Comparative oncology and clinical translation of glyco protein conjugated gold nano therapeutic agent (GA-198AuNP) [abstract]

Nanoscience Poster SessionAs part of our efforts toward clinical translation of GA-198AuNP, our studies are focused on therapeutic efficacy of nanoparticulate GA198AuNP agent in dogs with prostatic carcinoma. The overall goal is to gain clinical insights on therapeutic efficacy of GA198AuNP in a large animal model. We have performed a phase I clinical trial using GA-AuNP administered intravenously or intratumorally by injection or infusion. CT scans were performed prior to injection and 24 hours post injection in 3 of the 4 dogs. Following injections, dogs were allowed further treatment as recommended by the primary attending clinician. Four dogs have been treated to date. Complications related to GA-AuNP treatment were not observed, and all 4 dogs received adjunctive treatment with radiation therapy and/ or chemotherapy. These preliminary studies have clearly provided compelling evidence on the therapeutic potential of biocompatible GA-AuNP for their utility as novel therapeutic agents in treating various types of inoperable solid tumors. Intra-tumoral and intravenous administration of GA-AuNP is safe in dogs with spontaneously occurring tumors. As further therapeutic efficacy studies continue, the outcome of this clinical trial in a large animal model will generate therapeutic efficacy data which will be used for filing IND application for Phase I clinical trial studies. This clinical translation effort provides significant advances in terms of delivering optimum therapeutic payloads into prostate cancers with subsequent reduction in tumor volume, thus may effectively reduce/eliminate the need for surgical resection. This presentation will include details of clinical translation of GA198AuNP in prostate tumor bearing dogs

Dose escalation safety study of Nanotax® in dogs [abstract]

Comparative Medicine - OneHealth and Comparative Medicine Poster SessionThe goal of this project is to determine the suitability of CritiTech's existing formulation of fine-particle paclitaxel, Nanotax®, for the treatment of spontaneously-occurring cancer in dogs. The rationale behind this project is that paclitaxel is highly effective in the treatment of human cancers, but cannot be used in dogs because of their exquisite sensitivity to the solubilizing agents (e.g., CremophorEL®) used in commercially available formulations. Abraxane®, a paclitaxel coated with human serum albumin (HSA), is also unsatisfactory because the HSA induces an immune reaction in canines. CritiTech has demonstrated that Nanotax® increases overall survival in a mouse xenograft model of ovarian cancer and indeed has initiated Phase I human trials of the drug. To accomplish the objectives of this application, two specific aims will be pursued: (1) determine the maximally tolerated dose and assess the toxicities of Nanotax® administered to dogs by intravenous injection, and (2) determine the plasma pharmacokinetics of Nanotax® administered intravenously to dogs. Clinically normal dogs (n=3) were treated with increasing amounts of Nanotax® while monitoring clinical signs of toxicity via physical examination and laboratory evaluation. Serial plasma samples were collected and analyzed for paclitaxel content to determine pharmacokinetic parameters for each dose level. Preliminary evaluation suggests that pharmacokinetic parameters are dose-linear and that the drug is rapidly cleared from circulation. The circulating half life is short which may be a result of clearance by the reticuloendothelial system. Final postmortem evaluation will be performed to determine whether the drug has accumulated in any organ system. We will use data generated to determine an appropriate starting dose for a Phase I/II study of Nanotax® in tumor-bearing dogs to determine tolerability in a patient population and efficacy against various canine cancers

Y-chromosome DNA Is Present in the Blood of Female Dogs Suggesting the Presence of Fetal Microchimerism

