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Tumor, Normal Tissue, and Plasma Pharmacokinetic Studies of Fluorouracil Biomodulation With N-Phosphonacetyl-L-aspartate, Folinic Acid, and Interferon Alfa

From the Medical Research Council (MRC) Cyclotron Unit and the Cancer Research Campaign Positron Emission Tomography (CRC PET) Research Group, Section of Cancer Therapeutics, Imperial College School of Medicine, Hammersmith Campus, London, United Kingdom.

Address reprint requests to R.J.A. Harte, c/o Pat M. Price, MD, MRC Cyclotron Unit and CRC PET Research Group, Section of Cancer Therapeutics, Imperial College School Medicine, Hammersmith Campus, Du Cane Rd, London W12 0NN, United Kingdom

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: To evaluate the effect of N-phosphonacetyl-L-aspartate (PALA), folinic acid (FA), and interferon alfa (IFN-) biomodulation on plasma fluorouracil (5FU) pharmacokinetics and tumor and liver radioactivity uptake and retention after [¹⁸F]-fluorouracil (5-[¹⁸F]-FU) administration.

PATIENTS AND METHODS: Twenty-one paired pharmacokinetic studies were completed on patients with colorectal, gastric, and hepatocellular cancer, utilizing positron emission tomography (PET), which allowed the acquisition of tumor, normal tissue, and plasma pharmacokinetic data and tumor blood flow (TBF) measurements. The first PET study was completed when the patient was biomodulator-naive and was repeated on day 8 after the patient had been treated with either PALA, FA, or IFN- in recognized schedules.

RESULTS: TBF was an important determinant of tumor radioactivity uptake (r = .90; P < .001) and retention (r = .96; P < .001), for which radioactivity represents a composite signal of 5-[¹⁸F]-FU and [¹⁸F]-labeled metabolites and catabolites. After treatment with PALA, TBF decreased (four of four patients; P = .043), as did tumor radioactivity exposure (five of five patients; P = .0437), with no change in plasma 5FU clearance. With FA treatment, there were no differences observed in whole-body metabolism, plasma 5FU clearance, or tumor and liver pharmacokinetics. IFN- had measurable effects on TBF and 5-[¹⁸F]-FU metabolism but had no apparent affect on liver blood flow.

CONCLUSION: The administration of PALA and IFN- produced measurable changes in plasma, tumor, and liver pharmacokinetics after 5-[¹⁸F]-FU administration. No changes were observed after FA administration. In vivo effects may negate the anticipated therapeutic advantage of 5FU biomodulation with some agents.

	INTRODUCTION

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FLUOROURACIL (5FU) REMAINS the most widely prescribed anticancer drug for colorectal cancer, with response rates of 10% to 40% in cases of advanced disease.¹ Its biochemistry and pharmacology have been well characterized, confirming that thymidylate synthase (TS) is the key target enzyme involved.² Several approaches have been developed that aim to increase the therapeutic ratio, including biochemical modulation (biomodulation), dose and schedule modification, pharmacokinetically guided dosing, and regional drug-delivery systems.^3-7

Biomodulation is the pharmacologic manipulation of an anticancer drug by another compound with the goal of enhancing antitumor activity. The biomodulating agent may or may not have anticancer activity. Although a number of such interactions have been described with various antimetabolites, those with 5FU have most clinical importance.^8,9 The combination of 5FU and folinic acid (5FU/FA) has entered widespread clinical use; however, the translation of other biomodulating strategies from the laboratory has often failed to confirm in vitro or early clinical promise.

The 5FU/FA combination is based on the postulated folate depletion of tumor cells and evidence that increased intratumoral–reduced folates can overcome multiple mechanisms of 5FU resistance.¹ The mechanism requires that exogenous folate be metabolized to 5,10-methylene tetrahydrofolate, which acts as a methyl donor, stabilizing the reaction between fluorodeoxyuridylate (FdUMP) and TS and thus inhibiting the production of thymidylate, a key element in the synthesis and repair of DNA.^3,10,11 The greatest amount of clinical experience has accrued with 5FU/FA but in a wide range of dose/schedule combinations; the optimal dose/schedule combination remains uncertain.^12,13 The combinations used have increased response rates in advanced disease in comparison to 5FU alone. In a minority of studies, this has translated into a modest survival benefit.^14,15

