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Leiden/Amsterdam Center for Drug Research (L.A.C.D.R.), Division of Molecular Toxicology, Department of Pharmacochemistry, Vrije Universiteit Amsterdam, Amsterdam, the Netherlands
Abstract
Abstract I. Introduction A. General Introduction B. Prodrugs Designed to Increase the Bioavailability of Antitumor Drugs C. Prodrugs Designed to Increase the Local Delivery of Antitumor Drugs D. Prodrugs Activated by Enzyme Immunoconjugates and by Gene Therapy E. Aim and Scope of This Review II. Prodrugs Activated by Endogenous Enzymes A. Class 1 Oxidoreductases 1. Aldehyde Oxidase a. Enzymology of Aldehyde Oxidase. b. Localization of Aldehyde Oxidase. c. Activation of Prodrugs by Aldehyde Oxidase. d. Discussion of Aldehyde Oxidase as a Prodrug-Activating Enzyme. 2. Amino Acid Oxidase a. Enzymology of Amino Acid Oxidase. b. Localization of Amino Acid Oxidase. c. Activation of Prodrugs by Amino Acid Oxidase. d. Discussion of Amino Acid Oxidase as a Prodrug-Activating Enzyme. 3. Cytochrome P450 Reductase a. Enzymology of Cytochrome P450 Reductase. b. Localization of Cytochrome P450 Reductase. c. Activation of Prodrugs by Cytochrome P450 Reductase. d. Discussion of Cytochrome P450 Reductase as a Prodrug-Activating Enzyme. 4. DT-diaphorase a. Enzymology of DT-Diaphorase. b. Localization of DT-Diaphorase. c. Activation of Prodrugs by DT-Diaphorase. d. Discussion of DT-Diaphorase and Cytochrome P450 Reductase (Section II.A.3.) as Prodrug-Activating Enzymes. 5. Cytochrome P450 a. Enzymology of Cytochrome P450. b. Localization of Cytochrome P450. c. Activation of Prodrugs by Cytochrome P450. d. Discussion of Cytochrome P450 as a Prodrug-Activating Enzyme. 6. Tyrosinase a. Enzymology of Tyrosinase. b. Localization of Tyrosinase. c. Activation of Prodrugs by Tyrosinase. d. Discussion of Tyrosinase as a Prodrug-Activating Enzyme. B. Class 2 Transferases 1. Thymidylate Synthase a. Enzymology of Thymidylate Synthase. b. Localization of Thymidylate Synthase. c. Activation of Prodrugs by Thymidylate Synthase. d. Discussion of Thymidylate Synthase as a Prodrug-Activating Enzyme. 2. Thymidine Phosphorylase a. Enzymology of Thymidine Phosphorylase. b. Localization of Thymidine Phosphorylase. c. Activation of Prodrugs by Thymidine Phosphorylase. d. Discussion of Thymidine Phosphorylase as a prodrug-Activating Enzyme. 3. Glutathione S-Transferase a. Enzymology of Glutathione b. Localization of Glutathione c. Activation of Prodrugs by Glutathione d. Discussion of Glutathione 4. Deoxycytidine Kinase a. Enzymology of Deoxycytidine Kinase. b. Localization of Deoxycytidine Kinase. c. Activation of Prodrugs by Deoxycytidine Kinase. d. Discussion of Deoxycytidine Kinase as a Prodrug-Activating Enzyme. C. Class 3 Hydrolases 1. Carboxylesterase a. Enzymology and Localization of Carboxylesterase. b. Activation of Prodrugs by Carboxylesterase. 2. Alkaline Phosphatase a. Enzymology and Localization of Alkaline Phosphatase. b. Activation of Prodrugs by Alkaline Phosphatase. 3. {beta}-Glucuronidase a. Enzymology and Localization of b. Activation of Prodrugs by 4. Discussion of Hydrolase Enzymes as Prodrug-Activating Enzymes. D. Class 4 Lyases 1. Cysteine Conjugate {beta}-Lyase a. Enzymology of Cysteine Conjugate {beta}-Lyase. b. Localization of Cysteine Conjugate c. Activation of Prodrugs by Cysteine Conjugate d. Discussion of Cysteine Conjugate {beta}-Lyase as a prodrug-Activating Enzyme. III. Prodrugs Activated by Antibody-, Gene-, and Virus-Directed Enzyme Prodrug Therapy Approaches A. Nitroreductase 1. Enzymology of Nitroreductase. 2. Activation of CB 1954 by Nitroreductase. B. Cytochrome P450 1. Activation of Prodrugs by Nonhuman Cytochromes P450. C. Purine-Nucleoside Phosphorylase 1. Enzymology of Purine-Nucleoside Phosphorylase. 2. Activation of Prodrugs by Purine-Nucleoside Phosphorylase. D. Thymidine Kinase 1. Enzymology of Thymidine Kinase. 2. Activation of Ganciclovir by Thymidine Kinase. E. Alkaline Phosphatase 1. Activation of Prodrugs by Nonhuman Alkaline Phosphatase. F. {beta}-Glucuronidase 1. Activation of Prodrugs by Nonhuman {beta}-Glucuronidase. G. Carboxypeptidase 1. Enzymology of Carboxypeptidase. 2. Activation of Prodrugs by Carboxypeptidase. H. Penicillin Amidase 1. Enzymology of Penicillin Amidase. 2. Activation of Prodrugs by Penicillin Amidase. I. {beta}-Lactamase 1. Enzymology of {beta}-Lactamase. 2. Activation of Prodrugs by J. Cytosine Deaminase 1. Enzymology of Cytosine Deaminase. 2. Activation of 5-Fluorocytosine by Cytosine Deaminase. K. Methionine {gamma}-Lyase 1. Enzymology of Methionine {gamma}-Lyase. 2. Activation of Prodrugs by Methionine {gamma}-Lyase. IV. Concluding Remarks and Future Perspectives
The rationale for the development of prodrugs relies upon delivery of higher concentrations of a drug to target cells compared to administration of the drug itself. In the last decades, numerous prodrugs that are enzymatically activated into anti-cancer agents have been developed. This review describes the most important enzymes involved in prodrug activation notably with respect to tissue distribution, up-regulation in tumor cells and turnover rates. The following endogenous enzymes are discussed: aldehyde oxidase, amino acid oxidase, cytochrome P450 reductase, DT-diaphorase, cytochrome P450, tyrosinase, thymidylate synthase, thymidine phosphorylase, glutathione S-transferase, deoxycytidine kinase, carboxylesterase, alkaline phosphatase, 
-glucuronidase and cysteine conjugate -lyase. In relation to each of these enzymes, several prodrugs are discussed regarding organ- or tumor-selective activation of clinically relevant prodrugs of 5-fluorouracil, axazaphosphorines (cyclophosphamide, ifosfamide, and trofosfamide), paclitaxel, etoposide, anthracyclines (doxorubicin, daunorubicin, epirubicin), mercaptopurine, thioguanine, cisplatin, melphalan, and other important prodrugs such as menadione, mitomycin C, tirapazamine, 5-(aziridin-1-yl)-2,4-dinitrobenzamide, ganciclovir, irinotecan, dacarbazine, and amifostine. In addition to endogenous enzymes, a number of nonendogenous enzymes, used in antibody-, gene-, and virus-directed enzyme prodrug therapies, are described. It is concluded that the development of prodrugs has been relatively successful; however, all prodrugs lack a complete selectivity. Therefore, more work is needed to explore the differences between tumor and nontumor cells and to develop optimal substrates in terms of substrate affinity and enzyme turnover rates for prodrug-activating enzymes resulting in more rapid and selective cleavage of the prodrug inside the tumor cells.
