Retroviral transfer of human cytochrome P450 genes for oxazaphosphorine-based cancer gene therapy

Cyclophosphamide (CPA) and ifosfamide (IFA) are widely used anticancer prodrugs that are bioactivated in the liver by specific cytochrome P450 enzymes (CYPs). The therapeutic activity of these antitumor agents can be compromised by a low therapeutic index that is, in part, due to the systemic distribution of activated drug metabolites. Here, recombinant retroviruses were used to deliver six different CPA- or IFA-metabolizing human CYP genes to 9L gliosarcoma cells: 2B6, 2C8, 2C9, 2C18 (Met385 and Thr385 alleles), 2C19, and 3A4. Intratumoral cytochrome P450 expression conferred substantial sensitivity to CPA cytotoxicity, with the most dramatic effects seen with CYP2B6. Strong CPA chemosensitivity was also seen following transduction of CYP2C18-Met, despite a very low level of CYP protein expression (>60-fold lower than that of 2B6). In contrast to CPA, the cytotoxicity of IFA was greatest toward tumor cells transduced with CYP3A4, followed by CYPs 2B6 and 2C18-Met. A substantial further increase in chemosensitivity was achieved upon transduction of 2B6 or 2C18-Met-expressing tumor cells with P450 reductase, which provided for more efficient intratumoral prodrug activation and cytotoxicity at lower drug concentrations. With 2B6- plus P450 reductase-transduced tumor cells, CPA but not IFA conferred a strong cell contact-independent bystander cytotoxic effect on non-P450-expressing 9L cells. CPA treatment of tumors that were transduced with 2B6 or 2C18-Met together with P450 reductase and were grown s.c. in immunodeficient mice resulted in a large enhancement of the liver P450-dependent antitumor effect seen with control 9L tumors, with no apparent increase in host toxicity (growth delay of >25-50 days in P450-expressing tumors versus approximately 5-6 days without P450). CYP2B6 plus P450 reductase and CYP2C18-Met plus P450 reductase thus appear to be excellent gene combinations for use with CPA in P450/prodrug activation-based cancer gene therapy.     

Synergistic anticancer effects of ganciclovir/thymidine kinase and 5-fluorocytosine/cytosine deaminase gene therapies

BACKGROUND: A bacterial enzyme, Escherichia coli cytosine deaminase, which converts the prodrug 5-fluorocytosine into the toxic drug 5-fluorouracil, and a viral enzyme, herpes simplex virus thymidine kinase, which converts ganciclovir from an inactive prodrug to a cytotoxic agent by phosphorylation, are being actively investigated for use in gene therapy for cancer. The purpose of this study was to determine whether combining these prodrug-activating gene therapies might result in enhanced anticancer effects. METHODS: Rat 9L gliosarcoma cells were transfected with plasmids containing the E. coli cytosine deaminase gene (9L/CD cells), with plasmids containing the herpes simplex virus thymidine kinase gene (9L/TK cells), or with both expression plasmids (9L/CD-TK cells). The drug sensitivities of the cell lines were evaluated; in addition, the sensitivities of 9L and 9L/CD-TK cells mixed in varied proportions were measured. The effects of prodrug treatment on 9L/CD-TK tumor growth (i.e., size and volume) in nude mice were monitored. The isobologram method of Loewe and the multiple drug-effect analysis method of Chou-Talalay were used to measure the interaction between the two prodrug-activating gene therapies. To elucidate the mechanism of interaction, the phosphorylation of ganciclovir in 9L/CD-TK cells after varying prodrug treatments was studied. RESULTS AND CONCLUSIONS: The presence of transfected cytosine deaminase and thymidine kinase genes in 9L gliosarcoma cells reduced cell survival, both in vitro and in vivo, following treatment with the relevant prodrugs; the effects of the two components appeared to be synergistic and related mechanistically to the enhancement of ganciclovir phosphorylation by thymidine kinase following 5-fluorouracil treatment.     

