enzyme-catalyzed activation of anticancer prodrugs

8/17/2019 Enzyme-Catalyzed Activation of Anticancer Prodrugs

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Enzyme-Catalyzed Activation of Anticancer Prodrugs

MARTIJN ROOSEBOOM, JAN N. M. COMMANDEUR, AND NICO P. E. VERMEULEN

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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

A. General introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55B. Prodrugs designed to increase the bioavailability of antitumor drug . . . . . . . . . . . . . . . . . . . . . . . 56

C. Prodrugs designed to increase the local delivery of antitumor drugs . . . . . . . . . . . . . . . . . . . . . . . 56D. Prodrugs activated by enzyme immunoconjugates and by gene therapy . . . . . . . . . . . . . . . . . . . . 56

E. Aim and scope of this review. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

II. Prodrugs activated by endogenous enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 A. Class 1 oxidoreductases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

1. Aldehyde oxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

a. Enzymology of aldehyde oxidase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57b. Localization of aldehyde oxidase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58c. Activation of prodrugs by aldehyde oxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

d. Discussion of aldehyde oxidase as a prodrug-activating enzyme. . . . . . . . . . . . . . . . . . . . . . 582. Amino acid oxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

a. Enzymology of amino acid oxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58b. Localization of amino acid oxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

c. Activation of prodrugs by amino acid oxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60d. Discussion of amino acid oxidase as a prodrug-activating enzyme . . . . . . . . . . . . . . . . . . . . 62

3. Cytochrome P450 reductase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62a. Enzymology of cytochrome P450 reductase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

b. Localization of cytochrome P450 reductase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

c. Activation of prodrugs by cytochrome P450 reductase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63d. Discussion of cytochrome P450 reductase as a prodrug-activating enzyme . . . . . . . . . . . . 65

4. DT-Diaphorase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

a. Enzymology of DT-diaphorase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65b. Localization of DT-diaphorase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

c. Activation of prodrugs by DT-diaphorase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65d. Discussion of DT-diaphorase and cytochrome P450 reductase (Section II.A.3.) as

prodrug-activating enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 665. Cytochrome P450 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

a. Enzymology of cytochrome P450 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67b. Localization of cytochrome P450 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

c. Activation of prodrugs by cytochrome P450 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

d. Discussion of cytochrome P450 as a prodrug-activating enzyme. . . . . . . . . . . . . . . . . . . . . . 716. Tyrosinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

a. Enzymology of tyrosinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

b. Localization of tyrosinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71c. Activation of prodrugs by tyrosinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

d. Discussion of tyrosinase as a prodrug-activating enzyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73B. Class 2 transferases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

1. Thymidylate synthase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

Address correspondence to: Prof. Dr. Nico P. E. Vermeulen, Leiden/Amsterdam Center for Drug Research (L.A.C.D.R.), Division of Molecular Toxicology, Department of Pharmacochemistry, Vrije Universiteit Amsterdam, De Boelelaan 1083, Amsterdam, The Netherlands.E-mail: [email protected]

Article, publication date, and citation information can be found at http://pharmrev.aspetjournals.org.

DOI: 10.1124/pr.56.1.3.

0031-6997/04/5601-53–102$7.00PHARMACOLOGICAL REVIEWS Vol. 56, No. 1Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics 40104/1135361 Pharmacol Rev 56:53–102, 2004 Printed in U.S.A

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a. Enzymology of thymidylate synthase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73b. Localization of thymidylate synthase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

c. Activation of prodrugs by thymidylate synthase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74d. Discussion of thymidylate synthase as a prodrug-activating enzyme. . . . . . . . . . . . . . . . . . 75

2. Thymidine phosphorylase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75a. Enzymology of thymidine phosphorylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

b. Localization of thymidine phosphorylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

c. Activation of prodrugs by thymidine phosphorylase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76d. Discussion of thymidine phosphorylase as a prodrug-activating enzyme . . . . . . . . . . . . . . 773. Glutathione S-transferase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

a. Enzymology of glutathione S-transferase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77b. Localization of glutathione S-transferase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

c. Activation of prodrugs by glutathione S-transferase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77d. Discussion of glutathione S-transferase as a prodrug-activating enzyme . . . . . . . . . . . . . . 78

4. Deoxycytidine kinase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78a. Enzymology of deoxycytidine kinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

b. Localization of deoxycytidine kinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78c. Activation of prodrugs by deoxycytidine kinase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

d. Discussion of deoxycytidine kinase as a prodrug-activating enzyme . . . . . . . . . . . . . . . . . . 80C. Class 3 hydrolases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

1. Carboxylesterase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80a. Enzymology and localization of carboxylesterase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

b. Activation of prodrugs by carboxylesterase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 812. Alkaline phosphatase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

a. Enzymology and localization of alkaline phosphatase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82b. Activation of prodrugs by alkaline phosphatase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

3. -Glucuronidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83a. Enzymology and localization of -glucuronidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

b. Activation of prodrugs by -glucuronidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 834. Discussion of hydrolase enzymes as prodrug-activating enzymes. . . . . . . . . . . . . . . . . . . . . . . . 85

D. Class 4 lyases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

1. Cysteine conjugate -lyase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85a. Enzymology of cysteine conjugate -lyase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

b. Localization of cysteine conjugate -lyase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85c. Activation of prodrugs by cysteine conjugate -lyase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

d. Discussion of cysteine conjugate -lyase as a prodrug-activating enzyme . . . . . . . . . . . . . 86III. Prodrugs activated by antibody-, gene-, and virus-directed enzyme prodrug therapy approaches . . . . . 87

A. Nitroreductase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 871. Enzymology of nitroreductase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

2. Activation of CB 1954 by nitroreductase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87B. Cytochrome P450 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