For over 50 years chemotherapy has been used with varying success in the treatment of metastatic cancers.
Most chemotherapeutic agents were discovered empirically with no pre-existing knowledge of the biochemical mechanisms of action. More recently, a more rational approach to design prodrugs has been used, which is based on molecular targets that are responsible for cell transformation. However, this approach has been relatively ineffective against malignancies because knowledge about the responsible molecular targets for initiation and progression of cancer is still incomplete (Huang and Oliff, 2001
). A major problem with the use of many chemotherapeutic agents is their unacceptable damage to normal cells and organs, a narrow therapeutic index, a relatively poor selectivity for neoplastic cells, and multidrug resistance upon prolonged treatment due to up-regulation of efflux pumps, increased glutathione S-transferase expression, and enhanced DNA repair (Lowenthal and Eaton, 1996; Nielsen et al., 1996; Stavrovskaya, 2000).
A potential strategy to overcome the limitations of chemotherapeutic agents is the use of prodrugs. Prodrugs are compounds that need to be transformed before exhibiting their pharmacological action. The term prodrug was introduced in the late 1950s by Albert (1958). Prodrugs are often divided into two groups: 1) prodrugs designed to increase the bioavailability to improve the pharmacokinetics of antitumor agents and 2) prodrugs designed to locally deliver antitumor agents.
B. Prodrugs Designed to Increase the Bioavailability of Antitumor Drugs
Numerous antitumor drugs possess a limited bioavailability due to low chemical stability, a limited oral absorption, or a rapid breakdown in vivo (i.e., by first-pass metabolism) (Connors, 1986). To overcome these problems, various prodrugs that can be activated into antitumor drugs have been designed. In this case it is preferred if prodrugs are activated relatively slowly in the blood or liver, for example, thereby preventing acute toxic effects due to high peak concentrations of the antitumor drug (Connors, 1986). Therefore, prodrugs activated by enzymes with high catalytic efficiencies (kcat/Km), resulting in a rapid activation of the prodrug, are less attractive than prodrugs activated by enzymes with moderate catalytic efficiencies. An ideal prodrug designed to increase the bioavailability of an antitumor drug is slowly released. After this activation, the drug has to be transported via the bloodstream to the tumor site where it can execute its mode of action. However, due to the reactivity of most antitumor drugs, a limitation of this slow-releasing prodrug concept is that frequently nontumor tissues are also affected. Another drawback of the use of prodrugs activated by enzymes with low catalytic efficiencies may be the metabolism of these prodrugs by competing enzymes into inactive metabolites (Boddy and Yule, 2000).
C. Prodrugs Designed to Increase the Local Delivery of Antitumor Drugs
In this approach prodrugs are designed to achieve a high local concentration of antitumor drugs and to decrease unwanted side effects (Connors, 1986; Lowenthal and Eaton, 1996; Dubowchik and Walker, 1999). By this concept, referred to as targeting, organ-specific and tumor-specific prodrug activation can be achieved. To specifically activate prodrugs into a certain organ, either the enzyme involved in the prodrug activation must be selectively present in the target organ or the target organ should selectively take up the prodrug. In the case of tumor-specific targeting, the enzyme responsible for prodrug activation should be uniquely present in the tumor cell. Another possibility for tumor-specific targeting is by making use of hypoxic environments of solid tumors that can be treated with bioreductive prodrugs as described below (Lin et al., 1972; Begleiter, 2000). For both organ- and tumor-specific targeting, enzymes with high catalytic efficiencies are beneficial, enabling a rapid activation of the prodrug. A problem with tumor-specific targeting of prodrugs is that unlike bacteria and viruses, cancer cells do not contain molecular targets completely foreign to the host (Dubowchik and Walker, 1999).
D. Prodrugs Activated by Enzyme Immunoconjugates and by Gene Therapy
An alternative strategy to achieve local activation of prodrugs is the use of enzyme immunoconjugates. In this strategy, which is called antibody-directed enzyme prodrug therapy (ADEPT) or antibody-directed catalysis, antigens expressed on tumors cells are used to target enzymes to the tumor site (Fig. 1A). First, an enzyme-antibody conjugate is administered and allowed sufficient time to bind to tumor cells and to be cleared from the circulation. Subsequently, a prodrug is administered and selectively activated extracellularly at the tumor site. This concept was originally demonstrated by Philpott et al. (1973) and concerned generation of hydrogen peroxide from glucose catalyzed by glucose oxidase.
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Meanwhile, significant progress has been made with ADEPT approaches, since it is possible to design and use prodrugs that are not activated by human enzymes by using enzymes of nonhuman origin (Deonarain and Epenetos, 1994; Dubowchik and Walker, 1999; Syrigos and Epenetos, 1999). However, the scarcity of tumor-selective antigens is still a limitation in the applicability of ADEPT. Also, adverse immune effects may cause unwanted results. Another problem with this strategy is that the prodrug is activated extracellularly, and therefore, the antitumor drug that is released still must cross the cell membrane. Furthermore, the lack of effectiveness of ADEPT in humans so far is disappointing in view of the high efficacy observed in rodent models with immunoconjugates (Dubowchik and Walker, 1999).
