Metabolites of 5-Fluorouracil in Plasma and Urine, as Monitored by 19F Nuclear Magnetic Resonance Spectroscopy, for Patients Receiving Chemotherapy

with or without Methotrexate Pretreatment

William E. Hull, Rdiger E. Port, Richard Herrmann, et al. Cancer Res 1988;48:1680-1688.

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[CANCER RESEARCH 48, 1680-1688, March 15. 1988]

Metabolites

of 5-Fluorouracil

in Plasma and Urine, as Monitored by I9F Nuclear with

Magnetic Resonance Spectroscopy, for Patients Receiving Chemotherapy or without Methotrexate Pretreatment1
William E. Hull,2 Rdiger Port, Richard Herrmann,3 Brbel ritsch, and Werner Kunz E. B

German Cancer Research Center, Central Spectroscopy [W. E. H.J, Institute of Biochemistry [R. E. P., W. K.J, Neuenheimer Feld 280. D-6900 Heidelberg; and University of Heidelberg, Department of Medicine [R. H., B. B., -6900 Heidelberg; FRG D

ABSTRACT
"F NMR Spectroscopy at 470 MHz (11.7 Tesla) has been used to directly measure the levels of 5-fluorouracil (FU) and its fluorine-con taining catabolites in plasma and urine of colon cancer patients after i.v. infusion (10 min) of 60-230 /unol (8-30 mg) FU/kg, either with or without pretreatment with methotrexate (5.1-12.5 mg/kg). With a 1.5ml sample the minimum metabolite concentration than can be quantified is approximately 15 MMwithin 30 min and 3 1 MMwithin 12 h of 5 data acquisition. The first and second catabolites of FU, dihydrofluorouracil and a-fluoro-0-ureidopropanoic acid, exhibit steady-state behav ior with dose-dependent plasma concentrations of 5-40 MMfor approxi mately 10-90 min after infusion (12 patients, 16 treatments). The final cataboliti- a-fluoro-0-alanine (FBAL) was detected in plasma after 5-15 min, and the rate at which its concentration increased was independent of FU dose, while the maximum concentration reached at about the time FU disappeared (FU < 5 MMin 1-2 h) was dose-dependent. The area under the time curve for FU in plasma increased more than linearly with dose. Several patients showed elevated levels of free fluoride anin(!" ) in plasma (63 samples: median, 5 MM;maximum, 33 MM). In urine all of the above catabolites and 1- could be observed. In samples with pH .-.-- .3 (methotrexate patients, due to bicarbonate 7 infusion) \~tarbo\\-l- BAI, was also found in significant amounts. Uri nary excretion of FU and catabolites amounted to 2.6-30% of the dose within 2 h (14 patients, 18 treatments) and 60-66% within 24 h (three patients). The ratio FU/creatinine in 2-h urine increased more than linearly with FU dose. Urinary fluoride concentration reached a maximum during the first day after FU infusion and returned to normal background levels after 2-3 days (four patients). The pattern of FU catabolites observed in plasma or urine did not differ significantly between responders and nonresponders to therapy or between patients with FU monotherapy and patients with methotrexate pretreatment. Cytotoxic FU anabolites, i.e., nucleotides, were not detected in plasma or urine (i.e., are < 3 MM)- heir detection in tumor tissue will T be required for an assessment of individual responsiveness to FU. Possible toxic metabolic products derivable from FBAL, e.g., 2-fluoroacetate or 2-fluorocitrate, were not detected (i.e., are < 3 MM)in plasma or urine. This leads us to propose that the release of fluoride from FBAL results in the deactivation of the transaminase required for the i ata holism of FBAL.

(1), is still a widely used antineoplastic chemotherapeutic, mainly in the treatment of gastrointestinal and breast cancer. The metabolism of this drug has been extensively studied in tumor-bearing animals and in cultured cells, and a few metab olism studies have also been carried out in humans (2-8). Fig. 1 presents a scheme depicting the metabolic fate of FU, as determined by the earlier biochemical studies and augmented by these and other recent NMR investigations (9). FU cytotoxicity is believed to occur either by (a) incorporation into RNA (after conversion to 5-fluorouridine-5'-triphosphate) with sub sequent retardation of maturation of ribosomal RNA, or (b) by inhibition of thymidylate synthase via the anabolite FdUMP. Other mechanisms of FU cytotoxicity via 5-fluoro-2'-deoxyuridine-5'-triphosphate (10 and references therein) or fluoroacetate (from FBAL) (11, 12) have been discussed. The balance of anabolism versus catabolism in various tissues may explain the differences in their sensitivity to FU and may be a primary factor in determining therapeutic effectiveness versus toxic side effects. FU is only used as monotherapy in the treatment of colon cancer, where it produces remission in 15-20% of the cases (13). This limited response rate makes it desirable to detect nonresponsive patients in the early phase of treatment. 19F NMR Spectroscopy has been successfully used to detect FU metabolites noninvasively in vivo in tumor-bearing mice5 (14, 15), and similar measurements on humans now appear feasible (16). Encouraged by the initial work of Malet-Martino (17) involving I9F NMR studies of the metabolism of 5'-deoxy5-fluorouridine and in order to systematically prepare for clin ical applications in man, we began studying FU metabolites in human plasma and urine by 19F NMR Spectroscopy (18). The majority of our patients belonged to a phase III trial in which FU as a single agent was compared with FU preceded by MTX against colon cancer (19). In recent studies I9F NMR has been used to examine fluoropyrimidine metabolism in humans (9, 20, 21), to detect incorporation of FU into RNA in cell cultures (22, 23), and to study the structure of tRNA containing FU (24). Furthermore, the properties of the inhibitory FdUMP-thymidylate synthase complexes have been examined (25), and such a complex may have now been detected by NMR in cell cultures (26). We are also extending our investigations to include /// vivo NMR stud ies of tumor-bearing mice (15) and ex vivo studies of biopsy material from patients (27). Thus, I9F NMR will obviously be important in the future for elucidating the uptake and metabo lism of fluorine-containing drugs.

