Ames Data Submissions and Other Qualification Data for Impurities in Drug Substances

Transcription

1 Ames Data Submissions and Other Qualification Data for Impurities in Drug Substances Mark W. Powley, Ph.D. Pharmacologist FDA/CDER/Office of New Drugs

2 Outline Regulatory Background Ames Assay ICH M7 Submission of Qualification Data Case Studies Summary Disclaimer: This presentation represents the views of the speaker and not, necessarily, of the FDA. 2

3 Regulatory Background 3

4 Regulatory Background Non-Clinical Safety Assessment safety assessments conducted in support of drug development must be scientifically sound however, certain aspects of these assessments can differ for API vs. impurities assays or combinations of assays adherence to standard testing guidelines test article characteristics 4

5 Regulatory Background Regulatory Guidelines fortunately, there are numerous sources of regulatory recommendations covering impurity safety although FDA guidance documents exist, ICH guidelines take precedence Guideline Impurities Covered possibly involve testing not likely to involve testing ICH M7 ICH Q3A(R2) ICH Q3B(R2) ICH Q3C(R5) ICH Q3D low level mutagenic impurities impurities in drug substance impurities in drug product residual solvents elemental impurities 5

6 Regulatory Background Regulatory Guidelines guidelines more likely to involve testing ICH M7 (Q)SAR Ames Assay In Vivo Genotoxicity Studies Carcinogenicity Studies ICH Q3A/B Ames Assay In Vitro Mammalian Cell Assays General Toxicology Studies Other Studies Today s talk will focus on the Ames assay; however, other topics will be briefly covered. 6

7 Regulatory Background Regulatory Decision Making in addition to results of the safety assessment, additional factors are considered in the risk:benefit analysis severity of the clinical indication patient population duration of use availability of other drugs other 7

8 Regulatory Background Regulatory Decision Making specific considerations for impurities impurities in drugs intended for advanced cancer indications (i.e., serious and life threatening malignancies) are handled differently mutagenic impurities are less of a concern for an Ames positive API note: this is not necessarily true for an API that is positive in another genotoxicity assay (e.g., in vitro chromosomal aberration assay) 8

9 Regulatory Background Regulatory Decision Making specific considerations for impurities degree of known or potential exposure exposure is recommended limit exposure slightly > recommended limit less scrutiny of safety assessment exposure marginally > recommended limit exposure significantly > recommended limit or exposure is unknown more scrutiny of safety assessment 9

10 Ames Assay 10

11 Ames Assay Goal assess capacity to induce gene mutations 11 Mortelmans K., Zeiger E. (2000) Mut. Res. 455:29-60

12 Ames Assay Principle assay relies on Salmonella and/or E. coli strains with different combinations of genetic modifications his or trp mutation: ability to synthesize histidine (Salmonella) or tryptophan (E. coli) rfa mutation: defective lipopolysaccharide layer uvra or uvrb mutation: nucleotide excision repair pkm101 plasmid: error-prone DNA repair modifications maximize the likelihood that test article will reach target and that genetic damage will be 12 expressed as a mutation

13 Ames Assay Bacterial Tester Strain Characteristics Mutations Bacterial Tester Strain DNA Target Reversion Event his or trp Permeability Repair Plasmid Salmonella TA97 TA97a TA98 TA100 TA102 TA1535 TA1537 G:C G:C G:C G:C A:T G:C G:C frameshift frameshift frameshift base-pair substitution base-pair substitution + crosslinking base-pair substitution frameshift hisd6610 hisd6610 hisg46 hisg46 hisg428 hisg46 hisc3076 rfa rfa rfa rfa rfa rfa rfa uvrb uvrb uvrb uvrb - uvrb uvrb pkm101 pkm101 pkm101 pkm101 pkm101, paq1 - - E. Coli WP2 uvra WP2 uvra (pkm101) A:T A:T frameshift frameshift trpe trpe - - uvra uvra - pkm101 13

