Pharmacokinetics using fluorine NMR in vivo

PROGRESS IN NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY

ELSEVIER

Progress in Nuclear Magnetic ResonanceSpectroscopy 33 (1998) 1-56

Pharmacokinetics using fluorine NMR in vivo Peter Bachert Abteilung Medizinische Physik und Biophysik, Forschungsschwerpunkt Radiologische Diagnostik und Therapie, Deutsches Krebsforschungszentrum (dkfz, German Cancer Research Center), D-60120 Heidelberg, Germany Received 23 December 1997

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. A window to chemistry in living tissue . . . . . . . . . . . . . . . . . . . . . . 1.2. NMR properties of fluorine . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Applications of fluorine NMR in biomedicine . . . . . . . . . . . . . . . . . . . 2. The cytostatic drug 5-fluorouracil . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Clinical application of 5-fluorouracil . . . . . . . . . . . . . . . . . . . . . . . 2.2. Metabolism of 5-fluorouracil . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. The purpose of pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1. Monitoring pharmacokinetics by means of NMR . . . . . . . . . . . . . . . 3.1.2. Pharmacokinetic studies with positron emission tomography . . . . . . . . . . 3.2. Pharmacokinetic modeling - - theory . . . . . . . . . . . . . . . . . . . . . . 3.2.1. Linear kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2. Nonlinear kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Methods of fluorine NMR in humans . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Pulse experiments with surface coils . . . . . . . . . . . . . . . . . . . . . . . 4.2. Model solutions, data evaluation . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Measurement of relaxation times . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Spatial localization of fluorine NMR signals . . . . . . . . . . . . . . . . . . . 4.4.1. Fluorine N M R imaging . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.2. Volume-selective NMR spectroscopy . . . . . . . . . . . . . . . . . . . 4.4.3. Chemical shift imaging . . . . . . . . . . . . . . . . . . . . . . . . . 4.5, Magnetic double resonance . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5.1. Dynamic nuclear spin polarization - - theory . . . . . . . . . . . . . . . . 4.5.1.1. Truncated-driven 19F - - { IH} nuclear Overhauser effect . . . . . . . 4.5.1.2. Transient 19F - - { IH} nuclear Overhauser effect . . . . . . . . . . . 4.5.2. Proton spin decoupling . . . . . . . . . . . . . . . . . . . . . . . . . 0079-6565/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved PH S0079-6565(98)00016-8

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3 3 4 5 6 6 8 10 10 10

11 12 12 13 14 14 16

18 20 20 21 22 24 26 26 30 34

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P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

5. Biomedical applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Volume-selective fluorine N M R spectroscopy of the human liver . . . . . . . . . . . . . 5.2. Monitoring 5-fluorouracil catabolism with 19F - - { IH} double resonance . . . . . . . . . . 5.3. Clinical studies: 19F N M R during fluoropyrimidine chemotherapy . . . . . . . . . . . . . 5.4. Pharmacokinetic modeling: 5-Fluorouracil and o~-fluoro-/3-alanine in vivo . . . . . . . . . . 6. Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . .

36 36 39 41 48 51 52 52

Keywords: Fluorine; In vivo 19F NMR spectroscopy; Chemical-shift imaging; Double resonance; Dynamic nuclear polarization; Nuclear Overhauser effect; Spin decoupling; 5-Fluorouracil; t~-Fluoro-/3-alanine; Metabolism; Pharmacokinetics; Kinetic modeling; Nonlinear compartment model; Drug monitoring; Chemotherapy; Liver tumor

Nomenclature The abbreviations used in this article are as given later. Physics c~ t~E APS AQ B2 CSI d 2D Ap tr2 i FID FLASH FOV GP GS 1 15o

IR ISIS J~s NEX NOE PET RF p~ SAR SD o[s SIN STEAM TA TD

flip angle Ernst angle aperiodic pulse saturation acquisition decoupling field chemical-shift imaging slice thickness two-dimensional full line width at half maximum nuclear Overhauser enhancement free induction decay fast low-angle shot field of view phase-encoding gradient slice-selection gradient normalized NMR signal intensity normalized intensity in sum of spectra obtained during 50 min postinjection inversion recovery image-selected in vivo spectroscopy scalar spin-spin coupling constant number of excitations nuclear Overhauser effect positron emission tomography radio frequency direct dipolar longitudinal relaxation rate constant specific absorption rate standard deviation cross-relaxation rate constant signal-to-noise ratio stimulated-echo-acquisition mode measurement time interpulse delay

TE TG TI TM TR TS VOI W

echo time gradient time inversion time middle interval repetition time saturation time volume of interest transition probability

Chemistry CFBAL DFBP DHFU DHUDH dT/VIP FAC FBAL FdUDP FdUMP FdUrd FdUTP FGPA FHPA 5-FU FUDP FUMP FUPA 5-FUranuc FUrd FUTP NADPH PFC TFA

N-carboxy-c~-fluoro-fl-alanine 2,5-difluoro-benzophenone 5,6-dihydro-5-fluorouracil dihydrouracil dehydrogenase thymidine-5'-monophosphate 2-fluoroacetate c~-fluoro-~-alanine 5 -fluoro-2'-deoxyuridine-5 '-diphosphate 5 -fluoro-2'-deoxyuridine-5'-monophosphate 5-fluoro-2'-deoxyuridine 5 -fluoro-2'-deoxyuridine-5'-triphosphate a-fluoro-fl-guanidinopropanoic acid 2-fluoro-3-hydroxypropanoic acid 5-fluorouracil 5-fluorouridine-5'-diphosphate 5-fluorouridine-5'-monophosphate a-fluoro-/~-ureidopropanoic acid 5-fluorouracil-nucleosides and -nucleotides 5-fluorouridine 5-fluorouridine-5'-triphosphate nicotinamide adenine dinucleotide phosphate perfluorocarbon trifluoroacetic acid

Pharmacokinetics A amount of drug AUC area under curve b.s. body surface

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56 b.w. c

CL CLd CLe D kM Ro t]/2 t I/2,~

V Vt V2 V3 Vs v max

body weight concentration clearance distribution clearance elimination clearance dose Michaelis-Menten constant infusion rate half-life half-life during the terminal phase distribution volume central distribution volume (central compartment) peripheral distribution volume (peripheral compartment) distribution volume of catabolites steady-state distribution volume maximum velocity

I. Introduction The effective use of anticancer drugs requires an understanding of the biochemical and physiological processes that take place in the organism over the course of treatment. Usually these processes are assessed by studies of model systems, in vitro chemical analysis, and the evaluation of concentration-time profiles measured in body fluids. Because the response of tumors to antineoplastic drugs is often poor, monitoring of pharmacokinetics in the individual patient would be of great value. The measurement of drug and metabolite concentrations in the target organ permits the determination of the interindividual variability of local drug uptake and elimination, as well as the individual adjustment of the drug dose. The observation of drug pharmacokinetics and metabolism in the living organism (in vivo) became possible with the advancement of noninvasive detection techniques. These allow the measurement of the local disposition of administered drugs in the tissue with high spatial and temporal resolution, and selective monitoring of the kinetics of drugs and their conversion into metabolites. Maps of drug distribution in the human body are obtained by means of radiotracer methods, such as positron emission tomography (PET), which require the administration of compounds that carry a radioactive nucleus. A window to chemistry in vivo is created by nuclear magnetic resonance (NMR)

3

spectroscopy. In particular, fluorine NMR was applied in toxicology and medicine to study the pharmacokinetics of xenobiotics. The 19F nucleus is especially useful for this purpose, because of its favourable NMR properties and because of the large variety of fluorine-containing drugs that are in clinical use today. The aim of this article focuses on the application of fluorine NMR to studies in humans using whole-body scanners and on methods that enhance spatial, temporal and spectral resolution in 19F detection. Our primary interest is the pharmacokinetics of the anticancer agent 5-fluorouracil (5-FU), an analogue of the pyrimidine base thymine. Methodological developments and the basic concepts of pharmacokinetics, as well as clinical studies, are presented. A large number of in vitro and animal experiments with fluorine NMR were performed in the past, but these studies are not the topic of this article. 1.1. A window to chemistry in living tissue

In vivo NMR is a noninvasive detection method that uses nonionizing radiation which can be used for repeated measurements. In contrast to many other analytical techniques, NMR does not require prior knowledge of the nature of the metabolites. In vivo NMR can contribute to the solution of the following problems: • •

•

•

Biochemical analysis: detection of metabolites in living tissue. Quantitative analysis: determination of absolute metabolite concentrations in tissue and of intracellular magnesium ion concentration. Observation of chemical and physical processes: rates of enzyme-catalyzed and other chemical reactions, transport, diffusion, changes of metabolite concentrations and of intracellular pH. Pharmacokinetics: detection of drugs and labeled compounds after exogenous administration, monitoring drug distribution and elimination.

There are specific limitations of in vivo NMR spectroscopy, the most serious one being the poor sensitivity (owing to the low energy of - 2 × 10 -7 eV of the detected quanta):

4

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

•

•

•

•

Sensitivity: metabolite concentrations in tissue must be larger than 10-4M = 100 nmol/(g tissue) to be detected by means of in vivo NMR. Essentially, mobile compounds of low molecular mass can be observed. In some instances macromolecules are visible, for example, glycogen (mol. wt. = 107-109) by means of ~3C NMR [1]). Measurement time: the measurement time is restricted, which limits the signal-to-noise ratio (S/N) of in vivo NMR spectra. This is a particular problem in clinical studies, since most patients do not tolerate lying in the scanner for more than - 1 h. The total examination time of patients includes the time needed for positioning, preparatory proton NMR imaging, shimming and the measurement time of the spectra. Localisation: the application of NMR in vivo requires the acquisition of signals of selected regions within the examined tissue or organ. The low sensitivity limits the size of the detected volume. For example, the voxel size of localized in vivo 31p NMR spectroscopy of the human brain is >- 30 cm 3 in realistic measurement times. Spectral resolution: because the detected nucleus can be present in a variety of different metabolites, in vivo NMR spectra often show a complex superposition of resonances. In addition, physiological motion during signal accumulation, as well as susceptibility effects resulting from the heterogeneity of the tissue,

induce a broadening of in vivo NMR lines. The overlap of signals leads to problems with the quantitative evaluation of the spectra. Specific absorption rate (SAR): the irradiation of intense radiofrequency (RF) waves, for example in spin decoupling experiments, can lead to heating of the tissue. In vivo NMR was performed with nuclei present in tissue water (IH, 2H [2]), in free ions (23Na [3]), and in endogenous metabolites (1H [4-6], 13C [7-11], 31p [12-15]). Rare nuclei were detected in the tissue after exogenous administration of the free atom (hyperpolarized noble gases 3He [16], 129Xe [17]) or of biologically active compounds and drugs that carry a magnetic nucleus, e.g. 7Li [18], 13C [7], 15N [19], t 7 0 [20], 19F [21] and 31p [22]. In humans, these studies were performed with 3He [23,24], t29Xe [25], 7Li [26,27] and 13C [28-30]. In particular, fluorine has become an important probe for monitoring drug pharmacokinetics in humans [31-36].

