Research Article Received: 18 March 2008,
Revised: 19 January 2009,
Accepted: 19 January 2009,
Published online in Wiley InterScience: 3 March 2009
(www.interscience.wiley.com) DOI:10.1002/nbm.1375
A novel technique to monitor carboxypeptidase G2 expression in suicide gene therapy using 19F magnetic resonance spectroscopy Laura Mancinia,y, Lawrence Daviesb, Frank Friedlosb, Maria Falck-Miniotisa, Andrzej S Dzik-Jurasz a, Caroline J. Springer b, Martin O. Leach a and Geoffrey S. Paynea * 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; Km ¼ 1.71 mM; kcat ¼ 732 sS1), was measured with 19F 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 (Km ¼ 3.5 mM, kcat ¼ 747 sS1). 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 19F 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. Copyright ß 2009 John Wiley & Sons, Ltd. Keywords: GDEPT; carboxypeptidase G2;
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F MRS; molecular imaging; pharmacokinetics
INTRODUCTION The effective use of anticancer therapies is often limited by their toxicity to normal tissues. Suicide gene therapy or GDEPT (Gene-directed enzyme prodrug therapy; (1–6)) is designed to overcome this problem by administering a nontoxic prodrug that is activated only at the site of tumor. This selective activation is achieved by first transducing the tumor cells with a gene for the specific exogenous enzyme required for prodrug conversion. This strategy minimizes toxicity to normal tissues and is effective with various enzyme-prodrug systems in cell culture and animal tumor models. GDEPT has also been evaluated in several clinical trials (5). Several different enzyme-prodrug systems are being evaluated for use as GDEPT strategies. One example is bacterial carboxypeptidase G2 (CPG2) (7), which cleaves glutamate groups from appropriate nontoxic prodrugs to generate toxic compounds. It is becoming increasingly clear that clinical trials of new cancer drugs are significantly more efficient and effective when they include measurements of pharmacokinetic and pharmacodynamic behavior. Such measures enhance the rational selection of an optimal drug dose and schedule, aid decision making (such
* Correspondence to: Dr G. S. Payne, Cancer Research UK Clinical Magnetic Resonance Research Group, Royal Marsden Hospital, Downs Road, Sutton, Surrey SM2 5PT, UK. E-mail:
[email protected] a L. Mancini, M. Falck-Miniotis, A. S Dzik-Jurasz, M. O. Leach, G. S. Payne Cancer Research UK Clinical Magnetic Resonance Research Group, The Institute of Cancer Research and Royal Marsden NHS Foundation Trust, Sutton, Surrey SM2 5PT, UK b L. Davies, F. Friedlos, C. J. Springer Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, Sutton, Surrey SM2 5NG, UK y
Present address: Lysholm Department of Neuroradiology, National Hospital for Neurology and Neurosurgery, UCLH NHS Foundation Trust, London, UK. Contract/grant sponsor: Cancer Research UK [CUK]; contract/grant numbers: C309/A8274C1060/A5117. Abbreviations used: BSA, bovine serum albumin; CPG2, carboxypeptidase G2; CPMG, Carr-Purcell-Meiboom-Gill; DMEM, Dulbecco/Vogt modified Eagle’s minimal essential medium; FBS, fetal bovine serum; GDEPT, Gene-directed enzyme prodrug therapy; HRMS, High resolution mass spectrometry; SRB, sulforhodamine B; SW, spectral width (acquisition bandwidth).
