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.

Rectal carcinoma: MRI with histologic correlation before and after chemoradiation therapy.

OBJECTIVE: The purpose of this study was to use MRI to compare the morphologic features of rectal cancer before and 6 weeks after chemotherapy and radiation treatment to correlate the posttreatment MRI appearances with the histologic findings in resected tumors. MATERIALS AND METHODS: High-resolution T2-weighted MRI was performed before and immediately after a standardized 5-week course of chemoradiation therapy in the care of 30 patients with locally advanced adenocarcinoma of the rectum. Changes in morphologic features were evaluated with respect to primary tumor and nodal downstaging. The MRI findings after chemoradiation therapy were compared with the histologic findings in the resected specimens with respect to prediction of tumor stage and showing the relation between the tumor and the circumferential margin of resection. RESULTS: Tumor shrinkage > 30% was found in 19 (63%; 95% CI, 46-81%) of 30 patients, but changes in MRI T stage occurred in only five (17%; 95% CI, 3-30%) of 30 patients. Tumor regression from the circumferential resection margin was found in five patients, all findings confirmed at histologic examination. Nodal downstaging was observed in 13 (68%; 95% CI, 48-89%) of 19 patients; 11 patients were node free on the basis of both MRI findings and subsequent histologic results. Overall prediction of distance between tumor and circumferential resection margin was good, with a mean difference of -0.2 mm and an interclass correlation coefficient of 0.74. MRI was not useful for gauging disease activity of persistent abnormalities in mucinous tumors that often represented inactive mucin lakes. CONCLUSION: Decreases in tumor size and nodal downstaging can be seen on MRI after chemoradiation therapy in approximately two thirds of patients. The surgically more relevant parameter--distance between tumor and circumferential resection margin--can be accurately predicted. Errors were caused by the presence of considerable tumor, rectal wall fibrosis, and mucinous tumors.

Identification of biliary metabolites of ifosfamide using 31P magnetic resonance spectroscopy and mass spectrometry.

Biliary excretion is a significant component in the metabolism of many drugs, but remains difficult to detect and characterise non-invasively. A previous publication recently described the detection of metabolites of ifosfamide in gall bladder in a guinea pig model using in vivo 1H-decoupled 31P 3-D magnetic resonance spectroscopic imaging and a clinical 1.5 T MR scanner.. Here high-resolution 31P magnetic resonance spectroscopy (MRS) of extracted bile identifies peaks as parent ifosfamide (1.19+/-1.47 mM; mean+/-sd), carboxyifosfamide (2.04+/-1.04 mM) and a major contribution from a previously unreported peak at 16.0 ppm (4.05+/-2.38 mM). The unknown resonance was identified using liquid chromatography-mass spectrometry (LCMS) as the glutathione conjugate of ifosfamide (MW=531). This was confirmed by analysing products from the reaction of glutathione with ifosfamide using LCMS and MRS. These results demonstrate how combined in vivo and analytical MRS, together with mass spectrometry, can help identify visceral routes of drug metabolism, thereby aiding understanding of +/-drug disposition and mechanisms of action and toxicity. In particular, the distribution of ifosfamide and its metabolites into bile may be related to oxazophosphorine-related cholecystitis reported in patients.

The effect of Gd-DTPA on T(1)-weighted choline signal in human brain tumours.

The influence of Gd-DTPA on T(1)-weighted (T(1)W) proton MR spectra has been investigated in 19 patients with histologically verified low (n = 13) or high-grade (n = 6) gliomas. Repeat measurements were performed on 9 patients (7 low-grade and 2 high-grade), with 28 examinations performed in total. Comparison of spectra obtained before and after 0.2 mmol/kg Gd-DTPA showed contrast agent induced broadening of the choline signal without significant signal area change. Lack of enhancement of the choline signal with the T(1)-weighted acquisitions implies that the contrast agent and the trimethylamine-containing species do not undergo significant direct interaction. Contrast agent induced changes in the choline signal observed in this and previous studies may, therefore, be attributable to T2*/susceptibility-based effects.

Localized COSY and DQF-COSY 1H-MRS sequences for investigating human tibial bone marrow in vivo and initial application to patients with acute leukemia.