1.2. NMR properties of fluorine Early efforts to use fluorine as a source of the NMR signal were made in solids. In 1942, Gorter and Broer attempted, without success, to detect the resonance of KF at low temperatures [37]. The observation of fluorine chemical shifts was first reported by Dickinson in 1950 [38]. The 19F nucleus was used in a number of fundamental NMR experiments, e.g.

Table 1 Properties of selected in vivo NMR nuclei Nucleus

Spin (h)

Gyromagnetic ratio a

Larmor frequency at 1.5 T (MHz)

Natural abundance (%)

Abundance in tissue (%)

Relative NMR sensitivity × natural abundance

Biological sensitivity b

1H t3C 19F 31p

1/2 1/2 1/2 1/2

1.000 0.252 0.941 0.405

63.86 16.06 60.08 25.85

99.98 1.11 100 100

63.0 0.1 c 0.22

1.00 1.77 × 10 -4 0.83 6.63 × 10 -2

1.00 2.66 × 10 -5 -2.32 × 10 -4

a In units of 1H gyromagnetic ratio ~'H. b If all nuclei in the tissue can be detected by NMR. c Trace element.

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

the observation of the transient nuclear Overhauser effect (NOE) on anhydrous hydrofluoric acid [39] and the solid effect on LiF [40]. The sensitivity of a nucleus with spin quantum number 1 4:0 and gyromagnetic ratio 3, is proportional to l(l + 1) × 3'3 and to its abundance in the sample. Table 1 shows the properties of important in vivo NMR nuclei including the trace element fluorine. Naturally occurring fluorine consists almost entirely of 19F which is the only stable fluorine isotope (nuclear spin 1 = 1/2). Its sensitivity is (3,F/3,H)3 = 0.83 times that of the proton (~H), the most sensitive of all stable NMR nuclei. Hence equal concentrations of these nuclei produce comparable signal-to-noise ratios. While hydrogen is ubiquitous in the body with the major fraction bound in tissue water, the physiological concentration of 19F in biological systems is very low (less than 10 -6 M). Fluorine is mainly immobilized in solid structures. Its average fraction in the solid phase of teeth and bones is about 2 × 10-4. Since endogenous mobile metabolites do not contain fluorine in sufficient concentration to be detected by 19F NMR, signals must be derived from fluorinecontaining compounds that are administered exogenously. The advantage is that their resonances can be observed without interfering background signals. However, the concentration of administered fluorine in experimental studies of living organisms must be as low as possible to prevent toxic side effects and other physiological perturbations. The 19F chemical shift is very sensitive to changes in the molecular environment of the nucleus. The chemical-shift range is large ( > 200 ppm), which allows for the resolution of a number of resonances in studies of fluorinated drugs, even at the relatively low field strengths of whole-body NMR scanners. However, chemical-shift artefacts must be taken into account when spatial localization techniques are applied to 19F NMR (Section 4.4.2). To point out the differences to ~H NMR spectroscopy, localization methods based on the application of magnetic field gradients work well with protons owing to the narrow chemical-shift range of about 10 ppm. For example, a series of three slice-selective 90 ° RF pulses (STimulated-Echo-Acquisition Mode [STEAM]: 90°-TE/2-90°-TM-90°-TE/2-AQ) applied in the presence of orthogonal gradients

5

produces a stimulated spin-echo signal of nuclei that are localized in the selected region of threedimensional space (TE = echo time, TM = middle interval, AQ = acquisition period) [6]. However, in contrast to fluorine spectra, the 1H NMR signals of a multitude of endogenous metabolites superimpose and create a complex background signal. The strongest background signal originates from tissue water protons; it is at least 104 times stronger than the metabolite resonances and must be suppressed to avoid dynamic range problems.

1.3. Applications of fluorine NMR in biomedicine Fluorine is an important probe for high-resolution NMR studies of biological systems. The 19F nucleus is well-suited for selective labeling of biomolecules. For example, 4-perfluoro-tert-butyl-phenyliodoacetamide, a compound that contains nine fluorine atoms and provides a homogeneous and intense 19F NMR resonance, was used for labeling the sulfhydryl groups of the muscle protein actin [41]. Fluorine labels were applied to study the interaction of actin and myosin [42]. The detection of spectral changes of fluorine probes allows one to monitor conformational changes of macromolecules. A review regarding fluorine NMR of proteins was given by Gerig [43]. The use of fluorine as the source of the signal for NMR tomography started about 20 years ago. In experiments on model solutions, Holland et al. obtained the first fluorine NMR images [44]. The pioneering work on 19F NMR imaging in the gas phase was carried out by Heidelberger and Lauterbur [45]. They also obtained the first fluorine NMR images from biological tissue (in vitro and in vivo) using physiologically inert perfluorinated gases to visualize the distribution of the gas in the lungs [45]. The first example of in vivo 19F pulmonary ventilation images was generated in dogs using tetrafluoromethane (CF4) as the magnetically active agent [46]. Studies of perfluorocarbons (PFC), i.e. highly fluorinated organic liquids used as components in blood substitute formulations, have been carried out in animals using in vivo 19F NMR tomography [47,48]. Myocardial perfusion and cardiac function have been investigated by in vivo 19F NMR spectrosocpy and imaging at B0 = 1.9 T on rabbits using PFC and a water-soluble trifluoro-methyl sulfonic acid salt [49].

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

Another interesting aspect of 19F NMR of perfluorocarbons is the linear relationship between the partial pressure of oxygen (pO2) in the sample and the |gF spin-lattice relaxation rate (I/T0 of the individual resonances of these compounds [50]. Dissolved molecular oxygen is paramagnetic and reduces the nuclear spin relaxation times of PFC. Eidelberg et al. have estimated the oxygen tension in brain tissue from 19F parameter images [51]. Regional variations in tissue oxygenation, i.e. local pO2, were monitored in vivo by means of T~ measurements following administration of PFC emulsions [48,52-56]. Metabolites of organofluorine compounds in body fluids, tissues and excreta were identified and quantified by means of 19F NMR spectroscopy (e.g. Ref. [57]). Studies have been carried out using fluorinated anesthetics in circulating blood [58], in intact tissue [59], and in tumors [60]. Normal and tumor tissue in rats examined with 19F NMR of halothane showed different resonances. Because halothane is a hydrophobic and lipophilic probe, the effect was attributed to changes in the hydrophobic environments (membranes) in the tumor tissue [61-63]. In vivo 19F chemical-shift imaging (CSI, Section 4.4.3) was used to map the cerebral distribution of halothane and isoflurane in rats during uptake [64]. As outlined in Section 1.2, the 19F nucleus has favourable properties for the study of metabolism and pharmacokinetics of drugs in the organism. The potential of 19F NMR spectroscopy for this purpose was first investigated by Stevens et al. by measuring the cytostatic agent 5-FU and some of its metabolites in vivo [21]. Wolf and coworkers pioneered 19F NMR in humans [31]. In a 1.5 T whole-body scanner, they performed the first 19F NMR examinations of patients undergoing chemotherapy with 5-FU and demonstrated the noninvasive detection of a fluoropyrimidine and its major catabolite. This work also represents the first noninvasive NMR study of drugs in human patients.

They are classified as alkylating agents, antimetabolites (antifolates, purine analogues, pyrimidine analogues), inhibitors of mitosis, antibiotics and enzymes. 5-Fluorouracil (Fig. la), which belongs to the class of pyrimidine bases, is the most commonly used cytostatic agent in oncology. The compound was first synthesized and characterized as a cytostatic drug in 1957 by Heidelberger et al. [65]. The antitumor effect of 5-FU is based on the high demand of the tumors of nucleobases for replication and transcription of DNA and synthesis of RNA. 5-FU can be considered as a derivative of the nucleobase thymine where the methyl group at carbon atom 5 (C5) is replaced by a fluorine atom. The mode of action of 5-FU is inhibition of enzymes that synthesize thymine from uracil. There is also a catabolic pathway of 5-FU as is common for pyrimidine bases. 5-FU, which is degraded via this route, is no longer available for the antitumor activity. Some of the catabolites are suspected of causing the side effects of 5-FU chemotherapy. 2.1. Clinical application of 5-fluorouracil

5-Fluorouracil is used for pharmacotherapy of different malignancies including neck, breast and gastrointestinal cancer. 5-FU and its nucleoside derivative 5-fluoro-2'-deoxyuridine (FdUrd) are the preferred drugs for treatment of liver metastases of colorectal tumors. 5-FU is applied as a single drug, with modulating substances (e.g. methotrexate, interferon-u), or in polychemotherapy and radiochemotherapy regimes. The drug is either administered with bolus injeco II I

II

C 2 6C 0 ~ \i~ / \H

H H F 0 +1 I I / H--N-C--C--C

t

H

H H H

g.]

2. T h e c y t o s t a t i c d r u g 5 - f l u o r o u r a c i l

Cytostatic drugs are compounds that inhibit or substantially slow down the division of functionally active cells by interference of their metabolism.

a

b

Fig. 1. Chemicalstructureof 5-fluorouracil,5-FU(a), and ~x-fluoro~-alanine, FBAL(b).

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

tion, e,g. by intravenous infusion within 10 min, or continuously, e.g. by intravenous infusion for 24 h through several consecutive days. The latter mode of application leads to a constant level of the drug and its metabolites in plasma. Following bolus infusion, an exponential decay of the 5-FU concentra-

5-Fluorouracil

H,~ 3 ~

7

tion in plasma is expected. Administered daily doses of 5-FU typically range from 600 to 1000 mg/(m 2 body surface [b.s.]). The response rates of tumors to chemotherapy with 5-FU differ widely; in colorectal tumors it is below 20% of treated patients when 5-FU is used as a single

/F

I

/,

H

I Uracilphospho-

[ribosyltransferaseI

I Deoxyuridine phosphorylaseI

I Hx t

H.,~ I

F

F

HO--I?--Og:H2 , - , ~ I/"f "~'OH OH

I

OH

OH OH

FUrd

FdUrd

FUMP

5-Fluorouridine

5-Fluorouridine-5'-monophosphate

5-Fluoro-2'-deoxyuridine Thymidine kinase I

/ ~ l C.~idylate

FUDP

FdUMP -monophosphate hosphatereductase I 5-Fluoro-2'-deoxyuridine-5'

Ribonucleoside

5-Fluorouridine-5'-diphosphate

I Adenylal~

kinase ]

FUTP

hosphatereductase I

•~l-dTMP-kinase] FdUDP

5-Fluoro-2'-deoxyuridine-5'-diphosphate I

Thymidylate synthase

5-FIuorouridine-5'-triphosphate I Polymerase (RNA) II

FdUTP 5-Fluoro-2'-deoxyuridine5'-triphosphate

V

dTMP-synthase.FdUMP.MTHF[ Fig. 2. Intracellularanabolismof 5-FU.

