and Resulting Control Actions Class Definition Proposed Controls 1 Known mutagenic carcinogens Control < compound-specific acceptable limit 2 Known mutagens with unknown carcinogenic potential (Ames or other positive, no carc data) 3 Alerting structure unrelated to drug substance; no mutagenicity data Control <TTC Control <TTC or perform Ames (negative Class 5, positive Class 2 4 Alerting structure same as in drug substance bt which hihis non-mutagenic 5 No structural alerts, or alerts with sufficient data to demonstrate lack of mutagenicity Treat as non-mutagenic impurity Treat as non-mutagenic impurity 8
TTC Table 2: Acceptable intakes for an individual impurity Treatment duration <1 month 1-12 months >1-10 years >10 years Daily intake [µg/day] 120 20 10 1.5 Table 3: Acceptable intakes for total impurities Treatment duration <1 month 1-1212 months >1-10 10 years >10 years Daily intake [µg/day] 120 60 10 (30) 5 For clinical development up to 3 years. Similar principles could be applied to marketed products with justification. Higher or lower limits may be justified (case-by-case) 9
So you ve got a positive... Further hazard assessment of a Class 2 impurity (Ames or other in vitro positive) may be appropriate levels of the impurity cannot be controlled at an appropriate limit test the impurity in an in vivo gene mutation assay. Other in vivo genotoxicity assays should be justified based on mechanism of action of the impurity and site of contact (Note 3) 10
ICH M7 Note 3 Tests to investigate the in vivo relevance of in vitro mutagens (positive bacterial mutagenicity) In vivo test Transgenic mutation assays Mechanistic data to justify choice of test any Ames (+); justify tissue/organ Pig-a assay (blood) direct-acting Ames (+) Micronucleus test (blood/bm) direct-acting Ames (+) and clastogens Rat liver UDS test Ames (+) with S9 only; responsible liver metabolite generated in the test species; induces bulky adducts Comet assay chemical class specific; form alkaline labile sites or ss DNA breaks; justify tissue/organ Others with convincing justification 11
ICH M7 Note 3 Tests to investigate the in vivo relevance of in vitro mutagens (positive bacterial mutagenicity) In vivo test Transgenic mutation assays Mechanistic data to justify choice of test any Ames (+); justify tissue/organ Pig-a assay (blood) direct-acting Ames (+) Micronucleus test (blood/bm) direct-acting Ames (+) and clastogens Rat liver UDS test Ames (+) with S9 only; responsible liver metabolite generated in the test species; induces bulky adducts Comet assay chemical class specific; form alkaline labile sites or ss DNA breaks; justify tissue/organ Others with convincing justification 12
Historical Perspectives and Background Micronucleus Literally, small nucleus Historically i developed d and performed in rodent bone marrow Polychromatic Erythrocyte (PCE) Normochromatic Erythrocyte (NCE) Normal Maturation/expulsion of nucleus Normal NCE Stem Cell (erythroblast) Chromosomal Damage Micronuceated NCE 13
Historical Perspectives and Background Micronucleus Also allows determination of mechanism of action i.e. Clastogens i.e. Aneugens 14
Mechanism of Action: FISH and CREST CREST antikinetochore staining - antibodies FISH (fluorescent in situ hybridisation) 15
Mechanism of Action: FISH Uses fluorescent probes to detect centromeric DNA MN MN Negative Positive 16
Mechanism of Action: FISH Pancentromeric DNA probes for human and mouse 17
Mechanism of Action: CREST CREST - immunological detection of kinetochores (all species) 18
