Singh J¹., Zabka T¹., Uppal H¹,. Tarrant J¹., Clarke E²., Lin T¹., McCray B¹., La N¹., Nguyen T¹., Dhawan P¹., Doudement E¹., Kauss A¹., Dambach D¹., Misner D¹., Society of Toxicology 52nd Annual Meeting & ToxExpo 2013, Poster Abstract No.2179. 1 Genentech, South San Francisco, CA. 2 ReachBio Research Labs, Seattle, WA.

 INTRODUCTION

• Nicotinamide Adenine Dinucleotide (NAD) is a cofactor for glycolysis which is 50-100x increased in tumor cells (Warburg Effect)
• Nicotinamide Phospho Ribosyl Transferase (NAMPT) is the rate limiting enzyme in a salvage pathway of NAD generation
• NAMPT is upregulated in tumors
• Therapeutic rationale: NAMPT inhibition > ↓ NAD production > ↓ glycolysis > ↓ tumor growth
• NAD can be replenished by treatment with the precursor, nicotinic acid (NA)
• Two structurally diverse NAMPT inhibitors (NAMPTi), GMX-1778 and APO-866, failed to demonstrate clinical efficacy due to dose-limiting thrombocytopenia (Holen, 2008; Hovstadius, 2005)
• GMX-1778, APO-866 and additional NAMPTi were tested for toxicity on megakaryocytes (MK) in vitro. Toxicity was measured biochemically (cellular ATP levels) and functionally (colony forming capacity, or MK-CFC assay).
• Short-term (5-14) day rat toxicity studies were conducted to recapitulate clinical thromobocytopenia

MATERIALS and METHODS

MK viability:

MK precursor cell viability was measured by ATP (cell-titer glo), and differentiation was measured by colony formation in MK-CFC assays

Rat Toxicity studies:

• Rats (5 males per group) were dosed daily for 5- 14 days with GMX-1778 or APO-866, followed by terminal clinical pathology and anatomic pathology evaluation
• GMX-1778 was dosed by oral gavage at doses of at doses of 0 (vehicle control), 10, 30, or 100 mg/kg/day QD or bid
• APO-866 was dosed by intra-peritoneal injection at 0 (vehicle control) and 60 mg/kg bid

NAMPTi Tested

1. NAMPTi Reduce Colony Forming Capability of Human MK Cells

Similar effects were noted with monkey MK-CFC; mouse MK-CFC were less sensitive

2. NA Rescues NAMPTIi Toxicity on MK Cells

3. NAMPTi Reduce Cellular ATP During the 14-day Human MK Differentiation (Day 9 Data Shown)

NA rescued NAMPTi toxicity by preventing reduction in ATP

4. NAMPTi Decrease Bone Marrow Cellularity in Rats, with Decreased Circulating Reticulocytes, RBC, and Lymphocytes. Platelets Were Not Reduced

Bone Marrow from Control Rat                                                                         Bone Marrow from NAMPTi-Treated Rat

RESULTS

•GMX-1778 and APO-866 caused toxicity of MKs precursors in MK-CFC assays
•This manifested as reduction in ATP levels and reduced colony formation
•There was a species difference in sensitivity human and monkey > mouse (less sensitive)
• Effects were mitigated by NA co-treatment
•In 5-14 day rat toxicity studies, NAMPTi caused cellular depletion of hematopoietic tissues, with accompanying decreases in circulating reticulocytes, RBCs and lymphocytes. However, there was no significant reduction in circulating platelets
•Toxicity on MK is on-target; tracks with potency.
• Less potent NAMPTi had reduced toxicity on MK precursors in vitro and did not cause bone marrow depletion in rats
•MK cells may be susceptible to NAMPTi toxicity because of high metabolic active during the differentiation process
•Clinical safety considerations:
•The viability of NA to rescue NAMPTi-mediated thrombocytopenia has not been demonstrated
•Co-treatment with NA may mitigate NAMPTi-mediated thrombocytopenia to improve the risk-benefit profile
• Bone marrow toxicity is monitorable and reversible in the clinical setting
•Patient compliance to NA co-treatment will be an important consideration

REFERENCES

Holen K. et al. (2008). PK, toxicities of FK866, NAD biosynthesis Inhibitor. Invest New Drugs 26:45–51

Hovstadius P. et al (2005). Ph. I Study of CHS828 in Patients with Solid Tumor Malignancy. Clin. Cancer Res. 8: 2843–50

ACKNOWLEDGEMENTS

J. Hsu, T. O’Brien, D. Sampath, B. Liederer, M. Zak.