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

drug. The limited clinical effectiveness is attributed to an insufficient uptake of 5-FU by the tumor cells and/ or insignificant conversion of 5-FU into its cytotoxic anabolites. The low response rate requires new approaches of the treatment, such as individual adjustment of drug delivery and the modulation of 5-FU metabolism in combined chemotherapy. 2.2. Metabolism o f 5-fluorouracil

Biochemical and NMR studies of the metabolic fate of fluorinated pyrimidines have been carded out in cultured cells and in experimental animals, and several studies have also been completed in humans [21,31-33,66-81]. These investigations revealed a complex intracellular metabolism of 5-FU that participates, for the greater part, in the same pathways as uracil and its metabolites. The sequence of biochemical processes involving metabolic activation (anabolism) and degradation (catabolism) is illustrated in Figs 2 and 3. A review of 5-FU biochemistry and pharmacology is given in Refs. [82] and [83]. The cytotoxic activity of 5-FU requires anabolic conversion into nucleosides and nucleotides (5-FUranuc) such as 5-fluorouridine (FUrd), 5-fluoro-2'-deoxyuridine (FdUrd), 5-fluorouridine5'-monophosphate (FUMP), 5-fluorouridine-5'diphosphate (FUDP), 5-fluorouridine-5'-triphosphate (FUTP) and 5-fluoro-2'-deoxyuridine-5'-phosphates (FdUMP, FdUDP, FdUTP) [68]. The enzymes that promote the intracellular transformation of 5-FU into FUrd, FUMP and FdUrd are uridine phosphorylase (EC 2.4.2.3), uracil phosphoribosyl transferase (EC 2.4.2.9), uridine kinase (EC 2.7.1.48) and deoxyuridine phosphorylase (EC 2.4.2.23). FUMP is in equilibrium with the high-energy phosphate compounds FUDP and FUTP through the enzymes cytidylate kinase (EC 2.7.4.14) and nucleoside triphosphate adenylate kinase (EC 2.7.4.10). FUDP is deoxygenated to FdUDP (ribonucleoside diphosphate reductase, EC 1.17.4.1) which is in equilibrium with the deoxyuridine phosphates FdUMP and FdUTP through dTMP kinase (EC 2.7.4.9) and nucleoside diphosphate kinase (EC 2.7.4.6) (Fig. 2). Two mechanisms, attributed to the anabolites FdUMP and FUTP, are discussed in order to explain the anticancer effect of 5-FU: (1) inhibition of

thymidylate synthase (EC 2.1.1.45), a key enzyme of DNA synthesis (formation of ternary FdUMP.methylene-tetrahydrofolate.dTMP-synthase complexes [84]); and (2) the interference of maturation of ribosomal RNA because of the incorporation of 5-fluorouridine nucleotide units instead of uracil into RNA (RNA polymerase II, EC 2.7.7.6). As for other pyrimidine bases, 5-FU is degradated through enzymatic catabolism (Fig. 3). The major fraction of the administered 5-FU dose is converted into 5,6-dihydro-5-fluorouracil (DHFU) by liver metabolism (for this catabolite, antitumor activity by inhibition of thymidylate synthase was also demonstrated in Ehrlich ascites tumor cells [85]). The transaddition of two hydrogens to the C5-C6 double bond is catalyzed by the enzyme dihydrouracil dehydrogenase (DHUDH, EC 1.3.1.2 = dihydropyrimidine dehydrogenase) in the hepatocytes. This first step of enzymatic catabolism of 5-FU requires the highenergy cosubstrate reduced nicotinamide adenine dinucleotide phosphate (NADPH). After the reduction of 5-FU to DHFU, catabolism further proceeds through hydrolytic ring opening between carbon atoms C3 and Ca (dihydropyrimidinase, EC 3.5.2.2) leading to a-fluoro-/$-ureidopropanoic acid (FUPA). The major amount of FUPA is converted via decarboxylation and desamination (~-ureidopropionase, EC 3.5.1.6) into the low-toxicity amino acid analogue ct-fluoro-~-alanine (FBAL, Fig. lb), to carbon dioxide and ammonia. From CO2 and NH4+ , urea is formed by the urea cycle. Formation of FBAL from FUPA can also proceed via the intermediate u-fluoro-/~-guanidinopropanoic acid (FGPA) whereby urea is released. The major fraction of FBAL, which is the most important product of 5-FU catabolism, is excreted via the kidneys. However, in the presence of HCO3--, N-carboxy-ct-fluoro-/3-alanine (CFBAL) can be formed which is in equilibrium with FBAL in plasma and urine for pH -> 7.3 [74]. Release of small amounts of free fluoride anion (F-) from FBAL [74,86] after transaminase-catalyzed cleavage and transformation of FBAL into fluoroacetate (FAC) and 2-fluoro-3-hydroxypropanoic acid (FHPA) were also observed [87]. FAC is cardiotoxic and neurotoxic and could be responsible for some of the reported side effects of 5-FU treatment [88]. To illustrate the different drug and metabolite

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

9

5-Fluorouracil

o iL I H

I Dihydrouracil dehydrogenase

+NADPH/H*

-NADP÷

5,6-Dihydto-5-fluorouracil DI-IFU

H /

Dihydropyrimidinase ]

+ H20 1 - H+ a-lguoro-~-guanidinopropaaoicacid EGPA

a-Fluc¢o- [$-u~idoptopanoic acid

+ NH+

-

FUPA

+

H20 "--h.

H

[ lS-Ureidopropionas¢ I + H20/2H+

+ H20..'"

H

h r . o*

0 0 C~.~,H~ ~""

+n30+/ - HC~

t

+ HCO3"/ - HmO+

"OOC--'I~ H

+

CO 2 +

H

NH+÷, ,'

i v a-Fluoro-~-alanine

FBAL

N - C a r b o x y - a - f l u o r o - ~-alanine

CFBAL

4s

H2NCONH2 Urea

N

~,"

Fluoromalonic acid semialdehyde

"~

F"

Fluoroacetate

Fig. 3. Intracellularcatabolismof 5-FU. levels, Table 2 lists maximum concentrations of F-, 5-FU, FUPA, FBAL and DHFU measured with 19F NMR or HPLC in plasma of patients after systemic infusion of the cytostatic agent [74,89]. 5-FU catabolism mainly takes place in the liver (about 95% of the administered 5-FU dose is elimi-

nated on this route) as suggested by distribution images of 18F radioactivity in the tissue after intravenous injection of t8F-labeled 5-FU [90] and the distribution of DHUDH; the highest concentration of the enzyme is found in the liver parenchyma [67,91,92]. This agrees with the FBAL distribution

10

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

Table 2 Maximum concentrations of free fluoride anions (F-), 5-FU and 5-FU catabolites (FUPA, FBAL, DHFU) in plasma of patients after systemic infusion of different doses (D) of 5-FU (from Refs. [74,89])

D~_~u

c~_

c5_~

c%~,

c~

c~.~

8 mg/(kg b.w.) 20 mg/(kg b.w.) 320 mg/(m 2 b.s.) 960 mg/(m 2 b.s.)

30/zmol/l 10/~mol/kg ---

150 ~mol/l ~ >> 150 #mol/kg a 50 mg/1 = 385/xmol/l b 150 mg/l = 1150 tzmol/l b

20 tzmol/1 25/zmol/kg

40/zmol/l 100 #mol/kg

15 #mol/1 25/zmol/kg

--

--

--

--

--

--

NMR measurement [74]. b High-performance liquid chromatography (HPLC) measurement [89]. a 19 F

detected by means of 19F chemical-shift imaging in the liver of patients during 5-FU treatment (Section 5.1). In tumor tissue (Lewis lung carcinoma in mice) a much slower catabolism of 5-FU is observed and also anabolism to 5-FUranuc [21 ].

3. Pharmacokinetics

are studied that occur between the administration of a drug and its effect. The aim is to evaluate, model and interpret time courses of drug and metabolite concentrations in biological fluids and tissues and to assess drug bioavailability and toxic response [93]. Distribution and elimination are summarized with the term disposition; elimination includes the conversion into metabolites and excretion.

3.1. The purpose of pharmacokinetics 3.1.1. Monitoring pharmacokinetics by means o f NMR

Pharmacokinetics is_the quantitative analysis of the influence of the organism on drugs with respect to uptake, distribution and elimination (pharmacon is the Greek word for medicine and poison). Processes

NMR permits the direct and noninvasive observat i o n o f d r u g d i s p o s i t i o n in t h e t i s s u e . T h e p a r e n t c o m p o u n d a n d its m e t a b o l i c i n t e r m e d i a t e s c a n b e d i s t i n g u i s h e d o w i n g to t h e c h e m i c a l s p e c i f i t y o f N M R .

Table 3 Fluorine-19 NMR chemical shifts of 5-FU and 5-FU metabolites Compound 5-fluorouracil 5,6-dihydro-5-ftuorouracil c~-fluoro-/~-ureidopropanoic acid c~-fluoro-/3-alanine N-carboxy-ot-fluoro-13-alanine Fluoride anion 5-fluoro-2'-deoxyuridine 5-fluorouridine-5'-monophosphate 5-fluorouridine-5'-diphosphate 5-fluorouridine-5'-triphosphate 2-fluoroacetate 2-fluoro-3-hydroxypropanoic acid 3-fluoropyruvate 2-fluorocitrate 5-fluorouridine-3'-monophosphate 5-fluorouridine-2'-monophosphate 5-fluorouridine-5 '-diphosphate-N-acetylglucosamine 5-fluorouridine-5 '-diphosphate-glucose

~5(ppm) a 5-FU DHFU FUPA FBAL CFBAL FFdUrd FUMP FUDP FUTP FAC FHPA

-93.8 - 126.3 -111.0 - 112.8 -111.2 -44.0 -90.0 b - 89.2 -89.1 -89.1 - 141.4 - 113.6

[74] [741 [741 [74] [74] [74]

- 153.0

[74]

- 114.6

3'FUMP 2'FUMP

Reference

-89.6 -89.8 -89.0 -89.0

a Chemical shift versus trifluoroacetic acid (~ = 0); CFaCOOH in phosphate buffered saline (pH = 7) [74]. b Our own measurement.