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L. MANCINI ET AL. as whether to continue or close a drug development project), and may explain or predict clinical outcomes (8). Biomarkers that can be measured in a minimally invasive fashion are particularly advantageous. For the development of GDEPT, the ability to monitor the degree of targeted gene delivery and expression, as well as subsequent prodrug delivery and activation, would be of great value in both preclinical development of candidate enzymes and prodrugs and their clinical evaluation. The presence of fluorine in several of the prodrugs considered for activation by CPG2 suggests the use of 19F magnetic resonance spectroscopy (MRS) to monitor GDEPT therapy. Although MRS exhibits a lower detection sensitivity compared with radionuclide techniques, the 19F MRS chemical shift is extremely sensitive to the chemical environment, thus increasing the likelihood of a cleaved compound being distinguished from the parent. 19F is also advantageous because it has 100% natural abundance and has one of the highest detection sensivities of the MR nuclei (83% relative to 1H). The absence of background 19 F MR signals from organic compounds in living systems makes 19 F MR spectra relatively easy to interpret and quantify. Previous clinical applications of in vivo 19F MRS include the investigation of the metabolism of 5-fluorouracil and of the effects of co-administering other compounds (9,10), the monitoring of accumulation of antimicrobials in liver (11), and the development of compounds to report on tissue hypoxia (12). It has also been demonstrated that 19F MRS can monitor the conversion of 5-fluorocytosine to 5-fluorouracil using the GDEPT enzyme cytosine deaminase in xenografts (13). In this paper, we explore the use of 19F MRS in monitoring the cleavage by CPG2 of the mustard prodrug {4-[bis(2-chloroethyl) amino]-3,5-difluorobenzoyl}-L-glutamic acid (1) (14) and a stable hydroxyethyl analog with the aim of evaluating suicide gene delivery and expression, as well as drug delivery and activation.
METHODS Synthesis of compounds Prodrug 1, and the activated drug 1a (Figure 1) were synthesized as previously described (14). The prodrug analog 4-[bis(2hydroxyethyl)amino]-3,5-difluorobenzoyl-L-glutamic acid (2) was synthesized in two steps from the previously described 4-(bis(2-hydroxyethyl)amino)-3,5-difluorobenzoic acid (2a) (15). NMR spectra were determined in Me2SO-d6 using TMS or CFCl3 as internal standards on a Bruker Avance 250 spectrometer at 258C. High resolution mass spectrometry (HRMS) was performed using an Agilent 6210 time-of-flight mass spectrometer. Step 1: Diethyl [4-[bis(2-hydroxyethyl)amino]-3,5-difluorobenzoyl]L-glutamate
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Et3N (0.7 ml, 5 mmol) and 3,5-difluoro-4-[bis(2-hydroxyethyl) amino]benzoic acid (0.65 g, 2.5 mmol) were added to a solution of diethyl-L-glutamate hydrochloride (0.6 g, 2.5 mmol) in dry dimethylformamide (40 ml), followed by the addition of diethylcyanophosphonate (0.42 ml, 2.75 mmol). The mixture was stirred for 3 days, the solvent was evaporated, and the residue partitioned between EtOAc (125 ml) and H2O (100 ml). The organic layer was washed with citric acid (50 ml, 10%) and saturated sodium bicarbonate solution (50 ml), dried over MgSO4, and evaporated to dryness. Chromatography on silica gel using CH2Cl2/EtOH as an eluant gave 0.79 g (71%) of pure
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Figure 1. Structures of substrates for CPG2, {4-[bis(2-chloroethyl)amino]3,5-difluorobenzoyl}-L-glutamic acid (1) and 4-[bis(2-hydroxyethyl) amino]-3,5-difluorobenzoyl-L-glutamic acid (2), and of their cleaved products 4-(bis(2-chloroethyl)amino)-3,5-difluorobenzoic acid (1a) and 4-(bis (2-hydroxyethyl)amino)-3,5-difluorobenzoic acid (2a).