Gene therapy for malignant gliomas using replication incompetent retroviral and adenoviral vectors encoding the cytochrome P450 2B1 gene together with cyclophosphamide

Cyclophosphamide is an inactive prodrug which is converted by hepatic cytochrome P450 2B1 to cytotoxic metabolites which produce interstrand DNA cross-linking in a cell cycle-independent fashion. The limited ability of these metabolites to cross the blood-brain barrier contributes to the poor activity of cyclophosphamide against brain tumors. In this study we demonstrate that replication deficient retroviral and adenoviral vector-mediated gene transfer of cytochrome P450 2B1 into 9L glioma cells significantly increases the sensitivity of these tumor cells to cyclophosphamide in vitro, and prolongs the survival of animals bearing intracerebral 9L tumors treated with cyclophosphamide in vivo. Attempts to improve the effectiveness of retrovirally mediated transduction of the P450 2B1 gene by increasing the concentration of cyclophosphamide delivered to the tumors using intracarotid and intratumoral injections did not prolong animal survival, although survival was increased when a second treatment with P450-expressing retroviral vectors and cyclophosphamide was administered. These results suggest that in situ transduction of tumor cells with the P450 2B1 gene using retroviral and adenoviral vectors increases their sensitivity to cyclophosphamide and may have a potential role in the therapy of malignant gliomas.     

Diffusible cytotoxic metabolites contribute to the in vitro bystander effect associated with the cyclophosphamide/cytochrome P450 2B1 cancer gene therapy paradigm

Tumor cells become sensitive to the inert prodrug cyclophosphamide (CPA) after transfer of the gene encoding cytochrome P450 2B1. This enzyme activates CPA into 4-hydroxycyclophosphamide, which ultimately degrades into acrolein and phosphoramide mustard, the anticancer and DNA-alkylating metabolite. It is imperative that any prodrug-activating gene therapy strategy against cancer possess the capacity to affect the proliferation of tumor cells even when they do not express the transgene (bystander effect), because current methodologies cannot achieve gene transduction in all tumor cells. Prodrug-activating gene therapy schemes described to date exhibit a bystander effect that is not mediated by conditioned medium in culture and may depend on cell contact. In contrast, we find that CPA-sensitized, P450-expressing C6 glioma cells (C6-P450) transfer cytotoxicity to nonexpressing cells by releasing diffusible metabolites through the medium. A 3-h exposure to the prodrug is necessary and sufficient to achieve killing of the transfected cells, and medium conditioned by these cells can kill untransfected cells with similar potency. This bystander effect occurs in the presence of CPA even when only 10% of cells in culture express the P450 2B1 gene, and it is not reproduced by cells that have been irradiated. In an animal model of intracerebral brain tumors, expression of the P450 2B1 gene within the neoplastic cells enhanced significantly the antitumor effect of CPA, even when it was administered systemically. This study shows that CPA/P450 2B1 gene therapy represents a novel tumor-killing strategy that displays an expanded range of cytotoxic action both spatially and temporally within tumor cells and significantly potentiates the anticancer action of CPA when administered i.v.     

Regression of experimental brain tumors with 6-thioxanthine and Escherichia coli gpt gene therapy

The identification of transgenes with antitumor activity is critical to the development of gene therapy of cancer. Retrovirus-mediated transfer of the Escherichia coli gpt gene into rat C6 glioma cells without subsequent selection still inhibited the proliferation of this mixed polyclonal population upon addition of the prodrug, 6-thioxanthine, with an ID50 of 4.1 microM, whereas parental C6 cells were not affected at a concentration of 500 microM. In a time-course assay, effects of the prodrug on the mixed polyclonal cell proliferation required at least 10 days of exposure. In mixed co-cultures, a bystander effect was not present over the first 4 days of prodrug exposure, but required trypsinization of the co-cultures and replating at lower densities. This "modified" bystander assay thus revealed a 50% decrease in C6 cell proliferation, even when the initial ratio of gpt-expressing to parental C6 cells was as low as 1:19. In a nude mouse model of subcutaneous tumors, co-grafts of C6 glioma and gpt-retrovirus producer cells displayed retarded growth upon exposure to 6-thioxanthine (6-TX). In a nude mouse model of intracerebral tumors, grafting of the gpt-retrovirus producer cells leads to an 80% reduction in intracerebral tumor volumes after 6-TX treatment. This reduction results in a 28% increase in the mean time of survival of animals that harbor intracerebral tumors (p < 0.0005). These antitumor effects indicate that the gpt/6-TX enzyme/prodrug pair is a promising alternative to the thymidine kinase gene and ganciclovir combination in the gene therapy of cancer.     