[ 167] [ 167] [167] [74] [74] [74]

[167] [167] [167] [167]

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

With appropriate calibration, the peak area of the resonance gives a measure of the concentration of the corresponding metabolite. The potential role of ex vivo and in vivo NMR spectroscopy in monitoring pharmacokinetics of anticancer agents was reviewed [94-98]. High-field 19F NMR was applied to identify 5'deoxy-5-fluorouridine and metabolites [69,71] as well as 5-FU and metabolites [74] in the plasma and urine of patients receiving chemotherapy. An early investigation of intact cells was carried out on E. coli incubated with 5-FU [70]. An in vivo 19F NMR study, performed at 4.7 T in mice after the administration of 30 or 180 mg of 5-FU/(kg body weight [b.w.]), showed two resonances 17 and 19ppm upfield of the 5-FU signal; the latter was assigned to FBAL [21]. The same peaks were observed in the liver of the intact rabbit at 2.0 T [99]. Fluorine chemical shifts of 5-FU and its metabolites are listed in Table 3. In human patients, the conversion of administered 5-FU into the catabolite FBAL was monitored in timeresolved measurements at 8.5 min intervals after intravenous infusion of a therapeutic dose of 1.5 g of 5-FU [31]. Table 4 gives chemical shifts and assignments of 19F NMR signals detected in human liver in vivo during 5-FU treatment. 19F NMR was also used to study tissue pharmacokinetics (disposition) of fleroxacin in human liver and muscle in vivo [100]. The synthesis of a tracer is not needed, as in studies where fluorine-containing psychoactive drugs, such as the neuroleptics trifluoperazine and fluphenazine, are observed in patients [34-36]. Other nuclei can also be studied. For example, the kinetics of lithium after uptake of Li2CO3 was observed by localized 7Li NMR spectroscopy in Table 4 Chemical shifts and assignments of resonances resolved in 19F NMR spectra of the liver of patients receiving 5-FU chemotherapy Compound DHFU FBAL FUPA 5-FU 5-FUranuc a

c5(ppm)a - 126 -113 -111 -94 -89

Reference [79] [31] [81] [31] [33]

Chemical shift versus trifluoroaceticacid (t5 = 0).

11

brain and muscle of volunteers and in a patient with bipolar affective disorder [27]. Besides 5-FU, the pharmacokinetics of only a few anticancer agents were monitored in vivo by NMR spectroscopy, e.g. antifolates [101,102], iproplatin [103], 13C-labeled temozolomide [104], ifosfamide [22] and glucosylifosfamide mustard [105]. The 31p nucleus of ifosfamide resonates outside the chemicalshift range of endogenous phosphorus metabolites, which permits monitoring of the kinetics of the drug without interfering background signals. In addition to biochemical data provided by highresolution NMR spectroscopy, information on the local distribution of an administered compound or its metabolites in the body is needed for the study of drug action. A widely used technique is chemical-shift imaging (CSI, Section 4.4.3) [106-111]. With 19F CS1 the distribution and kinetics of the 5-FU catabolite FBAL could be observed in the human liver [80,112,113] (Section 5.1). The disadvantage of CSI is the limited spatial and temporal resolution, which is much lower than with conventional NMR imaging of water and lipid protons in the tissue. In comparison to radiotracer techniques such as PET, the sensitivity of NMR is smaller by a factor of 106-109. To estimate the potential of in vivo fluorine NMR with respect to these methods, a short account of PET will be given. 3.1.2. Pharmacokinetic studies with positron emission tomography

The principle of PET is the measurement of local activities of an administered tracer in the tissue with a coincidence technique. The tracer is a compound that carries a /3+-radioactive nucleus. The decaying nucleus emits a positron (e +) that scatters with electrons (e-) of the body tissues. Upon annihilation of the bound e + - e - system (positronium), the two particles in the spin-zero state convert into two 511 keV ~-quanta (the decay of the positronium spin-one state into three 3~-quanta is unimportant for the PET measurement). The two photons go out in opposite directions (180 ° _ 1°) and are recorded when they hit the detection system within the given time window. PET measures the location of the positron at the instant of annihilation. The method is insensitive to the chemical environment of the radioactive particles. Another disadvantage of PET is the

12

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

size of the required dose of radiopharmaceutical which is limiting for repeated examinations. The most important radionuclides for PET studies are llc, 13N, 150 andlSF. The corresponding stable isotopes have been detected in tissue by in vivo NMR either in endogenous metabolites (13C) or after administration of labeled compounds (13C, 15N, 170, 19F; see Section 1.1). Because of the high energy of the annihilation quanta (--1012 times the energy of RF photons), very low concentrations of - 1 0 - 1 2 M of the radiopharmaceutical can be detected [114]. This is far beyond the range of NMR. The spatial resolution of modern PET scanners is of the order of 5 mm in the three axes; the temporal resolution can be reduced to a few seconds (at the expense of spatial resolution). This is also not possible with NMR in vivo unless nuclei like protons in water and lipid molecules or hyperpolarized spin ensembles are observed. For PET studies of fluorinated drugs, the stable fluorine nucleus in the compound is replaced by 18F (this isotope is a relatively long-lived positron emitter with a half-life tu2 = 109.7 rain which decays via the reaction 18F---,~80+e+). 18F-labeled 5-FU (5-[18F]U) was used in PET studies in humans [90,115]. Before the/3+-decay, this radioactive compound will undergo the same biotransformations that are shown in Figs. 2 and 3. Since PET detects the total lSF pool in the tissue and cannot distinguish between the administered drug and the various labeled metabolites that are formed from the precursor, the combination with chemical-shift selective 19F NMR could be of interest [116]. 3.2. Pharmacokinetic modeling - - theory

In this section we briefly introduce some basic concepts of pharmacokinetics and discuss four pharmacokinetic models of different complexity (see Refs. [93,117]). The central issue is the measurement of concentration-time curves c(t), where c(t) represents the amount of a drug (or metabolite) per volume, c(t) = A(t)/V, at time t (dimension: mass/volume). The aim is to establish mathematical models that describe the observed profile c(t). Depending on the course of c(t) and the form of the corresponding solution of the model, linear and nonlinear systems can be distinguished.

3.2.1. Linear kinetics

The pharmacokinetics of a drug is linear when for any mode of administration the total area under the plasma concentration-time curve (AUC) is proportional to the dose D. A U C (dimension: concentration x time) is a measure of the "internal exposure" of the organism to the administered drug. Linear kinetics in the therapeutic dose range are desirable - - and also common. It implies the proportionality of transfer rates to concentrations, in particular first-order kinetics of drug distribution and elimination, and dose-independent input rate constants. The clearance CL (dimension: volume/time) is a pharmacokinetic parameter that measures the capacity of the organism to eliminate an administered drug. It can be thought of as the volume that is completely cleared from the drug per unit of time. In the case of linear kinetics, the clearance is a constant, independent of drug concentration. The observation of a constant half-life tl/2 of the plasma concentration-time curve c(t) after drug uptake is explained - - in a simple and abstract way by the assumption that the organism behaves like a linear one-compartment system. Following administration (R0 -----infusion rate, dimension: mass/time) the drug distributes in a compartment of volume V, from where it is eliminated with clearance CL. This is shown schematically in the diagram in Fig. 4. The drug distribution within V is assumed to be instantaneous, uniform and complete. Hence, with intravenous bolus injection, the drug concentration at time t = 0 is c(0) = D/V. It is also assumed that elimination from the compartment starts immediately. The model corresponds to a linear differential equation assuming that the velocity of drug - -

i

Ro

ICL

V

Fig. 4. Linear one-compartment model. After administration (infusion rate R0) into a compartment of volume V, the drug is eliminated with clearanceCL.

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

elimination is proportional to the actual drug concentration dc(t) - -dt

CL c(t) V

(1)

where the elimination constant k = CIJV. The solution of Eq. (1) is c(t) = c(0).exp( - kt) = ~.exp

- --~--t

(2)

with the half-life tin = In 2. __v CL" After intravenous bolus infusion an initial rapid decline of the plasma drug concentration is often observed. As a consequence, the half-life tin is not constant, even for linear systems. This behaviour can be described by means of a linear two-compartment model (Fig. 5). The drug is administered with infusion rate R0 and distributes into a central compartment (volume V1). Simultaneously, it exchanges between the central and a peripheral compartment (volume V2) with distribution clearance C L d. At the same time, the drug is eliminated from the central compartment (elimination clearance CLc). The mass balances for this model lead to the following pair of differential equations [93]: dcl (t) VI ~ = Ro - C L d ' [ C l (t) -

c2(t)]

-

C L e ' c I (t)

dc2(t) V2 ~ = CLd'[C 1(t) - c2(t)]

(3)

(4)

where c 1(0 and c2(t) are the drug concentrations in the distribution volumes V1 and V2, respectively. The solution of Eqs. (3) and (4) is biexponential in form with half-lives t~/2,~ and tl/2,~. Forward distribution and elimination occur during

R0

I

.c

v,

CLd

v~

ICLo Fig. 5. Linear two-compartment model. Central (V0 and peripheral compartments (V2) are connected by linear exchange processes in both directions (CLd). Elimination (CL~) occurs only from the central compartment.

13

the "distribution phase" which lasts until t ~ 3 × tl/2.~. During this phase, the half-life is constantly increasing and the absolute rate of elimination is a maximum - - at least after bolus injection. The distribution phase is followed by the "terminal phase" which reflects backward distribution and elimination (terminal half-life tl/2.~). The central compartment often comprises blood plasma and highly perfused organs such as heart, lungs, liver and kidneys, while the peripheral compartment may contain less perfused structures like bone and adipose tissue. It should be noticed that the concept of compartments is only an approximation that averages over a complex of macroscopic and microscopic properties of a biological system. The pharmacokinetic model is a set of coupled differential equations which describe the temporal evolution of the compartmentalized system [93]. Linear systems are modeled by a set of linear differential equations with solutions of the general structure c(t) = Z Li-exp( - ~kit )

(5)

i

3.2.2. Nonlinear kinetics Linear systems are characterized by the proportionality of administered dose and A UC. Otherwise the kinetics are nonlinear. Nonlinear kinetics are rare at therapeutic doses. Besides the effect of Michaelis-Menten elimination kinetics, the nonlinearity of binding processes, e.g. saturable binding at high doses, is a common cause of nonlinear pharrnacokinetics. 5-FU shows nonlinear kinetics within the therapeutic dose range, because of the saturation of 5-FU catabolism in the liver with increasing plasma concentration of the drug [118,119]. With higher doses a prolongation of terminal half-life tl/2,~ is observed. For any mode of administration the elimination clearance is CLe = Vmax/(ku + c), which decreases with increasing plasma concentration c. The pharmacokinetic parameter Vmaxis the maximum velocity of drug elimination (dimension: mass/time) and ku is the drug concentration in plasma where the elimination velocity equals ½Vmax(the substrate concentration at which the rate of a one-substrate enzyme-catalyzed

14

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

reaction is 50% of its maximum is called the Michaelis-Menten constant). The operation of Michaelis-Menten kinetics can be described by nonlinear compartment models. Based on earlier reports of nonlinear pharmacokinetics of 5-FU [118,120-122], these models were applied to describe 5-FU catabolism in the human liver as monitored by in vivo 19F NMR spectroscopy. In the nonlinear two-compartment model for 5-FU and FUPA + FBAL (Fig. 6) employed in Ref. [123], 5-FU is infused into a central compartment with volume VI which contains the liver. The drug is eliminated by saturable conversion (with maximum velocity Vmax and Michaelis-Menten constant kM) into, the catabolites FUPA + FBAL which have a common distribution volume V3 and a common elimination clearance CLe. The combination of the models in Figs 5 and 6 leads to the nonlinear three-compartment model shown in Fig. 7. The model corresponds to the following system of coupled differential equations [124]: dCl (t) Vl ~ = Ro -

CLd'[Cl (t) -

c2(t)]

Vmax C tt~ k M " ~ l ( t )" IK l

(6) dcz(t) V2 ~ = CLd.[C1(t) -- c2(t)]

(7)

dc3(t) V3 dt

(8)

Vmax kM+Cl(t) cl(t)-CLe'c3(t)

I

Ro

CLd

v,

v~

kM Vm~

ICL~ V3

Fig. 7. Nonlinear three-compartment model.

where cl(0, c2(0 and c3(t ) are the drug and metabolite concentrations in the distribution volumes V1, V2 and V3, respectively. Nonlinearity enters by way of the terms with cl(t) in the denominator. The pair of Eqs. (6) and (8), with CLd set equal to 0, corresponds to the two-compartment model depicted in Fig. 6. The nonlinear models of Figs. 6 and 7 were used to describe NMR signal-time curves in terms of pharmacokinetic parameters. These applications of kinetic modeling to data obtained in 19F NMR spectroscopy studies with human patients will be discussed in Section 5.4.