Alternative approaches designed to circumvent the limitations of ADEPT are gene-directed enzyme prodrug therapy (GDEPT) and virus-directed enzyme prodrug therapy (VDEPT) approaches (Deonarain et al., 1995; Singhal and Kaiser, 1998; Aghi et al., 2000; Smythe, 2000; Greco and Dachs, 2001). In these approaches, genes encoding prodrug-activating enzymes are targeted to tumor cells followed by prodrug administration (Fig. 1B). In GDEPT, nonviral vectors that contain gene-delivery agents, such as cationic lipids, peptides, or naked DNA, are used for gene targeting. In VDEPT, gene targeting is accomplished using viral vectors, with retroviruses and adenoviruses being the most often used viruses. For both GDEPT and VDEPT, the vector has to be taken up by the target cells, and the enzyme must be stably expressed in tumor cells. This process is called transduction. In addition, the prodrug must penetrate the cell membrane to be activated intracellularly. Because it is generally stated that gene targeting to every cell is impossible, the locally activated drug must also be able to kill nonexpressing cells, a phenomenon known as the "bystander effect."
GDEPT and VDEPT effectiveness has been limited to date by insufficient transduction of tumor cells in vivo; further research is needed to increase transduction. To overcome the common problems in ADEPT, protein engineering to humanize immunoconjugates, optimize their pharmacokinetics, and remove fractions that cause unwanted side effects is essential (Dubowchik and Walker, 1999).
E. Aim and Scope of This Review
Currently, much effort is made in the development of prodrugs that are activated by organ- or tumor-selective human enzymes, and because of the existing limitations of ADEPT, GDEPT, and VDEPT approaches, as illustrated above. To date, many enzymes have been evaluated for their ability to activate prodrugs of antitumor agents. This review provides a comprehensive inventory of these enzymes regarding their tissue distribution and presence in tumor cells. The enzyme kinetic parameters of the enzymes and their respective prodrugs will also be presented to classify each enzyme-prodrug system as a slow-release or organ-/tumor-specific strategy.
Section II describes the endogenous enzymes that are capable of activating antitumor prodrugs, their tissue distribution, and relative presence in tumor and normal tissue. Section III summarizes the most important enzymes and their respective prodrugs that are used in ADEPT, GDEPT, and VDEPT. Because several recent, detailed, and comprehensive reviews on ADEPT, GDEPT, and VDEPT have been published, this review will only give a brief overview of ADEPT, GDEPT, and VDEPT approaches to be able to put the former approaches into an appropriate perspective (Deonarain and Epenetos, 1994; Deonarain et al., 1995; Singhal and Kaiser, 1998; Dubowchik and Walker, 1999; Syrigos and Epenetos, 1999; Aghi et al., 2000; Smythe, 2000; Greco and Dachs, 2001; Huang and Oliff, 2001). In Section IV, the concluding remarks and future perspectives of enzymatic bioactivation of antitumor prodrugs are presented.
II. Prodrugs Activated by Endogenous Enzymes
Numerous enzymes have been used to activate prodrugs of antitumor agents. These enzymes belong to four International Union of Pure and Applied Chemistry classes. Enzymes from class 1 are the oxidoreductases, enzymes from class 2 represent the transferases, enzymes from class 3 are hydrolases, and enzymes from class 4 represent the lyases. The enzyme characteristics, their localization in normal and tumor tissue, and their prodrugs will be discussed in upcoming paragraphs.
1. Aldehyde Oxidase
a. Enzymology of Aldehyde Oxidase.
Aldehyde oxidases (EC 1.2.3.1) are FAD-, molybdenum-, and heme iron-containing enzymes, oxidizing aldehydes to the corresponding acids using molecular oxygen (Schomburg and Stephan, 1990-1998; Klaassen, 1996; Moriwaki et al., 1997). During this redox reaction, superoxide anions are also generated. In addition to aldehydes, these enzymes also catalyze the oxidation of pyrroles, pyridines, purines, pterins, and pyrimidines. Turnover numbers up to 4100 min-1 (2-methyl-butyraldehyde) and greatly varying Km values, i.e., from 0.002 mM (methylene blue) to 1.3 mM (N-methylnicotinamide), have been reported (Schomburg and Stephan, 1990-1998). Aldehyde oxidases are homodimeric proteins with a mol. wt. of 270 to 300 kDa depending on the species (Moriwaki et al., 1997).
b. Localization of Aldehyde Oxidase.
Aldehyde oxidase is widely distributed and is mainly located in the cytosolic fraction, although small amounts have been observed in the mitochondria of guinea pig liver (Moriwaki et al., 1997). Based on immunohistochemical staining of aldehyde oxidase in rat tissues, high concentrations of aldehyde oxidase were observed in the liver, esophagus, and lungs, whereas no staining was found in spleen and adrenal (Table 1) (Moriwaki et al., 1996). In human tissues, high immunostaining of aldehyde oxidase in liver and lung was observed. However, in contrast to the rat, aldehyde oxidase in humans was also present in high amounts in adrenal, testis, and prostate tissue (Table 2) (Moriwaki et al., 2001). In human tissues, aldehyde oxidase is not present in bladder, pancreas, ovary, thyroid, brain, skin, and heart. Although significant differences in tissue distribution of aldehyde oxidase occur among humans, rats, mice, and guinea, pig highest levels are present in the liver for all species (Beedham et al., 1987; Moriwaki et al., 1996, 2001; Kurosaki et al., 1999).
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Although little is known about the differences of aldehyde oxidase levels between normal and malignant tissues, it has been shown that the specific aldehyde oxidase activity in rat hepatoma cells is 3-fold higher than that observed in normal rat liver tissue (Harvey and Lindahl, 1982).
c. Activation of Prodrugs by Aldehyde Oxidase.