INTRODUCTION FU," first synthesized in 1957 by Heidelberger and coworkers

Received 5/13/87; revised 11/30/87; accepted 12/10/87. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This paper is dedicated to Professor Dr. Erich Hecker on the occasion of his 60th birthday and was supported in part by a grant from the Tumor Center, Heidelberg/Mannheim for reprints should be addressed. ' To whom requests (Colon Carcinoma Project). 3 Present address: Klinikum Charlottenburg der Freien Universitt, D-1000 Berlin 19, FRG. 4 The abbreviations used are: FU, 5-fluorouracil; CFBAL, N-carboxy-FBAL; DHFU, 5-fIuoro-5,6-dihydrouracil; FBAL, -fluoro-d-alanine; FdUMP, S-fluoro2'-deoxyuridine-5'-monophosphate; FdUTP, 5-fluoro-2'-deoxyuridine-5'-triphosphate; FGPA, a-fluoro-0-guanidinopropanoic acid; FUrd, S-fluorouridine; FUPA, a-fluoro-/3-ureidopropanoic acid (Af-carbamoyl-FBAL); -S/W, signal-tonoise ratio; F~, fluoride anin;AUC, area under the curve; MTX, methotrexate; NMR, nuclear magnetic resonance; HPLC, high-performance liquid chromatography.

MATERIALS

AND METHODS

Patients and Treatment. A total of 21 patients (11 males, age 46-70; 10 females, age 43-75) with metastatic gastrointestinal (mostly colon) or mammary carcinomas was investigated. In Group A four patients received FU once daily for 5 days (one cycle) at 61-99 ^mol (8-13
! W. E. Hull, R. E. Port, H. Osswald, and W. Kunz, manuscript in preparation.

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5-FLUOROURACIL METABOLITES IN PLASMA AND URINE

anbolIsm
FU

FUrd-

FUMP FUDP FUT?

F.tLJ

H/

FdUrd

FdjJIMP. **FdUDP FdUTP

-v
NADPH I
O

NADP-

enzymatic

catabolism H2NCNH2 -r urea

F-acetate, F-malonyl-CoA t F-malonic acid semialdehyde (transaminase)

A HN U *

/* C---F ...H* *

H*rH20HaNO^Vco2-CHFCOr,

NH,H20 H3N*-H2-CHF-C02F.BAL

P.HF_g

FJPA NH2 ' *H2N--NH-H2- CHF-COa-

-02C-NH-H2-CHF-C02 -

[1C-2]FU ["C-6JFU

JEfiEA

--H20

(plasma,

urine.

l,. ICFJAU pH 7.3)

equilibrium

H2 H2N_N + H3N*-H2-CHF-C02-

non-enzymatic

urea ZML Fig. 1. Schematic representation of the catabolic and anabolic pathways for 5-fluorouracil.Cytotoxic effects are caused by incorporation into RNA via 5fluorouridine-5'-triphosphateand by inhibitionof thymidylatesynthasevia the ternary complex involvingFdUMP and methylenetetrahydrofolate. HFU is formed D bytrans- addition of two hydrogenatoms (//*) (59). FBAL is in equilibriumwith the (V-carboxydduciCFBALformedbythe reactionwith bicarbonate.An additional a minor enzymaticpathwayto FGPA has been proposedby Heidelbergerand coworkers.The knownmetabolicfates of t--alanine indicated(53), but are apparently are inhibited by FBAL which may cause deactivationof the pyridoxal phosphate-dependenttransaminase by a mechanismthat releasesfluoride anin. he fate of the T commonly used I4Clabels has also been indicated.dTMP, thymidine-5'-monophosphate;FdUrd, 5-fluoro-2'-deoxyuridine; UDP, S-fluoro-uridine-S'-diphosphate; F FUTP,5-fluorouridine: MTHF, methylenetetrahydrofolate. mg)/kg, repeated every 3 weeks. In Group B one patient received FU once weekly at 110 /mol mg)/kg. In Group C four patients received (14 154-202 fimol (20-26 mg) FU/kg once every 3 weeks. In Group D 12 patients received a pretreatment of 11-28 ^mol (5.1-12.5 mg) MTX/ kg followed by 118-231 M"iol (15-30 mg) FU/kg, repeated once every 3 weeks. The number of treatments that individual patients had received prior to that treatment for which NMR analysis was performed varied greatly (1-14). In a few cases two treatments for a given patient were analyzed. A positive response to therapy (Figs. 5 and 6) was defined as regression or no change of tumor mass as verified by clinical examina tion or imaging procedures 6 weeks after the particular treatment for which NMR analysis was performed. FU Monotherapy (Groups A-C). FU (Fluroblastin, 50 mg FU/ml; Farmitalia Carlo Erba GmbH, Freiburg, FRG) was diluted with 5.25% glucose to give a total volume of 50 ml and was infused over a period of 10 min into a forearm vein by means of an infusion pump. MTX + FU Combination Therapy (Group D). Prior to MTX admin istration 250 ml of a 8.4% sodium bicarbonate solution was infused within l h to insure an alkaline urine pH. Beginning 7 h before FU administration, half of the methotrexate (MTX-Lederle, Cyanamid GmbH, Wolfratshausen, FRG) dose was given as i.v. bolus, followed by a 4-h i.v. infusion of the second half dose in a 500-ml electrolyte solution. After FU application as above patients received a 24-h infusion of 3000 ml of glucose and electrolyte solutions containing a total of 120 mval sodium bicarbonate. Sampling. Urine was taken from 20 patients during 27 FU treat ments. Samples were taken approximately 10 min before FU infusion, and total urine was then collected until 2 h after infusion (denned here as 0-2 h urine). In three cases urine was also collected 2-24, 24-48, and 48-72 h after infusion. Blood samples were taken from 12 patients during 16 FU treatments (in five cases with MTX pretreatment). A complete series of samples taken 1,5, 15, 30, 60, and 90 min after the end of FU infusion was analyzed for nine patients (nine treatments). At each time point, 2 ml of blood were aspirated from a vein of the forearm, contralateral to that where FU had been infused, into a glass tube containing 6 mg EDTA for anticoagulation. After the last blood sample was taken all samples were centrifuged at 3000 x g for 10 min and the plasma was aspirated. For one patient red blood cells were sampled by centrifuging anticoagulated blood and, after aspiration of plasma, washing the cells once with isotonic saline. All samples were stored at -20 or 60C until measurements were made. Creatinine Determination. Concentrations of creatinine in urine sam ples were determined using the Jaff reaction with kinetic measurement of dye product formation (28). In Figs. 6 and 7 concentrations of FU and metabolites, measured by NMR on 1.5-2 ml samples, are expressed relative to creatinine since total urine volume varied greatly (30-1100 ml) and had not been recorded for all patients. NMR Spectroscopy. Proton-coupled "F NMR spectra of urine and plasma samples (Figs. 2 and 3) were obtained at 470 MHz (11.7 Tesla) with a Bruker AM-500 FT NMR spectrometer using 5- or 10-mm diameter sample tubes (0.4- or 1.5-2.0-ml volumes), depending on the amount of sample available. It is a basic principle of Fourier transform NMR, which involves time-averaging or repetitive addition of the transient NMR signals, that the S/N ratio in the resulting spectra is proportional to \N or \MT, where A/ is the number of transients accumulated and Ml is the total measurement time. Thus, a factor 4 increase in N and A/7" is necessary to achieve a factor 2 improvement in S/N or equivalently to lower the detection threshhold (lowest detect-