4. Methods of fluorine N M R in humans

R0

J

v~ k M[ Vmax

v~ ICLe Fig. 6. Nonlinear two-compartment model. The drug is infused (R0) into a central compartment (V0 from which saturable elimination occurs (vmaxand kM) into another compartment (V3).

Studies in humans by means of 19F NMR spectroscopy have been performed in whole-body scanners with field strengths of B0 = 1.5-2.0 T [31-33,7781,112,125-127]. Surface coils, volume-selective acquisition techniques, and methods of magnetic double resonance were employed to enhance theamount of spectral information. Our own studies on patients are referred in the following to study A [112], B [126], C [33] and D [79]. 4.1. Pulse experiments with surface coils

Surface coils yield high sensitivity unless deep areas are examined [13]. The detected region (field of view, FOV) is approximately a half-sphere with volume ~2R 3, when R is the radius of the coil's loop. In experiments with these antenna systems,

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

fluorine NMR spectra are obtained with a one-pulseacquire sequence (et-AQ, ot = flip angle) unless localization and double resonance techniques are employed. The major problem with surface coils is the spatial inhomogeneity of the B 1-field, which means that the same flip angle cannot be obtained everywhere in the sensitive volume of the antenna (Fig. 8). The application of 90 ° and 180° pulses to spins in a larger region is impossible with these coils unless B 1-insensitive adiabatic RF pulses [128-130] are employed

L,_,/

NaC,

15

(rectangular, Gaussian and sinc-shaped RF pulses in the following sequence schemes are therefore indicated by "effective" flip angles c¢). Since inversion recovery (IR) and spin-echo sequences cannot be employed adequately, different approaches are needed to measure relaxation times with surface coils (Section 4.3). NMR images are obtained with gradient-echo techniques using excitation with low flip angle (FLASH, Fast Low-Angle SHot [131]). In examinations of the liver of patients undergoing 5-FU chemotherapy 19F NMR signals were acquired

_V Cod

wi c ®

c

X

Fig. 8. Spatial inhomogeneity of surface coil B rfield. (a) A small sample (diameter: 1.7 cm) filled with the model solution is placed at various positions relative to the coil (loop diameter: 8 cm) within a vessel filled with isotonic saline. (b) NMR signal intensity as a function of position (x,O,z) of the sample (distances in cm). The field profile strongly depends on the measurement parameter of the pulse sequence,

16

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

with circular surface coils with a diameter of the order of 1 5 c m [31-33,79,81]. In the examinations of patients with tumors in the liver, the FOV of the coil typically encompasses tumor tissue and a significant portion of normal liver tissue. The selective observation of drug pharmacokinetics in the tumor requires a fluorine spatial localization technique (Sections 4.4 and 5.1). To overcome the limited FOV of planar surface coils, curved resonators, e.g. flexible 30 × 17 cm 2 "figure-8" coils [80,81], were used for receiving N M R signals from the human liver. Fluorine signals of neck tumors in patients treated with 5-FU have been obtained with smaller surface coils (diameter: 5 cm). The antenna is used in transmit and receive mode at the 19F frequency. Measurements at the 1H Larmor frequency at the same field strength should also be possible to allow N M R imaging for control of localization as well as optimization of B0-homogeneity (shim) using the tissue water proton signal (in general, image quality is low with coils dedicated to N M R spectroscopy with nuclei other than 1H, hence diagnostic imaging will preferentially be carried out with volume coils). 19F - { IH} double-resonance techniques (Sections 4.5 and 5.2) were applied at 1.5 T in whole-body N M R scanners equipped with two RF systems [81,126]. Dual-tuned antenna systems of large diameter were constructed for examinations of the human liver. Gonen et al. used such a 30 × 17 cm 2 "figure-8" coil for 1H decoupling, and a 15 cm diameter single-turn 19F surface coil placed within the concave side of the I H antenna [81]. In another study, a planar surface coil system was employed that consists of two concentric loops of 14 cm diameter for

the 19Ffrequency (59.9 MHz) and 18 cm diameter for the 1H frequency (63.6 MHz) [126]. The problem with 19F-{1H} double resonance at low field strength is the small 19F/1Hfrequency difference that facilitates an electronic coupling of the two resonance circuits. To suppress this unwanted effect, the RF fields B1(19F) and B2(IH) of the concentric surface coil system are polarized orthogonal to each other. This orientation of the magnetic fields (B2(t), Bl(t) and B0 parallel to x, y and z directions, respectively) affords 40 dB of isolation between the t9F and 1H circuits. Cross talk is further reduced by a band-pass filter which provides an additional damping of 76 dB of the 1H frequency component into the 19F channel. 4.2. Model solutions, data evaluation

Model solutions are employed for several purposes, e.g. test of the antenna system, measurement of relaxation times, determination of optimum measurement parameters of the pulse sequences, tests of the localization in volume-selective N M R experiments, calibration of chemical shifts, signal intensities (peak integrals) and metabolite concentrations. Model solutions that were used for 19F NMR in whole-body scanners are found in Table 5. Fluorine resonances are assigned on the basis of their chemical shifts relative to a reference signal. Compounds that are often used in 19F N M R as an external reference standard are trifluoroacetic acid (CF3COOH, TFA, with chemical shift set to = 0.0 ppm), 2,5-difluorobenzophenone (2,5-DFBP, t5 = - 4 1 . 0 p p m ) , or sodium fluoride (NaF, t5 --- 4 4 . 0 ppm). For example, in clinical study D, a vial that contained a 1.2 M solution of TFA was mounted

Table 5 Fluorine-19 spin-lattice relaxation times of model solutions of TFA, 5-FU and FBAL Model solution

Volume (ml)

Mass (rag)

c (mM)

T1 (s) a

TFAb 5-FU (in NaC1) 5-FU (pH = 7.4d) FBAL (pH = 7.4d)

1 1010 50 50

139 250 325 268

1200c 1.9 50 50

2.51 __+0.06 3.50 _ 0.76 2.94 ± 0.23 2.72 _ 0.26

a Our own measurement with APS method at B0 = 1.5 T, mean 4- SD. b Chemical shift reference (5 = 0 ppm). c CF3COOH + H20/1:9. u PhysiologicalpH in liver.

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

17

Signal intensity

5-FU

5-FUranuc

I

-90

/

- 95

i

I

I

I

-100

-105

-110

-115

8 / pprn

Fig. 9. Fluorine-19NMR spectrum from the liver of patient no. 8 (studyC) obtained40-45 min after infusionof 5-FU (TR = 1 s, NEX = 256, TA = 4.3 min; surface coil of 15 cm diameter). The peaks are assignedto 5-FUranuc(~5= -89 ppm), 5-FU (6 = - 94 ppm) and FBAL (~5= - 113 ppm). The Lorentzianline fit of the resonancesis superimposedon the data (adapted with permissionfrom Radiology, 174, Semmleret al. [33], © 1990, RadiologicalSociety of North America). at the center of the surface coil. It was used as chemical shift as well as signal reference to correct for different coil loadings and to normalize peak integrals. In 19F NMR studies of 5-FU chemotherapy, the 5-FU resonance serves as internal chemical shift reference with 6 set to 0 ppm. In vivo 19F N M R spectra were evaluated quantitatively by means of routines that fit Lorentzian [33] or Gaussian [132] line shapes to the resonances. Fig. 9 gives an example. In this spectrum, which was obtained from the liver of a patient receiving 5-FU chemotherapy during 4 0 - 4 5 min after the beginning of the drug administration, least-squares fits of Lorentzian functions are superimposed on the detected peaks of total 5-FU nucleosides and nucleotides (5-FUranuc, resonating at 6 = - 8 9 ppm relative to TFA), 5-FU ( - 9 4 ppm) and FBAL ( - 1 1 3 ppm). To obtain normalized NMR signal intensities I, the fits of the detected peaks were integrated and divided by the peak area of the fit of the reference signal. Quantitation of the fluorine signals and a test of the sensitivity of the experiment is performed with the use of phantoms. A procedure that was employed in

clinical study D is outlined in the following. First, the concentrations expected in the tissue were estimated referring to a patient of 70 kg body weight who receives 5 5 0 m g 5-FU/(m 2 b.s.) via the ! [a.u.] 0.4 r ,[ 0.3~ 0.2

FBAL

.

f ~ [

J

J ~

,

,

~,,,

~ f

J

0.1

I 0

1

,

,

J

. . . .

2

¢ !mM Fig. 10. Normalized 19FNMR signalintensitiesof 5-FU and FBAL model solutions as a function of concentration (adapted with permission from Magnetic Resonance Imaging, 12, Schlemmer et al. [79], © 1994, ElsevierScience Ltd.).

18

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

intravenous route. This corresponds to a total dose of the drug of 1 g. Assuming a distribution volume of 5-FU of 28 1 the average concentration of the drug in plasma is 0.0357 g/l = 0.275 mmol/1 = 275/zM. Model solutions of 5-FU and FBAL were prepared accordingly (concentrations: CS-FU = 2 -n × 2.5 mM, CFBAL = 2 -n × 1.9 mM, n = 0,1 ..... 5) and filled in a 1.5 1 container. 19F NMR spectra were obtained with a 15 cm diameter surface coil in the measurement time TA = 17,1 min (repetition time TR = 1 s, number of excitations N E X = 1024). Fig. 10 shows the ratio I of integrated peak areas of 5-FU (or FBAL) and the reference TFA as a function of the concentration. The minimum concentrations that could be detected in this experiment were 160/xM of 5-FU and 120/zM of FBAL [79]. The data in Fig. 10 allow the estimation of the absolute concentrations of FBAL in tissue from in vivo 19F N M R spectra obtained with the same measurement technique (Section 5.3).

