Toxicity and the inability to eradicate all cancerous cells are major factors that impair chemotherapeutic cancer treatment. Enzyme/prodrug therapy seeks to overcome these problems through delivery of DNA encoding a sensitizing enzyme specifically to cancer cells followed by targeted delivery of a prodrug. Prodrugs are nontoxic compounds that kill cells upon activation by the sensitizing enzyme. Many nitroaromatic prodrugs kill both growing and nongrowing (quiescent) cells by activating an apoptotic cascade. This is advantageous as many cells within tumors are quiescent and resistant to many other types of cancer drugs. However, not all cells within the tumor are transformed by the delivered gene and do not express the sensitizing enzyme. Thus, the effectiveness of this approach necessitates that the activated drug diffuse to neighboring cells, a phenomenon termed the bystander effect. The long-term goal of this one-year Developmental Project is to develop nanoparticles (Nps) for delivery of a novel nontoxic cancer prodrug, 6-chloro-9-nitro-5-oxo-5H-benzo-(a)-phenoxazine (CNOB), and its activating enzyme, ChrRX, specifically to prostate tumors. ChrRX reduces CNOB to the toxic drug, 9-amino-6-chioro-5hibenzo[a]phenoxazine-5-one (MCHB) (Fig. E.1.1). Both CNOB and MCHB are fluorescent at emission wavelengths that permit differential noninvasive visualization. Several aspects of this prodrug regimen could therefore be determined bv simple fluorescence measurements: MCHB is a suspected DNA intercalating agent with an impressive bystander effect, and it can kill both growing and quiescent cells. Despite this property, CNOB, like other drugs, fails to fully penetrate solid tumors, highlighting a common problem in cancer chemotherapy, i.e., drug penetration barriers in solid tumors [(1);see C.1.1]. Improvements on four fronts are needed for translation of CNOB to the clinic. The first three (supported by existing/pending grants) are: decreasing the effective dose from the current level (lOmg CNOB/kg body weight) via enzyme and drug improvement;detailed pharmacokinetic/dynamic characterization;and removing the tumor penetration barriers to the gene (enzyme), the prodrug and MCHB (see C.1.1). The fourth element is the focus of this Developmental Project: developing a suitable vehicle for targeting the prodrug and sensitizing enzyme-encoding gene specifically to prostate tumors;no funding is currently available to pursue this goaL This one year development project is limited to developing Nps that can deliver the drug (CNOB) and the HchrRO gene specifically to prostate cancer in vitro. The term ChrRX is also used to refer to any improved enzyme (X) derived from the wild-type ?. coil enzyme ChrR. The improved version of the enzyme currently in use is termed ChrR6;it has been codon-optimized for efficient expression in mammalian cells and is referred to as HChrR6. In ongoing studies, the enzyme is being continually improved. We and others have utilized bacteria and viruses to deliver genes to tumors (2). However, the use of biological agents in treating cancer is problematic, as even attenuated bacteria can potentially harm weakened and often immunocompromised cancer patients. A detrimental immunologic reaction is another potential concern. Therefore we will develop biocompatible and biodegradable nanopartides as delivery agents. The Nps can protect the gene and prodrug from degradative/immunological processes, enhance prodrug solubility and permit facile manipulations to optimize delivery and biodistribution (see B.S, see C.3, Appendix 5.1.1). The Nps will be made visualizable by the use of CyS.5, the fluorescence of which can also be spectrally differentiated from both CNOB and MCHB (Fig. E.1.1). Fig. E.l.2. partially illustrates the advantages of the approach. Because of their differential fluorescence, it is possible to use live animals, rather than histology, for studies of intratumoral Nps distribution, release of CNOB from the Nps, CNOB conversion to MCHB and subsequent intratumoral MCHB distribution. Further, the use of vascular labels (e.g., Angiosense 750) will permit determination of how the intratumoral distribution of these entities relates to the vasculature. Identification of other penetration barriers will be similariy attempted. For example the extracellular matrix (ECM) is often responsible for the impaired intratumoral distribution of therapeutic components. Targeting of the stromal cells (Aim 1) and visualization of the resulting effect on the distribution of the therapy components will indicate the extent of stromal cell and ECM involvement in hampering effective distribution. """"""""Seeing"""""""" the components of the therapeutic regimen within the tumor will greatly facilitate identification of the problematic one (e.g., Nps, CNOB, and/or MCHB distribution) and measures for its improvement (see C.1.1). With transiational relevance in mind, we will use more clinically-relevant approaches to image the Nps (i.e. 64 Cuand Gd-DOTA) in future years of the project. The proposed approach is innovative not only in being visualizable (Fig. E.l.2), but also in other respects: use of novel biocompatible/biodegradable nanoparticles developed by Dr. Zare (Chair of Chemistry Dept.);use of tissue that maintains histological signatures to identify prostate cancer-specific markers (Dr. Peehl);involvement of a prostate surgeon and researcher (Dr. Presti);and ICMIC faculty for imaging expertise, who will provide expertise essential to this project. The specialized resources and expertise of these collaborators and that of Stanford ICMIC members are required for the proposed work: Zare's specialized Nps equipment;Peehl's microtome facility;and ICMIC Specialized Resource #2 bioluminescence/fluorescence imaging facilities (e.g.. Maestro, IVIS and FACS systems [see C.1.1]). In future years, ICMIC Specialized Resource #1 radiochemistry facilities will also be used. Collaboration letters are included.

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