Pharmacologic indicators of antitumor efficacy for oncolytic virotherapy.

Central to the development of oncolytic virotherapies for cancer will be a better understanding of the parameters that influence the outcome of virotherapy to treat disseminated cancer by i.v. administration versus regional disease by local treatment. Intratumoral administration of 01/PEME, an oncolytic adenovirus, required approximately 1000-fold less dose than i.v. administration to induce similar tumor growth inhibition. Despite the short (<10 min) circulating half-life of the virus DNA, we could monitor virus distribution to the tumor site and observed virus replication by >1000-fold increase in virus DNA copies over time. There were doses of 01/PEME for which the virus DNA concentration in the tumor increased over time but did not result in antitumor efficacy. Oncolytic virus replication at a tumor site may not be a relevant indication of antitumor efficacy. Efficient distribution to the tumor site may be one of the most critical parameters for antitumor efficacy with oncolytic virotherapy.

Successful adenovirus-mediated wild-type p53 gene transfer in patients with bladder cancer by intravesical vector instillation.

PURPOSE: To study safety, feasibility, and biologic activity of adenovirus-mediated p53 gene transfer in patients with bladder cancer. PATIENTS AND METHODS: Twelve patients with histologically confirmed bladder cancer scheduled for cystectomy were treated on day 1 with a single intratumoral injection of SCH 58500 (rAd/p53) at cystoscopy at one dose level (7.5 x 10(11) particles) or a single intravesical instillation of SCH 58500 with a transduction-enhancing agent (Big CHAP) at three dose levels (7.5 x 10(11) to 7.5 x 10(13) particles). Cystectomies were performed in 11 patients on day 3, and transgene expression, vector distribution, and biologic markers of transgene activity were assessed by molecular and immunohistochemical methods in tumors and normal bladder samples. RESULTS: Specific transgene expression was detected in tissues from seven of eight assessable patients treated with intravesical instillation of SCH 58500 but in none of three assessable patients treated with intratumoral injection of SCH 58500. Induction of RNA and protein expression of the p53 target gene p21/WAF1 was demonstrated in samples from patients treated with SCH 58500 instillation at higher dose levels. Distribution studies after intravesical instillation of SCH 58500 revealed both high transduction efficacy and vector penetration throughout the whole urothelium and into submucosal tumor cells. No dose-limiting toxicity was observed, and side effects were local and of transient nature. CONCLUSION: Intravesical instillation of SCH 58500 combined with a transduction-enhancing agent is safe, feasible, and biologically active in patients with bladder cancer. Studies to evaluate the clinical efficacy of this treatment in patients with localized high-risk bladder cancer are warranted.

Syn3 provides high levels of intravesical adenoviral-mediated gene transfer for gene therapy of genetically altered urothelium and superficial bladder cancer.

Using our model to grow superficial human bladder cancer in the mouse bladder, we have found that the polyamide compound, Syn3, when injected intravesically for 1 hour at 1 mg/mL on two consecutive days, markedly increases rAd-beta-gal intravesical gene transfer and expression. This enhanced transgene expression was much greater than obtain by the use of 22% ethanol, which had previously been shown to increase intravesical adenoviral gene transfer, whereas little or no gene expression was seen with exposure to only rAd-beta-gal. beta-Galactosidase staining was seen in virtually every normal urothelial and superficial tumor cell present, including tumors that express little or no coxsackie-adenovirus receptors when Syn3 was present. High adenoviral-mediated gene transfer was also documented in the pig bladder using Syn3 in a similar protocol. Therefore, Syn3 may overcome the limitations of adequate intravesical adenoviral-mediated gene transfer and, when combined with an appropriate adenoviral-mediated gene, could offer an effective approach to the treatment of superficial bladder cancer and perhaps even genetically altered precursor lesions.

Sustained intravesical interferon protein exposure is achieved using an adenoviral-mediated gene delivery system: a study in rats evaluating dosing regimens.