5-Ethynyluracil is a mechanism-based inhibitor of dihydropyrimidine dehydrogenase (DPD), thereby preventing the rapid breakdown of 5-fluorouracil (5-FU). The bioavailability of 5-ethynyluracil is greater than 60%, and the compound lacks organ selectivity. To improve the organ selectivity, 5-ethynyl-2(1H)-pyrimidinone was designed as a liver-specific prodrug (Fig. 2). 5-Ethynyl-2(1H)-pyrimidinone was activated to 5-ethynyluracil by aldehyde oxidase purified from rabbit liver. The prodrug itself did not affect DPD activity (Porter et al., 1994) (Table 3). The catalytic efficiency (kcat/Km) for 5-ethynyl-2(1H)-pyrimidinone oxidation was 60-fold higher than for N-methylnicotinamide, a well known aldehyde oxidase model substrate, and the Km value of aldehyde oxidase for 5-ethynyl-2(1H)-pyrimidinone was 50 µM. After oral administration of 5-ethynyl-2(1H)-pyrimidinone to rats (2 or 20 µg/kg), DPD activity was inhibited to a similar extent in liver, intestine, lung, spleen, and brain (Porter et al., 1994). Whether the lack of liver selectivity was due to a rapid distribution and/or clearance of 5-ethynyluracil or that other enzymes are involved in the bioactivation of 5-ethynyl-2(1H)-pyrimidinone remains unclear.
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To overcome the rapid breakdown of 5-FU in the gastrointestinal tract, 5-fluoro-2-pyrimidinone (5-FP) was synthesized as a 5-FU prodrug (Table 3, Fig. 2) (Guo et al., 1995). 5-FP is activated by rat liver aldehyde oxidase with a Km value of 220 µM and a Vmax of 8 nmol/min/mg. After oral or intravenous administration to mice, 5-FP was shown to be rapidly activated to 5-FU by aldehyde oxidase in the liver, whereas aldehyde oxidase activity was not present in the gastrointestinal tract. The half-life of 5-FP in plasma was at least 2-fold higher than that of 5-FU. Despite this interesting tissue selectivity, oral administration of 5-FP showed a similar cytostatic activity as 5-FU toward colon 38 tumor cells and P388 leukemia cells in mice (Guo et al., 1995).
5-Iodo-2'-deoxyuridine (IUdR) has been reported to be an effective radiosensitizer in vitro and in vivo (Kinsella, 1996). However, IUdR is rapidly metabolized by hepatic and extrahepatic enzymes, thereby limiting its bioavailability (Kinsella, 1996). Therefore, 5-iodo-2-pyrimidinone-2'-deoxyribose was developed as a prodrug and was activated by rat, mouse, and human hepatic aldehyde oxidase to IUdR (Table 3, Fig. 2). Allopurinol, a selective inhibitor of xanthine oxidase, did not alter bioactivation (Kinsella et al., 1994, 1998). The oxidation of 5-iodo-2-pyrimidinone-2'-deoxyribose in other rodent tissues including intestine, bone marrow, lung, brain, and kidney was more than 10-fold lower. The prodrug was further studied in rhesus monkeys and ferrets after oral and intravenous administration (Kinsella et al., 2000). Although its pharmacokinetics was satisfying, significant weight loss and gastrointestinal side effects were observed. However, no biochemical liver function abnormalities were demonstrated in serum. Based on these promising results, initial phase I clinical studies are in progress.
d. Discussion of Aldehyde Oxidase as a Prodrug-Activating Enzyme.
Aldehyde oxidase has been used to activate prodrugs. However, the wide distribution of this enzyme does not make it an ideal candidate for organ-selective targeting. This is illustrated by the fact that after oral administration of 5-ethynyl-2(1H)-pyrimidinone to rats' DPD activity, inhibited by released 5-ethynyluracil, was inhibited to a similar extent in liver, intestine, lung, spleen, and brain (Porter et al., 1994). Another problem with aldehyde oxidase is the large difference in substrate specificity between species (Johns, 1967; Guo et al., 1995). Therefore, animal models used to test the efficacy of aldehyde oxidase prodrugs might not be good models for humans. The best strategy is to first optimize prodrug activation by human aldehyde oxidase in vitro before testing the efficacy in vivo in animals expressing the human enzyme.
2. Amino Acid Oxidase
a. Enzymology of Amino Acid Oxidase.
Amino acid oxidases catalyze stereoselectively the oxidative deamination of amino acids to the corresponding 
-keto acids ammonia and hydrogen peroxide (Hamilton, 1985; Schomburg and Stephan, 1990-1998; Curti et al., 1992). In addition, amino acid oxidase from rat can dehydrogenate -hydroxy acids to their corresponding -keto acids. Amino acid oxidases have also been reported to catalyze -elimination reactions of some substrates, including -chloroalanine, -cyanoalanine, and selenocysteine Se-conjugates (SeCys conjugates), resulting in the formation of chloride, cyanide, and selenols, respectively, and concomitant production of pyruvate and ammonia (Walsh et al., 1971; Miura et al., 1980; Rooseboom et al., 2001b). Amino acid oxidases are flavoproteins that contain FAD as a cofactor, and they are involved in amino acid catabolism and inflammatory responses. L-Amino acid oxidase (EC 1.4.3.2) is a homodimeric flavoprotein with a mol. wt. between 85 and 150 kDa, whereas D-amino acid oxidase (EC 1.4.3.3) is somewhat smaller (mol. wt. 38-125 kDa). The latter has been purified both as a monomer (pig, Candida tropicalis) and a homodimer (Trigonopsis variabilis, Rhodotorula gracilis). Turnover numbers for L-amino acid oxidases up to 11,000 min-1 (L-arginine) and for D-amino acid oxidases up to 43,250 (D-alanine) have been reported. Km values are in the millimolar range, although lower Km values have been measured as well for some amino acids (50-100 µM) (Hamilton, 1985; Schomburg and Stephan, 1990-1998; Curti et al., 1992).
b. Localization of Amino Acid Oxidase.
