1. Nonclinical Pharmacology/ Toxicology Data for PROTOPIC  (Tacrolimus ointment for Atopic Dermatitis) Barbara Hill, Ph.D. Division of Dermatologic and Dental Drug Products

2. Outline Genotoxicity StudiesGenotoxicity Studies Photocarcinogenicity StudyPhotocarcinogenicity Study Dermal Carcinogenicity StudyDermal Carcinogenicity Study Overall SummaryOverall Summary

3. Genotoxicity Studies Ames mutagenicity test (Salmonella and E. coli)Ames mutagenicity test (Salmonella and E. coli) Mammalian in vitro mutagenesis assay (Chinese hamster lung-derived cells)Mammalian in vitro mutagenesis assay (Chinese hamster lung-derived cells) In vitro CHO/HGPRT assay of mutagenicityIn vitro CHO/HGPRT assay of mutagenicity In vivo clastogenicity assays performed in miceIn vivo clastogenicity assays performed in mice Findings:Findings: –No genotoxicity signal noted in any of the assay systems

4. Photocarcinogenicity Study Objective:Objective: –To determine in a hairless mouse model if dermal test article application combined with simulated sunlight exposure can reduce the time to formation of skin papillomas compared to simulated sunlight exposure alone

5. Photocarcinogenicity Study Findings:Findings: –Topical administration of the vehicle ointment enhanced photo co-carcinogenesis (shorten time to skin tumor formation; greater for male mice) –Topical administration of tacrolimus ointment had an additional small influence on skin tumor development beyond the vehicle effect (more prevalent in male mice)

6. Photocarcinogenicity Study Conclusion:Conclusion: –Based on the results of the nonclinical photocarcinogenicity assay, the sponsor proposed that a precaution be included in the label for patients to minimize or avoid exposure to natural or artificial sunlight during the use of 0.03% and 0.1% tacrolimus ointment

7. Dermal Carcinogenicity Study Objective:Objective: –To determine in a mouse model if daily dermal test article application can cause the formation of tumors at any organ site after two years of application

8. Dermal Carcinogenicity Study Findings:Findings: –High levels of mortality were exhibited in the 0.3%, 1% and 3% tacrolimus ointment dose groups –Statistically significant elevation in the incidence of pleomorphic lymphoma was noted in 0.1% tacrolimus ointment treated male (25/50) and female animals (27/50) compared to vehicle control male (2/50) and female animals (6/50)

9. Dermal Carcinogenicity Study Findings:Findings: –Statistically significant elevation in the incidence of undifferentiated lymphoma was noted in 0.1% tacrolimus ointment treated female animals (13/50) compared to vehicle control female animals (1/50)

10. Dermal Carcinogenicity Study Neoplastic Findings in Tacrolimus Ointment Treated Mice (n = 50) Note: Relatively high mortality was noted in the 0.1% dose group

11. Dermal Carcinogenicity Study The multiples of human systemic exposure levels ranged from 9 - 26 fold if the highest mean adult human AUC 0-24 hr value (0.1% tacrolimus ointment) is used for the calculationThe multiples of human systemic exposure levels ranged from 9 - 26 fold if the highest mean adult human AUC 0-24 hr value (0.1% tacrolimus ointment) is used for the calculation –Highest mean adult human AUC 0-24 hr value (0.1% tacrolimus ointment) = 20.4 ng·ml/hr –NOAEL mouse AUC 0-24 hr value = 189 ng·ml/hr –Lymphoma mouse AUC 0-24 hr value = 534 ng·ml/hr –NOAEL mouse/human exposure level = 189/20.4 = 9 –Lymphoma mouse/human exposure level = 534/20.4 = 26

12. Dermal Carcinogenicity Study However, the multiples of human systemic exposure levels are much less (3 - 9 fold) if the highest obtained adult human AUC 0-24 hr value (0.1% tacrolimus ointment) is used for the calculationHowever, the multiples of human systemic exposure levels are much less (3 - 9 fold) if the highest obtained adult human AUC 0-24 hr value (0.1% tacrolimus ointment) is used for the calculation –Highest adult human AUC 0-24 hr value (0.1% tacrolimus ointment) = 61.9 ng·ml/hr –NOAEL mouse AUC 0-24 hr value = 189 ng·ml/hr –Lymphoma mouse AUC 0-24 hr value = 534 ng·ml/hr –NOAEL mouse/human exposure level = 189/61.9 = 3 –Lymphoma mouse/human exposure level = 534/61.9 = 9

13. Dermal Carcinogenicity Study Conclusions:Conclusions: –The estimates of human systemic exposure data are highly variable and are dependent on the maximum body surface area that is treated in the atopic dermatitis patient –It is unclear what the ratio of mouse to human systemic exposure levels would be for pediatric patients since adequate AUC data are not available at this time

14. Dermal Carcinogenicity Study Conclusions:Conclusions: –Biologic plausibility of lymphoma formation in local lymph nodes can not be ruled out at this time –It is acknowledged that demonstrating this effect could be technically challenging

15. Summary The results of the photocarcinogenicity study suggest that tacrolimus ointment combined with simulated sunlight exposure shortens the time to skin tumor formation compared to simulated sunlight exposure aloneThe results of the photocarcinogenicity study suggest that tacrolimus ointment combined with simulated sunlight exposure shortens the time to skin tumor formation compared to simulated sunlight exposure alone

16. Summary Lymphomas were noted at the 0.1% tacrolimus ointment dose in the dermal mouse carcinogenicity studyLymphomas were noted at the 0.1% tacrolimus ointment dose in the dermal mouse carcinogenicity study The multiples of human systemic exposure ranged from 3 - 26 foldThe multiples of human systemic exposure ranged from 3 - 26 fold The multiple of human systemic exposure calculation is highly variable and is dependent on the systemic exposure level noted in humansThe multiple of human systemic exposure calculation is highly variable and is dependent on the systemic exposure level noted in humans –The best estimate would be obtained from maximum exposure conditions in pediatric patients