A Novel Therapeutic Approach In Acute Myeloid Leukemia (AML): In Vivo Preclinical Pharmacokinetic (PK), Pharmacodynamic (PD) and Antileukemia Activities Of Synthetic 2’-O-Methylphosphorothioate Mir-29b
2013
AML is one of the most common types of leukemia. Despite recent progress in understanding the mechanisms of leukemogenesis, molecular risk assessment and risk-adapted treatments, only 40% of younger ( 60 years) AML patients achieve long-term complete remission, indicating the need for novel therapeutic approach. microRNAs (miRs) are short non-coding RNAs and have been found deregulated in leukemia. In AML, aberrant downregulation of miR-29b leads to upregulation of Sp1 which complexes with NF κ B. This complex transactivates oncogenes such as DNMTs (DNA methyltransferases) that mediate DNA hypermethylation and epigenetic gene silencing, and FLT3 and KIT that encode receptor tyrosine kinases which mediate cell proliferation and survival. Thus, restoring normal levels of miR-29b with synthetic RNA oligonucleotides may downregulate the aforementioned oncogenes and represent a novel therapeutic approach in AML. In this study, we determined the PK and PD activities of a synthetic single-strand miR mimic, 2’-O-methylphosphorothioate (2’-MeOPS) miR-29b and demonstrated its effective dose and schedule to achieve antileukemia activity in vivo . PK studies of 2’-MeOPSmiR29b were performed at different dose levels (7.5, 16, 24, and 32 mg/kg) administered as intravenous (i.v.) or intraperitoneal ( i.p. ) dosing bolus in healthy CD2F1 mice. Plasma and major organ tissues were collected from 5 minutes to 48 hours following miR administration for determination of the miR concentrations. 2’-MeOPS miR-29b displayed a three-compartmental plasma PK profile with C 5min of 29.5 µM, area-under-the-curve ( AUC 0-∞ ) of 574.3 min*μM, alpha-half-life of 3.8 minutes, beta-half-life of 98.8 minutes, gamma-half-life of 21.5 hours, and a total body clearance of 0.007 L/min*kg when given i.v. at 24 mg/kg. I.v. and i.p. administrations showed similar PK behaviors. Tissue miR distribution showed slow elimination rate and substantially high concentrations throughout 48 hours in all tissues, including bone marrow (BM) and spleen, but not in brain. Plasma PK and tissue distribution lack dose-proportionality. Multiple daily dosing resulted in tissue accumulation. After four daily doses, BM and spleen miR-29b concentrations were twice higher than that of a single dose 6 or 24 hours following the last administration ( p PD studies showed time-dependent downregulation of DNMTs and Sp1 expression by 2’-MeOPSmiR-29b in BM of MV4-11 cells-engrafted NOD/SCID/Gamma mice, an aggressive murine model for AML. Those proteins were reduced ∼50% and ∼80%, respectively, 24 or 48 hours after a single bolus of 2’-MeOPSmiR-29b at 24 mg/kg. Based on the PK-PD data, two administration routes and schedules were tested one week after MV4-11 cells transplantation and followed longitudinally for disease progression and survival: ( a ) a dailyx4 i.v. doses in week 1 alternating with dailyx4 i.p. doses in week 2 which was repeated 3 cycles; ( b ) a dailyx4 i.p. doses every week for 8 weeks. 2’-MeOPSmiR-29b significantly improved the survival of AML mice. The median survival (ms) of saline and scrambled (sc) miR-treated AML mice were 28 and 29 days, respectively, post-transplantation (similar for a and b regimen) vs. 44 and 37 days of miR-29b-treated AML mice given a and b regimen, respectively ( p = 0.001). In order to increase the specificity of the synthetic miR delivery to leukemia cells in turn minimizing the potential host toxicity, we formulated a transferrin (Tf)-conjugated nanoparticle (NP) to encapsulate the 2’-MeOPSmiR to target MV4-11 cells that with enriched expression of Tf membrane receptor. In vivo i.p. administration of 2.4 mg/kg Tf-NP-2’MeOPSmiR-29b with b regimen prolonged the survival of the MV4-11-engrafted leukemic mice (ms = 43 days, p =0.01) as compared to the Tf-NP-2’MeOPS sc miR-treated mice (ms=30 days). Thus, the therapeutic activity of the Tf-NP-formulated 2’MeOPSmiR-29b was similar to that of naked 2’-MeOPSmiR-29b given at a 10-fold higher dose (2.4 vs. 24 mg/kg). To our knowledge, this is the first synthetic miR mimic to be evaluated for PK, PD activities and antileukemic efficacy in a murine leukemia model. The correlation of the PK and PD findings will facilitate the development a PK-PD model for designing optimal dose and schedule for future preclinical investigation and rapid transition to the clinic. Disclosures: Caligiuri: Innate Pharma: Membership on an entity’s Board of Directors or advisory committees.
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