Martijn Rooseboom

Enzyme-Catalyzed Activation of Anticancer Prodrugs

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, β-glucuronidase and cysteine conjugate β-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.

Induction of glutathione-S-transferase mRNA levels by chemopreventive selenocysteine Se-conjugates

Several selenocysteine Se-conjugates (SeCys-conjugates) prevent against chemically induced carcinogenesis. Bioactivation to selenols (RSeH) by beta-lyases is thought to be critical, but the mechanism of tumor suppression remains unclear. Induction of phase II biotransformation enzymes is a possible mechanism of chemoprevention. In this study, we evaluated the isoform-selective induction of glutathione-S-transferase (GST) at the mRNA level using a quantitative reverse transcriptase polymerase chain reaction assay. In cultured primary rat hepatocytes and H35 Reuber rat hepatoma cells, SeCys-conjugates time-dependently increased mRNA levels of GST Alpha isoforms and GST Pi, but not of GST Mu isoforms. Se-allyl-L-selenocysteine, the most potent chemopreventive SeCys-conjugate so far known, was also the most active GST inducer. After exposure for 24hr, it elevated GSTA2, GSTA3, GSTA5, and GSTP mRNA levels in primary hepatocytes 3.2+/-0.4-, 1.9+/-0.1-, 4.3+/-0.3-, and 2.9+/-0.3-fold, respectively. Se-allyl-D-selenocysteine was significantly less active, suggesting that stereoselective conversion of SeCys-conjugates to selenols is involved in GST induction. In H35 Reuber hepatoma cells, where conversion of SeCys-conjugates to selenols was 2-6-fold lower than in primary hepatocytes, GST induction was also much lower than in primary hepatocytes. SeCys-conjugates did not induce cytochrome P450 1A1, 2B1/2, or 3A1. This indicates that SeCys-conjugates are monofunctional inducers of phase II biotransformation enzymes. The present results suggest that induction of GST expression contributes to the chemopreventive activity of SeCys-conjugates.

Chemistry and Biological Activity of Novel Selenium-Containing Compounds

A large number of animal studies and epidemiological studies which have been performed in the last decades has revealed that a number of selenium (Se)-containing compounds possess chemopreventive activity. In a meta-analysis from a number of studies comparing the significance of serum Se, retinol, beta-carotene and vitamin E, Se emerged as the factor with the most consistent protective effect (Comstock et al., 1992). On the other hand, geographic analysis and several prospective and case-control studies showed that people with low blood Se had an increased risk of cancer. Recently, a placebo-controlled double-blind study involving 1312 patients with a history in skin cancer showed a much lower prevalence of developing and dying from lung, colon or prostate cancer in the group receiving supplementation of 200 µg Se per day (Clark et al., 1996). A selenized yeast-extract was given to the people of this study. Relative risk of cancer incidence in lung, colon and prostate was reduced to 0.54 (P=0.04), 0.37 (P=0.002) and 0.42 (P=0.03), respectively. The Se-compounds responsible for the chemopreventive effect of selenized yeast still are not completely characterized yet. Selenomethionine accounts for 20% of the Se-containing compounds (Bird et al., 1997). Other compounds identified include selenocystine, Semethylselenocysteine and selenoethionine, representing approximately 20%. However, still 40-50% of the total Se is unidentified.

Studies on the inhibition of human cytochromes P450 by selenocysteine Se-conjugates

1. To investigate whether cytochrome P450 (P450) inhibition can contribute to the chemopreventive activity of selenocysteine Se -conjugates (SeCys conjugates), 21 SeCys conjugates were screened for their inhibitory potency towards seven of the most important human P450s. 2. The majority of the SeCys conjugates produced near complete inhibition of CYP1A1 at a concentration of 250 µm. The most potent inhibitor, Se -benzyl- l -selenocysteine, displayed an IC 50 of 12.8 ± 1.2µm. CYP2C9, -2C19 and -2D6 were moderately (50-60%) inhibited by the SeCys conjugates. CYP1A2, -2E1 and -3A4 were least inhibited. 3. Studies on the susceptibility of CYP1A1 to SeCys conjugates implicated a thiol-reactive intermediate, as evidenced by reduced inhibition levels in the presence of glutathione and N -acetyl cysteine. Uncoupling of the P450-catalytic cycle was of no importance as ROS scavengers did not influence inhibition levels. 4. P450 inhibition by two physiologically relevant metabolite classes of SeCys conjugates was also studied. N -acetylation of SeCys conjugates consistently increased the inhibitory potency towards CYP1A2, -2C19, -2E1 and -3A4. Beta-lyase catalysed bioactivation of alkyl-substituted SeCys conjugates or Se -benzyl- l -selenocysteine produced little or no additional inhibition of P450 activity. For Se -phenyl- l -selenocysteine, however, significant increases in P450 inhibition were obtained by beta-lyase pre-incubation. 5. It is concluded that the potent and relatively selective CYP1A1 inhibition exerted by SeCys conjugates may contribute to their chemopreventive activity.

Uptake-toxicity relationships of a series of N-substituted N'-(4-imidazole-ethyl)thiourea in precision-cut rat liver slices

A previous study showed that the cytotoxicity of a series of N-p-phenyl-substituted N′-(4-imidazole-ethyl)thiourea in precision-cut rat liver slices increased with increasing electron-withdrawing capacity of the p-substituent and may be related to the Vmax/Km values of bioactivation of the thiourea-moiety by hepatic flavin-containing monooxygenases (FMOs). However, differences in the uptake of xenobiotics into precision-cut liver slices can also have consequences for the rates of metabolism of xenobiotics. In the present study, therefore, we investigated the rate and nature of uptake of 9 N-substituted N′-(4-imidazole-ethyl)thiourea into precision-cut rat liver slices. It was found that a five-fold difference exists among a series of N-substituted N′-(4-imidazole-ethyl)thiourea both in the initial rate of uptake and in the steady-state levels ultimately achieved in the precision-cut rat liver slices. It appeared that the most cytotoxic compounds were also the most readily absorbed compounds. The concentration-dependent initial rate of uptake could be described by a carrier-mediated saturable component and a non-saturable component. At cytotoxic concentrations, the non-saturable component accounted for more than 95% of the total uptake. From this study, it is concluded that differences in rate of uptake of thiourea-containing compounds may be a contributing factor to the differences in bioactivation by FMOs as the basis of the structure–toxicity relationships observed in precision-cut rat liver slices.

Enzymatic pathways of beta elimination of chemopreventive selenocysteine Se conjugates.

Publisher Summary Three naturally occurring selenocysteine Se conjugates (SeCys conjugates), Se-methyl-DL-selenocysteine, Se-(n-propyl)-DL - selenocysteine, and Se-allyl-DLselenocysteine, are relatively potent chemopreventive agents in methylnitrosourea and dimethylbenz[a]anthracene rat mammary tumor models. Enzymes involved in the formation of pharmacologically active selenium metabolites from SeCys conjugates and the assays to measure their respective enzyme activities are described in this chapter. The chapter describes experimental procedures used to investigate the β elimination of SeCys conjugates by cysteine conjugate β-lyases. The chapter also demonstrates that SeCys conjugates are metabolized by at least three different enzyme systems—namely, cysteine conjugate β-lyase/GTK, L-AAO, and FMO. Four biologically active products can be formed from SeCys conjugates by these enzymes: selenols, selenenic acids, selenoxides, and hydrogen peroxide. Theoretically, seleninic acids, of which methylseleninic acid displays chemopreventive activity, can also be formed via oxidation of selenenic acids by hydrogen peroxide.