Adult AML Slide Deck Master Clofarabine for T-cell Lymphomas Not as yet preliminary results from a phase I/II trial Steven M Horwitz Assistant Attending Memorial Sloan Kettering Cancer Center New York, New York

clofarabine structure Adult AML Slide Deck Master clofarabine structure 12/6/2018 5:49 AM Clofarabine is a Hybrid Purine Nucleoside Analog N H N H N N H H N H 2 2 2 2 2 N N N N N N N N N N N N N N N N C l N F N N C l N H O O H O O H H O O O O H O O H O F Clofarabine is a next generation nucleoside analog Clofarabine, FLU (fludarabine) and CLAD (cladribine) are halogenated cogneners of deoxyadenosine. Addition of Fluorine in the arabinose position on the C2 position of the ribose sugar imparts resistance to phosphorolysis H O H O O H H O O H O deoxyadenosine Cladribine Fludarabine Clofarabine Montgomery, J. Med. Chem. 1992, 35: 397-401

Addition of chlorine provides resistance to deamination Adult AML Slide Deck Master 12/6/2018 5:49 AM clofarabine stability Addition of chlorine provides resistance to deamination N H 2 N N Addition of fluorine provides resistance to phosphorylytic cleavage N C l N H O O This slide reviews the structural features of Clofarabine that impart the inherent stability of the compound Addition of Chlorine makes Clofarabine resistant to deamination (click to advance animation) Addition of Fluorine in the arabinose position makes Clofarabine resistant to phosphorylytic cleavage and stable at acid pH F H O Montgomery, J. Med. Chem. 1992, 35: 397-401 Xie KC, Plunkett W, Cancer Research. 1996, 56:3030-37

comparison of metabolism Adult AML Slide Deck Master 12/6/2018 5:49 AM comparison of metabolism Fludarabine Cladribine Clofarabine Glycosidic bond cleavage pH < 3 stable labile stable Major metabolites in cells TP MP+TP MP+TP Triphosphate Half-life hrs in CLL >24 13 >24

Adult AML Slide Deck Master stereospecificity for cytotoxicity The arabino configuration is important Parker, Mol Pharmacol. 55, 515, 1999

Mechanism of Action: Clofarabine Adult AML Slide Deck Master Mechanism of Action: Clofarabine 12/6/2018 5:49 AM moa Inhibits ribonucleotide reductase (RnR), resulting in decreased nucleotide pools Substrate for DNA polymerases to prevent DNA strand elongation Clofarabine disrupts the mitochondrial membrane. dNTP pool dNTP pool dNTP pool dNTP pool dNTP pool DNA incorporation Inhibition of DNA synthesis/ repair RNR incorporation P P P P P P P P P P P P Polymerase Polymerase Polymerase Polymerase Polymerase a a a a a , , , , , ε ε ε ε ε Clofarabine Inhibition of DNA synthesis/ Mitochondria ΔΨ ΔΨ ΔΨ ΔΨ ΔΨ 5 5 5 ’ ’ ’ ’ NT NT NT dCK dCK dCK dCK m m m m m Cytochrome C Cytochrome C Cytochrome C Cytochrome C Clofarabine is a prodrug which enters the cell through both equilibrative and concentrative nucleoside transporters. Once in the cell, Clofarabine is initially phosphorylated by deoxycytidine kinase or dCK to Clofarabine-Monophosphate and is subsequently phosphorylated by other kinases to its active form, Clofarabine-Triphosphate. dCK is upregulated in transformed cells Clofarabine acts through several mechanisms to lead to cell death. Inhibition of DNA synthesis by clofarabine is due to the inhibition of ribonucleotide reductase activity and inhibition of chain elongation by DNA polymerases dysruption of mitochondrial membrane integrity leading to the release of pro-apoptotic mitochondrial proteins , cytochrome C and apoptosis-inducing factor. Clofarabine Release Release Release Release Transporter CELL DEATH Clofarabine Parker, Cancer Res 1991, 51: 2386-2394 Xie and Plunkett, Cancer Res 1996;56:3030-3037 Genini, Blood 2000, 96: 3537-43