Cell surface chondroitin sulfate proteoglycans in tumor cell adhesion, motility and invasion

Genes that encode enzymes that convert inactive "prodrugs" into anticancer metabolites may be therapeutically useful against brain tumors. Unlike other genes tested to date in brain tumor models, the Escherichia coli gpt gene is unique in that it not only sensitizes cells to the prodrug 6-thioxanthine (6TX) but also encodes resistance to a different regimen (mycophenolic acid, xanthine, and hypoxanthine), thus providing a means to select for gpt-positive cells. In the present study, rat C6 glioma cells were infected with a retrovirus vector that transduces this gene. A clonal line (C6GPT-7) was derived that exhibited significant 6TX susceptibility in vitro with an ID50 of 2.5 mumol/L, whereas 50% growth inhibition of parental C6 cells was not achieved at concentrations tested (up to 50 mumol/L). This line also exhibited significant sensitivity to 6-thioguanine (6TG), with an ID50 of 0.05 mumol/L, whereas 50% growth inhibition of parental C6 cells was achieved at 0.5 mumol/L. In a "bystander" assay, C6GPT-7 tumor cells efficiently transferred 6TX sensitivity to C6 cells at ratios as low as 1:9 (C6GPT-7:C6). This in vitro bystander effect was abrogated when C6GPT-7 and C6 cells were separated by a microporous membrane, suggesting that it was not mediated by highly diffusible metabolites. In vivo both 6TX and 6TG significantly inhibited the growth of subcutaneously transplanted C6GPT-7 cells but not that of C6 cells in athymic mice. In an intracerebral model, both 6TX and 6TG exhibited significant antiproliferative effects against tumors formed by C6GPT-7 cells. These findings provide a basis for exploring further gene therapy strategies based on in vivo transfer of the E coli gpt gene to provide chemosensitivity against 6TX and 6TG.     

Cancer chemotherapy using suicide genes

Suicide gene therapy is a unique form of drug delivery system that allows for negative selection of malignant cells using a prodrug approach. Malignant cells are transduced with a gene encoding an enzyme that can metabolize an otherwise nontoxic prodrug into a toxic metabolite. The prototype of this system is the herpes simplex virus thymidine kinase gene (HSV-tk). Suicide genes may be introduced into tumor cells either by viral vectors or nonviral methods. Current work is underway to fine tune both the delivery systems and optimize the efficacy of the production of the toxic metabolites. Suicide gene therapy is an exciting strategy currently in clinical trial in the treatment of a number of tumors.     

The choice of prodrugs for gene directed enzyme prodrug therapy of cancer

Prodrugs are chemicals that are pharmacodynamically and toxicologically inert but which can be converted to highly active species. In cancer chemotherapy, enzyme activated prodrugs have been effective against certain animal tumours. However, in the clinic it has been found that human tumours containing appropriately high levels of the activating enzymes were rare and not associated with any particular type of tumour. Gene directed enzyme prodrug therapy (GDEPT) attempts to overcome this problem by killing tumour cells by the activation of a prodrug after the gene encoding for an activating enzyme has been targeted to the malignant cell. Here we summarise the various enzyme/prodrug systems that have been proposed for cancer therapy and comment on their suitability for GDEPT. This is because systems developed for other applications such as antibody directed enzyme prodrug therapy (ADEPT) may not be suitable for GDEPT. What is required are nontoxic prodrugs that can be converted intracellularly to highly cytotoxic metabolites that are not cell cycle specific in their mechanism of action. The active drugs released should also be readily diffusible and exert a bystander effect. Alkylating agents best meet these criteria. An example of a suitable enzyme/prodrug system may be a bacterial nitroreductase that can convert a relatively nontoxic monofunctional alkylating agent to a difunctional alkylating agent that is some ten thousand times more cytotoxic.     