N-Phosphonacetyl-L-aspartate (PALA) and the interferons have been studied with 5FU. PALA is an aspartate transcarbamylase inhibitor that interrupts the de novo pyrimidine synthesis pathway.¹⁶ By a decrease in the amount of uridine nucleotide pools, the incorporation of 5FU into RNA is facilitated. Inhibiting pyrimidine synthesis decreases concentrations of deoxyuridine monophosphate and therefore decreases competition with FdUMP for TS binding.^17,18 Clinical trials with 5FU/PALA have demonstrated the activity of the combination, but there appears to be no clear advantage of this combination over 5FU regimens, even with optimal scheduling.^19-22

Several mechanisms of interaction between 5FU and the interferons have been identified, including thymidine phosphorylase regulation (leading to increased intracellular levels of FdUMP), inhibition of adaptive upregulation of TS expression, and an increase in DNA damage.^23-28 Additionally, some pharmacokinetic studies have shown a decrease in 5FU clearance after IFN treatment, in contrast to FA and PALA treatment, for which there is no evidence of plasma pharmacokinetic effects on 5FU.^29-31 Phase III clinical trials utilizing 5FU ± IFN and 5FU/FA ± IFN provide mixed evidence of survival advantage with IFN, suggesting that sequence and schedule are critical in explaining the apparent inconsistencies.^23,32,33

The rational development of biomodulation strategies would be facilitated by having means for their evaluation in vivo. Nuclear magnetic resonance (NMR) spectroscopy and positron emission tomography (PET) are complementary techniques for the in vivo assessment of tumor and tissue pharmacokinetics, and 5FU is the most extensively investigated chemotherapy agent for both modalities. NMR spectroscopy lacks the sensitivity and spatial resolution of PET but can distinguish between chemical forms (ie, PET cannot differentiate labeled parent drug from labeled metabolite).^34,35 Evidence has been accumulating from [¹⁹F]-NMR spectroscopy that knowledge of intratumoral 5FU kinetics assists our understanding of the determinants of clinical response.^36-39 Similarly, PET studies have focused on pharmacokinetic analysis as a means of predicting chemotherapy response.^39,40 The aims of this PET study were (1) to evaluate the effect of biomodulation by PALA, FA, and IFN on plasma, tumor, and liver 5FU pharmacokinetics, (2) to investigate the determinants of any changes observed, and (3) to consider whether in vivo pharmacokinetic data helped to explain activity data in clinical trials.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Patient Selection
Twenty patients from the Department of Clinical Oncology, Hammersmith Hospital, were studied at the MRC Cyclotron Unit over a 20-month period (Table 1). All were scheduled to receive 5FU-based chemotherapy as either adjuvant or palliative therapy for histologically confirmed colorectal (18 patients), gastric (one patient), or hepatocellular (one patient) cancer. Eighteen patients were chemotherapy-naive, and the remaining two had had no chemotherapy in the preceding 3 weeks. One patient had an FA study 14 months after a PALA study (studies no. 13 and 1, respectively). Twelve male and eight female patients were studied, with ages ranging from 42 to 80 years (median, 59 years). Eligibility criteria included a life expectancy of greater than 12 weeks, performance status of 0 to 2, and adequate renal and hepatic function (ie, serum creatinine, < 125 µmol · L^-1; bilirubin, < 17 mmol · L^-1; and aspartate transaminase, < 35 mmol · L^-1). All gave written, informed consent to the study, which had Hammersmith Hospital Research Ethics Committee and Administration of Radioactive Substances Advisory Committee (UK) approval.

Study Design
Patients underwent two pharmacokinetic studies 1 week apart: the first on day 1, while biomodulator-naive, and the second on day 8, after the administration of either PALA, FA, or IFN (IFN-2a or -2b) before the study. PALA (250 mg · m^-2 intravenously [IV]) was administered in 100 mL N saline over 20 minutes on day 7; FA (30 mg IV bolus) was given on days 1 through 4, 7, and 8 (days 5 and 6 were the weekend); and IFN (3 millon units subcutaneously) was administered on days 1, 4, 7, and 8. FA and IFN were given after the PET study on day 1 and 30 minutes before the 5-[¹⁸F]-FU injection on day 8. Scans were carried out at approximately the same time of day (injection times are listed in Table 1). Patients were advised to have only a light breakfast on the day of scanning.