product. NMR dH 1.2 (2t, 6H, CH3), 2.05 (m, 2H, CH2CH), 2.44 (t, 2H, CH2CO2, J ¼ 7.5 Hz), 3.3 (m, 4H, CH2N), 3.45 (m, 4H, CH2O), 4.09 (2q, 4H, CH2CH3), 4.4 (m, 1H, CH), 4.53 (t, 2H, OH), 7.54 (d, 2H, H2 þ H6, JHF ¼ 10 Hz), 8.69 (d, 1H, NH, J ¼ 7 Hz); dF 117.92 (d, 2F, F3 þ F5, JHF ¼ 10 Hz). Step 2: 4-[bis(2-hydroxyethyl)amino]-3,5-difluorobenzoyl-L-glutamic acid (2) Diethyl [4-[bis(2-hydroxyethyl)amino]-3,5-difluorobenzoyl]-L-glutamate (0.446 g, 1 mmol) was dissolved in a mixture of ethanol (3 ml) and 1N NaOH (3 ml) and stirred for 3 h. The solution was partitioned between EtOAc (50 ml) and a mixture of H2O (18 ml) and 1N HCl (2 ml). The organic layer was dried over MgSO4 and evaporated to dryness to give 0.30 g (80%) of colorless glassy product. NMR dH 1.9 þ 2.1 (m, 2H, CH2CH), 2.34 (t, 2H, CH2CO2, J ¼ 7.5 Hz), 3.3 (m, 4H, CH2N), 3.46 (dt, 4H, CH2O, J ¼ 6 þ 11.5 Hz), 4.4 (m, 1H, CHCH2), 4.48 (t, 2H, OH, J ¼ 5 Hz), 7.53 (d, 2H, H2 þ H6, JHF ¼ 7.5 Hz), 8.55 (d, 1H, NH, J ¼ 8.5 Hz), 12.5 (s br, 2H, CO2H); dF 117.96 (d, 2F, F3 þ F5, JHF ¼ 10 Hz). HRMS: (M þ Hþ) calculated for C16H21F2N2O7: 391.13113 found 391.13147. In vitro MR The stability and MRS properties of the two 19F-containing substrates of CPG2 (1 and 2), and of their corresponding cleaved compounds 1a and 2a, shown in Figure 1, were studied using 19 F-MRS on a Bruker Avance spectrometer at 11.74 T and at a temperature of 310 K (378C). Compounds were dissolved in 30 ml of DMSO, and then diluted to a final concentration of 1.5 mM with a buffer made with 100 mM TrisDCl at pH 7.3, 260 mM ZnCl2 and 20% D2O. Since the widths of the resonances in MR spectra (and hence their amplitude and resolution) are determined not only by magnetic field homogeneity but also by broadening effects from binding to proteins, the prodrug/drug pair 1 and 1a were also diluted in buffers that additionally contained 0.2, 1, and 4% w/v of bovine serum albumin (BSA), or 10% fetal bovine serum (FBS). Similarly,
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F-MRS DETECTION OF CPG2 ACTIVITY IN GDEPT
the substrate/product pair 2 and 2a were also diluted in the buffer that contained 10% FBS. 19 F MR data were acquired with and without 1H decoupling, with TR ¼ 2 s, acquisition spectral width (SW) ¼ 10 ppm, NS ¼ 16 or 32 as specified in the figure captions, flip angle ¼ 908. Data were acquired with 8 k points, subsequently zero-filled to 16 k points, apodized with an exponential function (line broadening 1 Hz), Fourier transformed and phase corrected. The 19F chemical shifts are specified with respect to trichlorofluoromethane at 0 ppm (based on a measurement in a separate capillary). For measurements using repeated acquisition to follow the time course of enzymatic cleavage, the 19F MRS signals were quantified using peak integration, normalized to the area of the peak in the first spectrum. The stability of the compounds in the solution was assessed by measuring the peak amplitudes of proton-decoupled 19F-spectra acquired at regular intervals (every 10, 15, or 20 min depending on the compounds). The relaxation times T1 and T2 of the compounds were measured, together with their half-lives and line widths. T1 measurements used an inversion recovery sequence without 1 H decoupling (TR ¼ 6 s, SW ¼ 3 ppm, NS ¼ 16) and with inversion times TI ¼ 1, 200, 500, 700, 1500, 5000 ms. The relaxation times were calculated from the peak amplitudes (measured with the Bruker package) using the Origin software (Origin Labs, Northampton, MA, USA), fitting with the function Mz(TI) ¼ M0-(M0-Mz0) exp(-TI/T1), where M0 (the equilibrium magnetization), Mz0 (the magnetization along the z axis immediately after the inversion pulse) and T1 were the fitted parameters. T2 measurements used a CPMG sequence