1. Activation of prodrugs by nonhuman cytochromes P450 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88C. Purine-nucleoside phosphorylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

1. Enzymology of purine-nucleoside phosphorylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

2. Activation of prodrugs by purine-nucleoside phosphorylase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88D. Thymidine kinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

1. Enzymology of thymidine kinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

2. Activation of ganciclovir by thymidine kinase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89E. Alkaline phosphatase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

1. Activation of prodrugs by nonhuman alkaline phosphatase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89F. -Glucuronidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

1. Activation of prodrugs by nonhuman -glucuronidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90G. Carboxypeptidase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

1. Enzymology of carboxypeptidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 902. Activation of prodrugs by carboxypeptidase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

H. Penicillin amidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

1. Enzymology of penicillin amidase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

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2. Activation of prodrugs by penicillin amidase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90I. -Lactamase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

1. Enzymology of -lactamase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 912. Activation of prodrugs by -lactamase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

J. Cytosine deaminase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 921. Enzymology of cytosine deaminase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

2. Activation of 5-fluorocytosine by cytosine deaminase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

K. Methionine -lyase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 931. Enzymology of methionine -lyase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 932. Activation of prodrugs by methionine -lyase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

IV. Concluding remarks and future perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

Abstract——The rationale for the development of prodrugs relies upon delivery of higher concentra-tions of a drug to target cells compared to administra-tion of the drug itself. In the last decades, numerousprodrugs that are enzymatically activated into anti-cancer agents have been developed. This review de-

scribes the most important enzymes involved in pro-drug activation notably with respect to tissuedistribution, up-regulation in tumor cells and turn-over rates. The following endogenous enzymes are dis-cussed: aldehyde oxidase, amino acid oxidase, cyto-chrome P450 reductase, DT-diaphorase, cytochromeP450, tyrosinase, thymidylate synthase, thymidinephosphorylase, glutathione S-transferase, deoxycyti-dine kinase, carboxylesterase, alkaline phosphatase,-glucuronidase and cysteine conjugate -lyase. In re-lation to each of these enzymes, several prodrugs arediscussed regarding organ- or tumor-selective activa-tion of clinically relevant prodrugs of 5-fluorouracil,

axazaphosphorines (cyclophosphamide, ifosfamide,and trofosfamide), paclitaxel, etoposide, anthracy-clines (doxorubicin, daunorubicin, epirubicin), mer-captopurine, thioguanine, cisplatin, melphalan, andother important prodrugs such as menadione, mito-mycin C, tirapazamine, 5-(aziridin-1-yl)-2,4-dinitro-

benzamide, ganciclovir, irinotecan, dacarbazine, andamifostine. In addition to endogenous enzymes, anumber of nonendogenous enzymes, used in anti-body-, gene-, and virus-directed enzyme prodrug ther-apies, are described. It is concluded that the develop-ment of prodrugs has been relatively successful;however, all prodrugs lack a complete selectivity.Therefore, more work is needed to explore the differ-ences between tumor and nontumor cells and to de-velop optimal substrates in terms of substrate affinityand enzyme turnover rates for prodrug-activating en-zymes resulting in more rapid and selective cleavageof the prodrug inside the tumor cells.

I. Introduction

A. General Introduction

For over 50 years chemotherapy has been used with varying success in the treatment of metastatic cancers.

Most chemotherapeutic agents were discovered empiri-

cally with no pre-existing knowledge of the biochemical

1 Abbreviations: ADEPT, antibody-directed enzyme prodrug ther-

apy; GDEPT, gene-directed enzyme prodrug therapy; VDEPT, virus-

directed enzyme prodrug therapy; DPD, dihydropyrimidine dehydro-

genase; 5-FU, 5-fluorouracil; 5-FP, 5-fluoro-2-pyrimidione; IUdR,

5-iodo-2-deoxyuridine; SeCys conjugates, selenocysteine Se-

conjugates; GSH, glutathione; GS

, glutathione anion; tirapazamine(SR 4233), 3-amino-1,2,4-benzotriazine-1,4-dioxide; EO9, 3-hydroxy-

methyl-5-aziridinyl-1-methyl-2[1H-indole-4,7-dione]prop-2-en-1-ol);

NQO1, NADPH-quinone oxidoreductase-1; NQO2, NADPH-quinone

oxidoreductase-2; CB 1954, 5-(aziridin-1-yl)-2,4-dinitrobenzamide;

dacarbazine (DTIC), 5-(3,3-dimethyl-1-triazeno)imidazole-4-

carboxamide; HMMTIC, 5-(3-hydroxymethyl-3-methyl-triazen-1-

yl)imidazole-4-carboxamide; MTIC, 5-(3-methyltriazen-1-yl)imida-

zole-4-carboxamide; AQ4N, 1,4-bis-{[2-(dimethylamino- N -

oxide)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione; AQ4, 1,4-

bis-{[2-(dimethylamino)ethyl]amino-5,8-dihydroxyanthracene-9,10-

dione; 4- S-CAP, 4- S-cysteaminylphenol; N -Ac-4- S-CAP, N -acetyl-4-

S-cysteaminylphenol; GHB, -L-glutaminyl-4-hydroxybenzene;

I-GHB, -L-glutaminyl-4-hydroxy-3-iodobenzene; BVdUMP, ( E)-5-(2-

bromovinyl)-2-deoxyuridine 5-monophosphate; NB1011, ( E)-5-(2-

bromovinyl)-2-deoxy-5-uridyl phenyl-L-methoxyalaninylphosphor

amidate; 5-DFUR, 5-deoxy-5-fluorouridine; GST, glutathione S-

transferase; TER286, -glutamyl--amino-(2-ethyl- N , N , N , N -

tetrakis(2-chloroethyl)phosphorodiamidate)-sulfonyl-propionyl)-( R)-

()phenylglycine; S-CPHC-glutathione, S-( N - p-chlorophenyl- N -

hydroxycarbamoyl)glutathione; PTA, ci s-3-(9H-purin-6-

ylthio)acrylic acid; 6-MP, 6-mercaptopurine; CPT-11 (irinotecan),

7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin;