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5-FLUOROURACIL

METABOLITES

IN PLASMA AND URINE

;6F6 FU FBAL

for the spectrometer, and the C6F6 signal served as the chemical shift reference [approximately -87.48 ppm, whereby 10 mM trifluoroacetic acid in standard phosphate-buffered saline (pH 7) = 0 ppm; positive and negative relative shifts are termed downfield and upfield, respec tively] and as integration reference after calibration against standard FU solutions of known concentration. The capillary was doped with a small amount of the paramagnetic relaxation agent chromium(III) acetylacetonate, to insure that the '/', relaxation time of the reference signal was shorter than that of any of the metabolites. The biological samples themselves were not doped or manipulated in any way. The criteria and techniques used for determining absolute metabolite concentrations via NMR are described in detail elsewhere (9, 29). All spectra were taken without 'H-decoupIing using a short radio frequency pulse with small flip angle (e.g., 3 s 10) nd sufficiently long ~ a repetition time (0.5-1 s) so that fully relaxed spectra were obtained and peak areas were directly proportional to concentrations. The integral of the C6F6reference peak was calibrated against a 10-mM FU solution prepared from a weighed amount of FU stock (Roche) and checked against independent solutions of 100 and 400 /AI FU in 150 mM NaCl or in blood donor plasma, whereby the concentrations determined by NMR deviated by less than 10% from the "true" values. Thus, for our measurements the precision and reproducibility of the NMR concen tration determinations are generally on the order of 5-10% for concen trations > 50 M. lower concentrations the precision decreases and For depends upon the measurement time and the resulting S/N in the spectra. For example, quantitative analysis at a level of approximately 3 1 fiM requires 12-h data acquisition with a 10-mm tube. For practical reasons data acquisition was usually terminated when the detection threshhold reached approximately 5-10 AI. The spectral width for data acquisition was generally set to 80 KHz (65,536 data points), covering the shift range of approximately 0 to ppm. Before Fourier transformation the NMR free-induction 160 decays were treated with an exponential multiplication to optimize sensitivity, giving a line broadening of typically 20 Hz for plasma samples or 1-2 Hz for urine. After phase correction the various signal regions in the spectra were baseline corrected using the polynomial routines of the Bruker graphic display software, and the spectra were digitally integrated via software after setting the integral of the (.'!' peak to its calibration value. In plasma samples FU showed significant line broadening (100-150 Hz) and occasionally a very broad upfield shoulder, necessitating a more severe exponential multiplication and extended integration limits for accurate quantitation. AUCs and Regression Analysis. The areas under the plasma concen tration versus time curves (AUCs, Fig. 5) were calculated according to
CFBAL FU

f;
PPM

DHFU FURA
.

CONC,

IN

LIMOLAR

-110 -120 -130 PPM Fig. 2. 470 MHz "F NMR spectrum of 5-FU and catabolites in plasma. The patient (female, age 64, colon carcinoma with liver mtastases)eceived 202 /mol r FU (26 mg)/kg as a io-min infusion i.v. The plasma sample was obtained 30 min after end of infusion. The < 1,. signal conies from a coaxial reference capillary. Metabolite concentrations in /<\i were determined by integration of the spectrum. inset, signal from fluoride (4 JIM) at approximately 43.8 ppm. A 2-ml plasma sample was measured in a 10 mm tube for 14 h at 28'C using the following parameters: spectral width, 71428 Hz; 65,536 data points; 5-fis pulse width (approximately 11* flip angle); 1-s repetition time; 50.000 transients. 20-Hz
-80 -90

-100

exponential line broadening.

able metabolite concentration) by a factor of 2. The use of a IO inni instead of a 5-mm tube improves the S/N for a given measurement by a factor 4. This means there is a factor 4 reduction in detection threshhold for a given measurement time, or a factor 42 = 16 reduction in MT to achieve a given S/N. The biological fluids were examined directly without any pretreatment, in most cases at 28C in a few or cases at 4"(". We independently confirmed the identity of signals for all the known catabolites using authentic samples; an earlier report (14) had mistakenly assigned DHFU to the FUPA signal. For the purpose of calibrating chemical shifts and for providing a reference signal for integrating peak areas to determine concentrations, a concentric capillary containing a low concentration of (',,!',. benin zene-d* was employed (2-mm diameter in 5-mm tubes, 3-mm in 10mm tubes). The deuterated solvent provided the field/frequency lock

FBAL
FBAL

C6F6

FBAL

Fig. 3. 470-MHz "F NMR spectrum of 5FU and catabolites in urine (pH 8.53). The patient (female, age 50, colon carcinoma with liver and bone mtastases) eceived 125 /unol r FU (16 mg)/kg as a IO min infusion i.v. fol lowing pretreatment with MTX (2.6 mg/kg). A large amount of CFBAL (4.8 mM) and small amounts of the acid-labile unknowns I ', and I : (inset) are present at high pH. The fine structure of the signals (expanded insets) arises from 'H-"F spin-spin coupling. A 0.3-ml urine sample was measured in a 5-mm tube for 3.3 h at 28'C using the following parameters: spec tral width, 71428 Hz; 65,536 data points zerofilled to 131,072, 3-iis pulse width (22" flip angle); 1.5-s repetition time, 8000 transients; 1-Hz exponential line broadening. The chem ical shift scale is offset 0.2 ppm compared to the standard used for the data of Table 1.

JLfcJM ^JtrU^^^uJL ^MFW^^f^^f**

-110.0

-111.0 PPM

-112.0

-113.0

-126.0 PPM

FUPA-

3 CONC.

IN MMOLAR -SO -SO -60

-80
PPH

-110

-120

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5-FLUOROURAClL METABOLITES IN PLASMA AND URINE

Table ! "F'NMR parameters for 5-FU and its metabolites at 470 MHz (11.7 Tesla) and 28'C inRBC-93.68ppm) shift" ( MetaboliteFU DHFU FUPA FBAL pCFBAL* U,' Ut'Plasma-93.80 couplings (Hz) urine5.0 in

0.1 46.8. 14.2, 11.2* -126.27 + 0.15 AK>20 Hz' -11 1.07 0.05 19.1*51.2,27.8,23.3*52, 50.7, 28.2, -112.77 0.05 -43.82 0.2 -111.24 + 0.02 27.9, 24.4/ -110.52 _g -113.07 0.05'H-"F " Relative to trifluoroacetate in phosphate buttered saline (pH 7) = 0 ppm; deviations () epresent range of observed values for pH = 5-8. r * pH 7.84. e Couplings not resolved, line broadening apparently due to a chemical exchange equilibrium of the ureido group. * Acid-labile product of FBAL and bicarbonate found in urine with pH > 7.3 following bicarbonate infusion (MTX patients). ' Acid-labile unknowns in urine with pH > 7.3; probably derivatives of FBAL. fpH 8.53. 'Three couplings (one geminai and two vicinal), not accurately determined.