4.3. Measurement o f relaxation times

Knowledge of the relaxation times of the excited spins is needed in order to make the appropriate choice of pulse delay times. In particular, the ratio of repetition time and spin-lattice relaxation time, TR/T1, is an important parameter. Fig. 11 shows the result of experiments with model solutions of 5-FU and FBAL, where the 19F NMR signal was measured with different TR (one-pulse-acquire sequence, surface coil of 14 cm diameter). To optimize S/N per unit time of slowly relaxing spin systems, a short TR relative to T1 and a large number of excitations must be used [133]. Small TR/T] means a small Ernst angle CtE = arccos[exp(-TR/Tl)], that is, the flip angle which yields maximum signal for a given TR/T]. The relaxation times are also needed as input parameters for absolute quantitation. For example, T~ of FBAL in vivo was used for the quantitative determination of catabolite concentrations in the liver of

Signal intensity

Signal intensity

o 125

~o

x~o ~o TR I ms

~o

2500

~oo TR I ms

SIN

t25

SIN

C tOO-

75-

25-

2

o

~

i~0 r~ TR I ms

z~

z~o

o

o

~0

~

r~

~

v~

TR I ms

Fig. 11. Optimization of signal gain. 19F NMR signal intensities (a, b) and corresponding SIN (c, d) of 5-FU (a, c) and FBAL (b, d) model solutions as a function of repetition time TR.

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

19

lit 2

O~ 1 19F

time ms

512 I

256 I

128 I

I

....

8

--

I

t I

Fig. 12. Pulse sequence for APS experiment. Duration of time intervals in ms. patients receiving 5-FU infusions [132]. The problem is the reliable measurement of fluorine T~ in the tissue. The application of the IR sequence ( 1 8 0 ° - T I - 9 0 ° AQ, TI = inversion time) with surface coils is hampered by B rinhomogeneity (Section 4.1). Alternative approaches for the measurement of spin-lattice relaxation times are progressive saturation (variation of TR in successive measurements [31], Fig. 11), aperiodic pulse saturation (APS) [134] and the variable nutation angle method [ 135,136]. APS is a saturation recovery technique where the 180 ° pulse of the IR sequence is replaced by a B ~insensitive 90 ° pulse. For example, Fig. 12 shows a train of RF pulses with decreasing interpulse delay TD applied at time t prior to the readout pulse of flip angle a2. The resulting signal intensity is l(t) = Io × sin (~2 × [1 - (1 - cos c~L) × exp( - t/TO], where C~l is the "effective" flip angle of the saturating pulse train and Io is the signal of the equilibrium magnetization. The factor 1 - cos a~ takes into account imperfect flip angles (equal to 1 in the case of perfect saturation). Fig. 13 shows the result of an APS measurement of fluorine TI on a model solution of 5-FU (room temperature, 50 mM) at 1.5 T. Eight excitation pulses with delays TD ---- 512, 256 ..... 8 ms were applied; t was varied from 0.5 to 5.0 s in steps of 0.5 s. From this experiment T 5-vU ----2.94 _ 0.23 s was determined by least-squares fit of l(t) to the measured data. Table 5 also gives T~ values of model solutions of FBAL and TFA obtained with the same method. Spin-lattice relaxation times in the ranges o f T15-FU = 2.9-3.8 s a n d TIFBAL = 1.7-2.7 s were measured at B0 = 1.5 T and room temperature in different experiments on aqueous model solutions of 5-FU (Fluroblastin ®, Farmitalia, Carlo Erba GmbH, Freiburg, Germany, or Fluorouracil Roche ®, HoffmannLaRoche, Grenzach-Wyhlen, Germany) and FBAL (purchased from Paesel & Lorelei GmbH and Co.,

Frankfurt/Main, Germany) [79,126]. These Tl values are comparable to high-field data from human plasma (T 5-FU = 3.4 s at B0 = 11.7 T [W.E. Hull, dkfz]) and isolated perfused mouse liver (T [BAL = 2.5 s at 7.0 T [137]). Li et al. report somewhat longer spin-lattice relaxation times of their 5-FU model solution of T~-FU = 4.2 s (measured by the variable nutation angle method) and T~ -FU = 4.6 s (inversion-recovery method) [132]. In the former experiment, the 19F N M R signal intensity I was recorded as a function of the nutation angle 0 which was varied from 10° to 90 °. The plot of I(O)/sin 0 versus l(O)/tan 0 gives a straight line with the slope of exp( - TR/TO. The value of TI was obtained by linear regression analysis. The determination of T1 and T2 relaxation times of fluorine in administered compounds and their metabolites in vivo is a difficult task (in particular for T2), because of low sensitivity, B 1-inhomogeneity, Signal intensity [a.u.] 700 600 500 400 300 200 100

o

;,

5

tls

Fig. 13. Measurement of 19F spin-lattice relaxation time of 5-FU (model solution, 50 mM) with APS at 1.5 T. The fitting of the function l(t) = A × (1 - B × exp[ - t/T~]) to the data yields Tt = 2.94 +_ 0.23 s.

20

P. BacherCProgressin NuclearMagnetic Resonance Spectroscopy 33 (1998) 1-56

and changing concentrations in the course of drug disposition. Using the variable nutation angle method, Li et al. determined the in vivo spin-lattice relaxation time of FBAL in the human liver at 1.5 T during the plateau phase of FBAL at about 40 rain after 5-FU infusion [132]. Their result of T ~ AL = 1.6 _ 0.2 s (mean ___ standard deviation [SD]) agrees with T FBAL = 1.73 ___ 0.13 s measured with APS on a 50 mM aqueous solution of FBAL [126]. When the concentration of the detected compound changes rapidly during the experiment, an additional signal variation is introduced which produces a systematic error in Tl. In principle, TI can also be measured in this situation using APS with interleaved data acquisition. As usual the recovery time t is varied from one spectrum to the next, but now each spectrum is obtained in a time that is short compared to the period of the concentration changes and the whole series is repeated several times. Since the signal variations between repeated measurements are related by a simple proportionality, the slope of the recovery curve (Fig. 13) is not compromised. The ranges of values of T~, as determined from resonance line widths (full width at half maximum = A~'l/2(19F)), of 5-FU and FBAL in the human liver in vivo are T~5-Fu -~ 5-11 ms and T~FBAL --~ 4 - 7 ms [79]. The corresponding T2 values have not been reported so far. 4.4. Spatial localization of fluorine NMR signals To discriminate the signal contributions of different regions within an organ or tissue, a volume-selection technique must be employed. Spatial localization means that NMR signals are obtained from a volume of interest (VOI), while signal contributions of regions outside the VOI (signal contamination) are suppressed. The ideal localization technique produces 0% signal loss and 0% contamination. Sensitivity and abundance of the NMR nucleus in the tissue determine the minimum size of the VOI that yields a detectable signal (neglecting the sensitivity of the RF antenna). These properties are favourable for in vivo ~H NMR: the concentration of water protons in the tissue is of the order of 40 M (e.g. gray matter water content of 80% ~ 44.5 mol/l [138]) which yields a significant NMR signal from a 1 mm 3 VOI (or smaller) with a single excitation. However, rare

and insensitive nuclei and low metabolite concentrations require large detection volumes resulting in the poor spatial resolution of localized NMR spectroscopy in comparison to conventional 1H NMR imaging. 4.4.1. Fluorine NMR imaging Fluorine localization was first achieved by means of NMR imaging techniques with unresolved detection of the total signal of 19F in the sample [44-46]. The sensitivity was sufficient to obtain at B0 = 0.1 T fluorine images at 7.5 mm spatial resolution of rabbit lungs filled with CF4 [45]. The imaging technique employed in this study was two-dimensional (2D) projection-reconstruction encoding (TR = 5 ms, 31 projections, 128 free induction decay [FID] signals were averaged for each angle). Fluorine 2D spin-echo images were obtained at 1.44 T in solutions of fluorinated anesthetics and in vivo of rat liver following administration of the t9F-containing blood substitute Fluosol-DA ® [47]. The measurement time of the in vivo experiment was TA = 60 min (TR = 900 ms, TE = 15 ms, slice thickness d = 6 mm). Because of the short T 2 of many of the fluorine resonances of this compound the spinecho signal observed at 15 ms echo time contains primarily a single resonance. When the spectrum encompasses more than one resonance, e.g. that of 5-FU and FBAL, NMR imaging is compromised by spatial dispersion in slice-selection, and readout direction with chemical shift (see next section). To circumvent these problems in 19F NMR imaging during 5-FU treatment, a chemical-shift selective RF pulse [139,140] is included in the 2D FLASH sequence to presaturate either the 5-FU or the FBAL resonance [116]. This method was applied in a whole-body NMR scanner at 1.5 T to acquire 19F NMR images from tumor-bearing rats in vivo after administration of 200 mg 5-FU/(kg b.w.). A spatial resolution of 10 x 10 x 15 mm 3 was achieved in a measurement time TA = 40 min for one selective 19F NMR image (TR = 100 ms, TE = 2.7 ms, 16 X 16 image matrix, NEX = 1500; RF antenna: volume resonator of 10 cm diameter). The 5-FU image (obtained with excitation frequency set on the Larmor frequency of 5-FU) was acquired at 5-45 min after infusion of the drug, followed by the acquisition of the FBAL image at 5 0 - 9 0 m i n postinjection.

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1 56 4.4.2. Volume-selective N M R spectroscopy Volume-selective 19F N M R spectroscopy in human patients is a challenge, because o f the low abundance of fluorinated metabolites in the tissue. An average F B A L concentration of 0.92 ___ 0.26 mmol/(kg liver tissue) (mean - SD) for the first 50 min after drug infusion was estimated in a study with patients who received 5-FU doses varying from 750 to 2000 mg [79]. Using fluorine TI determined in vivo at 1.5 T and spatial localization, Li et al. measured CFBAL = 1.0 -- 0.2 m M in the liver of patients at 60 _ 10 min after administration of 5-FU [132]. Both studies indicate a ratio of in vivo concentrations of fluorine and protons of CFBAL/CH20 = - 2 . 5 x 10 -5. Therefore, a tremendous number of excitations of the order of (--0-~) 4×104 2 = 2 . 3 X 10 9 is needed to obtain an usable 19F N M R signal of F B A L from a 1 m m 3 volume in the human liver. In contrast, an in vivo fluorine signal can be expected with a single excitation when the detection volume is increased to approximately I mm3 = (3.6 cm) 3 This estima0.83x2.5× 10- 5 tion indicates the feasibility of 19F N M R studies in humans. Spatial localization of in vivo N M R spectroscopy was achieved with " f o c u s e d " B0 or B 1-fields (e.g. topical NMR, rotating-frame spectroscopy, depth

21

pulses [ 141,142]), but now volume-selective methods adapted from spin-warp imaging [143] are more common. Using gradient-phase encoding in combination with slice-selective excitation, these techniques permit a reliable localization and are easily implemented on N M R scanners. The application of magnetic field gradients G(x,y, z, t), which are superimposed on the homogeneous static field B0 for a period TG in a fixed temporal sequence, enables the localization of N M R signals either via the Larmor frequencies W~---"~l(G'-~-'~ B0) or via the different phases of the FID signals ~ = 3'i(G'x + B0)-TG. For example, in a magnetic field gradient Gz the spins take the same Larmor frequency in each slice orthogonal to the z axis, but different Larmor frequencies in parallel slices with relative shift 6z. Selective excitation of a spin species localized in a slice of width d is achieved by irradiation in the frequency range A~o = .Yi.Gz.d about the carrier frequency. Sliceselective excitation of F1D or echo signals in three dimensions is used by single-voxel localization techniques such as ISIS (Image-Selected In vivo Spectroscopy [144]) or STEAM [6]). Both methods have (to our knowledge) not been applied to volume selection in 19F NMR spectroscopy in whole-body scanners.