SN-38, 7-ethyl-10-hydroxycamptothecin; amifostine (WR-2721), S-2-

(3-aminopropylamino)-ethylphosphorothioic acid; WR-1065, ; 3-AP,

3-amino-pyridine-2-carboxaldehyde thiosemicarbazone; HMR 1826, ;DNR-GA3, N -[4-daunorubicin- N -carbonyl-(oxymethyl)-phenyl] O--

glucuronyl carbamate; DOX-GA3, N -[4-doxorubicin- N -carbonyl-

(oxymethyl)-phenyl]O--glucuronyl carbamate; PC, S-(6-purinyl)-L-

cysteine; GC, S-(guanin-6-yl)-L-cysteine; MeP-dR, 9-(-2-deoxy-

erythropentofuranosyl)-6-methylpurine (6-methylpurine-2-

deoxyribonucleoside); HSV-TK, herpes simplex virus type 1

thymidine kinase; CMDA, 4-[ N -(2-chloroethyl)- N -[2-(mesyloxy)eth-

yl]amino]benzoyl-L-glutamic acid; DPO, doxorubicin- N - p-hydroxy-

phenoxyacetamide; MelPO, melphalan- N - p-hydroxyphenoxyacet-

amide; NHPAP, N -(4-hydroxyphenylacetyl)palytoxin; LY 266070, ;

C-DOX (BMY 46633), ; CM, 7-(phenylacetamido)-cephalosporin mus-

tard; CCM, 7-(4-carboxybutanamido)-cephalosporin mustard; 5-FC,

5-fluorocytosine; WR 1065, S-2-(3-aminopropylamino)ethane thiol;

HMR 1826, N -[(4- R, S)-4-ethoxy-4-(1-O--D-glucopyranuronate)-

butyl]daunorubic in sodium salt.

ENZYME-CATALYZED ACTIVATION OF PRODRUGS 55


4/50

mechanisms of action. More recently, a more rational ap-proach to design prodrugs has been used, which is based on

molecular targets that are responsible for cell transforma-tion. However, this approach has been relatively ineffec-

tive against malignancies because knowledge about theresponsible molecular targets for initiation and progres-

sion of cancer is still incomplete (Huang and Oliff, 2001). A

major problem with the use of many chemotherapeuticagents is their unacceptable damage to normal cells andorgans, a narrow therapeutic index, a relatively poor se-

lectivity for neoplastic cells, and multidrug resistance uponprolonged treatment due to up-regulation of efflux pumps,

increased glutathione S-transferase expression, and en-hanced 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. Pro-drugs are compounds that need to be transformed before

exhibiting their pharmacological action. The term pro-drug was introduced in the late 1950s by Albert (1958).

Prodrugs are often divided into two groups: 1) prodrugsdesigned to increase the bioavailability to improve the

pharmacokinetics of antitumor agents and 2) prodrugsdesigned to locally deliver antitumor agents.

B. Prodrugs Designed to Increase the Bioavailability of Antitumor Drugs

Numerous antitumor drugs possess a limited bioavail-

ability due to low chemical stability, a limited oral ab-sorption, or a rapid breakdown in vivo (i.e., by first-pass

metabolism) (Connors, 1986). To overcome these prob-

lems, various prodrugs that can be activated into anti-tumor drugs have been designed. In this case it is pre-

ferred if prodrugs are activated relatively slowly in theblood or liver, for example, thereby preventing acute

toxic effects due to high peak concentrations of the an-titumor drug (Connors, 1986). Therefore, prodrugs acti-

vated by enzymes with high catalytic efficiencies (kcat / K m), resulting in a rapid activation of the prodrug, are

less attractive than prodrugs activated by enzymes withmoderate catalytic efficiencies. An ideal prodrug de-

signed to increase the bioavailability of an antitumordrug 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 limita-tion of this slow-releasing prodrug concept is that fre-

quently nontumor tissues are also affected. Anotherdrawback of the use of prodrugs activated by enzymes

with low catalytic efficiencies may be the metabolism of these prodrugs by competing enzymes into inactive me-

tabolites (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 de-

crease unwanted side effects (Connors, 1986; Lowenthaland Eaton, 1996; Dubowchik and Walker, 1999). By this

concept, referred to as targeting, organ-specific and tu-mor-specific prodrug activation can be achieved. To spe-

cifically activate prodrugs into a certain organ, eitherthe 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 caseof tumor-specific targeting, the enzyme responsible forprodrug activation should be uniquely present in the

tumor cell. Another possibility for tumor-specific target-ing is by making use of hypoxic environments of solid

tumors that can be treated with bioreductive prodrugsas described below (Lin et al., 1972; Begleiter, 2000). For

both organ- and tumor-specific targeting, enzymes withhigh 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 targetscompletely foreign to the host (Dubowchik and Walker,

1999).

D. Prodrugs Activated by Enzyme Immunoconjugatesand 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 enzymeprodrug therapy (ADEPT) or antibody-directed cataly-

sis, antigens expressed on tumors cells are used to targetenzymes to the tumor site (Fig. 1A). First, an enzyme-

antibody conjugate is administered and allowed suffi-

cient time to bind to tumor cells and to be cleared fromthe circulation. Subsequently, a prodrug is administered

and selectively activated extracellularly at the tumorsite. This concept was originally demonstrated by Phil-

pott et al. (1973) and concerned generation of hydrogenperoxide from glucose catalyzed by glucose oxidase.

