Differential cytotoxicity and bystander effect of the rabbit cytochrome P450 4B1 enzyme gene by two different prodrugs: Implications for pharmacogene therapy

Frank, Susanne; Steffens, Sabine; Fischer, Ute; Tlolko, Aurelia; Rainov, Nikolai G.; Kramm, Christof M.

doi:10.1038/sj.cgt.7700422

Cited by 15 publications

(19 citation statements)

References 26 publications

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“…Similar results were seen in a range of human hepatocellular carcinoma cell lines (Hep3B, HuH-7, and HepG2) transduced with rabbit CYP4B1 and treated with 4-IM, except that bystander effects seemed to be cell-specific [97]. In a further comparative study [98], 4-IM treatment of CYP4B1-transduced 9L glioma cells showed very little bystander effect (much less than that shown by GVC/HSV-Tk). Thus the utility of 4-IM for GDEPT needs further clarification.…”

Section: Prodrugs For Cyp Enzymessupporting

confidence: 52%

Prodrugs for Gene‐Directed Enzyme‐Prodrug Therapy (Suicide Gene Therapy)

Denny

2003

BioMed Research International

105

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This review focuses on the prodrugs used in suicide gene therapy. These prodrugs need to satisfy a number of criteria. They must be efficient and selective substrates for the activating enzyme, and be metabolized to potent cytotoxins preferably able to kill cells at all stages of the cell cycle. Both prodrugs and their activated species should have good distributive properties, so that the resulting bystander effects can maximize the effectiveness of the therapy, since gene transduction efficiencies are generally low. A total of 42 prodrugs explored for use in suicide gene therapy with 12 different enzymes are discussed, particularly in terms of their physiocochemical properties. An important parameter in determining bystander effects generated by passive diffusion is the lipophilicity of the activated form, a property conveniently compared by diffusion coefficients (log P for nonionizable compounds and log D7 for compounds containing an ionizable centre). Many of the early antimetabolite-based prodrugs provide very polar activated forms that have limited abilities to diffuse across cell membranes, and rely on gap junctions between cells for their bystander effects. Several later studies have shown that more lipophilic, neutral compounds have superior diffusion-based bystander effects. Prodrugs of DNA alkylating agents, that are less cell cycle-specific than antimetabolites and more effective against noncycling tumor cells, appear in general to be more active prodrugs, requiring less prolonged dosing schedules to be effective. It is expected that continued studies to optimize the bystander effects and other properties of prodrugs and the activated species they generate will contribute to improvements in the effectiveness of suicide gene therapy.

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Section: Prodrugs For Cyp Enzymessupporting

confidence: 52%

Prodrugs for Gene‐Directed Enzyme‐Prodrug Therapy (Suicide Gene Therapy)

Denny

2003

BioMed Research International

105

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“…Paramount for the intended use as pro-drug, however, is that the 5-day infusion regimens used in these trials readily achieved 4-IPO serum levels of up to 90 µM [22, 23], a concentration at which effective killing of human transduced HepG2 and primary T-cells expressing the P+12 mutant protein occurs already at 24 h and by >90% after 72 h (Figure 6 and 8). As there is no bystander effect of CYP4B1-activated 4-IPO for neighboring cells in vitro and 4-IPO mediates a proliferation-independent elimination of cells [50], we will next determine the elimination kinetics of circulating and also organ-infiltrating murine and human T-cells expressing the P+12 CYP4B1 in acute graft-versus-host disease (GvHD) models in vivo in mice. A key focus of these preclinical studies will be to determine how quickly the acute GvHD will clinically resolve after 4-IPO administration.…”

Section: Discussionmentioning

confidence: 99%

Identification of amino acid determinants in CYP4B1 for optimal catalytic processing of 4-ipomeanol

Wiek

Schmidt

Roellecke

et al. 2014

Biochemical Journal

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Mammalian CYP4B1 enzymes are cytochrome P450 monooxygenases that are responsible for the bioactivation of several exogenous pro-toxins including 4-ipomeanol (4-IPO). In contrast to the orthologous rabbit enzyme, we show here that native human CYP4B1 with a serine at position 427 is unable to bio-activate 4-IPO and does not cause cytotoxicity in HepG2 cells and primary human T-cells that overexpress these enzymes. We also demonstrate that a proline residue in the meander region at position 427 in human CYB4B1 and 422 in rabbit CYP4B1 is important for protein stability and rescues the 4-IPO bioactivation of the human enzyme, but is not essential for the catalytic activity of the rabbit CYP4B1 protein. Systematic substitution of native and p.S427P human CYP4B1 with peptide regions from the highly active rabbit enzyme reveals that 18 amino acids in the wild-type rabbit CYP4B1 protein are key for conferring high 4-IPO metabolizing activity. Introduction of 12 of the 18 amino acids that are also present at corresponding positions in other human CYP4 family members into the p.S427P human CYP4B1 protein results in a mutant human enzyme (P+12) that is as stable and as active as the rabbit wild-type CYP4B1 protein. These 12 mutations cluster in the predicted B–C loop through F-helix regions and reveal new amino acid regions important to P450 enzyme stability. Finally, by minimally re-engineering the human CYP4B1 enzyme for efficient activation of 4-IPO, we have developed a novel human suicide gene system that is a candidate for adoptive cellular therapies in humans.

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“…Other prodrugs have thus been evaluated for P450 mediated suicide gene therapy, such as the furanoterpenoid 4-ipomeanol, the nucleoside antimetabolite ftorafur, which is converted to 5-fluorouracil, the doxorubicin derivative MMDX, the estrogen receptor antagonist tamoxifen as well as the prodrugs 2-aminoantracene, dacarbazine and procarbazine. The therapeutic effects of 4-ipomeanol in a GDEPT approach using the rabbit P450 isoform 4B1 have been shown in vitro in different hepatocellular carcinoma and glioma cell lines as well as in gliosarcoma cell line xenografts in nude mice (Frank et al, 2002;Mohr et al, 2000;Rainov et al, 1998). A comprehensive overview of the existing P450 prodrugs, including their mechanism of conversion and the function of the actual active drug is given by Rooseboom et al (2004) and Chen and Waxman (2002).…”

Section: Cytochrome P450/cytochrome P450 Reductasementioning

confidence: 99%