Eradication of rat malignant gliomas by retroviral-mediated, in vivo delivery of the interleukin 4 gene

Overexpression of interleukin 4 (IL-4) can impair the tumorigenicity of glioma cells, but direct evidence of its antitumor efficacy after in vivo gene transfer into malignant gliomas has not been provided. To test this, we first injected into the brain of Sprague Dawley rats a 1:1 mixture of C6 rat glioblastoma cells and psi2.L4SN20 or E86.L4SN50 retroviral producer cells (RPCs), secreting 20 and 50 ng of IL-4/5 x 10(5) cells/48 h, respectively. Twenty-seven and 56% of rats receiving injections with these low- or medium-level IL-4 RPCs, respectively, survived tumor injection, whereas control rats died in about 1 month. E86.L4SN50 RPCs coinjected with 9L gliosarcoma cells into syngeneic Fischer 344 rats yielded similar results. A novel IL-4 RPC clone expressing higher levels of IL-4, E86.L4SN200, coinjected with 9L cells increased to 75% the fraction of long-term survivors and induced tumor regression in 50% of rats when injected into established 9L gliosarcomas. Cured rats developed an immunological memory because they rejected a challenge of wild-type 9L cells into the contralateral hemisphere. Magnetic resonance imaging was used to monitor 9L and C6 gliomas and gave direct evidence for tumor rejection in treated rats. Immunohistology showed inflammatory infiltrates in IL-4-treated tumors in which CD8+ T lymphocytes were more abundant, although CD4+ T lymphocytes, B lymphocytes, and macrophages were also present. Overall, these findings suggest that IL-4 gene transfer is a new, promising approach for treating malignant gliomas.     

Toward antibody-directed enzyme prodrug therapy with the T268G mutant of human carboxypeptidase A1 and novel in vivo stable prodrugs of methotrexate

Antibody-directed enzyme prodrug therapy (ADEPT) has the potential of greatly enhancing antitumor selectivity of cancer therapy by synthesizing chemotherapeutic agents selectively at tumor sites. This therapy is based upon targeting a prodrug-activating enzyme to a tumor by attaching the enzyme to a tumor-selective antibody and dosing the enzyme-antibody conjugate systemically. After the enzyme-antibody conjugate is localized to the tumor, the prodrug is then also dosed systemically, and the previously targeted enzyme converts it to the active drug selectively at the tumor. Unfortunately, most enzymes capable of this specific, tumor site generation of drugs are foreign to the human body and as such are expected to raise an immune response when injected, which will limit their repeated administration. We reasoned that with the power of crystallography, molecular modeling and site-directed mutagenesis, this problem could be addressed through the development of a human enzyme that is capable of catalyzing a reaction that is otherwise not carried out in the human body. This would then allow use of prodrugs that are otherwise stable in vivo but that are substrates for a tumor-targeted mutant human enzyme. We report here the first test of this concept using the human enzyme carboxypeptidase A1 (hCPA1) and prodrugs of methotrexate (MTX). Based upon a computer model of the human enzyme built from the well known crystal structure of bovine carboxypeptidase A, we have designed and synthesized novel bulky phenylalanine- and tyrosine-based prodrugs of MTX that are metabolically stable in vivo and are not substrates for wild type human carboxypeptidases A. Two of these analogs are MTX-alpha-3-cyclobutylphenylalanine and MTX-alpha-3-cyclopentyltyrosine. Also based upon the computer model, we have designed and produced a mutant of human carboxypeptidase A1, changed at position 268 from the wild type threonine to a glycine (hCPA1-T268G). This novel enzyme is capable of using the in vivo stable prodrugs, which are not substrates for the wild type hCPA1, as efficiently as the wild type hCPA1 uses its best substrates (i.e. MTX-alpha-phenylalanine). Thus, the kcat/Km value for the wild type hCPA1 with MTX-alpha-phenylalanine is 0.44 microM-1 s-1, and kcat/Km values for hCPA1-T268G with MTX-alpha-3-cyclobutylphenylalanine and MTX-alpha-3-cyclopentyltyrosine are 1.8 and 0.16 microM-1 s-1, respectively. The cytotoxic efficiency of hCPA1-268G was tested in an in vitro ADEPT model. For this experiment, hCPA1-T268G was chemically conjugated to ING-1, an antibody that binds to the tumor antigen Ep-Cam, or to Campath-1H, an antibody that binds to the T and B cell antigen CDw52. These conjugates were then incubated with HT-29 human colon adenocarcinoma cells (which express Ep-Cam but not the Campath 1H antigen) followed by incubation of the cells with the in vivo stable prodrugs. The results showed that the targeted ING-1:hCPA1-T268G conjugate produced excellent activation of the MTX prodrugs to kill HT-29 cells as efficiently as MTX itself. By contrast, the enzyme-Campath 1H conjugate was without effect. These data strongly support the feasibility of ADEPT using a mutated human enzyme with a single amino acid change.     

