Current Pharmaceutical Design - Volume 9, Issue 26, 2003

The problems associated with conventional anticancer agents that exert their actions by cytotoxicity are well known and often are dose limiting with regard to the therapeutic opportunity. Identification of strategies that rely on tumour selective drug activation is a very attractive approach to minimising host tissue toxicity and in principle can allow drug dosing regimens that are ultimately curative. Prodrug activation is central to the concept of tumour selective drug activation. Prodrugs sometimes do not find favour in the design of new drugs because of the possible biological variability in target tissue activation and consequent predictability of response. However in the treatment of cancers, which are invariably life-threatening, the prodrug offers a major opportunity for selective treatment whether it exploits the presence of a naturally occurring metabolic system or is coupled to a platform technology e.g. antibodies, genes, viral vectors and synthetic polymers all of which facilitate tumour specific activation. Both of these approaches were first identified in a previous issue of Current Pharmaceutical Design [1-7]. The present issue further elaborates prodrug activation that exploits tumour environment and delivery systems. Richard Knox and colleagues describes the use of NQO1 (DT- diaphorase) and nitroreductases as target enzymes for bioreductive activation of cytotoxic prodrugs. A fascinating component of this work is the discovery of NQO2, a latent nitroreductase that is present in some human tumours including colorectal cancers and hepatomas NQO2 in combination with the non-biogenic compound dihydronicotinamide riboside will reductively activate an aziridinyldinitrobenzamide (CB 1954) to a potent cytotoxin in vitro and in vivo [8]. The second article is by Brian Marples and colleagues and highlights the value of harnessing radiation therapy to control of the expression of a suicide gene delivered to the tumour prior to prodrug administration. Marples also discusses the treatment of tumours refractive to radiotherapy by employing oxygen-dependent promoters to produce selective therapeutic gene expression and prodrug activation in hypoxic cells [9]. A relatively new area, reviewed by Yoshisuke Nishi, is the use of catalytic antibodies (abzymes) that are genetically engineered to possess minimal immunogenicity and to selectively activate prodrugs that are not substrates for endogenous enzymes [10]. Adam Patterson and co-workers further elaborate on the theme of improving radiation therapy through GDEPT mediated prodrug activation and address the need for an effective bystander effect in which the cytotoxic prodrug metabolites redistribute efficiently into radiation responsive areas to enable radio sensitisation [11]. Lutz Tietze and T. Feuerstein elaborate further on enzyme activated prodrugs; specifically antibody-enzyme conjugates targeted to tumour associated antigens. Furthermore they discuss proton catalysed hydrolysis of prodrugs by harnessing the increased concentration of hydronium ions in malignant tissue under hyperglycaemic conditions [12]. References [1] McNally VA, Patterson AV, Williams KJ, Cowen RL, Stratford IJ, Jaffar M. Antiangiogenic bioreductive and gene therapy approaches to the treatment of hypoxic tumours. Curr Pharm Design 2002; 8: 1319-1333. [2] Patterson LH, Murray GI. Tumour cytochrome P450 and drug activation. Curr Pharm Design 2002; 8: 1335-1347. [3] Denny WA. Nitroreductase-based GDEPT. Curr Pharm Design 2002; 8: 1349-1361. [4] Wardman P. Indole-3-acetic acids and horseradish peroxidase: A new prodrug / enzyme combination for targeted cancer therapy. Curr Pharm Design 2002; 8: 1363-1374. [5] Searcey M. Duocarmycins-natures prodrugs? Curr Pharm Design 2002; 8: 1375-1389. [6] de Graaf M, Boven E, Scheeren HW, Haisma HJ, Pinedo H.M. Beta-glucuronidase-mediated drug release. Curr Pharm Design 2002; 8: 1391-1403. [7] Chen L, Waxman DJ. Cytochrome P450 gene-directed enzyme prodrug therapy (GDEPT) for cancer. Curr Pharm Design 2002; 8: 1405-1416. [8] Knox RJ, Burke PJ, Chen S, Kerr DJ. CB 1954: From the Walker tumour to NQO2 and VDEPT. Curr Pharm Design 2003; 9(26): 2091-2104. [9] Marples B, Greco O, Joiner MC, Scott SD. Radiogenetic therapy: Strategies to overcome tumour resistance. Curr Pharm Design 2003; 9(26): 2105-2112. [10] Nishi Y. Enzyme / Abzyme prodrug activation systems: Potential use in clinical oncology. Curr Pharm Design 2003; 9(26): 2113-2130. [11] Patterson AV, Saunders MP, Greco O. Prodrugs in genetic chemoradiotherapy. Curr Pharm Design 2003; 9(26): 2131- 2154. [12] Tietze LF, Feuerstein T. Enzyme and proton activated prodrugs for a selective cancer therapy. Curr Pharm Design 2003; 9(26): 2155-2175.