<div><p>Fetal microchimerism has been suggested to play contradictory roles in women’s health, with factors including age of the recipient, time elapsed since microchimerism occurred, and microchimeric cell type modulating disease. Both beneficial and harmful effects have been identified in wound healing and tissue regeneration, immune mediated disease, and cancer. This area of research is relatively new, and hindered by the time course from occurrence of fetal microchimerism to the multi-factorial development of disease. Dogs represent an excellent model for study of fetal microchimerism, as they share our environment, have a naturally condensed lifespan, and spontaneously develop immune-mediated diseases and cancers similar to their human counterparts. However, fetal microchimerism has not been described in dogs. These experiments sought preliminary evidence that dogs develop fetal microchimerism following pregnancy. We hypothesized that Y chromosomal DNA would be detected in the peripheral blood mononuclear cells of female dogs collected within two months of parturition. We further hypothesized that Y chromosomal DNA would be detected in banked whole blood DNA samples from parous female Golden Retrievers with at least one male puppy in a prior litter. Amplification of DNA extracted from five female Golden Retrievers that had whelped within the two months prior to collection revealed strong positive bands for the Y chromosome. Of banked, parous samples, 36% yielded positive bands for the Y chromosome. This is the first report of persistent Y chromosomal DNA in post-partum female dogs and these results suggest that fetal microchimerism occurs in the canine species. Evaluation of the contributions of fetal microchimeric cells to disease processes in dogs as a model for human disease is warranted.</p></div

Dog Characteristics and Gel Analysis.

<p>Animal number: Corresponds to the gel lanes in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068114#pone-0068114-g002" target="_blank">Figure 2</a>; Result: ‘+’ denotes FMC positive; blank denotes negative; Intensity: relative intensity of Y chromosomal band to background gel; Months since parturition: duration of time passed since last litter containing male offspring to date of blood draw; Male sibling if no litters prior: presence of male sibling in birth litter if nulliparous at time of blood draw.</p

Nested PCR of dilutions of male: female blood, with male to female ratios of 1∶1 to 1∶90,000.

<p>Bands of Y chromosomal DNA are detected in as small as 1∶60,000-fold M:F diluted samples, but not in 1∶90,000-fold diluted samples.</p

Immune response to C. novyi-NT immunotherapy

Abstract Clostridium novyi-NT (CVN-NT) spores germinate in hypoxic regions of tumors and have successfully cured induced neoplasia in mouse models and resulted in objective tumor responses in naturally developing neoplasia in the dog. The objective of this pilot, descriptive, prospective, clinical investigation, was to evaluate and describe the immune response to CNV-NT spores to better understand which immune pathways might play a role in the response to this bacteriolytic immunotherapy. Intratumoral injection of CNV-NT spores result in increased phagocytosis and NK cell-like function after treatment. Intravenous injection of CNV-NT spores resulted in increased LPS-induced TNF-α production, LTA-induced IL-10 production and NK cell-like function post-treatment. Increased NK cell-like function was sustained to 28 (intratumoral) or 56 (intravenous) days post-treatment, and increased phagocytic function was sustained to 28 days post-treatment suggesting that CNV-NT spores induce longer-term immune cell function changes. Future investigations evaluating long-term immune system changes and associations between immune function and tumor remission rates should include evaluation of these pathways

Presence of 320 bp segments of Y-chromosomal DNA following nested PCR amplification performed on banked female Golden Retriever whole-blood DNA samples.

<p>Initial PCR was performed with 650 bp amplicon primers followed by 320 bp nested primer amplification. The PCR products were electrophoresed in a 1% agarose gel with Tri-borate containing Gel-Red and visualized with Bio-Doc-UVA Imaging System. (L) = 100 bp DNA ladder; (+) = male DNA positive control; (−) = female nulliparous DNA negative control; (W) = water template control; (black) numbers = female samples positive for the presence of 320 bp Y-chromosome DNA segments; (gray) numbers = female samples negative for the presence of 320 bp Y-chromosome DNA segments.</p

PCR reactions for positive and negative controls.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068114#pone-0068114-g001" target="_blank">Figure 1A</a> represents the native primer for dog Y-specific DNA fragment of 650 bp, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068114#pone-0068114-g001" target="_blank">Figure 1B</a> represents the nested primer or ∼320 bp within the 650 bp fragment.</p