Role of CYP3A4 in human hepatic diltiazem N-demethylation: inhibition of CYP3A4 activity by oxidized diltiazem metabolites

The antihypertensive agent diltiazem (DTZ) impairs hepatic drug metabolism by inhibition of cytochrome P450 (CYP). The accumulation of DTZ metabolites in serum occurs during prolonged therapy and leads to decreased DTZ elimination. Thus, DTZ metabolites may contribute to CYP inhibition. This study assessed the role of human CYPs in microsomal DTZ oxidation and the capacity of DTZ metabolites to inhibit specific CYP activities. DTZ N-demethylation varied 10-fold in microsomal fractions from 17 livers (0.33-3.31 nmol/mg of protein/min). DTZ oxidation was correlated with testosterone 6beta-hydroxylation (r = 0.82) and, to a lesser extent, tolbutamide hydroxylation (r = 0.59) but not with activities mediated by CYP1A2 or CYP2E1. CYP3A4 in lymphoblastoid cell microsomes catalyzed DTZ N-demethylation but CYP2C8 and CYP2C9 were also active (approximately 20% and 10% of the activity supported by CYP3A4); seven other CYPs produced little or no N-desmethyl DTZ from DTZ. The CYP3A4 inhibitors ketoconazole and troleandomycin decreased microsomal DTZ oxidation, but inhibitors or substrates of CYP2C, CYP2D and CYP2E1 produced no inhibition. Some inhibition was produced by alpha-naphthoflavone, a chemical that inhibits CYP1As and also interacts with CYP3A4. In further experiments, the capacities of DTZ and three metabolites to modulate human CYP 1A2, 2E1, 2C9 and 3A4 activities were evaluated in vitro. DTZ and its N-desmethyl and N,N-didesmethyl metabolites selectively inhibited CYP3A4 activity, whereas O-desmethyl DTZ was not inhibitory. The IC50 value of DTZ against CYP3A4-mediated testosterone 6beta-hydroxylation (substrate concentration, 50 microM) was 120 microM. The N-desmethyl (IC50 = 11 microM) and N,N-didesmethyl (IC50 = 0.6 microM) metabolites were 11 and 200 times, respectively, more potent. From kinetic studies, N-desmethyl DTZ and N,N-didesmethyl DTZ were potent competitive inhibitors of CYP3A4 (Ki = approximately 2 and 0.1 microM, respectively). CYP3A4 inhibition was enhanced when DTZ and N-desmethyl DTZ underwent biotransformation in NADPH-supplemented hepatic microsomes in vitro, supporting the contention that inhibitory metabolites may be generated in situ. These findings suggest that N-demethylated metabolites of DTZ may contribute to CYP3A4 inhibition in vivo, especially under conditions in which N-desmethyl DTZ accumulates, such as during prolonged DTZ therapy.     

MR virtual endoscopy of the pancreaticobiliary tract

PURPOSE: Studying the metabolism of cytotoxic drugs has become increasingly necessary to predict clinically significant drug-drug interactions and to understand the basis of interindividual variations in the pharmacokinetics of anticancer agents. The aim of this study was to determine the feasibility of using V79 Chinese hamster fibroblasts, which are stably transfected with cytochrome P450 (CYP) cDNAs, to study the metabolism of cytotoxic drugs in vitro. METHODS: The 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine cell survival after incubation with drugs. Gas chromatography/mass spectroscopy was used for the quantitation of metabolites of cyclophosphamide and ifosfamide in culture medium. The coculture technique was used to study the generation of cytotoxic metabolites in culture medium. RESULTS: After treatment with either cyclophosphamide or ifosfamide (100 microM to 1 mM) cytotoxicity was demonstrated in only cytochrome CYP2B1- and cytochrome CYP3A4-expressing cells. Treatment of parental nontransfected cells that were cocultured with CYP-expressing cells with cyclophosphamide resulted in increased sensitivity to this drug. All active and inactive metabolites of cyclophosphamide and ifosfamide were detected in the culture medium. Cyclophosphamide-induced cytotoxicity in CYP2B1- and CYP3A4-expressing cells was abrogated by metyrapone and midazolam/ troleandomycin, respectively. Paclitaxel showed greater cytotoxicity against parental V79 cells than against the CYP2BI-, 2E1-, or 3A4-expressing cells, which was also influenced by cotreatment with CYP inhibitors. CONCLUSIONS: Stable expression of CYP cDNAs by V79 cells provided an in vitro system to study cytotoxic drug metabolism. Cell viability and metabolite assays were used to determine the differential metabolism and effects in different CYP-transfected cell lines treated with cytotoxic drugs. The potential use of this V79 cell expression system is in studying enzymes involved in the metabolism of cytotoxic drugs, especially early in drug development. In addition, this system may be used to determine drug interactions that may influence the outcome of therapy in patients with cancer.     