Historical Perspectives and Background Chrom Abs Structural or numerical changes in karyotype Similar il treatment/harvest t t/h t of bone marrow/peripheral blood, but score individual cells at metaphase Polyploidy Endoreduplication 19
Historical Perspectives and Background Comet Single Cell Gel Electrophoresis Assay Detects t ss/ds DNA breaks and alkali-labile li l sites Skin Bladder Spleen Kidney Brain Intestine Lung Bone Marrow Peripheral Blood Lymphocytes Liver Stomach 20
Historical Perspectives and Background Comet Level of DNA damage is correlated to length and amount of fragmented DNA migrating out of the nucleus (comet tail) 21
Historical Perspectives and Background TRM Big Blue models available in mice and rats Measure gene mutation ti in ANY tissue in the body Utilizes transgenic rodents that carry multiple copies of the viral vector in EVERY cell Measure temperature sensitive mutations in lambda cii gene 22
Historical Perspectives and Background TRM Dose animals Usually 28 days Collect/freeze tissues Day 31 and/or Day 77 Extract high MW DNA Excise shuttle vector Package into capsid (empty phage envelope) Infect E. coli Plate for plaque formation 24ºC selective for mutants 37ºC all plaques form (denominator) Count plaques and evaluate 23 Lambert et al. Mut. Res. 2005
Historical Perspectives and Background Pig-a Phosphatidylinositol N-acetylglucosaminyltransferase, subunit A X-linked gene involved in first step of producing anchor proteins for surface markers/antigens (e.g., CD59, CD24) 24 GPI-anchor EA P Man GLcN I P Protein I P EA P GLcN Man Man P EA CO EA P Man NH 2 Membrane : Phosphoethanolamine : Mannose :Glucosamine : Inositolphosphate DPM1 DPM2 Dol-P-Man syntase Phosphatidylinositol Acyl-CoA +NAc- Gln Dolicolphospatemannose Phospho- ethanolamine Protein UDP-N-acetylglucosamine (deacetylation) +mannose +ethanolamine Transamidase GPI-anchored Protein PIG-A PIG-C PIG-H GPI1 PIG-L PIG-B PIG-F GPI8 GAA1
Historical Perspectives and Background Pig-a Wild-type Cell fluorescent labeled antibodies against GPI-anchored proteins Pig-a Mutant Cell GPI Pig-a FCM analysis FCM analysis CD59 Fluorescent positive Fluorescent negative Genotoxin 25
Historical Perspectives and Background Pig-a A: Instrument Calibration Standard B: ENU Treated Rat, PRE Column C: ENU Treated Rat, POST Column Nucleic Acid Dye Fluorescen nce Anti CD59 PE Fluorescence ENU 40 mg/kg/day, g 3X UL = mutant phenotype RETs UR = wt RETs LL = mutant phenotype NCEs LR = wt NCEs 26
Sample Data: Integrated Studies Various genotoxicity endpoints can be integrated into classical in vivo subchronic toxicology studies micronucleus assay in peripheral blood or bone marrow chromosome aberration assay in peripheral blood or bone marrow Comet assay in peripheral blood or virtually any other tissue Pig-a gene mutation in peripheral blood 27
Sample Data: Stage III Pig-a 4-NQO 28-day subchronic study with 4NQO (Stankowski, et al., 2011) Dosing occurs on Days 1 28 (plus Day 29 since tissue Comet is included) Day: 11 1 2 3 4 15 29 42 28 n = 5 per group Vehicle Control 5 dose groups Positive Control Pig a Pig a Pig a & PBL Comet, PBL Comet, CAb and flow MN & PBL Comet, CAb and flow MN CAb and flow MN & Tissue Comet - Save additional Comet tissue samples for possible histological evaluation if warranted - No DSA, No TK
Sample Data: Stage III Pig-a 4-NQO 4NQO @ 0, 1.25, 2.50, 3.75, 5.00 and 7.50 mg/kg/day, 29X 400 350 300 Vehicle Control - Satellite Vehicle Control 1.25 mg/kg/day 2.50 mg/kg/day 3.75 mg/kg/day 5.00 mg/kg/day g 7.50 mg/kg/day (g) 250 200 150 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Day of Study 29