Amino acid oxidases occur in many species and are mainly located in peroxisomes. These cytosolic enzymes are present in various organs, and the tissue distribution of D- and L-amino acid oxidase is very similar (Tables 1 and 2). In mammals, amino acid oxidases are mainly present in the kidney, with liver containing somewhat fewer. However, the enzyme is not present in mouse liver (Konno et al., 1997). Significant levels have also been found in brain, nerve, leukocytes, adrenal cortex, intestine, heart, lung, tongue, skin, stomach, spleen, muscle, and fat tissue (Hamilton, 1985). The ability of L-amino acid oxidase to also oxidize -hydroxy acids is peculiar to the rat kidney, where the enzyme is frequently designated -hydroxy acid oxidase. Amino acid oxidase activity in hog has been observed in kidney, liver, and brain (Table 2) (Katagiri et al., 1991). Based on an enzyme immunoassay, the enzyme was detected in low amounts in lung, although no enzyme activity could be detected. In heart tissue from hog amino acid oxidase, activity was not observed (Katagiri et al., 1991). In humans amino acid oxidase is less widely distributed than in rat and hog and was found to a similar extent in kidney and liver, whereas low levels have been observed in the brain (Table 2) (Holme and Goldberg, 1982). Amino acid oxidase activity was not observed in human lung, spleen, heart, and serum (Holme and Goldberg, 1982). Differences in amino acid oxidase concentrations between normal and tumor cells have not been investigated so far.
c. Activation of Prodrugs by Amino Acid Oxidase.
D-Alanine was used as a prodrug to induce oxidative stress in brain tumor cells in vitro based on local bioactivation to hydrogen peroxide by D-amino acid oxidase (Stegman et al., 1998) (Table 4, Fig. 3). cDNA encoding D-amino acid oxidase of R. gracilis was mutated to remove the peroxisomal targeting sequence to prevent a possibly rapid breakdown of hydrogen peroxide by peroxisomal catalase. Exposure of brain tumor cells to D-alanine resulted in an elevated cytotoxicity mediated by oxidative stress when compared with parental cells. The Km value for oxidative deamination of D-alanine for the mutated protein was 0.7 mM, which is comparable with the wild-type protein (0.8 mM) (Stegman et al., 1998).
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SeCys conjugates were recently proposed as kidney-selective prodrugs of pharmacologically active selenols (Andreadou et al., 1996; Rooseboom et al., 2000). These compounds have been shown to be potent chemopreventive and antitumor agents (Ip, 1998; Ip et al., 1999) (Table 4). The compounds induce apoptosis in cell lines with wild-type or nonfunctional p53; these effects were not attributable to DNA damage (Ip et al., 2000; Zhu et al., 2000). Although the precise molecular mechanism of apoptosis induction remains to be elucidated, bioactivation to selenols is thought to be critical (Ip, 1998). SeCys conjugates were recently reported to be bioactivated by mammalian amino acid oxidases from rat and hog kidney and L-amino acid oxidase from snake venom to hydrogen peroxide and the corresponding -keto acid (Rooseboom et al., 2001b) (Fig. 3). Km values were in the micromolar range, and the catalytic efficiencies (kcat/Km) were comparable with that of L-phenylalanine, known as a good substrate for amino acid oxidases.
In addition to oxidative deamination, amino acid oxidases also catalyze -elimination of SeCys conjugates similarly, resulting in the formation of selenols, pyruvate, and ammonia (Fig. 3) (Rooseboom et al., 2001b). The bioactivation of SeCys conjugates by amino acid oxidase is stereoselective, indicating that the corresponding enantiomers might be used in antitumor and chemopreventive experiments, based on the presence of L-amino acid oxidase or D-amino acid oxidase in those systems. Currently, only racemates are used in such studies (Ip et al., 1999). The concomitant production of selenols and hydrogen peroxide from SeCys conjugates may have an advantage over the above-described hydrogen peroxide generation from D-alanine, because selenols also possess antitumor activity.
In addition to amino acid oxidases, two other enzymes, i.e., cysteine conjugate -lyases (EC 4.4.1.13; see Section II.D.1.) and flavin-containing monooxygenases (EC 1.14.13.8) are involved in the -elimination of SeCys conjugates (Commandeur et al., 2000; Rooseboom et al., 2001a). Therefore, the relative contribution of these enzymes will determine the organ-selective activation of these prodrugs. From in vitro studies, the kidney seems to be the major organ of prodrug activation, and the organ selectivity was comparable with S-(1-chloro-1,2,2-trifluoroethyl)-L-cysteine, which is known to cause selective nephrotoxicity in rodents (Commandeur et al., 1995; Rooseboom et al., 2000, 2002).
d. Discussion of Amino Acid Oxidase as a Prodrug-Activating Enzyme.
Amino acid oxidase has been used to activate some prodrugs. Amino acid oxidases in humans are mainly present in liver and kidney with low levels in the brain, thus implicating possible organ-selective targeting. Interestingly, the enzyme is not present in lung, spleen, heart, and serum. Amino acid oxidases demonstrate high turnover numbers toward appropriate substrates and are not genetically polymorphic in European populations (Barker and Hopkinson, 1977), in contrast to many other enzymes. The activation and toxicity of amino acid oxidase-dependent prodrugs have so far only been evaluated in vitro; the in vivo efficacy remains to be established.
3. Cytochrome P450 Reductase
a. Enzymology of Cytochrome P450 Reductase.
Cytochrome P450 reductase (EC 1.6.2.4; NADPH-ferrihemo-protein oxidoreductase, cytochrome c reductase) is localized in the endoplasmic reticulum and catalyzes the reduction of cytochrome P450s (P450s) using NADPH (Schomburg and Stephan, 1990-1998; Klaassen, 1996). This flavoprotein functions as an electron donor for P450, because electrons are transferred from NADPH to P450 via its FMN and FAD cofactors. The enzyme is able to reduce aldehydes and quinones directly or via P450s. Aldehydes are reduced to the corresponding alcohols, whereas in the case of quinones the one-electron reduction results in the formation of semiquinone free radicals. Semiquinone radicals are readily auto-oxidizable in the presence of oxygen, resulting in the formation of the parent quinone and superoxide anion, of which the latter can be converted to hydrogen peroxide and hydroxyl radicals, thereby initiating lipid peroxidation. Typical Km values are in the micromolar range, and turnover numbers up to 6100 min-1 (cytochrome c) have been reported (Schomburg and Stephan, 1990-1998; Klaassen, 1996).
b. Localization of Cytochrome P450 Reductase.
