Toxicity of Intravenously Administered ARCAs in the Absence or Presence of Docetaxel

As a prerequisite for a Phase I trial, we evaluated the biodistribution, persistence, and toxicity of CV787 following intravenous administration. Cotton rats were chosen over the mouse for Cotton rats are semipermissive for adenoviral replication [32, 94]. The objective of the first study was to determine the toxicity of CV787, after a single tail vein intravenous administration into the male Cotton rat, followed by a 3-day observation period. CV787 was administered at 3 x 1010 particles, 1 x 1011 particles, and 3 x 1011 particles per animal. There were no deaths or treatment-related clinical observations over the study period. In addition, there were no effects of treatment noted on body weight, food consumption, or clinical pathology or at the macroscopic pathological examination at all dose levels. As 3 x 1011 particles per animal was the highest achievable dose, this dose was considered the no-effect level when administered as a single bolus intravenous injection to the Cotton rat.

To investigate the biodistribution and persistence of CV787 following intravenous administration, we conducted a 21-day study with Cotton rats.

Twenty-seven male Cotton rats received either vehicle, replication-defective Ad5 CV730, an E1A deleted adenovirus mutant, and CV787 at 3 x 1011 particles per animal in a single intravenous tail vein injection on day 1. Three animals from each group were sacrificed on days 3, 8, and 22. DNA was extracted from plasma, testes, brain, spleen, liver, heart, lungs, kidneys, and bone marrow. The DNA obtained from tissues was quantified by optical density measurement and 100 ng was subjected to quantitative PCR using a TaqMan procedure specific for the Ad5 packaging site present in both CV730 and CV787.

There were no ill effects seen upon intravenous delivery of 3 x 1011 particles of CV787 per Cotton rat. As similar distribution of adenovirus seen in other rodents [94], CV787 accumulated primarily in the liver and spleen, and to a lesser extent in the heart, kidney, lung, and bone marrow. Viral DNA levels were very low in the testes and undetectable in the brain. The tissue distribution was similar for Ad5 CV730, a replication-defective control virus that was detected primarily in the spleen, liver, and kidney. The potential for nonspecific replication of CV787 was found to be very low, resulting in no more than a two- to fivefold increase in viral copy number during the course of the study.

A third study was designed to compare the acute toxicity and inflammatory potential of CV787 and CV706 by subcutaneous and intradermal administration in immunocompetent mice, C57BL/6. Four groups of five mice each were treated with a single dose of 1 x 1011 particles of CV787 or CV706 by subcutaneous (sc) or intradermal (id) injection on day 1. Four control groups were treated either intradermally or subcutaneously with the vehicle for CV787 or the vehicle for CV706. Two additional groups were treated intravenously (iv) with either virus at the same dose to compare the acute liver toxicity between CV787 and CV706. All animals were scarified on day 4. Blood was collected for serum chemistry analysis. Macroscopic examination for signs of toxicity was performed at necropsy. Portions of the liver, lung, spleen draining lymph nodes, and sc or id injection sites were collected in buffered formalin and examined for histopathological effects.

All animals survived until the scheduled day of sacrifice. No signs of toxicity were observed after treatment or during macroscopic examination in any of the id or sc groups. Serum chemistry analysis showed no significant changes following subcutaneous or intradermal administration of either virus preparation. With both viruses, histopathological analysis revealed a mild chronic inflammation in the areas surrounding the subcutaneous injection site in some of the animals, but no effects on any of the body organs were observed. Some inflammation was observed in the dermas following intradermal administration with both viruses, but the inflammation remained localized to the injection sites and the overall effects were no more severe than those observed after subcutaneous treatment. As expected, intravenous delivery of the virus at a dose of 1 x 1011 particles per animal resulted in significant liver toxicity, as evidenced by 5- to 100-fold increase of ALT and AST enzyme levels. The ALT and AST response was more pronounced for CV706 than for CV787. Intravenous treatment with CV706 also resulted in statistically significant effects on BUN, serum cholesterol, glucose, and calcium levels, which were not as apparent following the intravenous administration of CV787.