0.05 -126.27 0.02 -11 1.020.05 -112.77 0.03 -43.97 0.05 -111.22Chemical

0.03 -125.82-112.62Urine-93.77

the trapezoidal rule with linear interpolation for catabolites and loga rithmic for FU. When FU was no longer detectable, its concentration was set to zero with linear interpolation back to the previous data point. The data of Figs. 5 and 6 were subjected to least-squares regression analysis with FU dose as independent variable x. For Fig. SA a linear regression of the form y = bx was calculated. For the apparently nonlinear functions (Figs. 5, B and C, and 6/4), a linear regression of the logarithmically transformed data was calculated, log y = a + b\ogx.

RESULTS FU Metabolites in Plasma. As shown in Fig. 2, FU, its three expected catabolites, and in several cases, small amounts of fluoride anin, could be observed and quantitated in untreated plasma samples by means of "F NMR. Pertinent NMR param eters are summarized in Table 1. In no case could FU anabolites or other fluorine-containing metabolites such as 2-fluoroacetate (chemical shift, 6 = 141.16 ppm), 2-fluorocitrate (114.6 ppm), or 3-fluoropyruvate (-152.95 ppm) be observed (lowest detection limit ca. 2 IM samples measured for approxi for mately 12 h). Repeated measurements made over a period of many hours at 28Cdemonstrated that all observed species were quite stable in plasma, with the exception that occasionally some spontaneous conversion of DHFU to FUPA occurred. The catabolites DHFU, FUPA, and FBAL exhibited signal line widths only slightly greater than observed in saline or urine. On the other hand FU showed substantial line broadening in plasma which made quantitation more difficult. The observed line widths (Av) were usually 30-170 Hz and varied from patient to patient and from sample to sample. In several cases at high FU concentrations a second, much broader FU signal compo nent (5 ~ 94ppm, AV ~ 1000 Hz) appeared, amounting to 10-20% of the observed FU. This line broadening phenomenon was not observed in any urine samples nor in a series of red blood cell samples from one patient. For one plasma sample the FU parameters measured before/after heat treatment (80C for 10 min) were: 8 = -93.S3/-93.74 ppm, AK= 170/56 Hz, concentration = 260/285 pM. Thus, FU appears to associate with plasma proteins. The initial FU plasma concentration 1 min after the end of a 10-min infusion was 200-700 n\t for the dosage range used. At low dosage (Fig. 4A) FU decreased rapidly to <5 /M after 30 min, while at higher doses (Fig. 4B) FU became undetectable only after approximately 90 min. Semilogarithmic plots of FU concentration versus time (not shown) indicated monoexponential behavior at the lower FU doses and a tendency toward biexponential behavior and reduced slope of the /3-elimination phase at the higher doses, similar to results obtained with i.v. bolus injection (30). A more detailed analysis of FU plasma

15

30

45

60

75

90

time (min) after FU infusion

time (min) after RJ infusion

Fig. 4. Dose dependence of the pharmacokinetics of FU and its catabolites in plasma after a 10-min infusion of 5-FU (monotherapy). Metabolite concentrations in tM determined by "F NMR are plotted versus time after end of infusion for as two particular cases: at left (a) male patient, age 63, colon carcinoma with liver mtastases,61 ^mol (8 mg) FU/kg; at right (b) male patient, age 54, rectal carcinoma, liver and retroperitoneal lymph node mtastases.154 urnol (20 mg) FU/kg.

kinetics for these and other patients will be presented else where.6 The first catabolite DHFU could be observed (> 10 /M) within 5 min after infusion and at the higher FU doses even at the I min time point (Fig. 4).FUPA and FBAL appear 5-10 min after infusion and all three catabolites are generally observable from 15-90 min. The intermediates DHFU and FUPA show essentially steady-state behavior over the time period 15-90 min, with plasma concentrations of approximately 10-40 I*M for the range of FU doses used. The presumed end product of catabolism, FBAL, was the dominant FU metabolite after 30 min. As a rule at low FU doses (Fig. 4/4) FBAL reached a maximum concentration (pla teau) at the time when FU disappeared from plasma. At higher FU doses FBAL continued to increase as long as FU was present, and higher levels of FBAL were achieved (up to 170 /M). Interestingly, the initial rate at which the concentration of FBAL in plasma increases is virtually independent of FU dose, as seen in Fig. 4 and evidenced by the fact that for 10 of 13 treatments with doses of 61-210 ^mol FU/kg the FBAL con centration at 15 min fell in the narrow range of 19-33 ^M. In Fig. 2 FUPA gives a somewhat broadened signal without resolution of the 'H-'9F coupling constants JHp that can be resolved for the other catabolites. This is also true for spectra of urine (compare with Fig. 3). In a few cases plasma spectra (90-min time point, FBAL > 100 ti\\) showed an additional
* R. E. Port, B. Britsch, and R. Herrmann, manuscript in preparation.

