Forodesine Preclinical Studies

1. Forodesine Preclinical Studies Varsha Gandhi Professor M. D. Anderson Cancer Center Oct. 23, 2006 T-Cell Lymphomas - Bologna

2.  Purine Nucleoside Phosphorylase Mammalian PNPase Substrates inosine deoxyinosine guanosine deoxyguanosine PNPase dGuo

3. PNPase Deficiency & T-Lymphopenia Gua PNPase dGuo dGMP dGDP Giblett, Lancet 1975 dGTP Carson, PNAS 1977 Mitchell, PNAS 1978 Carson, PNAS 1979 T-lymphopenia

4. Approach Inhibitors of PNPase Observation dGuo-mediated cytotoxicity is specific to T-lymphoblasts Rationale PNPase serves as a target for drug development

5. Major PNPase Inhibitors • Hypoxanthine Ki 10 µM • Guanine Ki 22 µM • 6-methoxy-purine Ki 600 µM • 6-mercapto-purine Ki 73 µM • 6-thio-guanine Ki 110 µM • Allopurinol Ki 90 µM • 8-amino-G (analog) Ki 0.2 - 290 µM • 9-deaza-G (analog) Ki 0.08 - 8 µM • Peldesine (BCX34) Ki 30 nM (IC50) • Forodesine (BCX1777) Ki 40 pM

6. H H H Forodesine - Immucillin H 6 [(1S)-1-(9-deaza-hypoxanthine-9-yl)1,4-dideoxy-1,4-imino-d-ribitol] 9 Immucilin H BCX-1777 Forodesine Fodosine 4 1 Schramm, V. Biochemistry, 37:8615, 1998

7. Properties of Forodesine • Transition state inhibitor of PNPase • Very potent inhibitor: Ki = 73 pM • Tight binding: t½ of dissociation = 8 min • T-cell selective • Toxicity requires deoxycytidine kinase • Generates plasma dGuo levels similar to those in PNP deficient patients Kicska PNAS 2001

8. dGTP dCDP dUDP dCTP dTTP CDP UDP Proposed Mechanism of Action Cell Death by PNP Inhibition Ribonucleotide Reductase Kicska PNAS 2001

9. Mechanism of Action • Requires dGuo for cytotoxicity • Accumulation of dGTP • No decrease in other dNTPs • Stabilization of p53; phospho –ser 15 • Activation of caspase 9, 8, 3 • Hallmark features of apoptosis Bantia J Immunophar 2001; 2003; Balakrishanan Blood 2006

10. 1. Forodesine build up in plasma? Forodesine Investigations 2. Can it inhibit PNP in humans? 3. Accumulation of plasma dGuo? 4. Cellular dGTP accumulation?

11. Forodesine in T-Cell MalignanciesTreatment Plan Day 1 ___Forodesine__________________________ 0 hr Day 2 ____Forodesine_________Forodesine________ 24 hr 36 hr Forodesine = 40 mg/m2 Day 2-5 Identical

12. Plasma PharmacologyForodesineDeoxyguanosine

13. Plasma Pharmacokinetics During Therapy Forodesine median 5.4 µM (range 4 – 8 µM)

14. Plasma Pharmacokinetics During Therapy dGuo median 15 µM (range 3 – 34 µM)

15. dGuo dGMP dGDP dGTP PNPase Inhibition Intracellular Phosphorylation of dGuo

16. Intracellular Pharmacodynamics of dGTP During Forodesine Therapy Patient 2- co 1

17. Intracellular Pharmacodynamics of dGTP During Forodesine Therapy Pt 5 Pt 2 Pt 4 Pt 1 Pt 3 Gandhi, Blood 2005

18. PK/PD During Therapy Effect of dose in same patient mg/m2 dGuo mM 40 17 40 16 60 20 90 37

19. Forodesine in T-Lymphoid Malignancies Intracellular Pharmacodynamics of dNTPs dGTP dATP dTTP dCTP Forodesine

20. Summary • Appropriate levels of drug in plasma • Effective levels of dGuo in plasma • Accumulation of dGTP in leukemia cells • Saturation of dGTP accumulation at 40 mg/m2 dose • Cellular pharmacokinetics predict cytoreduction • No decrease in other dNTPs

21. Observations & Implications • Drug t½ ~10 hrs in plasma no need for bid • Saturation of cellular dGTP 40 mg/m2 dose MBD • Cytoreduction only for 14 days need protracted infusion • Drug tolerated could be used in combination

22. Mary Ayres K. Balakrishnan, PhD Rina Bhagat Lisa Chen, PhD Min Du, R.N. Jennifer Frey Fabiola Gomez Rama Nimmanapalli, PhD Cornel Phillip Tina Stellrecht, PhD Scott Teakell Brenita Tyler The Team Michael Keating, MD Debbi Thomas, MD Farhad Ravandi, MD

23. Thank You!