Cytochrome P450 reductase is located in many tissues (Table 1). In rat tissues the highest activity was found in adrenal gland followed by intestine (89% of adrenal activity), liver (70% of adrenal activity), kidney (47% of adrenal activity), and lung (31% of adrenal activity) (Benedetto et al., 1976). Rat testis and brain register relatively low activity, which is only 13% of the adrenal cytochrome P450 reductase activity. The lower cytochrome P450 reductase activity in lung and kidney than in liver (29% and 28%, respectively, of hepatic activity) was also observed by others (Litterst et al., 1975).
The distribution of cytochrome P450 reductase in humans is less well established than that in the rat. Based on immunological staining, the enzyme was shown to be present in a variety of human tissues (Baron et al., 1983; Hall et al., 1989). Strong staining was observed in the liver, lung, and small intestine, whereas the intensity of staining in the stomach and colon was considerably less (Hall et al., 1989). Presence of cytochrome P450 reductase was also shown in pancreas, gall bladder, appendix, adrenal gland, skin, breast, and prostate (Baron et al., 1983; Hall et al., 1989). The tissue distribution in human liver, lung, pancreas, adrenal gland, and gastrointestinal tract is similar to laboratory animals (Hall et al., 1989). However, cytochrome P450 reductase in the human kidney is more widely distributed than in the kidney of rat, rabbit, and minipig (Hall et al., 1989). The distribution is thought to be correlated with P450s, an enzyme system that is widely distributed in mammals (see Section II.A.5.b. and Tables 1 and 2).
Cytochrome P450 reductase is present in a variety of tumor cell lines including cells from leukemia and melanoma and central nervous system, breast, colon, lung, ovarian, prostate, and renal tumors (Yu et al., 2001). However, the level of activity in these tumor cells does not necessarily reflect that of the corresponding tumor tissue due to loss of enzyme activity as a result of cell culturing. Data on the levels of cytochrome P450 reductase between normal and tumor cells are diverse. In general, cytochrome P450 reductase activity is lower in tumor tissue than in the corresponding normal tissue and correlates with P450 activity (Forkert et al., 1996). Based on a study performed with human lung and breast tumors, only a small variation in cytochrome P450 reductase activity in tumor tissues versus normal tissues was observed (Lopez de Cerain et al., 1999). Recently, it was shown that the specific activity of Cytochrome P450 reductase may increase up to 1.8-fold in human liver cancer tissue compared with normal tissue (Plewka et al., 2000).
c. Activation of Prodrugs by Cytochrome P450 Reductase.
Several bioreductive prodrugs have been developed as anticancer agents (Table 5, Fig. 4) (Ross et al., 1996; Begleiter, 2000). The rationale for the development of bioreductive prodrugs is the presence of low levels of oxygen in solid tumors compared with normal tissues (Lin et al., 1972). Bioreductive prodrugs are being activated into their radical intermediates, which are oxidized to the prodrug under aerobic conditions. However, under the hypoxic conditions (e.g., solid tumors), this oxidation is much slower and thus usually results in higher levels of toxic radical intermediates, resulting in a solid tumor-selective therapy. In general, hypoxic cells in low oxygen tension regions are more resistant to treatment with radiotherapy, requiring a 2- to 3-fold higher radiation dose, indicating the importance of bioreductive drugs (Denny and Wilson, 2000).
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Menadione (vitamin K3; 2-methyl-1,4-naphthoquinone), a synthetic derivative of vitamin K1, displays antitumor activity against a variety of tumor cells, such as tumors from liver, colon, lung, stomach, and prostate and leukemia (Nutter et al., 1991; Jamison et al., 2001). Menadione also significantly reduced the growth rate of solid prostate tumors in nude mice, without any significant toxicity to bone marrow, changes in organ weight, or pathological changes to these organs (Jamison et al., 2001). Menadione is activated by cytochrome P450 reductase via a one-electron reduction resulting in the formation of a semiquinone radical, which subsequently may reduce molecular oxygen to superoxide anion (Table 5) (Misaka and Nakanishi, 1965). This oxidative stress induces a variety of effects, including depletion of glutathione (GSH), induction of single-stranded DNA breaks, and apoptosis. Menadione is also metabolized via a two-electron reduction by DT-diaphorase and carbonyl reductase (Jamison et al., 2001). In this case the nontoxic hydroquinone is generated, and therefore, the balance between the one- and two-electron reduction determines the extent of oxidative stress response (Nutter et al., 1991; Jamison et al., 2001).
Mitomycin C is a naturally occurring, clinically used, bioreductive alkylating prodrug that is activated by several enzymes, including cytochrome P450 reductase and DT-diaphorase (Bachur et al., 1979; Keyes et al., 1984; Tomasz, 1995; Hargreaves et al., 2000) (Table 5 and Section II.A.4.c.). After reductive activation of the quinone moiety, a series of spontaneous rearrangements results in the opening of the aziridine ring and the formation of a quinone methide intermediate, which alkylates the DNA (for a review, see Tomasz, 1995). Other DNA-alkylating agents have also been identified (Spanswick et al., 1998). Mitomycin C has been shown to kill preferentially hypoxic tumor cells (Rockwell and Sartorelli, 1990) and increases cure rates in head and neck cancer patients when used as an adjuvant to radiotherapy (Haffty et al., 1993). Enzymes involved in the activation of mitomycin C and its analog porfiromycin are cytochrome P450 reductase, DT-diaphorase, xanthine dehydrogenase, and xanthine oxidase. However, based on studies in Chinese hamster ovary cell lines, Cytochrome P450 reductase plays a predominant role in the hypoxic activation of these prodrugs (Belcourt et al., 1998). In this study Chinese hamster ovary cells were transfected with cDNAs of cytochrome P450 reductase or DT-diaphorase. In comparison with the parental cell line, the cells transfected with either cDNA-encoding cytochrome P450 reductase or DT-diaphorase were more sensitive toward mitomycin C and its analog porfiromycin, indicating an important role for both enzymes. However, no difference in cytotoxicity of mitomycin C and porfiromycin was observed under both hypoxic and aerobic conditions for DT-diaphorase-expressing cells, whereas for cytochrome P450 reductase-expressing cells, cytotoxicity was greater under hypoxic conditions than under aerobic conditions (Belcourt et al., 1998). In another study, it was also shown that mitomycin C is mainly activated by cytochrome P450 reductase to DNA binding adducts in COS1 cells expressing human Cytochrome P450 reductase. Only at high concentrations of mitomycin C did DT-diaphorase play a role in the activation (Joseph et al., 1996).