without 1H decoupling. Parameters used were TR ¼ 3.5 s, SW ¼ 3 ppm, NS ¼ 32, TE ¼ 4, 10, 40, 100, 200, 400, 800 ms (echo delay ¼ 2 ms) for 1 and 1a in buffer and for compound 2 in 10% FBS; TE ¼ 8, 20, 80, 200, 400, 800, 1600 ms (echo delay ¼ 2 ms) for 2a in buffer and in 10% FBS; TE ¼ 4, 8, 12, 16, 20, 24 ms (echo delay ¼ 2 ms) for 1 and 1a in 0.2% and 1% BSA and in 10% FBS; TE ¼ 0.4, 0.8, 1.2, 1.6, 2.0, 2.4 ms (echo delay ¼ 0.2 ms) for 1 in 4% BSA. The peak amplitudes were fitted with the function Mxy(TE) ¼ M0 exp(-TE/T2), where M0 and T2 were the fitted parameters. The cleavage of the substrate by CPG2 was assessed by adding CPG2 enzyme to make a final concentration of 0.015 unit ml1 in 1.5 mM of compound in buffer. 1H-decoupled 19F spectra were acquired continuously until no further reaction was observed (TR ¼ 2 s, SW ¼ 10 ppm, NS ¼ 16 or 32 as specified in the figure captions, flip angle ¼ 908, 8 k points acquired, zero-filled to 16Yk points). Cell measurements The pharmacokinetics of 2 was assessed in two separate cell lines, human MDA MB 361 breast carcinoma cells and human WiDr colon cancer cells, which had been engineered to express approximately 0.6 unit g1 (units per gram of wet weight when the cells are grown as a xenograft) and 4 unit g1, respectively, of the enzyme CPG2 in the cytoplasm (7). Confluent monolayers of cells were grown in 25 cm2 tissue culture flasks in Dulbecco/Vogt modified Eagle’s minimal essential medium (DMEM) with 10% FBS. For the MRS experiment the monolayers (containing approximately 7.8 105 MDA MB 361 cells and 3.1 106 WiDr cells) were rinsed three times, and the growth medium was replaced by serum-free DMEM (5 ml). 0.5 ml of a 10 mM solution of 2 in the same medium was added to the cells, giving an overall concentration of 2 of 0.91 mM.
The cells in the cell culture flask were measured in a 7 T Bruker Biospec spectrometer (Ettlingen, Germany) using a 1 cm diameter transmit/receive 19F surface coil. Temperature was maintained at approximately 34.58C by raising the temperature of the water in the gradient coils within the magnet bore. This was done 12 h before the experiment in order to achieve a stable temperature of the system. The temperature was measured with a thermocouple close to the flask, but far enough from the RF receiver coil to avoid signal artifacts. The cell flask was first positioned in the scanner to perform the shim; the temperature at this time was measured to be 32.28C. The sample was then extracted from the magnet to add compound 2 and put back in exactly the same position. This process for MDA MB 361 cells caused the temperature to drop to 29.18C. It subsequently rose to a stable temperature of 34.5 0.18C after 238 min. For WiDr cells the initial temperature drop was minimal (from 32.2 to 32.08C) and a stable temperature of 34.7 0.18C was reached after 198 min. 19F spectra without 1 H decoupling were acquired continuously for approximately 26 h, using a 908 Gaussian excitation pulse (calibrated using the scanner software), TR ¼ 1.6 s, SW ¼ 100 ppm and 256 acquisitions (giving a time resolution of 7 min). Data were acquired with 32 k points, subsequently zero-filled to 64 k points, apodized with an exponential function (line broadening 1 Hz), Fourier transformed and phase corrected. After the MRS experiment, the medium was centrifuged and the supernatant analyzed for product 2a by HPLC. Aliquots (10 ml) were injected onto a phenyl reverse phase column (Synergi Polar RP, Phenomenex UK; 250 4.6 mm) and eluted by a 0–50% acetonitrile gradient over 15 min buffered at pH 7 by 100 mM ammonium acetate, and monitored by optical absorption at 300 nm. Under these conditions, substrate 2 and product 2a eluted as resolved peaks at 3.3 and 4.9 min, respectively.