Fig. 14. Spatial dispersion with chemical shift. 19F2D FLASH image (a) of FBAL and 5-FU model solutions. The readout direction is from top to bottom and the carrier frequency was set on the FBAL resonance (TR = 20 ms, TE = 5 ms, NEX = 120, 64 x 128 image matrix, TA = 2.6 min, d = 20 mm, FOV = 200 mm, readout bandwidth = 72.5 Hz/pixel corresponding to a readout gradient time TG = 13.8 ms). The 19F frequency difference of 1140 Hz of 5-FU and FBAL at 1.5 T produces a relative shift of 16 pixel in readout direction. (b) t H NMR image at the same slice position (256 x 256 image matrix).

22

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

For nuclei that show the effect of chemical shift, encoding via the frequency leads to different spatial assignments for the various resonances, even for nuclei located at the same position. In the presence of a magnetic field gradient, e.g. in the z direction, the frequency difference a owing to the chemical shift is interpreted as spatial shift AZ (chemical-shift artefact). With c0(z, a) = 7i(1 - a)(Gz'z + B0)

frequencies of the detected nucleus can also lead to a misregistration in the imaging plane. The frequency encoding of one spatial coordinate produces a shift by TG × At• pixel of the location of one species against the other in the readout direction, where TG is the duration of the readout gradient and Av is the frequency difference (in Hz). This is demonstrated in Fig. 14a with a relative shift of 16 pixel (TG = 13.8 ms, At, = l l 4 0 H z ) between the 19F N M R images of two vials that contain aqueous solutions of 5-FU and FBAL, respectively. Fig. 14b shows an artefact-free proton image at the same slice position.

(9)

and the relation ~0(Az,0) = oa(0,a) the apparent shift is

az= tr

B

(10 4.4.3. Chemical-shift imaging

For 19F nuclei in 5-FU and F B A L with chemical-shift difference a = 19 ppm, the relative shift of the excited slices at B0 = 1.5 T amounts to Az = 28.5 mm for G z = 1 mT/m and Az = 1.4 mm for G z = 20 mT/m slice-selection gradient strength. Hence, the slicemisselection of nuclei with a large chemical-shift range can be reduced when strong gradients are available. In spin-warp NMR imaging, different resonance

CSI permits the detection of chemically shifted NMR signals from different VOIs simultaneously [106-111]. The multi-voxel method is also called spectroscopic or metabolic imaging. Since the FID signal is measured in the absence of a readout gradient, CSI does not face problems of misregistration in the readout direction. CSI in two dimensions (2D CSI) requires one slice-selection (GS) and two phase-encoding gradients (GP) in orthogonal

0[!

[

I ~ 19F

AQ

]

S=IH

-.....

cs

GP1

GP2

time lis

I

500

I

315

400

I

768

I

400

I

400

I

120

I

400

I

51200

I

Fig. 15. Pulse sequence for 19F 2D CSI, as employed in study A [112]. Following slice-selectiveexcitation of 19F spins with RF pulse oq in the presence of slice-selection gradient GS, two phase-encodinggradients (GP1, GP2) are applied before the detection (AQ) of the 19F NMR signal. An additional RF pulse as is irradiated at the Larmorfrequencyof ~H spins to induce a 19F NMR signal enhancement owing to transient NOE of dipolar coupled 19F-1H spins. Duration of time intervals in #s.

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1 - 5 6

directions (the pulse sequence in Fig. 15 is an example). For the detection of a cubic array of voxels (3D CSI) three orthogonal phase-encoding gradients are applied. Phase encoding of spatial information is based on the following principle. An ensemble of identical spins (neglecting chemical shifts as a first approximation) in a homogeneous static field B0 is excited with a RF pulse of spatially homogeneous amplitude. The NMR signal is the integral of the signals of spin packets at points } in three-dimensional space: S(t) = ~S(.~, t)d3x

(11 )

with S(}, t) = M± (~).e - t/r~.e -

ic°Ot.e - i~pO

number of phase-encoding steps, and ~wc the width of the detected voxel (analogously for the y direction). Discrete sampling is taken into account by the substitution fe

2~ri~.~.d3 X ~

g

^ " t) = JS(}, t).e - i~o(2,TG).d3x S(k,

=Zexp(-27qrilmpp, ,,

1

A violation of these relations leads to phase errors known as "aliasing" artefacts. For example, when Akx.Atx > 1/(Nx) the phase difference resulting from two successive phase-encoding steps A~o=27rAkx.Ax.Nx=2rAkx.FOV x exceeds 2r. The effect is a misregistration of the x position of the detected nuclei. With use of Eq. (13) the discrete version of Eq. (12) is obtained

T -1 Z P=T

= fS(Yc, t).e - 2rik.~.d3x

"sampling

(14)

-4

t

(13)

1

Akx.AX = Nx

s(m'n)(t)=

~ G(t').}dt').dax

-~xq-n-yynql)

The equality holds because of the theorem' ':

t+TG

=~S(~c,t).exp(-i'y,

~ exp( - 27ri[mpAkxAx + nqAkyAy]) p,q

Aky.Ay=

and transversal magnetization M±(2) immediately after the RF pulse, damping time constant T~, Larmor frequency ~00,and constant phase offset ~o0.The application of a magnetic field gradient G(t) for the time TG produces a phase shift ~o(2, TG) of the precessing magnetization that depends on the position ~ of the spin packet. The resulting phase-encoded signal reads:

23

T-' Z

~'q)(t)

-4 q= 2

(12) •exp [-- 27ri (~xP + - ~ ) ]

where k = (3'i/270 ftt+ ra G(t')dt'. Eq. (12) shows that S(k, t) and S(~, t) are Fourier pairs. This allows one to reconstruct the desired spatial signal distribution from the measured quantity S(k, t);. Continuous sampling of S(k, t) is not possible in practice. In the real experiment, the gradient strength is varied in discrete steps, while TG is kept constant to avoid signal changes due to relaxation. In the following we focus on the case of a 2D CSI experiment with slice-selection in the z direction and phase-encoding ir~m, n)the x-y plane with running k-values k = (mAkx, nAky, 0), where m = - -~..... -~ - 1 . Ny N~ , anon = - 2, "", ~- - I. Akx is the increment of the phase-encoding gradient in the x direction, Nx the

(15)

Discrete 2D Fourier transformation of ~(m,n/(t ) yields the signal S(P'q)(t) of nuclei located in voxel (p,q). In the real experiment, localization errors known as "voxel-bleeding" or point spread effects arise, because the spin packets are inhomogeneously distributed (or move) in the sample instead of being fixed at N~ × Ny equidistant points .Tc(P'q)m(pzXx, qAy, O). Voxel bleeding artefacts are signal variations in one voxel owing to the superposition of positive or negative signals from other (in particular the surrounding) voxels. So far ideal, i.e. noise-free signals have been considered. To include noisy signals, the acquired NMR

24

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

signal for phase-encoding step (m,n) is approximated as a superposition of the "true" signal ~s(m'n)(t) and the noise amplitude ~.(m.n).The noise is determined by receiver bandwidth and other factors, but is independent of the gradient strength. While s('n'n)(t) is proportional to the number of excitations at one phase-encoding step, the noise amplitude increases with the square root of NEX. Fourier transformation of Eq. (15) gives the NMR signal intensity S~'q)(t) of voxel (p,q): 1

Z[~(m,n)(t).gEX

S~'q)(t) ~- N x X IVy m.n [

(rap

-~- ~ . ( m , n ) . ~ ]

nq)]

•exp L27ri \ ~--+x Ny

(16)

Let N = Nx = Ny. The phase factors of ~4m,n)can be discarded because of incoherence of noise:

s~'q)(t) = NEX N2 m~,,n ~ s(m'n)(t)'exp [27ri( ~xx mp + -~y)] +

-~

x...f (m'")

(17)

m,n For Poisson-distributed noise the sum ~ , , , , ~'("'")=N.~. Then Eq. (16) transforms into S(t~ ' q) (t)

X~/NEX z. = NEX.S (p'q)(t) "~- T " ~

is

(18)

Thus the signal-noise ratio of the localized spectrum of voxel (p,q) is (S~°'q)(t)/~).N.~. The sum of all N x N FIDs obtained in the 2D CSI experiment q) ) g,ves ~ P'q S(~ ' (t) = NEX.~ P"q S(e'q (t) + ~ . ~ , ~ N where again y~p/2-1u/2 g-(p,O,=N-~'. Accordingly, the total S[N=(£p, q S(P'q)(t)]~).~ is independent of the number N of phase-encoding steps. For the application of CSI in vivo, the time needed to acquire the data must be considered. The total measurement time of (isotropic) 2D CSI is TA = TR X N ~- x NEX. Enhancement of spatial resolution by increasing N, e.g. by a factor of),, while leaving TA and the repetition time TR constant, reduces the S/N of the sum spectrum of all N x N detected voxels by a factor of 1/X. This agrees with the results in Fig. 16, which shows sum spectra from model solutions for N = 2, 4 and 8 (for fixed FOV and TA). The signalnoise ratio of the localized spectrum of voxel (p,q) is reduced by a factor of 1/X2. The effect is even stronger

for 3D CSI; it can be weakened by modified k-space sampling [145]. In any case, the sensitivity of the CSI experiment drops with increasing number of phase-encoding steps (for constant FOV). This parameter is related to spectral quality, spatial resolution and measurement time, and must therefore be chosen carefully. Fluorine CSI was applied to studies in human patients receiving 5-FU chemotherapy. 19F 2D CSI data of the liver were obtained with a voxel size of 6 x 6 x 4 cm 3 in a measurement time TA ----12.8 min (TR = 60 ms, NEX = 12 800) [112]. This time resolution was sufficient for monitoring the kinetics of the catabolite FBAL (Section 5.1). Li et al. obtained localized 19F NMR spectra of the human liver in vivo by means of 3D CSI with a voxel size of 4 x 4 X 4 c m 3 a n d T A = 8 . 5 m i n ( T R = ls, NEX = 512) [132] and TA = 45 min (TR = 260 ms, NEX = 10240) [80]. VOIs of 3 X 3 X 3 cm 3 for FBAL detection in a measurement time of TA = 45 min were possible with the use of 19F-{1H} double resonance [81 ] (Section 4.5.2). However, time frames TA >--40 rain preclude determination of the kinetics of 5-FU biotransformation. The improvement of time resolution while maintaining the spatial resolution (and vice versa) requires methods that enhance the sensitivity. In the following sections, double resonance techniques are discussed that amplify the 19F NMR signal, particularly in CSI experiments.

