Neutrally charged phosphorodiamidate Morpholino antisense oligomers: In vivo bioavailability in solid tumors

Abstract

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The phosphorodiamidate Morpholino oligomers (PMO) are a new class of neutral antisense agents that possess a unique structure, in which the deoxyribose moiety of DNA is replaced with a 6-membered morpholine ring and the charged phosphodiester internucleoside linkages are replaced with neutral phosphorodiamidate linkages. The PMO antisense action is not mediated by RNaseH mediated degradation of the RNA:DNA heteroduplexes but the mechanism of action includes steric blockade of ribosomal assembly at the translational start site and missplicing of target pre-RNA when PMO is targeted to the splice donor or acceptor sites, thus blocking translation of the selected mRNA. We have conducted a detailed characterization of the various factors that affect in vivo biodistribution and correlate in vivo efficacy via different routes of administration (local and parentral) in multiple xenograft, syngeneic, orthotopic and angiogenesis murine models. Administration of 5-20 mg/kg PMO in these models revealed specific inhibition of target protein levels demonstrated by immunoblot analysis in each case. PMO bioavailability was demonstrated by in situ photomicrography and/or flow cytometry in tumors, lung, liver, and kidney. HPLC-based quantitative analysis of tumor tissue post intraperitoneal (15 mg/kg) administration revealed 50-400 nM PMO concentration in the subcutaneous tumors which correlated with functional efficacy. Kidney and liver are primary sites of PMO distribution which is nearly equivalent to phosphorothioate oligonucleotides (PS-ON). However, different PMO sequences influence the degree of tissue accumulation and the relative ratios of organ distribution. The plasma concentration versus time is best fit with a two-compartment model as it is with PS-ON, however, unlike the PS-ONs which have sequence-independent pharmacokinetics, the PMOs demonstrate sequence-specific pharmacokinetic behavior. A Phase I clinical study was conducted to address the issue of PMO bioavailability in surgically excised malignant tumors from patients with adenocarcinoma of prostate and breast after intravenous administration of a single dose of 90 mg PMO targeted against c-myc (AVI-4126). The study objectives were to evaluate the safety, determine AVI-4126 concentration in tissue samples of the tumors, and the distribution of AVI-4126 (margin vs. tumor core). Significant concentrations of intact PMO similar to the animal models were detected in both human prostate and breast tumor tissue with increased distribution in the tumor core in the case of the vascular breast tumors. No serious adverse events (graded according to NCI-CTC) were reported. These studies demonstrate PMO bioavailability in tumor tissue and establish the feasibility of using PMOs targeting specific genes in human cancer clinical trials.