Another clinically used prodrug, tirapazamine, is a bioreductive agent that is activated by a one-electron reduction by cytochrome P450 reductase to a cytotoxic nitroxide radical intermediate before further reduction to the nontoxic metabolite SR 4317 (3-amino-1,2,4-benzotriazine-1-oxide) (Walton et al., 1989; Brown, 1993; Denny and Wilson, 2000) (Table 5). Because the nitroxide radical is quenched by molecular oxygen under aerobic conditions, it has been shown to cause selective toxicity by both DNA alkylation and formation of hydroxyl radicals in hypoxic cells (Brown, 1993). Although tirapazamine appears to be predominantly bioactivated by cytochrome P450 reductase, other enzymes such as P450 and DT-diaphorase may play a role as reviewed by Patterson et al. (1998). Tirapazamine is reduced by DT-diaphorase via four-electron reduction to the inactive benzotriazine SR 4330 (3-amino-1,2,4-benzotriazine) (Walton et al., 1992). Tirapazamine was active against human breast tumor cells and human nonsmall-cell lung cancer cells; cytochrome P450 reductase activity correlated with cytotoxicity in both kinds of cells (Patterson et al., 1995a; Chinje et al., 1999). Furthermore, cells overexpressing human cytochrome P450 reductase were more sensitive toward tirapazamine than parental cells, and cells with a lower cytochrome P450 reductase activity were less sensitive (Patterson et al., 1997; Saunders et al., 2000b). These promising preclinical studies with tirapazamine have resulted in clinical evaluation for treatment against lung, head, and neck cancer (Rodriquez et al., 1996; Lee et al., 1998), and as such this prodrug is currently in phase II and III clinical trials as an adjunct to cisplatin-based chemotherapy or radiation.
The activation of another clinically used prodrug EO9 (3-hydroxymethyl-5-aziridinyl-1-methyl-2[1H-indole4,7-dione]prop-2-en-1-ol) has been shown to be catalyzed by cytochrome P450 reductase and DT-diaphorase (see Section II.A.4.c.) (Walton et al., 1991). Based on studies performed with breast cancer cells expressing Cytochrome P450 reductase, it was shown that both under aerobic and hypoxic conditions cytochrome P450 reductase contributes to the bioactivation of EO9 (Saunders et al., 2000a) (Table 5). Furthermore, using purified rat cytochrome P450 reductase it was shown that the formation of a free radical- and DNA-damaging species from EO9 was catalyzed by this enzyme (Bailey et al., 2001) The relative contributions of the enzymes Cytochrome P450 reductase and DT-diaphorase remain unclear, however.
d. Discussion of Cytochrome P450 Reductase as a Prodrug-Activating Enzyme.
Because several bioreductive drugs (e.g., mitomycin C) are activated by both Cytochrome P450 reductase and DT-diaphorase, the use of cytochrome P450 reductase as an enzyme to activate prodrug will be discussed in Section II.A.4.d.
4. DT-diaphorase
a. Enzymology of DT-Diaphorase.
DT-diaphorase [EC 1.6.99.2; NAD(P)H dehydrogenase (quinone)] catalyzes the two-electron reduction of several substrates, including quinones such as vitamin K, using NAD(P)H (Schomburg and Stephan, 1990-1998; Ross et al., 1994; Klaassen, 1996). DT-diaphorase is a flavoprotein containing FAD and FMN as cofactors, and it reduces quinones to the corresponding hydroquinones (diols). This reductive pathway is considered a detoxification pathway since it is generally not associated with oxidative stress, unlike one-electron reduction reactions of quinones by cytochrome P450 reductase, although there are exceptions (see below) (Ross et al., 1994). DT-diaphorase is a homodimeric protein with subunits of 27 kDa. Mouse, rat, and human DT-diaphorase appear to possess two, three, and four forms of this enzyme, respectively (Riley and Workman, 1992). The enzyme NADPH-quinone oxidoreductase-1 (NQO1) accounts for the majority of DT-diaphorase activity present in human tissues. Another DT-diaphorase enzyme known as NADPH-quinone oxidoreductase-2 (NQO2) is polymorphic expressed in humans (Jaiswal et al., 1990). Turnover numbers for DT-diaphorase are often high and have been reported to be up to 700,000 min-1 (menadione), and Km values are often in the micromolar range (Maerki and Martius, 1960).
b. Localization of DT-Diaphorase.
DT-diaphorase, which is located in the cytosol, is present in virtually all mammalian tissues, including liver, brain, heart, lung, kidney, small intestine, and mammary gland (Tables 1 and 2). In the rat the highest DT-diaphorase activity is with menadione as a substrate present in the liver, followed by lung, colon, and kidney (Schlager and Powis, 1990). However, using dichloroindophenol as a substrate, the highest activity was observed in the lung closely followed by the liver (90% of lung activity), whereas colon (64% of lung activity) and kidney (16% of lung activity) were of less importance (Schlager and Powis, 1990). Whether this is due to the involvement of other enzymes in the metabolism of menadione compared with dichloroindophenol is not known.
The tissue distribution of DT-diaphorase in humans is significantly different from that in rats (Table 2) (Schlager and Powis, 1990). Highest DT-diaphorase activity levels in humans are found in the stomach with lower activity levels in kidney (30% of stomach activity), breast (13% of stomach activity), colon (4% of stomach activity), liver (4% of stomach activity), and lung (3% of stomach activity) (Schlager and Powis, 1990). High mRNA levels of NQO1 in humans have been found in skeletal muscle, whereas lung, liver, and kidney contain lower amounts, and a very low expression is observed in heart tissue (Jaiswal, 1994). Expression of NQO2 mRNA is highest in kidney. Lower amounts of NQO2 mRNA have been found in skeletal muscle, liver, and lung, whereas heart and brain amounts are even lower (Jaiswal, 1994).
