Vikram Arora

A novel antisense inhibitor of MMP-9 attenuates angiogenesis, human prostate cancer cell invasion and tumorigenicity.

Androgen deprivation therapy causes a paradoxical elevation of matrix metalloproteinases (MMPs) including MMP-9 resulting in aggressive tumor phenotype in many patients with prostate cancer. In this study, we have evaluated a novel antisense phosphorodiamidate Morpholino oligomer (PMO) targeted against MMP-9 in models of angiogenesis and in human prostate xenograft in athymic mice. The treatment of androgen-independent DU145 human prostate cells with a 21-mer MMP-9 antisense PMO caused a dose-dependent inhibition of cell proliferation compared to scrambled or MMP-2 antisense PMO at similar concentrations. This was associated with decreases in MMP-9 expression, gelatinolytic activity and increased stability of the insulin-like growth factor-binding protein (IGFBP-3), a proapoptotic factor and MMP-9 substrate. In vitro invasion assays revealed a 40–60% inhibition of DU145 cell invasion in the presence of 25 μM MMP-9 antisense PMO. A significant decrease in endothelial cell migration and vascularization was observed in the Matrigel plug assay in mice when treated intraperitoneally with 300 μg/day MMP-9 antisense for 21 days. In the highly vascular DU145 tumor xenografts, MMP-9 inhibition caused decreased tumor growth with regression in 50% of the animals. Histological analysis revealed increased apoptosis and fibrous tissue deposits in the MMP-9 antisense-treated tumors compared to the scrambled and saline controls. No apparent toxicity or mortality was associated with the MMP-9 PMO treatment. In summary, the MMP-9 antisense PMO inhibited in vitro prostate cancer cell proliferation, invasion and in vivo angiogenesis. These data establish the feasibility of developing a site-directed, nontoxic antisense therapeutic agent for inhibiting local invasion and metastasis.

In vivo Bioavailability and Pharmacokinetics of a c-MYC Antisense Phosphorodiamidate Morpholino Oligomer, AVI-4126, in Solid Tumors

Phosphorodiamidate morpholino oligomers (PMO) inhibit targeted gene expression by preventing ribosomal assembly, thereby preventing mRNA translation. AVI-4126, a PMO targeted against c-MYC, has been extensively characterized in multiple cancer and other disease models and is currently in human clinical trials. A phase I clinical study was conducted to address the issue of PMO bioavailability in malignant tumors surgically excised from patients with adenocarcinoma of prostate and breast 1 day after i.v. administration of a single dose of 90 mg AVI-4126 PMO. The study objectives were to evaluate safety, to determine AVI-4126 concentration in tissue samples of the tumors, and to examine the distribution of AVI-4126 (margin versus tumor core). Significant concentrations of intact PMO similar to the animal models were detected in both human prostate and breast tumor tissues with increased distribution in the tumor core for the vascular breast tumors. No serious adverse events (graded according to National Cancer Institute Common Toxicity Criteria) were reported. Another phase I study was conducted in normal human volunteers to assess AVI-4126 plasma pharmacokinetics following single i.v. administration of 90 mg AVI-4126. Data from both human studies indicated similar plasma concentration-time profile. These studies show PMO bioavailability in tumor tissue and establish the feasibility of using PMO targeting specific genes in human cancer clinical trials.

Neutrally Charged Phosphorodiamidate Morpholino Antisense Oligomers: Uptake, Efficacy and Pharmacokinetics

Antisense technology constitutes development of sequence-specific DNA or RNA analogs that can block the activity of selected single-stranded genetic sequences and offer the potential of high specificity lacking in many current drug treatments. The sequencing of the human genome has greatly increased the potential of this approach. Antisense oligonucleotides, the most commonly used antisense approach, are unmodified or chemically modified single stranded RNA or DNA molecules specifically designed to hybridize to corresponding RNA by Watson-Crick binding. Phosphorodiamidate Morpholino oligomers (PMO) are a novel class of non-ionic antisense agents that inhibit gene expression by binding to RNA and sterically blocking processing or translation. PMOs have shown excellent efficiency and safety profile via various routes of administration in multiple animal and human studies. This review will summarize the preclinical studies with PMOs on the road to their development as therapeutic agents with particular emphasis on in vivo biodistribution and pharmacokinetics.