RESULTS AND DISCUSSION Prodrug 1 and drug 1a in solution 1
H-decoupled 19F MR spectra of prodrug 1 and the corresponding activated product 1a each showed just one peak (at 118.0 and 119.0 ppm, respectively), since in both cases the two fluorine nuclei occupy chemically equivalent positions (Figure 1). For typical magnetic field homogeneities achievable in vivo (usually 360 660 12 150 110 7 203 29 337 14 >24 h >24 h >24 h
T2 ¼ 1/(p line-width); ND ¼ not done. Note that in the presence of protein an ‘effective’ relaxation time constant is measured, owing to fast exchange between the bound and free forms. For T2 the result also depends on the sequence used and pulse spacing. All measurements were made without 1H decoupling.
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F-MRS DETECTION OF CPG2 ACTIVITY IN GDEPT
Figure 3. The kinetics of cleavage of 1.5 mM 2 (squares) to 2a (circles) in buffer with 10% FBS following addition of CPG2 enzyme (final concentration 0.015 unit ml1) as measured with 1H-decoupled 19F MRS at 11.74 T, with TR ¼ 2 s and 16 transients per data point. For clarity only a subset of time points is shown.
Figure 4. 19F MR spectra obtained after addition of 0.91 mM 2 to the culture flask containing cultured MDA MB 361 cells expressing CPG2, showing cleavage to 2a. Relative concentrations were measured by 19 F MRS at 7 T with TR ¼ 1.6 s, 256 transients and no 1H decoupling. The time to the right of each spectrum indicates the midpoint of the spectral acquisition. For clarity spectra acquired only at specific intervals are shown in the figure, while all data were included in the analysis. The slow time-dependent drift in chemical shifts was due to the temperature equilibration within the magnet bore (see Methods).
drop described in the Methods section is probably responsible for the change in the chemical shift positions of both compounds at early time points (this spectrometer is not equipped with a lock channel). Cleavage from substrate 2 to product 2a was approximately linear in time, as in FBS solution. Quantitatively the rate of decrease of substrate 2 (0.098 0.002% min1, falling to 50% of its initial concentration after about 470 min) was faster than the apparent rate of increase of the product 2a (0.076 0.001% min1). This rate difference is probably due to greater partial saturation at this TR (2s) of product 2a (T1 ¼ 1016 ms in 10% FBS) compared with substrate 2 (T1 ¼ 563 ms in 10% FBS). If the T1 values in FBS are used to correct for the signal intensities in the cell suspension, then one would expect a rate of appearance of product 2a of 0.074% min1, which is very close to the observed value of 0.076% min1. HPLC analysis of the medium at the end of the experiment detected 758 mM of 2a, corresponding to 83% of the total compound initially present in the medium. This is in good agreement with the MR measurement of 88%. Separate HPLC analyses, at 378C also showed that substrate 2 reached 50% of its initial concentration after approximately 500 min (Figure 5b), in accordance with the MRS results (Figure 5a; 470 min). Similar measurements were performed with CPG2-expressing WiDr cells. In this case, the MR signal was detected from a region containing approximately 2.2 105 cells. The conversion half-life for 2 of 326 min was shorter than that for the MDA MB 361 cells (470 min). This difference may be due to the higher expression of enzyme in the WiDr cells, or other factors affecting intracellular uptake of the prodrug such as diffusion and membrane transport.