During each complete pharmacokinetic study, two PET scans were performed: (1) a C¹⁵O₂ scan to measure tissue perfusion, and (2) a 5-[¹⁸F]-FU scan to measure tumor (ie, hepatic metastasis) and liver 5FU pharmacokinetics. Additionally, 5-[¹⁸F]-FU plasma pharmacokinetics were measured. For six studies, perfusion measurements were not obtained, and for five studies, plasma kinetic data measurements were not obtained, because of technical difficulties. For 15 of the paired studies (nine PALA, three FA, and three IFN), only the radiolabeled 5-[¹⁸F]-FU was injected (ie, tracer studies, delivering 5FU doses of between 1.4 and 10.8 mg · m^-2). In six studies (three FA and three IFN), the 5-[¹⁸F]-FU was coinjected with 375 to 400 mg · m^-2 unlabeled 5FU (ie, full-dose studies; Table 1).

Patient Repositioning
Patients were repositioned in the scanner on successive weeks, using anatomical landmarks and a pilot positioning scan. Because this method is not exact, the second images were shifted and rotated (rigid transformation) using a program that corrected for minor PET to PET misalignment to ensure that identical tissue volumes were sampled on successive weeks.⁴¹

Tissue-Perfusion Measurement
Tissue-perfusion scans were conducted immediately before the 5-[¹⁸F]-FU scans. For tissue-perfusion scans, the patient inhaled C¹⁵O₂ that had been synthesized on-site and delivered via a light face mask at an activity of 4 MBq · mL^-1 and a constant flow rate of 500 mL · min^-1 for 3.5 minutes. For 10 minutes, dynamic measurements were made of the radioactivity in arterial blood, tumor, and normal liver, and from these data, tumor blood flow per unit volume of tissue (TBF)—a measure of tumor perfusion—was calculated using established methodology.^42-44 In these calculations, the fraction of the tumor volume into which H₂¹⁵O diffuses, ie, the fractional volume of distribution, was also estimated (ie, a value of between 0 and 1). Because similar calculations for hepatic perfusion have not been validated, liver-perfusion measurements were restricted to a qualitative measurement, ie, Flow index = mean tissue radioactivity concentrationmean blood radioactivity concentration

{smtxt}with the mean concentrations calculated over the duration of the 3.5-minute infusion. The FI value will range from 0 to the fractional volume of distribution, with higher-perfusion regions having higher values. The short half-life of ¹⁵O (2.04 minutes) allowed the 5-[¹⁸F]-FU scan to follow 10 minutes after the C¹⁵O₂ scan.

5-[¹⁸F]-FU Data Acquisition and Processing
Data acquisition, processing, and analysis followed previously developed protocols.⁴⁵ A rapid and efficient synthesis of 5-[¹⁸F]-FU was developed using high-performance liquid chromatography for purification.⁴⁶ In the 42 5-[¹⁸F]-FU syntheses, the median radiochemical purity was 98.1% (range, 84.2% to 100.0%), with uracil the only identified impurity. The median injected radioactivity dose was 321 MBq (range, 117 to 437 MBq), associated with a median 5FU dose of 5.3 mg · m^-2 (range, 0.6 to 10.8 mg · m^-2).

The 5-[¹⁸F]-FU solution, either alone or mixed with a therapeutic dose of 5FU, was injected via a venous line inserted into the right antecubital fossa. After the injection, the venous line was flushed with 5 mL of saline to ensure that all of the 5FU had entered the patient; this procedure was completed within 60 seconds.

After 5-[¹⁸F]-FU injection, dynamic PET data were acquired for 1 hour, using an ECAT 931-08/12 PET scanner (CTI, Knoxville, TN), with data collected into discrete time frames ranging from 30 to 600 seconds. This resulted in images detailing the biodistribution of radioactivity in the camera's field of view for each time frame. The image-analysis software program Analyze (Mayo Clinic, Rochester, MN) was used to define sample volumes in tumor and normal liver.⁴⁷ This was done in conjunction with computed tomography or ultrasound films, and to minimize sampling error, only tumor deposits greater than 3 cm in diameter were sampled. Because of this effect and the 10.8-cm axial field of view of the PET camera, tumor data were acquired from 12 of 17 patients who had computed tomographic evidence of disease, and normal liver data were acquired from all patients. Mean radioactivity in the sample volumes for each of the time frames was then calculated, creating representative radioactivity versus time curves (ATCs) for the tumor and liver. These ATCs were then corrected for radioactive decay and radioactivity dose injected using the formula Corrected ATC = ATC (MBq |mZ ml-1) x etRadioactive dose (MBq)

{smtxt}where = 0.0001053 · s-1 is the decay constant of ¹⁸F and t is the midframe time, measured from injection time. Areas under these corrected ATCs were then calculated: AUC500, during the first ₅₀₀ seconds, and AUC_3,600, during the entire 1-hour scan (3,600 seconds).