O6-alkylguanine-DNA alkyltransferase inactivation by ester prodrugs of O6-benzylguanine derivatives and their rate of hydrolysis by cellular esterases

To modulate the bioavailability and perhaps improve the tumor cell selectivity of O6-alkylguanine-DNA alkyltransferase (AGT) inactivators, pivaloyloxymethyl ester derivatives of O6-benzylguanine (BG) were synthesized and tested as AGT inactivators and as substrates for cellular esterases. The potential prodrugs examined were the 7- and 9-pivaloyloxymethyl derivatives of O6-benzylguanine (7- and 9-esterBG), and of 8-aza-O6-benzylguanine (8-aza-7-esterBG and 8-aza-9-esterBG) and the 9-pivaloyloxymethyl derivative of 8-bromo-O6-benzylguanine (8-bromo-9-esterBG). The benzylated purines were all potent inactivators of the pure AGT and of the AGT activity in HT29 cells and cell extracts. Each ester was at least 75 times less potent than the corresponding benzylated purine against the pure human AGT. In contrast, the activities of esters and their respective benzylated purine were similar in crude cell extracts and in intact cells. The increase in potency of esters in cellular extracts could be explained by a conversion of the respective prodrug to the more potent benzylated purine in the presence of cellular esterases. The apparent catalytic activity (Vmax/Km) of liver microsomal esterase for 8-azaBG ester prodrugs was 70-130 times greater than for BG prodrugs and 10-20 times greater than for 8-bromo-9-esterBG. Tumor cell hydrolysis of the esters varied considerably as a function of cell type and prodrug structure. These data suggest that these or related prodrugs may be advantageous for selective AGT inactivation in certain tumor types.     

Drug metabolism in hepatocyte sandwich cultures of rats and humans

Adult hepatocytes from rat and man were maintained for 2 weeks between two gel layers in a sandwich configuration to study the influence of this culture technique on the preservation of basal activities of xenobiotic-metabolizing phase I and phase II enzymes. The response of these enzyme activities to an enzyme inducer was investigated using rifampicin (RIF). Basal levels of cytochrome P-450 (CYP) isozymes were characterized by measuring ethoxyresorufin O-deethylation (EROD), ethoxycoumarin O-deethylation (ECOD), and the specific oxidation of testosterone (T). In hepatocytes from untreated rats, CYP isozyme levels, including the major form CYP 2C11, increased during the first 3 days in culture. After this period of recovery, the levels of CYP 2C11, CYP 2A1, and CYP 2B1 decreased, whereas CYP 3A1 increased. In contrast to these dynamic changes, CYP activities such as CYP 1A2 and the major isozyme CYP 3A4 were largely preserved until day 9 in cultures of human hepatocytes. In measuring phase II activities, a distinct increase in glucuronosyltransferase (UDP-GT) activity toward p-nitrophenol (PNP) was found for rat and human hepatocytes over 2 weeks in culture. Sulfotransferase (ST) activity toward PNP showed an initial increase, with a maximum at day 7 and day 9 in culture, respectively, and then decreased until day 14. Glutathione S-transferase (GST) activity decreased constantly during the time of culture. Effects of the enzyme-inducing drug rifampicin on phase I and phase II enzymes were investigated using cultured human hepatocytes. Rifampicin treatment (50 micromol/L) for 7 days resulted in a 3.7-fold induction of CYP 3A4 at day 9 in culture. ECOD activity was increased sixfold and phase II ST activity increased twofold compared to the initial value at day 3. No effect of rifampicin on CYP 3A was found in cultures of rat hepatocytes. These results demonstrate that rat and human hepatocytes preserve the major forms of CYP isozymes and phase II activities and respond to inducing drugs such as rifampicin. The novel hepatocyte sandwich culture is suitable for investigating drug metabolism, drug-drug interactions and enzyme induction.     