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 byusing enzymes of nonhuman origin (Deonarain and

Epenetos, 1994; Dubowchik and Walker, 1999; Syrigosand Epenetos, 1999). However, the scarcity of tumor-

selective antigens is still a limitation in the applicability

of ADEPT. Also, adverse immune effects may cause un-wanted results. Another problem with this strategy isthat the prodrug is activated extracellularly, and there-

fore, the antitumor drug that is released still must crossthe cell membrane. Furthermore, the lack of effective-

ness of ADEPT in humans so far is disappointing in viewof the high efficacy observed in rodent models with im-

munoconjugates (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,

56 ROOSEBOOM ET AL.


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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-de-livery agents, such as cationic lipids, peptides, or naked

DNA, are used for gene targeting. In VDEPT, gene tar-geting is accomplished using viral vectors, with retrovi-

ruses and adenoviruses being the most often used vi-ruses. For both GDEPT and VDEPT, the vector has to be

taken up by the target cells, and the enzyme must bestably expressed in tumor cells. This process is called

transduction. In addition, the prodrug must penetrate

the cell membrane to be activated intracellularly. Be-cause it is generally stated that gene targeting to everycell is impossible, the locally activated drug must also be

able to kill nonexpressing cells, a phenomenon known asthe “bystander effect.”

GDEPT and VDEPT effectiveness has been limited todate by insufficient transduction of tumor cells in vivo;

further research is needed to increase transduction. Toovercome the common problems in ADEPT, protein en-

gineering to humanize immunoconjugates, optimizetheir 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 illus-trated above. To date, many enzymes have been evalu-

ated for their ability to activate prodrugs of antitumor

agents. This review provides a comprehensive inventoryof these enzymes regarding their tissue distribution andpresence in tumor cells. The enzyme kinetic parameters

of the enzymes and their respective prodrugs will also bepresented 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 tissuedistribution, and relative presence in tumor and normal

tissue. Section III summarizes the most important en-zymes 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 reviewwill only give a brief overview of ADEPT, GDEPT, and

VDEPT approaches to be able to put the former ap-proaches into an appropriate perspective (Deonarain

and Epenetos, 1994; Deonarain et al., 1995; Singhal andKaiser, 1998; Dubowchik and Walker, 1999; Syrigos and

Epenetos, 1999; Aghi et al., 2000; Smythe, 2000; Grecoand Dachs, 2001; Huang and Oliff, 2001). In Section IV ,

the concluding remarks and future perspectives of enzy-matic bioactivation of antitumor prodrugs are pre-

sented.

II. Prodrugs Activated by Endogenous Enzymes

Numerous enzymes have been used to activate pro-

drugs of antitumor agents. These enzymes belong to fourInternational Union of Pure and Applied Chemistry

classes. Enzymes from class 1 are the oxidoreductases,enzymes from class 2 represent the transferases, en-

zymes from class 3 are hydrolases, and enzymes fromclass 4 represent the lyases. The enzyme characteristics,

their localization in normal and tumor tissue, and theirprodrugs will be discussed in upcoming paragraphs.

A. Class 1 Oxidoreductases1. Aldehyde Oxidasea. Enzymology of Aldehyde Oxidase. Aldehyde oxi-

dases (EC 1.2.3.1) are FAD-, molybdenum-, and hemeiron-containing enzymes, oxidizing aldehydes to the cor-

responding acids using molecular oxygen (Schomburg and Stephan, 1990 –1998; Klaassen, 1996; Moriwaki et

al., 1997). During this redox reaction, superoxide anionsare also generated. In addition to aldehydes, these en-

zymes also catalyze the oxidation of pyrroles, pyridines,purines, pterins, and pyrimidines. Turnover numbers up

to 4100 min1 (2-methyl-butyraldehyde) and greatly

varying K m values, i.e., from 0.002 mM (methylene blue)

FIG. 1. Principles of drug targeting, using ADEPT, VDEPT, andGDEPT. A, in ADEPT an enzyme antibody is administered that binds tothe surface of antigen-presenting target cells. Subsequently, a prodrug isadministered that is activated by the enzyme, resulting in the formationof a toxic drug. In most cases the toxic drug has to penetrate the cellmembrane to enable cell death. In some cases the drug can cause celldeath without penetration through the cell membrane (e.g., palytoxin). B,a DNA construct containing an enzyme-encoding gene is delivered to thetumor cells using nonviral (GDEPT) or viral (VDEPT) vectors. The geneis transcribed, and the generated mRNA is translated to yield the func-tional enzyme. The enzyme that is subsequently expressed activatesintracellularly a nontoxic prodrug into a toxic drug that causes cell death.



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to 1.3 mM ( N -methylnicotinamide), have been reported

(Schomburg and Stephan, 1990–1998). Aldehyde oxi-

dases 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 oxi-

dase is widely distributed and is mainly located in the

cytosolic fraction, although small amounts have beenobserved in the mitochondria of guinea pig liver (Mori-

waki et al., 1997). Based on immunohistochemical stain-

ing of aldehyde oxidase in rat tissues, high concentra-

tions 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 oxi-

dase in liver and lung was observed. However, in con-

trast 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 tis-

sues, aldehyde oxidase is not present in bladder, pan-creas, 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; Ku-

rosaki et al., 1999).

Although little is known about the differences of alde-

hyde oxidase levels between normal and malignant tis-

sues, it has been shown that the specific aldehyde oxi-

dase 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 dihy-

dropyrimidine dehydrogenase (DPD), thereby prevent-

ing 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 / K m) for 5-ethynyl-2(1H)-pyrimidinone oxidation was 60-fold higher than

for N -methylnicotinamide, a well known aldehyde oxi-

dase model substrate, and the K m value of aldehyde

oxidase for 5-ethynyl-2(1H)-pyrimidinone was 50 M.

