title: A novel technique to monitor carboxypeptidase G2 expression in suicide gene therapy using 19F magnetic resonance spectroscopy.
abstract: Development and evaluation of new anticancer drugs are expedited when minimally invasive biomarkers of pharmacokinetic and pharmacodynamic behaviour are available. Gene-directed enzyme prodrug therapy (GDEPT) is a suicide gene therapy in which the anticancer drug is activated in the tumor by an exogenous enzyme previously targeted by a vector carrying the gene. GDEPT has been evaluated in various clinical trials using several enzyme/prodrug combinations. The key processes to be monitored in GDEPT are gene delivery and expression, as well as prodrug delivery and activation. {4-[bis(2-chloroethyl)amino]-3,5-difluorobenzoyl}-L-glutamic acid, a prodrug for the GDEPT enzyme carboxypeptidase-G2 (CPG2; K(m) = 1.71 microM; k(cat) = 732 s(-1)), was measured with (19)F magnetic resonance spectroscopy (MRS). The 1 ppm chemical shift separation found between the signals of prodrug and activated drug (4-[bis(2-chloroethyl)amino]-3,5-difluorobenzoic acid) is sufficient for the detection of prodrug activation in vivo. However, these compounds hydrolyze rapidly, and protein binding broadens the MR signals. A new CPG2 substrate was designed with hydroxyethyl instead of chloroethyl groups (K(m) = 3.5 microM, k(cat) = 747 s(-1)). This substrate is nontoxic and stable in solution, has a narrow MRS resonance in the presence of bovine and foetal bovine albumin, and exhibits a 1.1 ppm change in chemical shift upon cleavage by CPG2. In cells transfected to express CPG2 in the cytoplasm (MDA MB 361 breast carcinoma cells and WiDr colon cancer cells), well-resolved (19)F MRS signals were observed from clinically relevant concentrations of the new substrate and its nontoxic product. The MRS conversion half-life (470 min) agreed with that measured by HPLC (500 min). This substrate is, therefore, suitable for evaluating gene delivery and expression prior to administration of the therapeutic agent.
authors.affiliation: Cancer Research UK Clinical Magnetic Resonance Research Group, The Institute of Cancer Research and Royal Marsden NHS Foundation Trust, Sutton, Surrey SM2 5PT, UK.
authors.forename: Laura
authors.initials: L
authors.lastname: Mancini
authors.forename: Lawrence
authors.initials: L
authors.lastname: Davies
authors.forename: Frank
authors.initials: F
authors.lastname: Friedlos
authors.forename: Maria
authors.initials: M
authors.lastname: Falck-Miniotis
authors.forename: Andrzej S
authors.initials: AS
authors.lastname: Dzik-Jurasz
authors.forename: Caroline J
authors.initials: CJ
authors.lastname: Springer
authors.forename: Martin O
authors.initials: MO
authors.lastname: Leach
authors.forename: Geoffrey S
authors.initials: GS
authors.lastname: Payne
mesh_terms: D002021:Buffers; D045744:Cell Line, Tumor; D005461:Fluorine / Q000737:chemistry*; D041722:Genes, Transgenic, Suicide; D015316:Genetic Therapy; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D011355:Prodrugs / Q000737:chemistry; D012996:Solutions; D011623:gamma-Glutamyl Hydrolase / Q000378:metabolism*
chemical_list: D002021:Buffers; D011355:Prodrugs; D012996:Solutions; D005461:Fluorine; D011623:gamma-Glutamyl Hydrolase