Figure 5. Pharmacokinetics of cleavage of 2 (squares) to 2a (circles) in a confluent monolayer of MDA MB 361 cells expressing CPG2: (a) relative concentrations measured with 19F MRS (integrated peak areas from the spectra in Figure 4), following addition of 0.91 mM of 2 to the cell culture medium and (b) Experiment as in (a) but with measurement of absolute concentrations with HPLC.
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L. MANCINI ET AL. Since the compounds do not contain reactive mustard groups, they are expected to be nontoxic. Both 2 and 2a gave no growth inhibition at 1 mM in a sulforhodamine B (SRB) assay (17) using either CPG2 expressing or non-expressing WiDR cells (data not shown). The properties of the new substrate 2 seem well suited to following GDEPT enzyme expression in vivo with 19F MRS. The compound remains stable until cleaved by the enzyme CPG2, and the resonances are relatively sharp (approximate linewidth 28 Hz). The concentrations used (0.9 mM) are clinically relevant and yielded 19F MR spectra with a good signal-to-noise ratio in a few minutes. In principle a similar methodology could be applied to the monitoring of a range of other gene therapy systems.
CONCLUSIONS Two fluorine-containing substrates of CPG2 have been synthesized, which are detectable in cell culture systems (monolayers) by 19 F MRS, and demonstrate a chemical shift change upon cleavage of approximately 1 ppm, which should be sufficient for resolution in vivo. The nontoxic analog 2 has certain MRS characteristics that are advantageous compared with the prodrug 1. In particular, 2 is stable in solution and exhibits a single narrow MR resonance which is not significantly broadened by the presence of bovine and fetal bovine serum albumin. Therefore, at a given concentration the 19F MR signals for 2 will have a higher signal-to-noise ratio compared with 1, and will therefore, lead to larger signals for a given concentration than 1. In solution, compound 2 is rapidly cleaved by CPG2 to give the well-resolved product 2a with linear kinetics. In cell lines expressing CPG2, compound 2 can be easily detected by 19F MRS at clinically relevant concentrations (0.9 mM), and cleavage to form 2a can be readily monitored. Therefore, compound 2 has potential for noninvasive monitoring of the expression of CPG2 in GDEPT therapy using 19F MRS. However, a linear relationship between the observed reaction rate and expression levels of CPG2 has yet to be demonstrated. Similar methodology may be applied to monitoring a range of other therapy systems.
Acknowledgements We thank Prof Paul Workman and Dr Dan Niculescu-Duvaz for helpful discussions and support.
REFERENCES 1. Niculescu-Duvaz I, Cooper RG, Stribbling SM, Heyes JA, Metcalfe JA, Springer CJ. Recent developments in gene-directed enzyme prodrug therapy (GDEPT) for cancer. Curr. Opin. Mol. Ther. 1999; 1: 480–486.