The subcutaneous and intradermal effects observed in this study suggest that CV787 poses no additional risk of inflammation over CV706. Results from intravenous treatment of C57BL/6 mice indicate that CV787 may show less systemic toxicity than CV706. Also, it does not appear that Cotton rats add any new information on toxicity compared to the use of more common and easier to use laboratory mouse strains such as C57BL/6.

A synergistic antitumor efficacy was observed when CV787 was combined with taxane in the prostate tumor xenograft [88]. To examine the toxicity of CV787 in combination with the chemotherapeutic agent docetaxel, we conducted a fourth toxicology study in C57BL/6. The 4-day and 2 8-day effects were evaluated in mice given a single daily dose of dexamethasone (Decadron) each day for 3 consecutive days, a single injection of CV787 at low (1 x 108 particles per animal), medium (3 x 109 particle per animal), or high (1 x 1011 particles per animal) dose, and a single dose of the chemotherapeutic agent, docetaxel (100 mg/m2). Six different treatment groups were established using identical treatment regimens for animals in subgroups sacrificed at 4 days and at 28 days. Each treatment subgroup consisted of eight randomly assigned mice. The ARCA virus vehicle and all three doses of virus were given intravenously via tail vein injection. All virus injections were given on day 1 at 1-2 h prior to Decadron administration. Dexamethasone and docetaxel were both given separately by intraperitoneal injection. At the end of the 4-day and 28-day treatment periods, respectively, blood samples were taken for complete blood count (CBC), including platelet count, and serum chemistry analysis just prior to sacrifice. Tissues were taken for organ weights and histopathologic evaluation of liver, lung, spleen, kidney, brain, heart, mesenteric lymph nodes, bone marrow, any enlarged lymph nodes, and any gross lesions.

Acute virus-associated changes at day 4 were characterized by hepatocellular necrosis and elevated serum leakage (liver) enzymes (ALT, AST) seen in the groups who received a high dose of CV787 alone or high dose of CV787 in combination with docetaxel, as well as an increasing incidence of lymphoid hyperplasia from mid-dose (3 x 109 particles per animal) to highdose (1 x 1011 particles per animal) virus subgroups. These findings indicate dose-related viral effects without evidence of augmentation by docetaxel in combination with dexamethasone. In contrast, the severity of the lymphoid hyperplasia in the high-dose virus subgroup may have been ameliorated some by the docetaxel combination. Evidence of acute (high-dose) virus-associated hepatocellular toxicity was resolved by day 28 and replaced by a viral dose-dependent increase in mild multifocal hepatic mononuclear cell inflammatory infiltrates in subgroups who received CV787 at a dose of 3 x 109 particles per animal or 1 x 1011 particles per animal with or without docetaxel. These inflammatory changes are most likely to be associated with chronic effects of viral replication and antigen stimulation and do not appear to be ameliorated by docetaxel combination.

There was acute mild/moderate bone marrow necrosis at day 4 from high-dose virus plus docetaxel combination in two of eight mice, but not from high-dose virus alone or from lesser-dose virus plus docetaxel combination. There were no bone marrow lesions seen in any animals at day 28, so if the effect is real, it appears to be a reversible effect of the high-dose virus plus docetaxel combination. Acute mild to moderate bone marrow hypoplasia was found at day 4 in all eight mice only in the high-dose virus plus docetaxel combination. Hypoplasia was not seen at day 28. The occurrence of bone marrow necrosis (2/8) and hypoplasia (8/8) in the day 4 high-dose virus plus docetaxel combination subgroup constitutes evidence for an additive but reversible effect of the combination. The 25% bone marrow necrosis and 100% hypoplasia were the only additive toxic effects ascribed to the combination of docetaxel with virus. These toxicities appeared to be dose-dependent with respect to the virus and are reversible.

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