Sample Data: Stage III Pig-a 4-NQO 4NQO @ 0, 1.25, 2.50, 3.75, 5.00 and 7.50 mg/kg/day, 29X CD59 NEG RETs (x10-6 +SEM) 50 p < 0.05 40 30 20 95% UCL 10 0 - - - Day - -1- - - - - - - Day - 15 - - - - - - - Day - 29 - - - Dose mg/kg/day 0.00 1.25 2.50 3.75 5.00 7.50 30
Sample Data: Stage III Pig-a 4-NQO 4NQO @ 0, 1.25, 2.50, 3.75, 5.00 and 7.50 mg/kg/day, 29X CD59 NEG RBCs (x 10-6 +SEM) 50 40 30 20 10 p < 0.05 95% UCL 8 6 4 2 0 rage No. of RETs (% +/- SEM) 0 - - - Day - -1- - - - - - - Day - 15 - - - - - - - Day - 29- - - -2 Ave Dose (mg/kg/day) 0.00 1.25 2.50 3.75 5.00 7.50 31
Sample Data: Stage III Pig-a 4-NQO 4NQO @ 0, 1.25, 2.50, 3.75, 5.00 and 7.50 mg/kg/day, 29X mn nrets (% +SD) 0.35 0.28 0.21 p = 0.094 p = 0.079 0.14 0.07 p<005 0.05 4.25 2.5 0.75-1 -2.75 Av verage No. of RETs (% +/ /- SD) 0 - - - Day - 4- - - - - Day - 15 - - - - - Day - 29 - - - -4.5 Dose (mg/kg/day) 0.00 1.25 2.50 3.75 5.00 7.50 %RETs 32
Sample Data: Stage III Pig-a 4-NQO 4NQO @ 0, 1.25, 2.50, 3.75, 5.00 and 7.50 mg/kg/day, 29X CAbs w/ /o Gaps (% +SEM) 33 7.5 6.0 4.5 30 3.0 1.5 0.0 p < 0.05 95% UCL CAb - - - - Day 4 - - - - - Day - 15 - - - - - Day - 29 - - - Dose (mg/kg/day) 0.00 1.25 2.50 3.75 5.00 7.50 3.0 1.5 0.0-1.5 15-3.0-4.5 SEM) Avera age Mitotic Index (% +/-
Sample Data: Stage III Pig-a 4-NQO 4NQO @ 0, 1.25, 2.50, 3.75, 5.00 and 7.50 mg/kg/day, 29X 3 5 Tail Intensity (Median % + SD) 2.4 1.8 1.2 0.6 95% UCL PBL 0-5 -10-15 Ave erage No. of Clouds (% +/- SD) 0 - - - Day - 4- - - - - - - Day - 15 - - - - - - - Day - 29- - - Dose (mg/kg/day) /d -20 0.00 1.25 2.50 3.75 5.00 7.50 % Clouds 34
Sample Data: Stage III Pig-a 4-NQO 4NQO @ 0, 1.25, 2.50, 3.75, 5.00 and 7.50 mg/kg/day, 29X 7.5 9 (Median % +SD) Tail Intensity 6 4.5 3 1.5 0 Mean VC - Stomach 95% UCL - Liver -Day-15 Liver - - - - Day - 29 - Liver - - - - - Day - 29 Stomach - - - - 2-5 -12-19 -26 +/-SD) Average e Number of Clouds (% Dose (mg/kg/day) 0.00 1.25 2.50 3.75 5.00 7.50 35
Sample Data: Stage IV Pig-a Urethane Vehicle control Urethane @ 25, 100 and 250 mg/kg/day, 29X Ethyl methanesulfonate (EMS) at 200 mg/kg/day Days 3, 4, 13, 14, 15, 27, 28 and 29 n = 10 (treated) 6/group analyzed for gene tox endpoints 10/group analyzed for classical tox endpoints and necropsy males only GLP-compliant 36
Sample Data: Stage IV Pig-a Urethane Pig-a High Throughput Protocol mnret (flow) PBL Comet and chromosome aberration Comet in various other tissues mnpce (manual) ~100 300 µl samples pre-dose Day 4 Day 15 Day 29 clinical chemistry 37
Urethane Stage IV: Endpoints Pig-a High Throughput Protocol mnret (flow) PBL Comet and chromosome aberration Comet in various other tissues mnpce (manual) ~100 300 µl samples pre-dose Day 4 Day 15 Day 29 clinical chemistry 38
Pig-a Scoring: High Throughput Method Wild type (WT) cells labeled with anti CD59 PE Add Anti PE particles and Counting Beads Cells plus anti PE paramagnetic particles & Counting Beads Key Anti CD59 PE positive erythrocytes (WT) Anti CD59 PE negative erythrocytes (mutant) Counting Beads Anti PE paramagnetic particles Majority towards magnetic column separation Small fraction towards nucleic acid dye staining & analyze for: %RET RBC:Bead ratio RET:Bead ratio Eluate highly depleted of wild type cells Spin step Concentrated eluate Nucleic acid dye staining Analyze for: Mutant RBC:Bead ratio Mutant RET:Bead ratio 39
40 Pig-a Scoring: High Throughput Method
Pig-a Scoring: High Throughput Method A: Instrument Calibration Standard B: ENU Treated Rat, PRE Column C: ENU Treated Rat, POST Column Nucleic Acid Dye Fluorescen nce Anti CD59 PE Fluorescence ENU 40 mg/kg/day, g 3X UL = mutant phenotype RETs UR = wt RETs LL = mutant phenotype NCEs LR = wt NCEs 41