Plasma Sampling and Assay
Continuous sampling of arterial blood radioactivity concentrations were performed simultaneously with PET data acquisition.⁴⁸ Additionally, up to six discrete samples were taken for measurement of (1) the ratio of radioactivity in plasma/radioactivity in whole blood and (2) the contribution of 5-[¹⁸F]-FU to the total radioactivity in plasma (which is a composite signal of 5-[¹⁸F]-FU and [¹⁸F]-metabolites). The plasma/whole-blood radioactivity ratio was determined by measurement in a well counter after the blood was centrifuged for 2 minutes at 2000 x g. High-performance liquid chromatography analytical techniques were used to calculate the contribution of 5-[¹⁸F]-FU to the total activity in plasma. The discrete measurements were then extrapolated to the entire duration of the study by regressing a delayed exponential function through data points of radioactivity in plasma/radioactivity in whole blood and by regressing a sigmoid-type function through data points of the fraction of activity in plasma attributed to 5-[¹⁸F]-FU. These functions were chosen because they best described the population data and assumed no a priori knowledge. Application of these continuous corrections to the continuous measurements of radioactivity in blood produces the ATC of 5-[¹⁸F]-FU in plasma. After decay correction, areas under these curves were calculated numerically, from which estimates of the clearance of 5FU from plasma could be derived using the formula Clearance (L |mZ min-1 |mZ m-2) = Radioactive dose (MBq)AUC (MBq |mZ min |mZ L-1 x Body surface area (m2)

{smtxt}No extrapolation of the time activity curves was required because the levels of 5-[¹⁸F]-FU in plasma were negligible by the end of the 1-hour data-acquisition period.

Statistical Analysis
The effects of biomodulation on TBF, hepatic flow index (HFI), liver and tumor AUCs, whole body clearance, plasma/whole-blood radioactivity partitioning, and plasma 5-[¹⁸F]-FU/radioactivity partitioning were examined. Statistically significant changes in these parameters were examined using paired t tests. Data were also pooled (combining PALA, FA, and IFN data) for the examination of relationships between pharmacokinetic parameters, and correlation coefficients (Pearson's) were calculated. Significant relationships were then determined by rejecting the null hypothesis that there is no relationship.⁴⁹

	RESULTS

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Typical 5FU radioactivity ATCs for liver and tumor for both tracer and full-dose studies are detailed in Fig 1, where radioactivity represents a composite signal of 5-[¹⁸F]-FU and [¹⁸F]-labeled metabolites and catabolites. These demonstrate that curves are both tissue- and 5FU-dose–dependent. The range of values of TBF, HFI, tumor, and liver AUCs and whole-body clearance for all studies are listed in Table 2, and the percentage change in each of these parameters with biomodulation is listed for all studies in Table 3.

View larger version (17K): [in this window] [in a new window]   Fig 1. Graphs of ATCs for (A) tracer studies and (B) full-dose studies. ——, mean liver ATC; – – – –, colorectal metastases, mean value (tracer studies) and individual values (full-dose studies); – · – hepatocellular tumor ATC.

Tumor Data
Figure 2 demonstrates relationships between the AUC_3,600 value for individual metastases in studies with more than one metastasis sampled and the mean value calculated from the multiple metastases sampled. Similar results were obtained for TBF and AUC₅₀₀ data. They confirm that different tumor samples give similar results especially when the tumor samples are large. This suggests that which tumor is sampled in an individual is unimportant because the changes observed are similar. The combined mean values were used for patients with multiple hepatic tumor samples.

View larger version (16K): [in this window] [in a new window]   Fig 2. Graph of mean tumor AUC3,600 versus tumor AUC3,600 for individual metastases. x, Metastasis regions of greater than 500 pixels (16.8 cm3); , metastasis regions smaller than 500 pixels; ——, line of identity.