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5-FLUOROURACIL METABOLITES IN PLASMA AND URINE

signal at approximately 110.5ppm (approximately 30 overlapping with the FUPA signal (see "Discussion"). Free fluoride (F~) was observed (>2 ^M) in about one-fourth of the samples that were appropriately examined, i.e., 63 sam ples: median, 5 ^M; max, 33 M 4A). The signal is generally (Fig. broad indicating the effect of some weak association equilib rium, but it is expected that fluoride strongly bound to proteins or otherwise tightly complexed will give a signal too broad to be detectable at these low concentrations. For nine treatments the areas under the plasma concentration versus time curves (AUC) were calculated for the time interval 15-90 min after infusion. A plot of the AUC for FU plus all catabolites versus FU dose is shown in Fig. 5A. A reasonably good linear correlation is obtained (correlation coefficient, r = 0.86). The corresponding plot for the AUC of FU alone (Fig. 5B) shows clearly that the amount of FU circulating in plasma after 15 min tends to increase more than linearly with increasing FU dose. The AUCs for the sum of all catabolites show the opposite trend (Fig. 5C), increasing in a less than linear manner with FU dose. For one patient (FU = 202 imol/kg,no MTX) RBCs were also collected and measured in the same manner as for plasma. The FU concentration in the RBC suspension was found to be uniformly a factor 3.7 0.2 less than in plasma at all time points. DHFU was detected at approximately 10 fiM for 15, 30, and 60 min, while FUPA was not detected (<4 M). Small amounts of FBAL were detected at 30 and 60 min (10 and 18 MM,respectively, compared to 51 and 113 /M plasma). in FU Metabolites in Urine. In the 0-2-h urine samples >97% of the total observed fluorine was in the form of FU and its known catabolites DHFU, FUPA, FBAL plus CFBAL and F~ (up to 2%). CFBAL and two unidentified compounds Ui and U2 (Fig. 3, Table 1) appeared consistently in urine with pH > 7.3 (MTX patients). CFBAL could be easily generated in urine containing FBAL by adding bicarbonate and increasing the pH to above 7.3. When the pH of alkaline urine is lowered to <7 by adding dilute HC1, CFBAL disappears and there is a corre sponding increase ir FBAL. We also confirmed the formation of CFBAL in plasma, as reported by Malet-Martino et al. (9), by adding FBAL to a plasma sample at pH 7.8, 28C, hereby w the characteristic "F multiplet signal for CFBAL appeared at -111.2 ppm (FBAL, 3.2 IHM;CFBAL, 0.2 HIM). The compounds Ui and U2 depend on pH in a manner similar to CFBAL. They never amount to more than 1% of the FU dose and are most likely also adducts of FBAL with other components of urine. As mentioned above, plasma spectra (pH 7.5-7.8) occasionally show an additional signal at the posi tion corresponding to Ui.

In general the levels of DHFU in urine were quite low (total excretion < 0.5%). Repeated measurements of a given sample over a period of several hours showed that the rate of sponta neous conversion of DHFU to FUPA was not high enough to cause significant errors in the analysis. For the treatments studied 2.6-30% of the FU dose was excreted in the 0-2-h urine (median, 10.4%; n = 18), with 0.914% of dose appearing as unmetabolized FU (median, 3.8%). The results of two separate treatments for the same patient differed by <5% of the dose for total excretion of FU and catabolites in 0-2-h urine and by <2.3% of the dose for excre tion of unmetabolized FU (four patients). For three patients receiving FU monotherapy (152-202 ^mol/kg) urine was col lected for 3 days. In the first 24 h 60-66% of the dose was excreted as fluorine compounds (5-8% as FU). In the second and third 24-h periods only 1.1-1.6% and 0.2-0.4% of dose, respectively, appeared in urine as primarily FBAL plus F~ and FUPA. The concentrations of FU and FBAL + CFBAL in 0-2-h urine, expressed relative to creatinine (see "Methods"), have been plotted versus FU dose in Fig. 6. As was found for the FU plasma AUCs, the ratio FU/creatinine in urine increases more than linearly with increasing dose (compare Fig. 5B with 6/4). The (FBAL + CFBAL)/creatinine ratio (Fig. 6B) shows large scatter and no correlation with dose (correlation coefficient, r = 0.03).For those patients where 0-2-h urine was sampled at two separate treatments, only the results of the earlier treatment have been entered in Fig. 6. Figs. 5 and 6 demonstrate that there is no clear distinction between the data observed for responders and for nonresponders. Similarly, there are no differences evident for patients receiving FU monotherapy or pretreatment with MTX. For most patients free fluoride (F~) was found in 0-2-h urine with widely varying concentrations (19-300 M). after Even normalization to creatinine levels, fluoride excretion showed no clear dose dependence (linear regression analysis, r = 0.2). In four cases urine samples taken just prior to FU infusion were examined and approximately 10-30 ^M F~ could be de tected, and a number of urine samples from normal volunteers showed similar concentrations of F~. After FU infusion F~ levels in urine were significantly elevated up to 24 h after infusion and returned to pretreatment levels after 48-72 h (Fig. 7). The total amount of F~ excreted in urine represented 0.51.3% of the FU dose. The commercial FU stock solutions (Fluorblastin, 50 mg FU/ml, pH 8.93) were also examined by "F NMR. Signals from approximately 40 different, largely unidentified, sub stances (none of which were DHFU, FUPA, or FBAL) could

20000-,

20000-,

Fig. 5. Area under plasma concentration curve (AUC) for FU and its catabolites for the time period 15-90 min after end of a 10-min infusion plotted as a function of FU dose. Representative data for a group of nine pa tients show: A, sum of FU and catabolites; B, FU alone; C, sum of catabolites (largely FBAL). Symbols, patients with positive (A) or negative (V) responses to therapy or (O) when no response assessment could be made; solid symbols, pretreatment with MTX. Equations of regression lines: A,y = 19.1 x (r = 0.86); B, logy = -2.96 + 2.93 log* (r = 0.94); C, \ogy = 2.61 + 0.601 log* (r = 0.83).

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b)
A response to therapy v tumor progression o no assessment of response open symbols: FU only solid symbols: MTX-f FU 2-

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Using 5-mm sample tubes and the relaxation agent chromium(III) acetylacetonate the Martino group was able to achieve a metabolite detection threshhold of approximately 10 /M approximately 12 h of data acquisition. With 10-mm for sample tubes (2-ml sample) and without relaxation agent our instrumentation allows a detection threshhold of 1-2 /<Min 12 h. For quantitative work reasonable limits are approximately 3 1 fiM for 12 h acquisition and approximately 15 /JM for 5 30-min acquisition.7 In the course of our work we had independently identified F"