Pig-a Scoring: High Throughput Method Old Basic Protocol 18 23 minutes/sample ~0.3 x 10 6 RETs 1 x 10 6 RBCs New High Throughput Protocol 6 7 minutes/sample 3 7 x 10 6 RET equivalents 100 x 10 6 RBC equivalents Fewer zeros for mutant RETs No zeros for mutant RBCs Significantly greater power to detect small increases Negative results are negative 42
Sample Data: Stage IV Pig-a Urethane Dosing occurs on Days 1 28 (plus Day 29 as tissue Comet is included) Day: 1 1 2 3 4 15 29 42 n = 6 per group Vehicle Control 3 dose groups Positive Control Pig a PBL Comet, CAb and flow MN Pig a & PBL Comet, CAb and flow MN - Save additional Comet tissue samples for possible histological evaluation if warranted - No DSA, No TK Pig a & PBL Comet, CAb and flow MN; Tissue Comet and BM MN 43
Sample Data: Stage IV Pig-a Urethane 35 30 0-6 RE ET CD59- x 10 25 20 15 10 5 0 Day -3/-5 Day 15 Day 29 000 0.00 25.0 100 250 PC (mg/kg/day) /d 44
Sample Data: Stage IV Pig-a Urethane 35 30 25 0-6 RB BC CD59- x 10 20 15 10 5 0 Day -3/-5 Day 15 Day 29 000 0.00 25.0 100 250 PC (mg/kg/day) /d 45
Sample Data: Stage IV Pig-a Urethane 10.0 9.0 8.0 7.0 %RETs 6.0 5.0 40 4.0 3.0 2.0 1.0 0.0 Day -3/-5 Day 15 Day 29 0.00 25.0 100 250 PC (mg/kg/day) 46
Sample Data: Stage IV Pig-a Urethane 1.2 1.0 0.8 mnret (%) 0.6 0.4 0.2 0.0 Day 4 Day 15 Day 29 0.00 25.0 100 250 PC (mg/kg/day) 47
Sample Data: Stage IV Pig-a Urethane 0.8 0.6 mnpce (%) 0.4 0.2 0.0 Day 29 0.00 25.0 100 250 PC (mg/kg/day) 48
Sample Data: Stage IV Pig-a Urethane 30.0 25.0 Tail DNA (%) - PBL 20.0 15.0 10.0 5.0 E 0.0 Day 4 Day 15 Day 29 0.00 25.0 100 250 PC (mg/kg/day) 49
Sample Data: Stage IV Pig-a Urethane 50.0 40.0 Ta ail DNA (%) 30.0 20.00 10.0 E E 0.0 Liver Brain Spleen Kidney Lung 0.00 25.0 100 250 PC (mg/kg/day) 50
Sample Data: Stage IV Pig-a Urethane 51 Fold-Increase Endpoint Day 4 Day 15 Day 29 RET CD59- nd 74 24 RBC CD59- nd 4.2 8.1 mnret 66 6.6 40 4.0 53 5.3 PBL CAb - - - mnpce nd nd 5.0 Comet - PBL - E - - Liver nd nd E - Brain nd nd - - Spleen nd nd E - Kidney nd nd - - Lung nd nd - nd = not determined - = negative response E = equivocal response ( 2.0-fold increase)
Sample Data: Big Blue TRM - B(a)P and ENU 28-day subchronic study with B(a)P Dosing occurs on Days 1 28 Day: 1 2 3 4 28 31 311 n = 5 6 per group Vehicle Control B(a)P @ 50 mg/kg/day x 28 ENU @ 40 mg/kg/day x 3 Pig a & flow MN (PBL) & cii mutation (liver and bone marrow) 52
Sample Data: Big Blue ENU/B(a)P Treatment (mg/kg/day) x days Mut Freq (x10-6 ) Pig-a Mut Freq (x10-6 ) Liver cii BM cii RBC RET mnnce mnret (%) (%) Olive Oil (0.0) x 28 43.6±6.20 38.1 ±14.2 0.139±0.0419 0.106±0.131 0.327±0.0160 0.565±0.0552 BaP (50.0) x 28 213±36.3 674±75.1 14.0±5.00 65.3±16.6 0.691±0.0612 0.881±0.0725 Fold Increase 4.89 17.7 101 616 2.11 1.56 ENU(40.0) x 3 181±37.2 405±90.0 28.9±6.15 111±53.5 0.457±0.237 0.674±0.120 Fold Increase 4.15 10.6 208 1050 1.40 1.19 Significant increase (p< 0.001) 53
54 Back-up Slides
55 Sample Data - Stage III Pig-a: 4-NQO Dose Rate
56 Sample Data - Stage III Pig-a: 4-NQO Dose Rate
57 Sample Data - Stage III Pig-a: 4-NQO Dose Rate
58 Sample Data - Stage III Pig-a: 4-NQO Dose Rate
59 Sample Data - Stage III Pig-a: 4-NQO Dose Rate
Sample Data - Stage III Pig-a: 4-NQO Dose Rate Single/3X acute dose regimen Pig-a mutant t RBCs/RETs larger increases appear sooner and persist mnrets much larger increases on Day 3 PBL/Liver Comet positive 3 hours after treatment; does not persist 3X appeared to give larger responses than 1X exposure?? 60
Thank You Questions? leon.stankowski@bioreliance.com 61