1. Pre-Clinical Studies and Pharmacology of 13-Cis-Retinoic Acid in Neuroblastoma C. Patrick Reynolds, MD PhD Childrens Hospital Los Angeles University of Southern California Children’s Oncology Group

2. Control 10  M Retinoic Acid High-Dose, Pulse Retinoic Acid Induces Neuroblastoma Differentiation

3. Regulation of Transcription by Retinoids Transcriptional Repression RARE ATRA RAR CPB/p300 9-cis-RA ACTR Activation of Transcription RXR AGGTCA 5bp AGGTCA RARE RXRRAR SMRT or N-coR mSin3a HDAC1 AGGTCA 5bp AGGTCA

4. Naturally Occurring Retinoids all trans-retinoic acid retinol (vitamin A) retinal 9-cis-retinoic acid 13-cis-retinoic acid

5. 10  M Retinoic Acid Control Retinoic Acid Induces Prolonged Differentiation and Growth Arrest in SMS-LHN Day 28 (RA for 14 days, control passed x 4)

6. 13-cis-Retinoic Acid Causes Sustained Growth Arrest of Neuroblastoma Neuroblastoma cell line given two pulses (2 weeks each) of 5  M 13-cis-retinoic acid MYCN protein expression by immunoblotting was down- regulated Phase I study achieved levels of 7.2 + 5.3  M peak and 4.1 + 2.7  M trough

7. Pharmacokinetics of 13-cis-Retinoic Acid

9. 13-cis-RA Shows Stable AUC During Therapy

10. Formulation Sub-Optimal For Young Children Liquid in soft gelatin capsule (10, 20, 30, 40 mg) Can be pierced and chewed or children trained to swallow Often must be squeezed into food for young children causing increased dosing variability Out-of-capsule administration may be associated with decreased 13-cis-RA levels Veal et al, Br J Cancer 96:424, 2007 Extemporaneous liquid formulations have been associated with toxicity likely due to metabolism to ATRA

11. Drug Antagonism by 13-cis-RA in Neuroblastoma SMS-KCNR SMS-SAN

12. High-Risk Neuroblastoma Burden During Therapy 13-cis-RA ? LONG-TERM SURVIVAL

13. How can 13-cis-RA For Neuroblastoma Be Improved ? Isotretinoin labeling should include recommended dosing and approaches to use of the existing formulation in pediatric oncology Pharmacokinetic and possibly pharmacogenomic studies could allow for PK and/or PG-guided dosing Further study on administration route (in or out of capsule) effects on PK are needed A stable formulation suitable for young children and safe for handling by women of child-bearing potential would be optimal

14. Acknowledgements Clinical Trials Judy Villablanca Kate Matthay Bob Seeger Children’s Oncology Group Pharmacokinetics Vas Avramis Anis Khan The Patients and Parents who Participated in our Clinical Trials