as a product of FU catabolism ( 18) before learning of the results of the Martino group (20, 21). Here we present NMR data for three acid-labile substances not observed in urine by these workers. CFBAL and the unknowns Ui and U2 occur only in urine with pH > 7.3, a result of the bicarbonate infusion 50 WO 150 200 250 50 100 150 200 250 protocol for MTX pretreatment. Chemical shifts and coupling FU dose (tmoles/kg) FU dose (moles/kg) constants are consistent with all three being TV-substituted de Fig. 6. Concentration ratio of FU (A) and its major catabolite FBAL + CFBAL et al. (9) demonstrated at (B) to creatinine in 0-2 h urine following a IO min infusion of FU. Point, one rivatives of FBAL. Malet-Martino treatment for one patient, and the symbols have the same meaning as in Fig. 5. high FBAL concentrations the equilibrium in plasma between Regression line (A): iogy = -3.63 1.57 log* (r = 0.72). FBAL and CFBAL involving bicarbonate (Fig. 1); however, their observation of two separate signals for CFBAL remained unexplained. Our experiments with both urine and plasma demonstrate, as expected, a single CFBAL species with chem 0.03ical shift slightly upfield of FUPA. The additional signal downfield of FUPA observed by the Martino group appears at a | position that would correspond to Ui. In a few cases we also observed such an additional signal at approximately 110.4 0.02ppm in patient plasma containing >100 IM FBAL. The iden tities of Ui and U2 still remain to be determined. In general, we defined the integral of the broad signal in the region 110to 0.01 ppm for plasma as the FUPA concentration, but it may 112 include a contribution from CFBAL and possibly U,. In plasma only FU showed significant line broadening, and the effect was highly variable from patient to patient. The center 0.00 of the broadened FU resonance was generally shifted as much 0 -24 -48 -72 as 0.1 ppm upfield for line widths >100 Hz. This shift and time (hours) after FU infusin Fig. 7. Urinary excretion ot free fluoride (F~) following a 10-min infusion of broadening were greatly reduced after a short heat treatment FU. The fluoride concentration, normalized to creatinine, was determined by "F that precipitated proteins. This behavior is consistent with an NMR for patients receiving 85-190 imol/kgFU monotherapy. Solid symbols. association equilibrium of FU binding to plasma proteins and urine samples taken just prior to therapy; open sysmbols, samples of urine collected was not observed for a suspension of red blood cells. In the for 0-2, 2-24, 24-48, and 48-72 h after FU infusion. fast-exchange regime (e.g., weak binding) such an association equilibrium will generally lead to line broadening and possibly be observed at individual concentrations of approximately to a displacement of the observed chemical shift by amounts 0.002-0.2 mole % relative to FU. For example, a 5-month-old sample obtained from the clinic contained 0.2% F~ as the main that are proportional to the fraction of FU bound. In some impurity and 1% fluorine in all other impurities combined. The cases (FU ~ 0.5 HIM) we observed a second, very broad FU impurity levels increase with time, probably due to the high signal component displaced approximately 0.5 ppm upfield, pH. Values for F' and for all other impurities combined were, indicating that approximately 10-20% of the FU formed a tight FU-protein complex that is in the slow-exchange regime (ex respectively, 0.9 and 3% at 22 months and 2.6 and 6.8% at 57 months. Thus, a fraction of the observed F" excretion by change lifetime > 1 ms under our conditions). This is in patients can result from fluoride already present even in fresh agreement with an earlier study which found that 10% of FU binds tightly to plasma proteins (31). In their study of the FU stock. metabolism of 5'-deoxy-FUrd Malet-Martino and coworkers observed the "F NMR signal (without significant broadening) DISCUSSION for this substance in plasma and an additional broadened signal "F NMR Parameters of FU Catabolites. The NMR parame (Av 200 Hz) shifted 1.5 ppm upfield, which disappeared upon ters summarized in Table 1 show reasonable agreement with deproteinization (9, 17). This is indicative of rather tight comthose presented originally by Malet-Martino et al. (17). We plexation with plasma proteins (slow-exchange regime only). could obtain more precise measurements for the JHF coupling In a more detailed study (32) it was found that for 0.1-3.5 HIM 5'-deoxy-FUrd, the amount of complex observed was 0.05-0.2 constants of DHFU and FBAL, for example; however, we were not able to measure couplings for FUPA due to significant line HIM, similar to the amount that we found for the very broad FU signal component. Thus, 5'-deoxy-FUrd appears to bind broadening possibly caused by an intermediate rate of the protonation equilibrium for the ureido moiety or of a confor- more tightly than FU to plasma proteins so that, in contrast to mational equilibrium. All other catabolites as well as CFBAL, 7 A factor 5 reduction in concentration requires a factor 25 increase in mea surement time (see "Methods"). Ui, and U2 always gave highly resolved spectra.
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(45), and the results of in vivo I9F NMR experiments with mice5 (15). The data for the 0-2-h urinary excretion of FU catabolites Kinetics of FU and Major Catabolites. This study confirms (Fig. 6B) showed large scatter and no clear dose dependence. that a major portion of i.v. administered FU is excreted in urine Data for repeated treatments of individual patients showed less as catabolites, primarily FBAL, and that a significant amount scatter, and for the few cases where we examined 0-24-h urine of unmetabolized FU appears at the higher doses used. For much less variability among patients was observed. Thus, Fig. three of our patients where 0-24-h urine was collected, the 6B reflects significant interpatient differences in short-term observed excretion amounted to 60-66% of the FU dose, com renal elimination of FBAL. pared to values of 50-100% found in studies with i.v. bolus Our measurements of FU and catabolites in packed red blood injection (11, 20, 33-35). In their "F NMR study of a single cells indicate that FU is in equilibrium between plasma and patient receiving bolus injections of 750 mg (5.77 mmol) FU, erythrocytes. However, catabolism in erythrocytes is slow since Bernadou et al. (20) found that 90 10% of the dose was only small amounts of DHFU and FBAL were found. Similar excreted over 24 h and that 8-14.6% of the dose was excreted behavior has been reported for treatments with 5'-deoxy-FUrd as unmetabolized FU within 2 h after each treatment. For our (17). patients receiving similar doses infused over 10 min only 2-3% Other Metabolites. Malet-Martino et al. (9) have observed FU was excreted. In agreement with the French group we find the nonenzymatic formation of 7V-carboxy-FBAL in plasma at that after 24 h small amounts (<2% of dose) of FBAL, FUPA, FBAL concentrations > 100 ^M. For our patients receiving and F~ are still being excreted. MTX the accompanying bicarbonate infusion results in an Efficient catabolism and renal elimination mean that only a alkaline urine pH and the formation of large amounts of minor fraction of the applied FU dose can possibly be converted CFBAL and small amounts of the unknown substances Ui and to cytotoxic anabolites, and this occurs not only in tumors but U2 (Table 1). In the past FGPA has been discussed as a possible also in normal tissues (3, 27, 36, 37). The time course of FU intermediate in the FU catabolic pathway (Fig. 1) (8). When or related fluoropyrimidines and their catabolites in plasma FU was labeled with I4C in position 6, a small portion of the and urine, which we and others (9, 20) observe by 19F NMR, label appeared as a metabolite designated as FGPA in urine, cannot directly reflect the intensity of FU anabolism in tumors. liver, and kidney of the mouse (11). With I4C in position 2 of Thus, as has been shown for the pharmacokinetics of FU alone FU, small amounts of the label appeared as the presumed (30), we could find no useful correlation between the parameters FGPA, and significant amounts of label were found as urea, observable by NMR and tumor response to FU therapy. Such especially in the liver (3). However, in this experiment labeled studies may, however, provide useful information that can urea could arise via the urea cycle by conversion of labeled CO2 possibly be correlated with toxic side effects. that is released to form FBAL (Fig. 1), without the need to MTX pretreatment had no observable influence on the meas form FGPA. We examined the possibility that FGPA could be ured FU catabolism (Figs. 5 and 6). This is reasonable based produced in a nonenzymatic equilibrium of FBAL with urea or on the concept that MTX operates synergistically in the cyto FUPA with ammonia, but we could observe no such reaction toxic anabolic pathway of FU either by (a) interfering with de in urine, even at pH 9 (e.g., no increase in either Ui or U2). novo purine synthesis which results in an accumulation of 5- Thus, from our studies we can offer no new evidence for the phosphoribosy 1-1-pyrophosphate followed by enhanced forma existence of FGPA either as an obligatory or a minor FU tion of FU nucleotides (38, 39) or (b) interfering with thymi- metabolite. Appearance of Fluoride. Free fluoride anin (F~) has been dine-5'-monophosphate synthesis by inhibition of dihydrofolate reduc-aseand enhancement of the binding of FdUMP to described as a metabolite of FU (20) and 5'-deoxy-5-fluorouriFU, no significant kinetic (lifetime) line broadening of the free 5'-deoxy-FUrd signal is observed. thymidylate synthase (40). Mechanism a is supported by recent in vivo "F NMR studies of animal tumors, where increased dine (9) on the basis of NMR studies. Our work confirms these reports and demonstrates (Fig. 7) that urinary fluoride levels in our patients are elevated above normal "background" levels for 1-2 days following FU administration. Although commercial FU preparations are contaminated with small amounts of flu oride (typically 0.1-0.2 mole % for fresh material), this can account for only a fraction of the urinary fluoride observed. In agreement with other reports (9) we find that the time course of urinary fluoride excretion is similar to that of FBAL. The fraction of total metabolite excretion found in 0-2-h urine decreases in the order FU > DHFU + FUPA > FBAL + CFBAL > F~, suggesting that fluoride is the last catabolite to appear. Most convincing is the demonstration that F" is re