clofarabine as a single agent in relapsed/refractory adult AML Adult AML Slide Deck Master 12/6/2018 5:49 AM clofarabine as a single agent in relapsed/refractory adult AML Study n Regimen Response Therapeutic Area Safety Profile Kantarjian J.Clinical Oncology 2003 51 (32 AML) Phase I CLO 7.5- 55 mg/m2 /d x 5 ORR (AML/ALL) = 16% CR=6% Solid Tumors, Acute Leukemias, Myeloma, Lymphoma DLT= Transient Gd 3/4 elevations in bilirubin and transaminases Blood 2003 62 (31 AML) Phase II CLO 40 mg/m2/d x 5 CR(AML)=42% ORR(AML)=55% *ORR 56% in pts with unfavorable cytogenetics AML MDS,CML-BP, ALL Myelosuppression, infections, transient elevations in bilirubin and transaminases, rash, N/V *Foran Clo 221 ASCO 2003 Poster 32 (21 eval) 1 CRp, 1 PR Median age 63 years N/V, diarrhea, transient transaminitis, stomatitis *Genzyme-sponsored study

Adult AML Slide Deck Master anecdotes 23 y/o male with relapsed T-lymphoblastic lymphoma (s/p ALL-2) Biopsy at relapse showed NK/T-cell lymphoma (CD3+, CD56+, TdT-) Refractory Hyper-CVAD CR to first cycle of clofarabine, then proceeded to allo transplant In previous cloforabine studies 2 other patients with T-cell disase, T-ALL, both CR Dose and schedule for Phase 1/2 combination studies of clofarabine and Ara-C were based on in vitro studies.

clofarabine in combination Adult AML Slide Deck Master clofarabine in combination 12/6/2018 5:49 AM Study n Regimen Response Disease Safety Profile *Faderl Blood 2005 32 CLO 40 mg/m2/d x5 + Ara-C 1 g/m²/d x5 CR (AML) =28% ORR (AML) =40% AML, MDS, ALL, CML-BP Myelosuppression, infection, LFT, N/V, rash, diarrhea, PPE, mucositis, Faderl Blood 2006 60 CLO 40 mg/m2 + Ara-C 1g/m2 CR= 52% CRp=8% ORR=60% AML or High Risk MDS Myelosuppression, infections, diarrhea, N/V, headache, rash, PPE,, LFT Foran ASH 2005 Abstract 4 CLO 30 mg/m2/d x 5 + Ara-C 100 mg/m2/d x 7 CR = 50% AML > 60 years 2 infection related deaths Dose amended to 25% reduction Burnett British Soc Hem 2006 30 ClO 15-30 mg/m²/d x 5 + Daunorubicin 50g/m²/d x3 CR=83% Suitable for intensive chemo Not available

Adult AML Slide Deck Master rationale 12/6/2018 5:49 AM Anecdotal responses in T-cell and NK/T lymphoma Long intracellular half-life Moderate dose dependent toxicities Good penetration into tissues (extranodal disease) Potential for combination studies with other active drugs for T-cell lymphoma Dose and schedule for Phase 1/2 combination studies of clofarabine and Ara-C were based on in vitro studies.

objectives-phase I/II study Adult AML Slide Deck Master objectives-phase I/II study 12/6/2018 5:49 AM Determine the MTD and DLT for clofarabine on a q3 week cycle in relapsed/refractory T-cell lymphoma Preliminary assessment of Efficacy in T-cell lymphoma Explore basis for future combination studies Dose and schedule for Phase 1/2 combination studies of clofarabine and Ara-C were based on in vitro studies.

Adult AML Slide Deck Master design 12/6/2018 5:49 AM Phase I/II Study Relapsed/Refractory NK/T or T-cell lymphoma Post transplantation allowed clofarabine clofarabine 22 23 24 1 2 3 4 5 Dose and schedule for Phase 1/2 combination studies of clofarabine and Ara-C were based on in vitro studies. Accelerated titration design-start at 4 mg/m2 daily x 3 Grade 2 non-hematologic toxicity-traditional dose escalation 3-6 pt/cohorts Can modify to 5d regimen of q2week regimen Treat expanded cohort at MTD So far-2nd dose level (8mg/m2)