There were no dramatic changes in the shape of the tumor ATC with either biomodulation or 5FU dose. After PALA administration, TBF was reduced (four of four patients; P = .043), as was AUC₅₀₀ (five of five patients; P = .0078) and AUC_3,600 (five of five patients; P = .0437). There were no consistent changes in TBF and AUCs in patients who received FA. Conversely, in those patients receiving IFN, TBF increased (three of three patients; not significant), and AUC increased in two of three patients (no change in one patient; not significant).

Pooled data demonstrated significant correlations between TBF and AUC₅₀₀ (r = .90; P < .001) and AUC₅₀₀ and AUC_3,600 (r = .96; P < .001), indicating the importance of drug delivery in initial drug uptake and its intratumoral retention up to 1 hour after administration.

Normal Liver Data
There was a strong and apparently one-to-one relationship between the percentage change in tumor FI and percentage change in TBF (r = .9524; P < .001). This was accepted as evidence that the FI had general utility in measuring changes in tissue perfusion and suggested the appropriateness of using HFI to measure changes in hepatic perfusion.

Normal liver ATC shape differed among the 15 patients assessed with tracer doses of 5-[¹⁸F]-FU and the six who received concomitant unlabeled 5FU. There were no additional changes in shape that were attributable to biomodulation. The addition of PALA, FA, or IFN resulted in no apparent change in HFI, AUC₅₀₀, or AUC_3,600. However, after IFN, there was a nonsignificant decrease in liver AUC_3,600 (five of six patients). Pooled data demonstrated significant correlations between HFI and AUC₅₀₀ (r = .59; P = .02) and AUC₅₀₀ and AUC_3,600 (r = .65; P = .0014).

Plasma Clearance Data
As anticipated, there were differences in plasma 5-[¹⁸F]-FU clearance for the 11 tracer studies (range, 1.12 to 2.14 L · min^-1 · m^-2; n = 11) and the five full-dose studies (therapeutic bolus injection; clearance range, 0.41 to 0.84 L · min^-1 · m^-2; n = 5). There were no differences in 5FU clearance that were attributable to PALA or FA, but there was a nonsignificant decrease in whole-body clearance with IFN (four of five patients, with the remaining patient having a notable increase in HFI; see Discussion).

There was no change in 5-[¹⁸F]-FU/radioactivity in plasma partitioning that was attributable to PALA and FA. IFN studies showed an increased 5-[¹⁸F]-FU/radio-activity ratio for three of three therapeutic-dose studies and two of three tracer studies, with no change in the remaining tracer study (Fig 3).

View larger version (25K): [in this window] [in a new window]   Fig 3. Graphs of 5-[18F]-FU/radioactivity in IFN studies. Line types indicate patients; – – – –, patient no. 17. x, Data points for the first scan; , data points for the second (modulated) scan.

There was a significant correlation between percentage change in whole-body clearance and percentage change in HFI (r = .7791; P = .0017) (Fig 4).

View larger version (16K): [in this window] [in a new window]   Fig 4. Correlation between changes in HFI and whole-body clearance (r = 0.7791; P = .001694). *, PALA studies; , FA studies; x, IFN studies. Full-dose studies are circled. ——, line of best fit; – – – –, line of identity.

Evaluation of Results
Errors in the realignment of the second PET scan could cause bias because of liver contamination of the tumor signal. This could cause artifactual change in tumor AUC because regions are defined on one set of images and then applied to both sets. However, for the PALA data, almost identical results were produced with sample regions drawn on either the first or the realigned second scan images.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Increased understanding of intracellular pharmacokinetics and the mechanism of action of 5FU has allowed the development of biomodulating strategies; however, laboratory promise has not readily translated into clinical success. This may relate to alterations in plasma pharmacokinetics and tumor and liver drug delivery, which negate the anticipated therapeutic advantage from modulation of intracellular pharmacokinetics. The application of recently developed PET techniques in these pilot studies has allowed direct comparison of conventional plasma pharmacokinetic parameters, TBF, and tumor and normal tissue radioactivity data after 5-[¹⁸F]-FU administration in spontaneous human tumors in vivo in cancer patients.

