Astarabine, a Cytarabine Pro-Drug, Is Safe and Efficacious in the Treatment of Leukemia - Results of Animal Studies
Authors: Gengrinovitch Stela , Ben Yakar Ruth , Zuckerman Tsila , Krivoy Norberto , Rowe Jacob M. 
Published: October 2019

Abstract
The chemotherapy drug cytarabine is a standard of care in the treatment of Acute Myeloid Leukemia (AML) and relapsed Acute Lymphoblastic Leukemia (ALL), and is the backbone of induction and consolidation chemotherapy for AML. However, cytarabine is highly toxic, with severe side effects such as cerebellar toxicity and bone marrow suppression. Moreover, doses are significantly attenuated for older patients and patients with hepatic or renal dysfunction. Hence, the toxicity of cytarabine considerably limits its use.
Astarabine is a construct of cytarabine covalently bound to asparagine. Unlike cytarabine, Astarabine's toxicity was shown to be mostly specific to leukemia cells. Leukemia cells lack asparagine synthetase and are dependent on external source for asparagine, hence asparagine was shown to be selectively taken up by leukemia cells by active transport. Astarabine is taken up by leukemia cells, where it is metabolized into the cytotoxic cytarabine molecules, resulting in leukemia cell death.
The effect of Astarabine on 14 different cell lines was investigated. The studies demonstrated that most of the hematological cancer cell lines were sensitive to both Astarabine and cytarabine, while solid tumors cell lines were significantly less sensitive. Astarabine metabolite assay in human leukemia cell lines detected both Astarabine and free Cytarabine metabolites, indicating that Astarabine is a pro-drug of cytarabine.
Animal pharmacokinetics studies were performed in mice and pigs. Pharmacokinetics analysis in mice was preformed by Astarabine single intraperitoneal (IP) injection. Astarabine half-life t1/2 of distribution and elimination in mice plasma ranged between 0.645-1.105 hours. Pharmacokinetics analysis in pigs preformed by Astarabine single intraveneous (IV) injection indicated that 50% (t1/2) of Astarabine is cleared from the pigs blood within 17.5-22.8 hours, a significantly higher t1/2 compared to the reported Cytarabine half-life.
In vivo tolerability/toxicology studies determinedAstarabine IP repeated maximal tolerated dose (MTD) in IRC mice as 781 mg/kg/day, administrated for 7 consecutive days. IP repeated MTD of Astarabine in SCID mice was determined as 1275-1462 mg/kg/day, administrated for 7 consecutive days. IV repeated MTD in SCID mice was determined as 400 mg/kg/day, administrated for 14 days. Astarabine IV repeated MTD in young pigs was determinedto be 28 mg/kg/day, administrated for 6 consecutive days. Hence, it is concluded Astarabine improves drug tolerability and reduce toxicity in animals by 10-20 fold compared to the reported toxicity of cytarabine.
In vivo efficacy studies were performed in a leukemia mouse model (L1210). The L1210 mouse leukemia creates a semi-solid tumor in the abdomen of the mice. Leukemia transplanted mice (12 mice per group) received 14 consecutive daily IP doses of 312 mg/kg/day or 1250 mg/kg/day of Astarabine, or saline control. The control mice died or sacrificed on day 17 due to cancer growth. The abdominal diameter of mice in the 312 mg/kg/day dose Astarabine and the control groups increased during the study (by 41% and 57.8%, respectively), while the abdominal diameter of the 1250 mg/kg/day dose Astarabine group decreased by 8.8%. The abdominal diameter results correlated with the rate of tumor growth: tumors were found in necropsies of mice of both control and 312 mg/kg/day dose Astarabine, however, no tumors were found in necropsies of mice treated with the 1250 mg/kg/day dose Astarabine during recovery period and on termination day. The results present a dose response of Astarabine in leukemia models.
In conclusion, Astarabine may serve as a safer, non-toxic alternative to cytarabine for treatment of AML and ALL. Furthermore, Astarabine may offer effective treatment to older patients where current available treatment results in poor outcome. Studies in humans are ongoing.

Disclosures
Gengrinovitch: BioSight Ltd.: Employment, Equity Ownership, Patents & Royalties. Ben Yakar:BioSight Ltd.: Employment, Equity Ownership. Rowe:Amgen: Consultancy; BioSight Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx Ltd.: Consultancy.