After oral administration of 5-ethynyl-2(1H)-pyrimidi-

none to rats (2 or 20 g/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 clear-

ance of 5-ethynyluracil or that other enzymes are in-

volved in the bioactivation of 5-ethynyl-2(1H)-pyrimidi-

none remains unclear.

To overcome the rapid breakdown of 5-FU in the gas-trointestinal tract, 5-fluoro-2-pyrimidinone (5-FP) was

synthesized as a 5-FU prodrug (Table 3, Fig. 2) (Guo etal., 1995). 5-FP is activated by rat liver aldehyde oxidase

with a K m value of 220 M and a V max 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 activitywas 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 P388leukemia cells in mice (Guo et al., 1995).

5-Iodo-2-deoxyuridine (IUdR) has been reported to bean effective radiosensitizer in vitro and in vivo (Kinsella,

1996). However, IUdR is rapidly metabolized by hepaticand extrahepatic enzymes, thereby limiting its bioavail-

ability (Kinsella, 1996). Therefore, 5-iodo-2-pyrimidi-none-2-deoxyribose was developed as a prodrug and

was activated by rat, mouse, and human hepatic alde-hyde oxidase to IUdR (Table 3, Fig. 2). Allopurinol, a

selective inhibitor of xanthine oxidase, did not alter bio-activation (Kinsella et al., 1994, 1998). The oxidation of

5-iodo-2-pyrimidinone-2-deoxyribose in other rodenttissues 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 functionabnormalities were demonstrated in serum. Based on

these promising results, initial phase I clinical studiesare in progress.

d. Discussion of Aldehyde Oxidase as a Prodrug-Acti-

vating Enzyme. Aldehyde oxidase has been used to ac-

tivate prodrugs. However, the wide distribution of thisenzyme does not make it an ideal candidate for organ-

selective targeting. This is illustrated by the fact thatafter oral administration of 5-ethynyl-2(1H)-pyrimidi-

none to rats’ DPD activity, inhibited by released 5-ethy-nyluracil, 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 dif-ference in substrate specificity between species (Johns,1967; Guo et al., 1995). Therefore, animal models used to

test the efficacy of aldehyde oxidase prodrugs might notbe good models for humans. The best strategy is to first

optimize prodrug activation by human aldehyde oxidasein vitro before testing the efficacy in vivo in animals

expressing the human enzyme. 2. Amino Acid Oxidase

a. Enzymology of Amino Acid Oxidase. Amino acidoxidases catalyze stereoselectively the oxidative deami-

nation of amino acids to the corresponding -keto acids

ammonia and hydrogen peroxide (Hamilton, 1985;

58 ROOSEBOOM ET AL.


7/50

T A B L E 1

T i s s u e d i s t r i b u t i o n o f r a t e n z y m e s i n v o l v e d i n p r o d r u g a c t i v a t

i o n

L i v e r

K i d n e y

I n t e s t i n e

B r a i n

L u n g

A d r e n a l

T e s t i s

S k i n

S p l e e n

H e a r t

S e r u m

S t o m a c h

L y m p h n o d e

M u s c l e

A l d e h y d e o x i d a s e a

( 4 . 0 )

( 1 . 4 )

( 1 . 3 )

( )

( 0 . 4 )

N D ( )

( )

( )

N D ( 0 . 6 )

N D

- ( )

( )

- ( )

N D ( )

A m i n o a c i d o x i d a s e

b

l - A A O

d - A A O

( 1 6 . 2 )

( 1 7 . 1 )

( )

( 2 1 . 5 )

( )

( )

( )

( )

( )

( N D )

( N D )

( )

( )

( )

C y t o c h r o m e P 4 5 0 c

0 . 2 2 – 0 . 9 2

0 . 0 5 – 0 . 2 1

0 . 0 2 – 0 . 1 3

0 . 0 2 5 – 0 . 0 5 1

0 . 0 3 5

0 . 5 0

0 . 0 5 – 0 . 1 0

0 . 0 5

0 . 0 2 5

P 4 5 0 r e d u c t a s e d

3 7 . 1

2 5 . 1

4 7 . 4

6 . 1

1 6 . 4

5 3 . 1

7 . 1

D T - d i a p h o r a s e e

3 8 9

2 8

7 6

1 1 7

T h y m i d y l a t e s y n t h a s e f

0 . 1 8

0 . 0 7

0 . 0 4

0 . 1 1

0 . 3 9

1 . 4 2

3 . 3 2

0 . 1 4

N D

T h y m i d i n e p h o s p h o r y l a s e g

1 4

2 . 3

2 5

N D

G l u t a t h i o n e S - t r a n s f e r a s e h

C y t o s o l i c

1 4 0 0

3 3 6

4 2 9

1 9 0

7 9

2 5 3

3 8 5 0

5 6

9 3

M i c r o s o m a l

1 2 6

8 . 5

6 0

7 . 9

1 5

5 2

1 2 9

9

7 . 2

D e o x y c y t i d i n e k i n a s e i

0 . 0 3 2

0 . 0 9 1

C y s t e i n e c o n j u g a t e - l y a s e j

2 . 2

2 3 . 0

0 . 6

0 . 8

0 . 2

0 . 7

0 . 9

a B a s e d o n i m m u n o h i s t o c h e m i c a l s t a i n i n g . , e q u i v o c a l s t a i n i n g ; , l o w s t a i n i n g ; , m o d

e r a t e s t a i n i n g ( M o r i w a k i e t a l . , 1 9 9 6 ) . V a l u e s i n p a r e n t h e s e s i n d i c a t e s p e c i f i c a c t i v i t i e s i n g u i n e a p i g t i s s u e s e x p r e s s e d a s

n

m o l / m i n / m g o f p r o t e i n w i t h p h t h a l a z i n e ( B e e d h a m e t a l . , 1 9 8 7 ) . I n t e s t i n e v a l u e r e p r e s e n t s s u m

o f i l e u m , j e j u n u m , a n d d u o d e n u m ( B e e d h a m e t

a l . , 1 9 8 7 ) .