After PALA administration, TBF and tumor AUC_3,600 decreased, but there was no change in liver pharmacokinetics or whole-body 5FU clearance, suggesting that adverse changes in tumor 5FU delivery, uptake, and retention after PALA administration limit the likelihood of therapeutic advantage. However, although the PET techniques are good at characterizing early effects—ie, plasma clearance, tumor drug delivery, and initial uptake of radioactivity—they are unable to resolve small, later changes in tumor radioactivity levels or to resolve the chemical form of the tracer (ie, 5-[¹⁸F]-FU or [¹⁸F]-metabolite). Therefore, potentially beneficial changes in intracellular pharmacokinetics that occur after 1 hour will be missed. Although they might be sufficient to overcome the reduction in 5FU delivery to tumors that was observed in this PET study, these data suggest if the in vitro advantages of the 5FU/PALA combination are to be realized clinically, then use of the 5FU/PALA combination with a TBF modulator may be a more appropriate treatment method to study in future trials.

The 5FU/FA combination has the greatest demonstrated clinical efficacy, yet we did not observe any changes in plasma handling and clearance or tumor and normal liver pharmacokinetics that were attributable to biomodulation in the FA studies. However, the mechanism of action of FA would not necessarily predict changes in plasma or liver drug handling, and the lack of chemical resolution of the PET detection system prevents it from discriminating small but clinically important differences in the proportion of fluorinated nucleotides contributing to the total tumor signal.¹ The conclusion from these PET studies is that there is no in vivo reason that might limit the efficacy of the biomodulation strategy because of an unforseen pharmacokinetic disadvantage.

IFN had effects on tumor, liver, and plasma pharmacokinetics; however, the relationship was complex. There was evidence of inhibition of 5FU metabolism with no change in HFI, suggesting that IFN decreases 5FU metabolism by a means other than modulation of hepatic blood flow. Additionally, IFN increased TBF (P = .034 with a paired t test, but with n = 3) but caused a nonsignificant decrease in the plasma clearance and retention of radioactivity in liver. However, there was one patient (no. 17) for whom the ratio of 5-[¹⁸F]-FU/radioactivity in plasma was pushed to the right, indicating inhibition of 5FU metabolism (as with the other studies; Fig 3), yet plasma 5-[¹⁸F]-FU clearance rose, as did the liver AUC_3,600 (opposite to the other IFN studies). This apparently paradoxical result may be explained by the measured increase in HFI and inhibition of metabolism, whereby the disappearance of 5-[¹⁸F]-FU from plasma was more rapid, but the appearance of metabolites in plasma was slower. We have demonstrated variations in plasma pharmacokinetics, TBF, and liver retention, all of which might be anticipated to lead to increased efficacy; however, 5FU/IFN combinations have not entered widespread clinical practice. These data would suggest the importance of further development of the 5FU/IFN combination, focusing attention on the timing of IFN administration and the impact on TBF modulation, in addition to exploiting its intracellular advantage.

Finally, these studies confirm the importance of the 5FU dose in determining plasma clearance. 5FU is primarily eliminated by a saturable metabolic process with a K_m estimated at 15 µM, and these data suggest that PET tracer 5-[¹⁸F]-FU studies are kinetically analogous to prolonged venous infusion studies (as evidenced by the fact that the small dose of 5-[¹⁸F]-FU in the PET study failed to saturate metabolism).^50-53

These are pilot studies, hence there is the need to confirm observations and the opportunity to extend this study to other combinations of 5FU dose and modulator, eg, dihydropyrimidine dehydrogenase inhibitors.⁵⁴ The immediate clinical relevance of these studies is in demonstrating that detailed pharmacokinetic studies in limited numbers of patients which incorporate new technology allow insights that are unobtainable by other means. In the longer term, this approach, when combined with the complementary [¹⁹F]-NMR spectroscopy and increased understanding of molecular determinants of chemotherapy response, may contribute to the development of treatment strategies by (1) providing in vivo markers of some of the potential causes of treatment failure and (2) generating hypotheses for increasing treatment efficacy and providing a means for their evaluation.

	NOTES

 
The CRC PET Research Group is supported by Cancer Research Campaign grant no. SP2 193/0101. R.J.A.H. was supported by a grant from Schering Plough UK. Additional funding was provided by United States Bioscience Pharma and Roche Products.

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Submitted August 11, 1997; accepted January 6, 1999.

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