Intracellular Levels of Oblimersen Sodium and Target Downregulation Correlates to Clinical Activity of bcl-2 Antisense in Acute Myeoid Leukemia (AML).

Oblimersen sodium (Ob, Genasense™) is an 18 mer phosphorothioate antisense directed against bcl-2, an antiapoptotic protein that mediates chemoresistance in malignant cells. In order to assess whether detectable intracellular concentrations (ICs) of Ob are achievable in vivo, we have developed a sensitive and specific ELISA-based assay. This assay involves Ob hybridization to a 5′-end overhang of a 3′-biotinylated capture probe ligated to a digoxigenin (Dig)-labeled probe, and detection by an anti-Dig-alkaline phosphatase system. In K562 cell extract, the assay was linear within 50–2000 pM range with a limit of quantification (LOQ) of 50 pM (equivalent to 5.0 fmol/100 μL). The withtin-run coefficients of variation (CVs) in 4 spiked concentrations were between 3–7% in 6 replicates with accuracy values between 93–109%. The between-run CVs were between 6–12% and accuracy values between 97–102%. The specificity of the assay was demonstrated by low cross reactivity with mismatched oligonucleotides and putative 3′-end metabolites shortened by 1, 2 or 3 nucleotides. Validation of IC measurement was performed in vitro in K562 cells treated with fluorescent labeled Ob conjugated to oligofectamine. At Ob concentrations between 0.1 – 10 μM, Bcl-2 mRNA downregulation measured by real-time RT-PCR occurred efficiently. Nonlinear regression analysis of a dose-response curve showed that 50% Bcl-2 downregulation (IC₅₀) occurred at approximately 0.29 μM, corresponding to an Ob IC concentration of 37 pmole/mg protein. Cellular uptake was confirmed by microscopy and flow cytometry. To validate these results in vivo, we measured Ob IC in bone marrow samples from untreated AML pts aged > 60 yrs enrolled on the phase I study OSU 0164. These pts were induced with Ob 7 mg/kg/d CIVI on days 1–10, cytarabine 100 mg/m²/d CIVI on days 4–10 and daunorubicin administered iv at two dose levels (45 mg/m²/d IV and 60 mg/m²/d) on days 4–6. Among 21 pts assessable for clinical response and Bcl-2 levels, at pretreatment, Bcl-2 copy numbers (normalized to ABL) were higher among 12 pts who achieved a CR (median 85,325; range 19,120–149,100) than among 9 non-responsive (NR) pts (32,100 bcl-2 /abl copies; range 1,488–163,500) (P=.04; Mann-Whitney test). Following 72 hr Ob infusion, a decrease (−38%) in median Bcl-2/ABL mRNA copies in CR patients and an increase (+115%) in Bcl-2/ABL copies in NR pts (P=.002; Mann-Whitney test) were observed by real time RT-PCR. A trend in higher median IC of Ob was observed in CR pts (17.0 pmole/mg protein; range 1.5–30.0) as compared to NR pts (4.4 pmole/mg protein; range 0.33–28.0) (P=.06; Mann-Whitney test). Six of 7 pts with IC above the median obtained a CR. No differences were observed in the Ob plasma PKs between the CR pts [median steady state concentration (Css) 2.8 μg/mL, area-under-the-curve (AUC) 772 μg*hr/mL and clearance (Cl) 9.6 L/hr) and the NR pts (median Css 3.4 μg/mL, AUC 752 μg*hr/mL, Cl 6.4 L/hr). Although the number of samples analyzed was small, our data suggest that, despite interpatient variability of both Bcl-2 mRNA expression and Ob uptake, this antisense can be successfully delivered to pts and result in clinically relevant target downregulation. A Cancer and Leukemia Group B phase III AML study to characterize prospectively the interplay of IC levels of the Ob and Bcl-2 downregulation is in progress.