OBJECTIVES: To evaluate whether a recombinant replication-deficient adenovirus containing the secreted human interferon alpha-2b gene (rAd-IFN) could improve the tissue and urine levels of IFN protein by transducing the urothelium with the secreted human IFN-alpha gene. We also assessed whether varying the interval between rAd-IFN/Syn3 treatments would improve the duration and levels of gene expression. METHODS: The rats received intravesical administration of rAd-IFN at varying concentrations in a formulation containing Syn3, an agent identified that facilitates passage of the adenovirus through the protective barrier of the bladder. Urine was collected daily for 7 days, and human IFN was measured in the urine by enzyme-linked immunosorbent assay. For the redosing studies, the animals received a second dose at varying intervals ranging from 1 to 7 days after the first dose or at longer intervals (30, 60, or 90 days). RESULTS: Rats that received intravesical administration of rAd-IFN in a Syn3 formulation expressed levels of human IFN protein in their urine at peak concentrations of 50,000 to 100,000 pg/mL, but were undetectable by 7 days. Expression was localized to the bladder with only minimal systemic exposure to IFN. Short-term redosing marginally improved the IFN urine concentrations, with maximal levels achieved when a second dose was administered 3 days after a first dose. Although gene expression was attenuated when a second dose was given 5 to 7 days after the first treatment, the levels and duration of IFN expression recovered when the interval was increased to 90 days. CONCLUSIONS: Intravesical treatment with rAd-IFN facilitates high levels of IFN transgene exposure and may be a new approach to treating superficial bladder cancer.

Characterization of hemodynamic events following intravascular infusion of recombinant adenovirus reveals possible solutions for mitigating cardiovascular responses.

Intravascular administration of recombinant adenovirus (rAd) in cancer patients has been well tolerated. However, dose-limiting hemodynamic responses associated with suppression of cardiac output have been observed at doses of 7.5 x 10(13) particles. While analysis of hemodynamic responses induced by small-molecule pharmaceuticals is well established, little is known about the cardiovascular effects of rAd. Telemetric cardiovascular (CV) monitoring in mice was utilized to measure hemodynamic events following intravascular rAd administration. Electrocardiogram analysis revealed a block in the SA node 3-4 min postinfusion, resulting in secondary pacemaking initiated at the AV node. This was associated with acute bradycardia, reduced blood pressure, and hypothermia followed by gradual recovery. Adenovirus-primed murine sera with high neutralizing antibody (nAb) titers could inhibit CV responses, whereas human sera with equivalent nAb titers induced by natural infection were, surprisingly, not inhibitory. Interestingly, repeat dosing within 2-4 h of the primary injection resulted in desensitization, resembling tachyphylaxis, for subsequent CV responses. Last, depletion of Kupffer cells prior to rAd infusion precluded induction of CV responses. These inhibitory effects suggest that rAd interactions with certain cells of the reticular endothelial system are associated with induction of CV responses. Significantly, these studies may provide insight into management of acute adverse effects following rAd systemic delivery, enabling a broadening of therapeutic index.

Acute hepatotoxicity of oncolytic adenoviruses in mouse models is associated with expression of wild-type E1a and induction of TNF-alpha.

Replication competent adenoviruses with various E1 modifications designed to restrict their replication to tumor cells are being evaluated as oncolytic agents in clinical trials. In mouse models, we observed that such oncolytic adenoviruses showed greater hepatotoxicity than E1-deleted adenovirus vectors following intravenous administration. Additional studies in congenic BALB/c, nude, and beige/Scid mice demonstrated dose-dependent hepatotoxicity and indicated that beige/Scid was the most sensitive strain. Comparison of E1-containing viruses showed that hepatotoxicity correlated with expression of wild-type E1a in the liver. Pharmacokinetic analysis showed rapid increases in viral DNA levels in the liver with a virus containing wild-type E1a. This was correlated with rapid induction of TNF-alpha to high levels and with rapid elevation of serum ALT. Hepatotoxicity was significantly reduced for an adenovirus with deletions in the region E1a (dl01/07) or a virus lacking E1a. The results suggest a mechanism for hepatotoxicity involving virus-induced production of local TNF-alpha release and E1a-mediated sensitization of hepatocyte killing.