2. Blaese RM, Culver KW, Ishii H, Oldfield EH, Ram Z, Wallbridge S. Brain tumor treatment: significant contributions. Science 1992; 258: 1960. 3. Pandha HS, Martin LA, Rigg A, Hurst HC, Stamp GW, Sikora K, Lemoine NR. Genetic prodrug activation therapy for breast cancer: a phase I clinical trial of erbB-2-directed suicide gene expression. J. Clin. Oncol. 1999; 17: 2180–2189. 4. Niculescu-Duvaz I, Springer CJ. Introduction to the background, principles, and state of the art in suicide gene therapy. Mol. Biotechnol. 2005; 30: 71–88. 5. Dachs GU, Tupper J, Tozer GM. From bench to bedside for genedirected enzyme prodrug therapy of cancer. Anticancer Drugs 2005; 16: 349–359. 6. Portsmouth D, Hlavaty J, Renner M. Suicide genes for cancer therapy. Mol. Aspects Med. 2007; 28: 4–41. 7. Marais R, Spooner RA, Light Y, Martin J, Springer CJ. Gene-directed enzyme prodrug therapy with a mustard prodrug/carboxypeptidase G2 combination. Cancer Res. 1996; 56: 4735–4742. 8. Workman P, Aboagye EO, Chung YL, Griffiths JR, Hart R, Leach MO, Maxwell RJ, McSheehy PM, Price PM, Zweit J. Minimally invasive pharmacokinetic and pharmacodynamic technologies in hypothesistesting clinical trials of innovative therapies. J. Natl. Cancer Inst. 2006; 98: 580–598. 9. Findlay MP, Leach MO, Cunningham D, Collins DJ, Payne GS, Glaholm J, Mansi JL, McCready VR. The non-invasive monitoring of low-dose, infusional 5-fluorouracil and its modulation by interferon-alpha using in vivo 19F magnetic resonance spectroscopy in patients with colorectal cancer: a pilot study. Ann. Oncol. 1993; 4: 597–602. 10. Presant CA, Wolf W, Waluch V, Wiseman C, Kennedy P, Blayney D, Brechner RR. Association of intratumoral pharmacokinetics of fluorouracil with clinical response. Lancet 1994; 343: 1184–1187. 11. Payne GS, Collins DJ, Loynds P, Mould G, Murphy PS, Dzik-Jurasz AS, Kessar P, Haque N, Yamaguchi M, Atarashi S, Leach MO. Quantitative assessment of the hepatic pharmacokinetics of the antimicrobial sitafloxacin in humans using in vivo 19F magnetic resonance spectroscopy. Br. J. Clin. Pharmacol. 2005; 59: 244–248. 12. Seddon BM, Payne GS, Simmons L, Ruddle R, Grimshaw R, Tan S, Turner A, Raynaud F, Halbert G, Leach MO, Judson I, Workman P. A phase I study of SR-4554 via intravenous administration for noninvasive investigation of tumor hypoxia by magnetic resonance spectroscopy in patients with malignancy. Clin. Cancer Res. 2003; 9: 5101–5112. 13. Stegman LD, Rehemtulla A, Beattie B, Kievit E, Lawrence TS, Blasberg RG, Tjuvajev JG, Ross BD. Noninvasive quantitation of cytosine deaminase transgene expression in human tumor xenografts with in vivo magnetic resonance spectroscopy. Proc. Natl. Acad. Sci. U S A. 1999; 96: 9821–9826. 14. Davies LC, Friedlos F, Hedley D, Martin J, Ogilvie LM, Scanlon IJ, Springer CJ. Novel fluorinated prodrugs for activation by carboxypeptidase G2 showing good in vivo antitumor activity in genedirected enzyme prodrug therapy. J. Med. Chem. 2005; 48: 5321–5328. 15. Friedlos F, Davies L, Scanlon I, Ogilvie LM, Martin J, Stribbling SM, Spooner RA, Niculescu-Duvaz I, Marais R, Springer CJ. Three new prodrugs for suicide gene therapy using carboxypeptidase G2 elicit bystander efficacy in two xenograft models. Cancer Res. 2002; 62: 1724–1729. 16. Oconnor CJ, Denny WA, Fan JY, Gravatt GL, Grigor BA, McLennan DJ. Hydrolysis and alkylating reactivity of aromatic nitrogen mustards. J. Chem. Soc. Perkin Trans. 2 1991; 1933–1939. 17. Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, Warren JT, Bokesch H, Kenney S, Boyd MR. New colorimetric cytotoxicity assay for anticancer-drug screening. J. Natl. Cancer Inst. 1990; 82: 1107–1112.
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