14 Ames Assay Critical Aspects per OECD Test Guideline 471 and/or ICH S2(R1) combination of bacterial strains dose range metabolic activation controls data interpretation GLP vs. non-glp 14

15 Ames Assay Combination of Bacterial Strains OECD Test Guideline 471, ICH S2(R1), and ICH M7 recommend a 5-strain Ames assay as follows: phenotype should be confirmed 15

16 Dose Range Ames Assay doses should be spaced at half log intervals when establishing top dose, consider 1. toxicity (e.g., thinning of background lawn or reduction in background revertants) 2. solubility 3. limit dose for non-toxic, soluble test articles is 5000 µg/plate note: experiments typically conducted with triplicate plating 16

17 Ames Assay Metabolic Activation assays are conducted with and without metabolic activation routinely includes liver S9 from rats treated with known enzyme inducer to maximize metabolic activity allows evaluation of mutagenic potential associated with both parent compound and metabolite(s) CYP450 + O 17

18 Ames Assay Controls positive controls standardized for the various strains and metabolic conditions assure that assay is working as expected negative controls solvent/vehicle assure that assay is working as expected help with interpreting assay results 18

19 Ames Assay Data Interpretation 5 non-toxic doses should be analyzed control values should be consistent with historical ranges positive response typically requires dose-related increase in at least 1 strain with or without metabolic activation examples of relevant increases 2-fold revertants/plate vs. concurrent controls for TA98, TA100, and TA102 3-fold revertants/plate vs. concurrent controls for TA1535 and TA

20 Ames Assay GLP vs. non-glp GLP study features: Study Director serves as the single point of control study is conducted according to a written protocol, referencing appropriate SOPs quality assurance audit(s) of critical aspects of the study (e.g., addition of test article, incubations) documentation of test article characterization a final report signed by the Study Director GLP does not ensure good science a non-glp study could be better than a GLP study 20

21 ICH M7 21

22 ICH M7 Experimental Impurity Testing follow standardized testing protocol To assess the mutagenic potential of impurities, a single bacterial mutagenicity assay can be carried out with a fully adequate protocol according to ICH S2(R1) and OECD strain Ames assay 22

23 ICH M7 Experimental Impurity Testing deviating from standard 5-strain assay is permitted but requires justification the selection of bacterial tester strains may be limited to those proven to be sensitive to the identified alert. For impurities that are not feasible to isolate or synthesize or when compound quantity is limited bacterial mutagenicity testing could be carried out using a miniaturized assay format examples Ames II mini-ames (6-well) 23 micro-ames (24-well)

24 ICH M7 Experimental Impurity Testing test article should be neat impurity not a mixture To follow up on a relevant structural alert (Class 3 in Table 1), either adequate control measures could be applied or a bacterial mutagenicity assay with the impurity alone can be conducted. previously published guidelines suggest impurity present/spiked in API may be appropriate for Ames testing this is not a scientifically sound approach and should be avoided 24

25 ICH M7 Experimental Impurity Testing ideally, assays should be conducted GLP assays are expected to be performed in compliance with Good Laboratory Practices (GLP) regulations lack of full GLP compliance does not necessarily mean that the data cannot be used to support clinical trials and marketing authorization. 25

26 ICH M7 Evaluating Publically Available Data potential sources of data TOXNET databases (CCRIS, CPDB, etc.) NTP database PubMed EPA ATSDR WHO material safety data sheets (MSDS) other 26

27 ICH M7 Evaluating Publically Available Data existing data may be useful; however, the reliability of the study and results must be assessed discordant reports need to be addressed positive results unlikely to be scrutinized the opposite is true for reports of negative results is experimental design appropriate? does data interpretation adhere to current standards? detailed information is needed to address these questions 27

28 ICH M7 Evaluating Publically Available Data Is experimental design appropriate? example: combination of bacterial strains many studies will not use 5 bacterial strains limited # of bacterial strains may be acceptable with justification need to review available information to make sure strains cover your specific case 28