Involvement of human CYP1A isoenzymes in the metabolism and drug interactions of riluzole in vitro

Cytochrome P450 (CYP) and uridine diphosphate glucuronosyltransferase (UGT) isoenzymes involved in riluzole oxidation and glucuronidation were characterized in (1) kinetic studies with human hepatic microsomes and isoenzyme-selective probes and (2) metabolic studies with genetically expressed human CYP isoenzymes from transfected B-lymphoblastoid and yeast cells. In vitro incubation of [14C]riluzole (15 microM) with human hepatic microsomes and NADPH or UDPGA cofactors resulted in formation of N-hydroxyriluzole (K(m) = 30 microM) or an unidentified glucuroconjugate (K(m) = 118 microM). Human microsomal riluzole N-hydroxylation was most strongly inhibited by the CYP1A2 inhibitor alpha-naphthoflavone (IC50 = 0.42 microM). Human CYP1A2-expressing yeast microsomes generated N-hydroxyriluzole, whereas human CYP1A1-expressing yeast microsomes generated N-hydroxyriluzole, two additional hydroxylated derivatives and an O-dealkylated derivative. CYP1A2 was the only genetically expressed human P450 isoenzyme in B-lymphoblastoid microsomes to metabolize riluzole. Riluzole glucuronidation was inhibited most potently by propofol, a substrate for the human hepatic UGT HP4 (UGT1.8/9) isoenzyme. In vitro, human hepatic microsomal hydroxylation of riluzole (15 microM) was weakly inhibited by amitriptyline, diclofenac, diazepam, nicergoline, clomipramine, imipramine, quinine and enoxacin (IC50 approximately 200-500 microM) and cimetidine (IC50 = 940 microM). Riluzole (1 and 10 microM) produced a weak, concentration-dependent inhibition of CYP1A2 activity and showed competitive inhibition of methoxyresorufin O-demethylase. Thus, riluzole is predominantly metabolized by CYP1A2 in human hepatic microsomes to N-hydroxyriluzole; extrahepatic CYP1A1 can also be responsible for the formation of several other metabolites. Direct glucuronidation is a relatively minor metabolic route. In vivo, riluzole is unlikely to exhibit significant pharmacokinetic drug interaction with coadministered drugs that undergo phase I metabolism.     

Toward targeted "oxidation therapy" of cancer: peroxidase-catalysed cytotoxicity of indole-3-acetic acids

PURPOSE: The study aimed to identify suitable prodrugs that could be used to test the hypothesis that peroxidase activity in cells, either endogenous or enhanced by immunological targeting, can activate prodrugs to cytotoxins. We hypothesized that prototype prodrugs based on derivatives of indole-3-acetic acid (IAA), when activated by peroxidase enzymes (e.g., from horseradish, HRP) should produce peroxyl radicals, with deleterious biological consequences. METHODS AND MATERIALS: V79 hamster cells were incubated with IAA or derivatives +/- HRP and cytotoxicity assessed by a clonogenic assay. To assess the toxicity of stable oxidation products, prodrugs were also oxidized by HRP without cells, and the products then added to cells. RESULTS: The combination of prodrug and enzyme resulted in cytotoxicity, but neither indole nor enzyme in isolation was toxic under the conditions used. Although lipid peroxidation was stimulated in liposomes by the prodrug/enzyme treatment, it could not be measured in mammalian cells. Adding oxidized prodrugs to cells resulted in cytotoxicity. CONCLUSIONS: Although the hypothesis that prodrugs of this type could enhance oxidative stress via lipid peroxidation was not established, the results nonetheless demonstrated oxidatively-activated cytotoxicity via indole acetic acid prodrugs, and suggested these as a new type of substrate for antibody-directed enzyme-prodrug therapy (ADEPT). The hypothesized free-radical fragmentation intermediates were demonstrated, but lipid peroxidation associated with peroxyl radical formation was unlikely to be the major route to cytotoxicity.