Transdermal Use of Phosphorodiamidate Morpholino Oligomer AVI-4472 Inhibits Cytochrome P450 3A2 Activity in Male Rats

AbstractPurpose. To determine if dermal absorption of an antisense phosphorodiamidate Morpholino oligomers (PMO) can inhibit target gene expression in the liver in vivo. Method. Antisense PMO targeted to cytochrome P450 (CYP) 3A2 was applied topically to adult male rats at doses of 0.03, 0.3, and 3.0 mg. CYP3A enzyme activity in the underlying skin and liver was evaluated 24 h following application. Results. Systemic PMO bioavailability was determined by detection of full-length PMO in liver and fluorescence micrography in underlying skin. CYP3A enzyme activity were measured by hydroxylation of 7-benzyloxy-4-(trifluoromethyl)-coumarin and data were expressed as nanomoles of product/ 100 μg S9 protein/h. A topical dose of 0.03 mg inhibited enzyme levels from 576 ± 17 (vehicle) and 564 ± 20 (control PMO) to 432 ± 20 in the antisense-treated liver (p < 0.05). Increasing the dose to 0.3 mg further inhibited enzyme level to 278 ± 13 (p < 0.005). The inhibition did not increase further when the dose was increased to 3 mg. In the skin, starting enzyme levels were approximately one third of the liver (171 ± 9) and maximum inhibition was reached at a lower dose. Topical delivery of 0.03 mg led reduced skin enzyme levels in half to 89 ± 32 (p < 0.05). Increasing the dose to 0.3 mg and 3.0 mg did not produce any further inhibition, at 73 ± 8 and 72 ± 17 respectively. Conclusion. Topical application of antisense PMO in rats is a feasible delivery strategy for gene targets in liver and underlying skin.

Reduction in Tamoxifen-Induced CYP3A2 Expression and DNA Adducts Using Antisense Technology

Tamoxifen (TAM) is widely used in the treatment and prevention of breast cancer. There is clear evidence that cytochrome P450 (CYP) 3A enzymes play an important role in TAM metabolism, resulting in metabolites that lead to formation of TAM–DNA adducts. We have investigated the effect of CYP3A2 antisense (AVI‐4472) exposure on CYP3A2 transcription, enzyme activity, translation, and TAM–DNA adducts, in livers of rats administered TAM (50 mg/kg body weight [bw]/day) for 7 days. The study design included administration of 0, 0.5, 2.5, or 12.5 mg AVI‐4472/kg bw/day for 8 days, beginning 1 day before TAM exposure. The specific activity of CYP3A2 was increased after TAM administration, and decreased significantly (∼70%) in the presence of 12.5 mg AVI‐4472. CYP3A2 protein levels, determined by immunoblot analysis, showed a similar pattern. Hepatic TAM–DNA adduct levels were measurable in all TAM‐exposed groups. However, when rats were co‐treated with 2.5 and 12.5 mg AVI‐4472/kg bw/day, statistically significant (∼50%) reductions in TAM–DNA adduct levels (2.0–2.8 adducts/108 nucleotides) were observed compared to rats treated with TAM alone (5.1 adducts/108 nucleotides). Rat toxicology U34 arrays (Affymetrix) were used to investigate the modulation of gene expression patterns on co‐administration of TAM with AVI‐4472. Results indicated that several CYP genes were down regulated although no significant induction of CYP3A2 was observed in the TAM‐exposed rats co‐treated with AVI‐4472. Overall the data suggest the utility of antisense technology in the redirection of TAM metabolism thereby lowering TAM genotoxicity in rat liver. Published 2005 Wiley‐Liss, Inc.