b E x p r e s s e d a s m o l / m i n / m g o f p r o t e i n w i t h d - a l a n i n e a s a s u b s t r a t e i n h o g t i s s u e s ( K a t a g i r i e t a l . , 1 9 9 1 ) . I n l u n g t i s s u e t h e e n z y m e w a s d e t e c t e d w i t h a n e n z y m e i m m u n o a s s a y ; h o w e v e r , e n z y m e a c t i v i t y c o u l d n o t b e

d

e t e r m i n e d .

c E x p r e s s e d a s n m o l / m g o f m i c r o s o m a l p r o t e i n

( V a i n i o , 1 9 8 0 ) .

d

E x p r e s s e d a s n m o l / m i n / m g o f p r o t e i n w i t h c y t o c h r o m e c a s a s u b s t r a t e ( B e n e d e t t o e t a l . , 1 9 7 6 ) .

e E x p r e s s e d a s n m o l / m i n / m g o f p r o t e i n w i t h m

e n a d i o n e a s a s u b s t r a t e ( S c h l a g e r a n d P o w i s , 1 9 9 0 ) .

f E x p r e s s e d a s n m o l / h / m g o f p r o t e i n w i t h d U M

P a s a s u b s t r a t e ( H a s h i m o t o e t a l . , 1 9 8 8 ) . H i g h

a c t i v i t i e s w e r e o b s e r v e d i n t h y m u s ( 1 1 . 3 3 ) a n d

b o n e m a r r o w ( 2 . 7 0 ) .

g E x p r e s s e d a s m o l / h / g o f t i s s u e w i t h t h y m i d

i n e a s a s u b s t r a t e ( Z i m m e r m a n a n d S e i d e n b e r g

, 1 9 6 4 ) .

h

E x p r e s s e d a s n m o l / m i n / m g o f p r o t e i n w i t h 1

- c h l o r o - 2 , 4 - d i n i t r o b e n z e n e ( C D N B ) a s a s u b s t r a t e ( D e P i e r r e a n d M o r g e n s t e r n , 1 9 8 3 ) .

i E x p r e s s e d a s p m o l / m i n / m g o f p r o t e i n w i t h d e o x y c y t i d i n e a s a s u b s t r a t e ( C h a n e t a l . , 1 9 8 3 ) . I n a n o t h e r s t u d y , h i g h a c t i v i t y i s p r e s e n t i n t h y m

u s , b o n e m a r r o w , s p l e e n , a n d s k e l e t a l m u s c l e (

H a r k r a d e r e t a l . , 1 9 8 0 ) .

j E x p r e s s e d a s n m o l / m i n / m g o f p r o t e i n w i t h S - ( 2 - c h l o r o - 1 , 1 , 2 - t r i f l u o r o e t h y l ) - l - c y s t e i n e a s a s u b s t r a t e ( R o o s e b o o m e t a l . , 2 0 0 2 ) . S i m i l a r t i s s u e d i s t

r i b u t i o n w a s o b s e r v e d w i t h v a r i o u s S e C y s c o n j u g

a t e s a n d S - ( 1 , 2 - d i c h l o r o v i n y l ) -

l - c y s t e i n e ( J o n e s e t a l . , 1 9 8 8 ; R o o s e b o o m e t a l . , 2 0 0 2 ) . I n t e s t i n e a c t i v i t y r e p r e s e n t s t h e s u m o f l a r g e a n d s m a l l i n t e s t i n e .

N D , n o d e t e c t a b l e e n z y m e a c t i v i t y o r n o s t a i n

i n g o b s e r v e d ; , n o d a t a a v a i l a b l e .



8/50

Schomburg and Stephan, 1990 –1998; Curti et al., 1992).In addition, amino acid oxidase from rat can dehydroge-

nate -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 forma-

tion of chloride, cyanide, and selenols, respectively, andconcomitant production of pyruvate and ammonia(Walsh et al., 1971; Miura et al., 1980; Rooseboom et al.,

2001b). Amino acid oxidases are flavoproteins that con-tain FAD as a cofactor, and they are involved in amino

acid catabolism and inflammatory responses. L-Aminoacid 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 bothas a monomer (pig, Candida tropicalis) and a ho-

modimer (Trigonopsis variabilis, Rhodotorula gracilis).Turnover numbers for L-amino acid oxidases up to

11,000 min1 (L-arginine) and for D-amino acid oxidasesup to 43,250 (D-alanine) have been reported. K m values

are in the millimolar range, although lower K m valueshave been measured as well for some amino acids (50 –

100 M) (Hamilton, 1985; Schomburg and Stephan,1990 –1998; Curti et al., 1992).

b. Localization of Amino Acid Oxidase. Amino acidoxidases 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 inthe kidney, with liver containing somewhat fewer. How-

ever, the enzyme is not present in mouse liver (Konno etal., 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 acidoxidase 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 (Table2) (Katagiri et al., 1991). Based on an enzyme immuno-

assay, the enzyme was detected in low amounts in lung,

although no enzyme activity could be detected. In hearttissue from hog amino acid oxidase, activity was notobserved (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 oxi-

dase activity was not observed in human lung, spleen,heart, and serum (Holme and Goldberg, 1982). Differ-

ences in amino acid oxidase concentrations between nor-mal 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