Interestingly, DT-diaphorase levels are often strongly elevated in tumor tissues compared with normal tissues, making it an interesting target enzyme (Schlager and Powis, 1990; Riley and Workman, 1992; Spanswick et al., 1998). Elevated levels of DT-diaphorase in human tissues have been observed in primary tumors from lung (12- to 18-fold), liver (4- to 19-fold), colon (3- to 4-fold), and breast (3-fold) compared with normal tissue. However, 8- to 12-fold lower DT-diaphorase activity was found in kidney tumors, compared with nontumor tissue and stomach tumors having 2- to 3-fold lower DT-diaphorase activity, compared with nontumor tissue (Schlager and Powis, 1990).
c. Activation of Prodrugs by DT-Diaphorase.
Streptonigrin is an aminoquinone antibiotic isolated from cultures of Streptomyces flocculus and has been shown to possess antitumor activity against lymphomas, leukemias, and melanomas and is activated by DT-diaphorase (Kremer and Laszlo, 1966; Bolzan and Bianchi, 2001) (Table 6, Fig. 4). Although the active metabolite has not been identified so far, the quinone moiety is essential since the antitumor activity is lost when the quinone moiety is absent as in isopropylidine azastreptonigrin (Kremer and Laszlo, 1967). Streptonigrin was also cytotoxic toward human colon carcinoma cells, and this cytotoxicity was correlated to DT-diaphorase activity (Beall et al., 1996; Winski et al., 2001). Activation of streptonigrin causes cytotoxicity via DNA strand breaks and inhibits DNA and RNA synthesis as secondary effects to ATP depletion and formation of hydroxyl radicals and hydrogen peroxide (Kremer and Laszlo, 1966; Beall et al., 1996). The genotoxic effects of this antitumor prodrug have been reviewed recently (Bolzan and Bianchi, 2001).
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As described above, mitomycin C is a naturally occurring, clinically used, bioreductive alkylating prodrug that is activated by DT-diaphorase, xanthine oxidase, xanthine dehydrogenase, and cytochrome P450 reductase (Table 6 and Section II.A.3.c.) (Bachur et al., 1979; Keyes et al., 1984; Tomasz, 1995; Cummings et al., 1998; Spanswick et al., 1998; Hargreaves et al., 2000). The role of DT-diaphorase in the bioactivation of mitomycin C has been somewhat controversial. However, the use of cell lines with high and low DT-diaphorase activity showed a role for this enzyme, and DT-diaphorase from rat liver carcinoma cells was able to bioactivate mitomycin C to the DNA-alkylating intermediates (Suresh Kumar et al., 1997; Cummings et al., 1998; Spanswick et al., 1998). Human DT-diaphorase was shown to be much less effective than the rat enzyme, and it has been proposed to be less efficient than other mitomycin C-metabolizing enzymes (Cummings et al., 1995; Chen et al., 1997). Mitomycin C acts not as a substrate but as an inhibitor of purified human kidney DT-diaphorase, and mitomycin C is not a substrate for purified rat liver DT-diaphorase (Workman et al., 1989). On the other hand, most tumors overexpress DT-diaphorase, making it an interesting target enzyme for novel bioreductive drugs (Workman et al., 1989; Schlager and Powis, 1990; Spanswick et al., 1998).
5-(Aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) is activated to the cytotoxic metabolite 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide by both Escherichia coli nitroreductase and rat DT-diaphorase isolated from Walker cells (Knox et al., 1988, 1992) (Table 6, Fig. 4). The rat DT-diaphorase was able to reduce the 4-nitro group of CB 1954, whereas the E. coli nitroreductase could reduce either of the 2-nitro groups. CB 1954 has been shown to be reduced with four electrons by both rat and human DT-diaphorase, preferentially to the highly cytotoxic 4-hydroxylamine (Walton et al., 1992). However, the bioactivation of CB 1954 is too slow in human tumor cells (Boland et al., 1991). A novel antitumor prodrug therapy was developed (Knox et al., 2000). It was shown that the DT-diaphorase enzyme NQO2 was able to bioactivate CB 1954. This enzyme is present in human tumor cells but appeared to be inactive. Upon cell exposure to reduced nicotinamide riboside or other reduced pyridinium compounds, the enzyme NQO2 was activated, resulting in a 100- to 3000-fold increase in the cytotoxicity of CB 1954. NQO2 activity appeared to be related to the expression of NAD(P)H dehydrogenase (quinone) (DT-diaphorase; NQO1) (Knox et al., 2000). As described later (Section III.A.), ADEPT and GDEPT approaches have been developed for CB 1954 in combination with E. coli nitroreductase.
Diaziquone [2,5-bis(carboethoxyamino)-3,6-diaziridinyl-1,4-benzoquinone] is an antitumor prodrug (Fig. 4) that is bioactivated by DT-diaphorase purified from human colon carcinoma cells (HT-29). The cytotoxicity and DNA strand breakage induced by diaziquone in these cells were inhibited by the specific DT-diaphorase inhibitor dicoumarol (Ross et al., 1990; Siegel et al., 1990) (Table 6). The bioactivation of diaziquone via two-electron reduction catalyzed by DT-diaphorase results in the formation of the corresponding hydroquinone, which auto-oxidized in the presence of molecular oxygen leading to the semiquinone radical, superoxide, hydrogen peroxide, and hydroxyl radical, thereby inducing oxidative stress (Fisher and Gutierrez, 1991). Cytochrome P450 reductase also activates diaziquone to the semiquinone radical, superoxide, hydrogen peroxide, and hydroxyl radical but by a one-electron reduction pathway (Fisher and Gutierrez, 1991). Several analogs of diaziquone were synthesized and tested as reviewed recently (Hargreaves et al., 2000).
d. Discussion of DT-Diaphorase and Cytochrome P450 Reductase (Section II.A.3.) as Prodrug-Activating Enzymes.
Hypoxia, which often occurs in solid tumors, appears to be an attractive target as evidenced by the development of successful bioreductive prodrugs such as tirapazamine. Bioreductive prodrugs are activated to cytotoxic agents (often radicals' intermediates) that are rapidly oxidized back to the parent nontoxic prodrugs in tissues with high levels of oxygen, whereas in hypoxic tissues (e.g., solid tumors) quenching is less efficient, resulting in a solid tumor-selective therapy (Lin et al., 1972).