synthesis of FU nucleotides was observed (41). Our findings, that the FU plasma AUC (Fig. 5) and FU renal excretion (Fig. 6A) increase more than linearly with dose, are consistent with the concept that the metabolic elimination of FU is increasingly saturated within the therapeutic dose range (42). It has often been assumed that the reduction of FU to DHFU is rate limiting, and this appears to be true in liver and in many solid tumors but not in intestinal mucosa, pancreas, lung, or lymphocytes where the second step to FUPA appears to proceed very slowly (43). The liver enzyme dihydrouracil reductase demonstrates significant substrate inhibition for ar aci! > 125 MM(43). This may contribute to the saturation of leased after direct application of FBAL in the rat (21). FU catabolism (plasma clearance) that we observe for patients Typical free fluoride background levels in plasma are approx with FU doses > 90 /mol/kg, where plasma concentrations imately 1 MM(46). Levels observed in our patients following exceed 125 >M as long as 30 min. for FU application ranged from 5 to 33 ^M, high enough to lead The dose-independent rate of appearance of FBAL in plasma to inhibition of certain enzymes (47). Some of the clinical side (Fig. 4) and the less than linear increase in total catabolite effects of FU, e.g., vomiting and nausea (48), are also known plasma AUC (Fig. 5C) with FU dose may be due in part to in acute poisoning by inorganic fluoride (49), whereby docu saturation of catabolism, but transport of charged catabolites mented symptoms appeared when plasma levels of fluoride such as FBAL out of the cell could also be the rate-limiting were 180-820 ^M (50). It should be noted that fluoride contam process. The importance of the latter effect is supported by the ination in commercial FU preparations increases with storage observation of high intra-/extracellular catabolite gradients for time (more rapidly at higher concentrations with higher pH). rat hepatocytes (44), high liver/plasma catabolite ratios in rats Metabolism of FBAL. In view of the known neurotoxic side
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FU plasma concentration. However, HPLC requires significant sample preparation, experimentation with columns, eluents and flow rates, frequent calibration checks, and for many applica tions the use of radioactive labels (e.g., FU catabolites). On the other hand, "F NMR techniques allow the precise and repro our much higher instrumental sensitivity allows us to place ducible determination of all fluorine-containing substances si concentration limits of < 3 n\i for FBAL metabolites other multaneously with just a single calibration procedure and allow than fluoride. This is surprising in view of the report that - the detection and structure determination of previously un alanine is rapidly metabolized to acetate in the rat (52). The known products (e.g., CFBAL and F~). However, to improve first step in 0-alanine catabolism is a transamination reaction therapy regimens or to provide an early prognosis, such studies of biological fluids will probably not suffice. "F NMR tech to form malonic acid semialdehyde (53). The corresponding product from FBAL would be HC(O)CHFCO2~, which can niques allow one to make the necessary direct measurement of subsequently undergo decarboxylation to 2-fluoroacetaldehyde the levels of cytotoxic anabolites in normal and tumor tissue and conversion to 2-fluoroacetate. An extensive literature on biopsies (27) and even noninvasively by means of in vivo "F the inhibitory effect of -fluoro-a-amino acids on racemase and NMR (16). transaminase reactions (54) indicates that the Schiffs base intermediate formed with the pyridoxal phosphate cofactor of these emzymes can eliminate HF to form an encamine- which REFERENCES deactivates the enzyme. Recently a /3-alanine transaminase has 1. Heidelberger, O., Chaudhuri, N. K., Danneberg. P.. Mooren, D., Griesbach, been isolated from Streptomyces gr-seasand was found to be L., Duschinsky, R., Schnitzer, R. J., Pleven, E., and Scheiner, J. Fluorinated fully inhibited after incubation with FBAL (55). Since FBAL pyrimidines, a new class of tumor-inhibitor) compounds. Nature (Lond.). 4561: 663-666, 1957. also possesses the structural requirements for the elimination of HF (as H+ and F~), we propose that the transaminase 2. Chaudhuri, N. K., Montag. B. J., and Heidelberger, C. Studies on fluorinated pyrimidines III. The metabolism of 5-fluorouracil-2-'4C and 5-fluorooroticreaction leads to the F~ observed in the FU metabolism studies 2-MC acid in viro. Cancer Res., 18: 318-328. I958a. and to deactivation of the transaminase (Fig. 1). This would explain the release of F~ from FBAL in the rat (21 ) and why no significant catabolism of FBAL to 2-fluoroacetate is ob served. Malet-Martino et al. (9) have discussed the rationale behind the use of 5'-deoxy-FUrd instead of FU in terms of the lower plasma concentrations of FU attained and the expected reduc tion in toxic side effects. However, massive doses of 5'-deoxyFUrd were given (approximately 80 mmol), and approximately 50% of the drug appears in urine as FBAL, meaning that at least 40 mmol FU must have been generated intracellularly (approximately 15 mmol FU are applied in a typical high dose treatment). Furthermore, the reported steady-state plasma lev els of FBAL (200-400 UM) and F" (20-30 MM)over 6 h were as high