T A B L E 2

T i s s u e d i s t r i b u t i o n o f

h u m a n e n z y m e s i n v o l v e d i n p r o d r u g a c t i v

a t i o n

L i v e r

K i d n e y

I n t e s t i n e

B r a i n

L u n g

A d r e n a l

T e s t i s

S k i n

S p l e e n

H e a r t

S e r u m

S t o m a c h

L y m p h n o d e

M u s c l e

A l d e h y d e o x i d a s e a

/

N D

N D

N D

N D

A m i n o a c i d o x i d a s e b

l - A A O

0 . 3 3

0 . 3 5

N D

N D

N D

N D

N D

d - A A O

4 . 5 1

5 . 9 4

0 . 4 8

N D

N D

N D

N D

C y t o c h r o m e P 4 5 0 c

0 . 2 6 – 1 . 0 2

0 . 0 3

0 . 2 3 – 0 . 5 4

0 . 0 0 5

D T - d i a p h o r a s e d

1 7

1 1 7

1 7

1 0

3 8 7

T h y m i d i n e p h o s p h o r y l a s e e

1 . 3 7

0 . 2 2

0 . 4 2

1 . 4 4

1

. 1 2

0 . 3 5

0 . 2 3

1 . 5 1

N D

G l u t a t h i o n e S - t r a n s f e r a s e f

C y t o s o l i c

1 . 8 0

1 . 4 1

0 . 6 6

0 . 6 1

M i c r o s o m a l

1 0 0

3 4

2 3

2 4

1 5

1

1 4

3

D e o x y c y t i d i n e k i n a s e g

3 7

1 4

3 6

1 1

C y s t e i n e c o n j u g a t e - l y a s e

h

0 . 4 3

0 . 1 3

a B a s e d o n i m m u n o h i s t o c h e m i c a l s t a i n i n g . ,

e q u i v o c a l s t a i n i n g ; , l o w s t a i n i n g ; , m o d e r a t e s t a i n i n g ( M o r i w a k i e t a l . , 2 0 0 1 ) .

b E x p r e s s e d a s m o l / m i n / m g f o r f o u r i n d i v i d u a l s w i t h d - a l a n i n e a s a s u b s t r a t e ( H o l m e a n d G o l d b e r g , 1 9 8 2 ) .

c E x p r e s s e d a s n m o l / m g o f m i c r o s o m a l p r o t e i n

( V a i n i o , 1 9 8 0 ) .

d

E x p r e s s e d a s n m o l / m i n / m g o f p r o t e i n w i t h d

i c h l o r o i n d o p h e n o l a s a s u b s t r a t e ( S c h l a g e r a n d P o w i s , 1 9 9 0 ) . A s i m i l a r t i s s u e d i s t r i b u t i o n w a s o

b s e r v e d w i t h m e n a d i o n e a s a s u b s t r a t e .

e E x p r e s s e d a s n m o l / h / m g o f p r o t e i n w i t h t h y m i d i n e a s a s u b s t r a t e ; i n t e s t i n e a c t i v i t y r e p r e s e n

t s t h e s u m o f c o l o n a n d s m a l l i n t e s t i n e ( Y o s h i m

u r a e t a l . , 1 9 9 0 ) .

f C y t o s o l i c G S T e x p r e s s e d a s n m o l / m i n / m g o f p r o t e i n w i t h b e n z o a p y r e n e - 4 , 5 - o x i d e a s a s u b s t r a t e ( P a c i f i c i e t a l , 1 9 8 8 ) . M i c r o s o m a l G S T e x p r e s s e d a s p e r c e n t a g e o f l i v e r m R N A e x p r e s s i o n t h a

t i s k n o w n t o c l o s e l y c o r r e l a t e

w

i t h G S T p r o t e i n a n d e n z y m e a c t i v i t y ( B u e t l e r e t a l . , 1 9 9 5 ; E s t o n i u s e t a l . , 1 9 9 9 ) .

g E x p r e s s e d a s p m o l / m i n / m g o f p r o t e i n w i t h d

e o x y c y t i d i n e a s a s u b s t r a t e ( A r n e r e t a l . , 1 9 9 2 , # 5 5 9 ) . I n t e s t i n e v a l u e r e p r e s e n t s c o l o n , a n d h e a r t v a l u e r e p r e s e n t s v e n t r i c l e .

h

E x p r e s s e d a s n m o l / m i n / m g o f p r o t e i n w i t h S

- ( 2 - c h l o r o - 1 , 1 , 2 - t r i f l u o r o e t h y l ) - l - c y s t e i n e a s a s u b s t r a t e ( R o o s e b o o m e t a l . , 2 0 0 0 , 2 0 0 2 ) . S i m i l a r t

i s s u e d i s t r i b u t i o n w a s o b s e r v e d w i t h v a r i o u s S e C y s c o n j u g a t e s ( R o o s e b o o m e t

a

l . , 2 0 0 2 ) .

N D , n o d e t e c t a b l e e n z y m e a c t i v i t y o r n o s t a i n

i n g o b s e r v e d ; , n o d a t a a v a i l a b l e .

60 ROOSEBOOM ET AL.


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in brain tumor cells in vitro based on local bioactivationto hydrogen peroxide by D-amino acid oxidase (Stegman

et al., 1998) (Table 4, Fig. 3). cDNA encoding D-aminoacid oxidase of R. gracilis was mutated to remove the

peroxisomal targeting sequence to prevent a possiblyrapid breakdown of hydrogen peroxide by peroxisomal

catalase. Exposure of brain tumor cells to D-alanine re-sulted in an elevated cytotoxicity mediated by oxidative

stress when compared with parental cells. The K m valuefor 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).

