A novel technique to monitor carboxypeptidase G2 expression in suicide gene therapy using 19F magnetic resonance spectroscopy.
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.
Mancini
Laura
L
Cancer Research UK Clinical Magnetic Resonance Research Group, The Institute of Cancer Research and Royal Marsden NHS Foundation Trust, Sutton, Surrey SM2 5PT, UK.
Davies
Lawrence
L
Friedlos
Frank
F
Falck-Miniotis
Maria
M
Dzik-Jurasz
Andrzej S
AS
Springer
Caroline J
CJ
Leach
Martin O
MO
Payne
Geoffrey S
GS
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*
D002021:Buffers; D011355:Prodrugs; D012996:Solutions; D005461:Fluorine; D011623:gamma-Glutamyl Hydrolase