or higher than the maxima observed in our FU studies. Recently, /3-alanine has been shown to competitively inhibit transport of the inhibitory neurotransmitter 7-aminobutyric acid (56, 57). FBAL may have a similar effect. Furthermore, the possible deactivation of transaminases by FBAL, as dis cussed above, may be of significant consequence for the levels of 7-aminobutyric acid and 0-alanine, whose increase can lead to neurological disorders (53 and references therein). These findings, coupled with the acknowledged similar neurotoxic effects of both 5'-deoxy-FUrd (9, 58) and FU (51) should stimulate further investigation of the direct biological effects of FBAL. NMR Versus HPLC. Our studies and the work of Martino and coworkers have shown that "F NMR allows the pharmacokinetics of fluoropyrimidines and the time dependence of fluorine-containing metabolites in plasma and urine to be stud ied quantitatively, without resorting to any chemical treatment or Chromatographie techniques. Currently, our 19FNMR detec tion limit for a 12-h measurement is approximately 2 nmol of substance. The use of paramagnetic relaxation agents, higher "F radio frequency transmitter power (excitation bandwidth) and 'H-decoupling should be able to reduce this threshhold by at least a factor of 4. Commonly used HPLC techniques have lower absolute detection threshholds, e.g., in our case 200 M'of plasma were extracted and 10 i\ thereof were used for HPLC for a UV-detection limit corresponding to approximately l n\\
3. Chaudhuri, N. K., Mukherjee, K. L., and Heidelberger, C. Studies on fluor inated pyrimidines VIIThe degradative pathway. Biochem. Pharmacol., /: 328-341, 1958b. 4. Mukherjee. K. L., Boohar, J.. Wentland. D., Ansfield, F. J., and Heidelberger, C. Studies on fluorinated pyrimidines XVI. Metabolism of 5-fluorouracil-2I4C and 5-fluoro-2'-deoxyuridine-2-14C in cancer patients. Cancer Res., 23: 49-66, 1963. 5. Heidelberger, C. Fluorinated pyrimidines. Prog. Nucleic Acid Res. Mol. Biol., 4: 1-50, 1965. 6. Heidelberger. C. Fluorinated pyrimidines and their nucleosides. In: A. C. Sartorelli and D. G. Johns (eds.), Antineoplastic and Immunosuppressive Agents, part II, pp. 193-231. Berlin-Heidelberg-New York: Springer, 1975. 7. Heidelberger, C., Danenberg, P. V., and Moran. R. G. Fluorinated pyrimi dines and their nucleosides. Adv. Enzymol., 54: 58-119, 1983. 8. Myers, C. E. The pharmacology of the fluoropyrimidines. Pharmacol. Rev., 33: 1-15, 1981. 9. Malet-Martino, M. C., Armand, J. P., Lopez, A.. Bernadou, J., Bteille, J. P., Bon, M., and Martino, R. Evidence for the importance of 5'-deoxy-5fluorouridine catabolism in humans from ' '1 nuclear magnetic resonance spectrometry. Cancer Res., 46: 2105-2112, 1986. 10. Schuetz, J. D., Collins. J. M., Wallace, H. J.. and Diasio, R. B. Alteration of the secondary structure of newly synthesized DNA from murine bone marrow cells by 5-fluorouracil. Cancer Res., 46: 119-123, 1986. 11. Mukherjee. K. L., and Heidelberger, C. Studies on fluorinated pyrimidines IX. The degradation of 5-fluorouracil-6-14C. J. Biol. Chem., 235: 433-437, 1960. 12. Koenig, H., and Palei, A. Biochemical basis for fluorouracil neurotoxicity. The role of Krebs cycle inhibition by fluoroacetate. Arch. Neurol., 23: 155160, 1970. 13. Davis, H. L. Chemotherapy of large bowel cancer. Cancer (Phila.), 50: 26382646. 1982. 14. Stevens, A. N., Morris, P. G., lies, R. A., Sheldon, P. W.. and Griffiths, J. R. 5-Fluorouracil metabolism monitored in viro by "F-NMR. Br. J. Cancer, 50: 113-117. 1984. 15. Hull. W. E., Port, R., Osswald, H., Kunz. W.. Juretschke. H. P., and Schuff, N. In-vivo 19F-NMR study of 5-fluorouracil metabolism in liver and im planted tumors of the mouse. Abstracts of the 5th Annual Meeting, Society of Magnetic Resonance in Medicine. Montreal, p. 594. 1986. 16. Wolf. W., Albright, M.J.. Silver, M. S., Weber, H.. Reichardt, U., and Sauer, R. Fluorine-19 NMR spectroscopic studies of the metabolism of 5-fluorour acil in the liver of patients undergoing chemotherapy. Magn. Reson. Imaging, 5: 165-169. 1987. 17. Malet-Martino, M. C., Martino, R., Lopez, A.. Bteille. P.. Bon, M., J. Bernadou, J., and Armand, J. P. New approach to metabolism of 5'-deoxy5-fluorouridine in humans with fluorine-I9 NMR. Cancer Chemother. Phar macol., 13: 31-35, 1984. 18. Port. R. E., Hull, W. E., Herrmann. R., Britsch. B.. and Kunz, W. Metabolites of 5-fluorouracil in human plasma and urine as determined by "F NMR spectroscopy. J. Cancer Res. Clin. Oncol.. ///(Supple.).- 22. 1986. 19. Herrmann. R., Knuth, A., Kleeberg. U., Middecke, R.. and Abel. U. Random ized multicenler trial of sequential methotrexate (MTX) and 5-fluorouracil (FU) vs. FU alone in mctastatic colorectal cancer (CRC). Proc. Am. Soc. Clin. Oncol., 5:91, 1986. 20. Bernadou. J.. Armand, J. P., Lopez, A., Malet-Martino, M. C., and Martino, R. Complete urinary excretion profile of 5-fluorouracil during a six-day

effects of FU therapy (12, 51), we reckoned with the possibility of detecting 2-fluoroacetate or 2-fluorocitrate as metabolic products of FBAL. However, even at the highest FU doses used neither of these substances was detected. Similar negative re sults were reported for 5'-deoxy-FUrd treatments (9), whereby

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21. 22.

23. 24.

25.

26.

27.

28. 29. 30. 31.

32.

33.

34.

35.

36.

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