29 ICH M7 Evaluating Publically Available Data Is experimental design appropriate? example: top dose many studies will not use appropriate dose range if top dose is not 5000 µg/plate review to determine if toxicity or solubility was limiting 29

30 ICH M7 Evaluating Publically Available Data Is experimental design appropriate? example: antibacterial and other highly cytotoxic compounds testing in a bacterial system will not be appropriate for all impurities in cases where dosing is severely limited by toxicity, another in vitro assay may be needed» e.g., large colony formation in a mouse lymphoma assay 30

31 ICH M7 Evaluating Publically Available Data Does data interpretation adhere to current standards? example: use of qualifiers in describing results some reports will describe assay results as weakly positive however, the Ames assay is not designed to establish mutagenic potency weak positives are treated as positive for regulatory decision making 31

32 Submission of Qualification Data 32

33 Submission of Qualification Data Qualification Data submit detailed study Impurity Related Data reports and/or publications (Q)SAR vs. providing brief summary Ames Assay statements or simply citing In Vitro Mammalian Cell Studies references In Vivo Genotoxicity Studies provide detailed General Toxicology Studies calculations including Carcinogenicity Studies assumptions Other Studies submission must allow Calculations (e.g., PDE) reviewer to assess 33 reliability of the data

34 Case Studies 34

35 Case Studies #1: Insufficient Experimental Ames Testing impurity A was identified in drug substance with expected exposure slightly > ICH Q3A qualification threshold but < 1 mg/day [ICH M7 threshold for applying (Q)SAR] impurity A present at levels <1% in API batch used for GLP Ames testing the Sponsor suggested the negative result from this assay was sufficient for impurity qualification due to concerns with testing impurity + API, the assay was not considered an adequate assessment of 35 mutagenic potential

36 Case Studies #1: Insufficient Experimental Ames Testing because the impurity was difficult to synthesize, a (Q)SAR assessment was performed impurity A was predicted to be non-mutagenic Conclusion Although experimental Ames assay was inadequate, impurity A was considered nonmutagenic based on negative (Q)SAR assessment. 36

37 Case Studies #2: Use of Publically Available Data impurity B was identified in drug substance 2 published Ames assays found in public domain negative study (1981) assay conducted in TA98, TA100, TA1535, and TA1537 with and without metabolic activation top dose 10,000 µg/plate appropriate negative and positive control responses 37

38 Case Studies #2: Use of Publically Available Data 2 published Ames assays found in public domain weakly positive study (1980) assay conducted in TA98, TA100, TA1535, and TA1537 with and without metabolic activation top dose 10,000 µg/plate appropriate negative and positive control responses detailed review of data found no clearly positive results (e.g., very slight increases < 2-fold background only at 10,000 µg/plate) 38

39 Case Studies #2: Use of Publically Available Data Conclusion Data interpretation in 2 nd assay deemed questionable, impurity B considered to be non-mutagenic. 39

40 Case Studies #3: Evaluation of an Antibacterial Impurity impurity C was identified in drug substance (Q)SAR predictions were positive Sponsor conducted an OECD compliant Ames assay top dose limited to 7 µg/plate due to toxicity; therefore, Ames not considered appropriate assay for evaluating mutagenic potential Conclusion Impurity C deemed to possess mutagenic potential. Sponsor asked to control impurity to appropriate limit (e.g., TTC) or test in 40 another assay capable of detecting gene mutations.

41 Summary 41

42 Summary Compared with regulatory expectations for API safety assessment, there is significant flexibility for evaluating impurities. Default assumption is that Ames testing of impurities will adhere to standard protocols; however, deviations are possible with sufficient justification. Published data can be useful but only when adequate details are available. 42

43 Summary Regulatory submissions must include sufficient details so the reviewer can verify conclusions. Based on consideration of risk:benefit, sometimes a suboptimal assay will be good enough or negative enough. Regulatory decisions for a specific impurity are not always applicable across submissions context is critical. 43