CB 1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] has been the subject of continued interest for over 30 years. As an anti-cancer agent, it represents one of the very few examples of a compound that shows real anti-tumor selectivity. Unfortunately, for the treatment of human disease, this anti-tumor selectivity was seen only in certain rat tumors. The basis for the anti-tumor selectivity of CB 1954 is that it is a prodrug that is enzymatically activated to generate a difunctional agent, which can form DNA-DNA interstrand crosslinks. The bioactivation of CB 1954 in rat cells involves the aerobic reduction of its 4-nitro group to a 4-hydroxylamine by the enzyme NQO1 (DT-diaphorase). The human form of NQO1 metabolizes CB 1954 much less efficiently than rat NQO1. Thus human tumors are insensitive to CB 1954. In view of the proven success of CB 1954 in the rat system, it would be highly desirable to re-create its anti-tumor activity in man. This has led to the development of CB 1954 analogs and other prodrugs activated by nitroreduction such, as those based on a self-immolative activation mechanism. A gene therapy-based approach for targeting cancer cells and making them sensitive to CB 1954 and related compounds has been developed. VDEPT (gene-directed enzyme prodrug therapy) has been used to express an E. coli nitroreductase in tumor cells and human tumor cells transduced to express this enzyme are very sensitive to prodrugs activated by nitroreduction. CB 1954 is in clinical trial for this application. Recently it has been shown that a latent nitroreductase is present in some human tumors. This is NQO2 - an enzyme that requires for activity, the non-biogenic compound dihydronicotinamide riboside (NRH) as a cosubstrate. When active, NQO2 is 3000 times more effective than human DT-diaphorase in the reduction of CB 1954. NRH and reduced pyridinium derivatives that, like NRH, act as co-substrates for NQO2, produce a dramatic increase in the cytotoxicity of CB 1954 against human cell lines in vitro and its anti-tumor activity against certain human xenografts in vivo. NQO2 activity is substantially raised in tumor samples from colorectal and hepatoma patients (up to 14-fold). A phase I clinical trial of an NQO2 co-substrate with CB 1954 is scheduled.

The aim of cancer gene therapy is to selectively kill malignant cells at the tumor site, by exploiting traits specific to cancer cells and / or solid tumors. Strategies that take advantage of biological features common to different tumor types are particularly promising, since they have wide clinical applicability. Much attention has focused on genetic methods that complement radiotherapy, the principal treatment modality, or that exploit hypoxia, the most ubiquitous characteristic of most solid cancers. The goal of this review is to highlight two promising gene therapy methods developed specifically to target the tumor volume that can be readily used in combination with radiotherapy. The first approach uses radiation-responsive gene promoters to control the selective expression of a suicide gene (e.g., herpes simplex virus thymidine kinase) to irradiated tissue only, leading to targeted cell killing in the presence of a prodrug (e.g., ganciclovir). The second method utilizes oxygen-dependent promoters to produce selective therapeutic gene expression and prodrug activation in hypoxic cells, which are refractive to conventional radiotherapy. Further refining of tumor targeting can be achieved by combining radiation and hypoxia responsive elements in chimeric promoters activated by either and dual stimuli. The in vitro and in vivo studies described in this review suggest that the combination of gene therapy and radiotherapy protocols has potential for use in cancer care, particularly in cases currently refractory to treatment as a result of inherent or hypoxia-mediated radioresistance.

Clinically useful prodrug activation systems for cancer therapy can be applied in combination with the exogenous activating enzymes, by which masked prodrugs are able to unmask to exert cytotoxic effects on the target tumors. In essence, designing prodrugs not to be degenerated or activated by the endogenous enzymes is needed. Prodrug activation systems are to be delivered to the tumor site by delivery tools, including antibodies, genes, viral vectors and synthetic polymers, directed to the target tumors. Highly selective accumulation of the prodrug activation system at the tumor site is critically important for the efficacy of the prodrug activations. Genetic engineering of antibodies have made it possible to create a bispecific antibody and its derivatives, which are of special value to the functional antibodies with one arm to direct the target tumor tissues, and another to recruit the effector cells or molecules that can effectively kill the tumor cells. The technology has further opened the window for catalytic antibodies as a prodrug activating system. Catalytic antibodies have two distinct advantages over the enzymes: First, they can be selected to catalyze the reaction that is not catalyzed by the endogenous enzymes. Second, in order to minimize immunogenicity, humanization is applicable to catalytic antibodies. In viewing the concept and experimental data with a few clinical trials of recent approaches of prodrug activation systems, their potential utility in clinical oncology is further discussed.

Improvements in the radiotherapeutic management of solid tumors through the concurrent use of gene therapy is a realistic possibility. Of the broad array of candidate genes that have been evaluated, those encoding prodrug-activating enzymes are particularly appealing since they directly complement ongoing clinical chemoradiation regimes. Gene- Directed Enzyme-Prodrug Therapy (GDEPT) only requires a fraction of the target cells to be genetically modified, providing that the resultant cytotoxic prodrug metabolites redistribute efficiently (the bystander effect ). This transfer of cytotoxicity to neighboring non-targeted cancer cells is central to the success of any gene therapy strategy, irrespective of the therapeutic gene employed. In the context of genetic chemoradiotherapy, efficient prodrug metabolite diffusion will be a prerequisite for efficient radiosensitization. Some, but not all GDEPT approaches have been analysed in combination with radiotherapy. Examples of prodrugs of clinically established chemotherapeutic agents currently used in conjunction with radiotherapy include: 5-fluorocytosine (5FC), cyclophosphamide (CPA), irinotecan (CPT-11), gemcitabine (dFdC), capecitabine, mitomycin C (MMC) and AQ4N. Other GDEPT paradigms, such as ganciclovir (GCV) and Herpes Simplex thymidine kinase (HSV-tk), dinitrobenzamide (DNB) mustard or aziridinyl analogs and the E. coli nitroreductase (NTR), CMDA or ZP2767P with Pseudomonas aeruginosa carboxypeptidase G2 (CPG2), and indole-3-acetic acid (IAA) activated by horseradish peroxidase (HRP) have no clinically established chemotherapeutic counterpart. Each prodrug is discussed in this review in the context of GDEPT, with a particular attention to translational research and clinical utility in combination with radiotherapy.

This review is a survey of two approaches for a selective anticancer therapy that are based on a specific cleavage of specially designed non-toxic prodrugs with the liberation of a cytotoxic compound either by antibody-enzyme conjugates targeted to tumor-associated antigens or by acid-catalyzed hydrolysis of the prodrugs due to the increased concentration of hydronium ions in malignant tissue under hyperglycemic conditions. Herein, the design, synthesis and the biological testing of prodrugs are described.