PURPOSE: To develop 2D 1H-MRS measurement sequences for the evaluation of bone marrow lipids, and to assess these measurement sequences in healthy and diseased bone marrow. MATERIALS AND METHODS: Single-voxel localized variants of COSY and DQF-COSY 2D 1H-MRS sequences were developed for use at 1.5 T to investigate the biochemical composition of human bone marrow in vivo. The performance of each sequence was initially tested in vitro using lipid phantoms. An unsaturated lipid proton index was developed to interrogate the degree of unsaturation within the triacylglyceride (TAG) acyl chains. Localized 2D 1H-MRS data were obtained from the bone marrow of healthy controls (N = 6), patients presenting with acute leukemia (N = 6) and patients with acute leukemia in remission (N = 4). RESULTS: The COSY and DQF-COSY data recorded from all subject cohorts were similar, and the unsaturated lipid proton index did not reveal significant differences between patient groups. Variations in water content and measured relaxation times showed minor differences between the measurement groups. CONCLUSIONS: No significant differences were observed in the spectra obtained from bone marrow using the 2D 1H-MRS sequences. A novel unsaturated lipid proton index was developed.

Quantitative assessment of the hepatic pharmacokinetics of the antimicrobial sitafloxacin in humans using in vivoF magnetic resonance spectroscopy.

AIMS: To measure hepatic concentrations of the fluorine-containing antimicrobial, sitafloxacin, using in vivo(19)F magnetic resonance spectroscopy (MRS). METHODS: Data were acquired from eight healthy subjects at 2, 5, 8 and 24 h following doses of 500 mg day(-1) for 5 days using a (1)H/(19)F surface coil in a 1.5T clinical MR system. Tissue water was used as a reference. RESULTS: Estimated liver concentrations at 2 h were 15.0 +/- 4.0 microg ml(-1) (mean +/- 95% CI), compared with 3.54 +/- 0.58 microg ml(-1) in plasma (n = 6), and fell below threshold concentrations (2 microg ml(-1)) by 24 h. CONCLUSIONS: (19)F MRS is able to detect and quantify sitafloxacin in the liver. There was no evidence for the hepatic retention of the drug.

Human gallbladder bile: noninvasive investigation in vivo with single-voxel 1H MR spectroscopy.

Proton (hydrogen 1) magnetic resonance (MR) spectroscopy was used to study model and porcine bile in vitro. The method was subsequently developed to facilitate the acquisition of in vivo 1H MR spectra from the gallbladder bile of 10 human volunteers. Signals attributable to phosphotidylcholine and conjugated bile acid protons were observed in eight of the 10 volunteers. Phosphotidylcholine concentrations were estimated, and five values (mean = 35.8 mmol/L, SD = 9.8) were within the expected range of levels in human bile. Findings in this preliminary investigation indicate that human gallbladder bile can be qualitatively and quantitatively studied noninvasively with 1H MR spectroscopy.

Ifosfamide pharmacokinetics and hepatobiliary uptake in vivo investigated using single- and double-resonance 31P MRS.

MRS has considerable potential for the measurement of drug pharmacokinetics in vivo. In this study single- and double-resonance (31)P MRS was used to investigate the biodistribution, pharmacokinetics, and metabolism of ifosfamide following administration of 500 mg/kg ifosfamide in guinea pigs. (1)H-decoupling was found to nearly double the signal of detected peaks. However, in contrast to studies of ifosfamide in solution, the polarization transfer sequence gave no further signal enhancements. This was attributed to significantly reduced relaxation times in vivo. Chemical shift imaging (CSI) measurements showed that significant proportions of ifosfamide-related (31)P MRS signals arose from the liver, as expected, but also from the gall bladder, which was not predicted from the current literature. Signals were observed within 5 min of the end of administration. The halflife in liver was approximately 74 min, whereas in gall bladder there was no measurable signal decay during the 2.5-hr studies. High-resolution (31)P MRS of bile showed that the "ifosfamide" peak in vivo consists of at least two compounds. The lower-concentration peak is ifosfamide, and an investigation is under way to identify the higher-concentration peak. Other peaks observed in bile are tentatively assigned to carboxy-ifosfamide and dechloroethyl-ifosfamide. Overall, (1)H-decoupled (31)P MRS has proved to be a useful tool for investigating the metabolism of ifosfamide in vivo.

Diffusion MRI for prediction of response of rectal cancer to chemoradiation.

Prediction of tumour response before onset of treatment could have considerable clinical benefit. Since the apparent diffusion coefficient (ADC) of a tumour's water content can show the extent of necrosis, we looked for a possible correlation of ADC with response to treatment. We measured mean tumour water ADC before and after chemotherapy and chemoradiation in 14 patients with locally advanced rectal cancer, with a quantitative magnetic resonance diffusion imaging sequence. We found a strong negative correlation between mean pretreatment tumour water ADC and percentage size change of tumours after chemotherapy (r=-0.67, p=0.01) and chemoradiation (r=-0.83, p=0.001). Persistence of low ADC in responders after chemotherapy could represent loss of a non-viable fraction of the treated tumour.