SeCys conjugates were recently proposed as kidney-selective prodrugs of pharmacologically active selenols

(Andreadou et al., 1996; Rooseboom et al., 2000). Thesecompounds have been shown to be potent chemopreven-

tive 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 werenot attributable to DNA damage (Ip et al., 2000; Zhu et

al., 2000). Although the precise molecular mechanism of apoptosis induction remains to be elucidated, bioactiva-tion to selenols is thought to be critical (Ip, 1998). SeCys

conjugates were recently reported to be bioactivated bymammalian amino acid oxidases from rat and hog kid-

ney and L-amino acid oxidase from snake venom to hy-drogen peroxide and the corresponding -keto acid

(Rooseboom et al., 2001b) (Fig. 3). K m values were in themicromolar range, and the catalytic efficiencies (kcat / K m)

were comparable with that of L-phenylalanine, known asa good substrate for amino acid oxidases.

In addition to oxidative deamination, amino acid oxi-dases 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 acidoxidase is stereoselective, indicating that the corre-

sponding enantiomers might be used in antitumor andchemopreventive experiments, based on the presence of

L-amino acid oxidase or D-amino acid oxidase in thosesystems. 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 hydro-gen peroxide generation from D-alanine, because sel-

enols 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 (EC1.14.13.8) are involved in the -elimination of SeCys

conjugates (Commandeur et al., 2000; Rooseboom et al.,2001a). Therefore, the relative contribution of these en-

zymes will determine the organ-selective activation of these prodrugs. From in vitro studies, the kidney seems

FIG. 2. Activation of prodrugs by aldehyde oxidase.

TABLE 3 Prodrugs activated by aldehyde oxidases

Prodrug Drug Pharmacology Reference

5-Ethynyl-2(1H)-pyrimidinone 5-Ethynyluracil Mechanism-based inhibitor of DPD Porter et al., 1994

IPdR IUdR Radiosensitizer Kinsella et al., 19945-FP 5-FU Thymidylate synthase inhibitor/incorporated intoDNA and RNA

Guo et al., 1995

TABLE 4 Prodrugs activated by amino acid oxidases

Prodrug Drug Pharmacology Reference

d-alanine Hydrogen peroxide Oxidative stress Stegman et al., 1998

SeCys conjugates Selenols and hydrogen peroxide Apoptosis inducer and cancer preventive agent Rooseboom et al., 2001b



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to be the major organ of prodrug activation, and theorgan selectivity was comparable with S-(1-chloro-1,2,2-

trifluoroethyl)-L-cysteine, which is known to cause selec-tive 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 usedto activate some prodrugs. Amino acid oxidases in hu-

mans are mainly present in liver and kidney with low

levels in the brain, thus implicating possible organ-se-lective targeting. Interestingly, the enzyme is not

present in lung, spleen, heart, and serum. Amino acidoxidases demonstrate high turnover numbers toward

appropriate substrates and are not genetically polymor-phic in European populations (Barker and Hopkinson,

1977), in contrast to many other enzymes. The activa-tion and toxicity of amino acid oxidase-dependent pro-

drugs have so far only been evaluated in vitro; the in vivo efficacy remains to be established.

3. Cytochrome P450 Reductasea. Enzymology of Cytochrome P450 Reductase. Cyto-

chrome P450 reductase (EC 1.6.2.4; NADPH-ferrihemo-

protein oxidoreductase, cytochrome c reductase) is local-ized in the endoplasmic reticulum and catalyzes thereduction 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 toP450 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 reduc-tion results in the formation of semiquinone free radi-

cals. 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 lattercan be converted to hydrogen peroxide and hydroxyl

radicals, thereby initiating lipid peroxidation. Typical K m values are in the micromolar range, and turnover

numbers up to 6100 min1 (cytochrome c) have beenreported (Schomburg and Stephan, 1990 –1998; Klaas-

sen, 1996).b. Localization of Cytochrome P450 Reductase. Cyto-

chrome P450 reductase is located in many tissues (Table

1). In rat tissues the highest activity was found in adre-nal gland followed by intestine (89% of adrenal activity),

liver (70% of adrenal activity), kidney (47% of adrenalactivity), and lung (31% of adrenal activity) (Benedetto

et al., 1976). Rat testis and brain register relatively lowactivity, which is only 13% of the adrenal cytochrome

P450 reductase activity. The lower cytochrome P450reductase activity in lung and kidney than in liver (29%

and 28%, respectively, of hepatic activity) was also ob-served by others (Litterst et al., 1975).

The distribution of cytochrome P450 reductase in hu-mans 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 theliver, 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 reduc-

tase 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 humanliver, lung, pancreas, adrenal gland, and gastrointesti-

nal tract is similar to laboratory animals (Hall et al.,1989). However, cytochrome P450 reductase in the hu-

man kidney is more widely distributed than in the kid-

ney of rat, rabbit, and minipig (Hall et al., 1989). The

FIG. 3. Activation of prodrugs by amino acid oxidase. Reaction of oxidative deamination of SeCys conjugates (A), and reaction of -elimination of SeCys conjugates (B). Reaction (B) is also catalyzed by cysteine conjugate -lyase enzymes ( Section II.D.1.).

62 ROOSEBOOM ET AL.


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distribution is thought to be correlated with P450s, anenzyme 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 mel-anoma 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 doesnot necessarily reflect that of the corresponding tumortissue due to loss of enzyme activity as a result of cell

culturing. Data on the levels of cytochrome P450 reduc-tase between normal and tumor cells are diverse. In

general, cytochrome P450 reductase activity is lower intumor 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 cytochromeP450 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 cyto-

chrome P45

enzyme-catalyzed activation of anticancer prodrugs

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