Development of a formulation that enhances gene expression and efficacy following intraperitoneal administration in rabbits and mice.

We conducted a series of experiments to determine if intraperitoneal (IP) delivery of recombinant adenovirus (rAd)-based therapies is improved through carrier vehicle selection, and compared an icodextrin solution (a high molecular weight dextrin with a prolonged peritoneal cavity residence time) with a standardized phosphate buffered saline (PBS) delivery solution. In vitro, comparative adenovirus particle concentration determination (27 h) and bioactivity assay (24h) indicated equivalent compatibility with icodextrin or PBS. In vivo, rabbits treated IP (100 ml) with rAd-betagal 1 x 10(9) P/ml in icodextrin showed improved transgene expression throughout the peritoneal wall compared to rAd-betagal in PBS. In PC-3 tumor-bearing mice treated IP with 5 x 10(9) P/0.5 ml or 1 x 10(10) P/0.5 ml rAd-betagal, transgene expression was significantly enhanced (p < 0.01) with icodextrin compared to PBS in both tumor specimens and peritoneal wall. In subsequent studies we compared prolongation of survival in intraperitoneal PC-3 and MDAH-2774 human xenograft tumor models in nude mice using rAd-p53 in icodextrin or PBS in multi-dose ranging (1 x 10(8) to 1 x 10(10) P) experiments. The icodextrin formulation alone significantly increased rAd-p53 mediated survival (p < 0.05). In animals, these results show that IP rAd gene therapy can be improved with the use of icodextrin, and suggest that prolonged retention and distribution in the peritoneal cavity is an important factor.

Cyclodextrin-modified polyethylenimine polymers for gene delivery.

Linear and branched poly(ethylenimines), lPEI and bPEI, respectively, grafted with beta-cyclodextrin are prepared to give CD-lPEI and CD-bPEI, respectively, and are investigated as in vitro and in vivo nonviral gene delivery agents. The in vitro toxicity and transfection efficiency are sensitive to the level of cyclodextrin grafting. The cyclodextrin-containing polycations, when combined with adamantane-poly(ethylene glycol) (AD-PEG) conjugates, form particles that are stable at physiological salt concentrations. PEGylated CD-lPEI-based particles give in vitro gene expression equal to or greater than lPEI as measured by the percentage of EGFP expressing cells. Tail vein injections into mice of 120 microg of plasmid DNA formulated with CD-lPEI and AD-PEG do not reveal observable toxicities, and both nucleic acid accumulation and expression are observed in liver.

Intravesical Ad-IFNalpha causes marked regression of human bladder cancer growing orthotopically in nude mice and overcomes resistance to IFN-alpha protein.

We have produced prolonged, high local concentrations of interferon in vivo by intravesical instillation of adenoviruses encoding interferon-alpha (Ad-IFNalpha) together with the gene transfer-enhancing agent Syn3. We found sustained interferon protein levels for days, both in normal mouse urothelium and in human bladder cancer cells growing as superficial bladder tumors in nude mice using an orthotopic bladder model developed by us. Tumor burden in the bladder was determined utilizing cancer cells containing the green fluorescent protein. Marked tumor regression was observed following two 1-h exposures of Ad-IFNalpha/Syn3 and little or no cytotoxicity was detected in normal cells. Similar intravesical instillation of clinically relevant concentrations of IFN protein alone or Ad-IFNalpha without Syn3 was ineffective. Surprisingly, in vitro, Ad-IFNalpha also caused caspase-dependent death of bladder cancer cell lines that were resistant to high concentrations of IFN-alpha protein, including the cell line used in vivo. These findings demonstrate that Ad-IFNalpha can overcome resistance to IFN-alpha protein both in vitro and in vivo and support evaluation of intravesical Ad-IFNalpha/Syn3 for the treatment of superficial bladder cancer.