4.5. Magnetic double resonance Double resonance is the excitation and detection of the NMR spectrum of a nuclear spin species of interest (spins I) in the presence of additional irradiation in another frequency range (of spins S) in the same experiment. The consequences are signal intensity changes, or the removal of spin multiplets in the I-spin spectrum. The corresponding methods are called dynamic nuclear polarization (nuclear Overhauser effect, NOE [146]) and spin decoupling, respectively. Detailed studies devoted to the coupled system of I = 19F and S = 1H spins have shown that dipolar nuclear relaxation establishes a mechanism for amplification of the fluorine NMR signal. The transient NOE of the two-spin system of liquid HF was

25

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

TFA

F"

5-FU

6 /

FBAL

ppm b

m

m~

~

~

a~

a

/

d /

6

ppm

-&*

-~

-40

-~

~

-m

ppm

Fig. 16. Fluorine-19 2D CSI performed with differentnumbersN X N of detected voxels at constantfield of view (FOV = 50 × 50 cm2) and measurementtime (TA -- 4.2 min). The FOV covers three vials filled with solutionsof TFA (reference), 5-FU and FBAL, with dissociated F-. The sum spectra of all N x N voxels are shown for N = 2 (a), 4 (b) and 8 (c) (chemical shift scale:/t of 5-FU was set to 0 ppm). Measurement parameters: TR = 100 ms, NEX = 640 (a), 160 (b) and 40 (c). Quantitativeevaluationof the FBALsignalin the sum of all N x N spectra gives SIN = 101 (N = 2), 47 (N = 4) and 26 (N = 8). investigated in the classic work of Solomon [39]; a theoretical study of dipolar nuclear spin relaxation of 19F and of the 19F-{1H} NOE in multispin systems was undertaken by Hull and Sykes [147]. Methods of heteronuclear double resonance have first been applied to in vivo 31p and 13C NMR in whole-body scanners to improve sensitivity and spectral resolution [10,148-150]. However, low S / N is also a persistent problem of in vivo 19F NMR studies as discussed in Section 4.4.2. Often the signal of 5-FU is invisible, in particular when the drug is given intravenously [79]. The detection of other 5-FU

derivatives in vivo, such as cytotoxic anabolites (5-FUranuc) and the catabolic intermediates FUPA and DHFU is even more difficult [33,79,81]. Dynamic nuclear polarization of dipolar coupled t 9 F - t H spins in aqueous 5-FU and FBAL was studied [126]. The purpose was to provide pulse techniques that improve in vivo 19Fsensitivity without impairing the time-resolution of pharmacokinetic data. Double resonance was also applied to remove scalar 19F-IH s p i n - s p i n couplings [81,126]. This interaction is significant in FBAL and in DHFU [31,71,74] and thus produces broad peaks. Collapse

26

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

of J-multiplets by means of spin decoupling will be favourable for the detection of these compounds. These methods require a second RF field B2(t) oscillating at the Larmor frequency of protons (the "decoupling field") in addition to the fluorine detection field B l(t). The 19F-{ IH} double-resonance experiments discussed in the following were performed at B0 = 1.5 T in a Magnetom whole-body NMR scanner equipped with two RF systems. Information on the dual-tuned surface coil employed is given in Section 4.1.

- ¢0) ,Ts(t) -

(20)

(iz(t)) is the expectation value of the z component of the I-spin angular momentum operator and (Iz) is its thermal equilibrium value (analogously for S-spins). The temporal changes of I- and S-spin signal enhancements are described by a coupled system of linear differential equations [39]: ^0

,

.

d~h(t) = _ ~l(t)'Pl - ~s(t)'Ols" "Ys dt "YI

(21)

d~/s(t) 3,1 = - ~/s(t)'ps - ~/l(t)'trlS• ~ dt

(22)

4.5.1. Dynamic nuclear spin polarization - - theory

While the dipole-dipole interaction between I and S spins cannot directly influence the shape of the I-spin spectrum in the case of motional narrowing in liquids, it mediates an efficient relaxation mechanism. As a consequence, the perturbation of the S-spin system by selective RF irradiation changes the populations of the states of the I-spin system. The resulting alteration of NMR signal intensities is called the NOE [146]. The signal changes depend on the timing of the pulse sequence; therefore kinetic NOE studies must be performed to detect the measurement parameter for optimum enhancement. The effect is easily analyzed for a system of two dipolar coupled spins I = 1/2 and S = 1/2 in a rigid molecule in isotropic tumbling motion (neglecting scalar spin-spin interactions, Jts = 0). In an external magnetic field B 0 = (0, 0, B0), four Zeeman spin states result, that are classified using the z components of angular momentum operators of both spins. A possible representation is provided by the product states ota, /3/3, ~/3 and/3c~ where the 1-spin states ]I,m > are ]1/2,1/2 > = ot and ]1/2, - 1/2 > = /3. The spin system can undergo single-quantum transitions (otc~*-* c~/3, ¢~ot,--*/3c~,/3/3,--, o#3 and/3/3 ,--* /3o~, with transition probabilities per second Wu and Wls) and zero- and double-quantum transitions (or/3,--* /3or and otc~ *--,/3/3, probabilities Wois and W21s). In the real experiment there is a whole ensemble of these coupled I-S-spin systems. The zero- and double-quantum transitions mediate cross-relaxation processes that are responsible for changes of the magnetizations of I and S spins:

Qz(t))- Qz° ) rh(t) (~z)

(19)

The coefficients O~ = Wois + 2Wn + Wz~s and as = Wois 4- 2Wls 4- W2is are the direct dipolar longitudinal relaxation rate constants and ais = W2~s - W0xsis the cross-relaxation rate constant [146]. The rate constants p~, as and cris are functions of the molecular tumbling time ~'¢ (motional correlation time). In the case of vanishing dipole-dipole interactions, the rate a~s = 0, and Eqs. (21) and (22) are solved by 7h(t) = rh(0)-exp(-pit) and r/s(t) = ~/s(0).exp(-pst), respectively. For uncoupled spins, the damping constants p~ and Os correspond to the spin-lattice relaxation rate constants defined by the Bloch equations. Time-resolved NOE experiments, where the evolution of the I-spin signal enhancement is observed as a function of the driving period of the S spins or of the delay between S- and I-spin excitation, are of particular interest [146,151]. 4.5.1.1. Truncated-driven 19F-[I H} nuclear Overhauser effect. When the flip angle of the S-spin

magnetization is approximately constant during RF irradiation, the evolution of the I-spin magnetization is described by Eq. (21). Let TS indicate the time of irradiation at the Larmor frequency of S spins before readout of the I-spin signal. With the test function 7/I(TS) = c v e x p ( - p I T S ) + c 2 and the initial conditions tit(O)= T° and ~s(O)= ~/o the solution is [126] ~/I(TS) = =

~/°' ( I ' -

[ r + £ ] -exp[ - pITS])

~/°.r.(1 - exp[ - pl(TS -- T)I)

(23)

27

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

TS i . . . . . . . . . . . . . . . . . . . . . . . . .

AQ

I -----19F

S =IH

time

500

ItS

315 I 500

1 000 000

I

............

300

51200

_n_-_L, _._,?o . . . .

Fig. 17. Pulse sequence of truncated-driven [9F-{IH} NOE experiment. The driving period TS of IH spins is varied in subsequent measurements. Duration of time intervals in #s. where

r-=

1 .In ( 1 +

0

and r-

3's tr[s ~fl Pl

The resulting signal e n h a n c e m e n t is called the truncated-driven NOE. In the case of thermal equil i b r i u m of I spins (r~° = 0) and c o n t i n u o u s saturation of S spins d u r i n g the time interval TS (on average r/°(0) = - 1), Eq. (23) transforms into rh(TS) = I'.(1 - exp[ - piTS])

(24)

9

9

a

8 7

8- I

b

7q

5-FU

6-1

6 q

For long saturation periods the signal e n h a n c e m e n t approaches the m a x i m u m value 7h(~ ) = 'OI,max = r . N O E theory predicts the relation 01 -- 2als in the case of extreme n a r r o w i n g (~0.rc << 1) and w h e n the relaxation m e c h a n i s m is purely dipolar. Referring to Eq. (23), a m a x i m u m fluorine signal e n h a n c e m e n t of r/l,m,x = 7s/23q = 0.53 is then expected for dipolar coupled I = 19F and S = 1H spins in small, rapidly t u m b l i n g molecules. The presence of other magnetic interactions such as scalar coupling or chemical-shift anisotropy (as well as the violation o f the extreme narrowing condition) reduces the N O E factor [146]. For experimental verification of the truncateddriven 19F-{IH} NOE, the pulse sequence shown in Fig. 17 was employed. A series of n rectangular R F

5J

5

0

4-4

4

g

FBAL

3

2-t

2

,

1

of

o 5

0

-5

- ~0 6//pprn

- 15

-20

-25

.5

I 0

-5

--10 6/ppm

--15

-20

-25

Fig. 18. Fluorine- 19 NMR spectra of 5-FU and FBAL obtained without (a) and with (b) irradiation of the proton spin system before the detection period of the ttuorine signal (model solutions, 50 rnM). Pulse sequence, see Fig. 17, with n = 30, TR = 31 s and NEX = 4.19F_ {t H } NOE signal enhancements of --0.3 for 5-FU and of --0.5 for FBAL are observed (chemical shift scale: ~5of 5-FU was set to 0 ppm) (adapted with permission from Journal of Magnetic Resonance, B 108, Krems et al. [126], © 1995, Academic Press, Inc.).

28

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

ql 0.6

ql

a

0.5-

0.6

0.4-

|mll

•

|-II|m-

- wllWll

•

0.2-

0.2

0.1-

0.1

0.0-,

0.0

11

2b

}

i"

tl

}

It½!

I

~|

0.3

16

t

0.4

0.3-

o

b

!

0.5

21

3o

o

TSI s

16

11 TSIs

2b

3o

Fig. 19. Truncated-driven 19F- { l H } NOE enhancement r/I of 5-FU (a) and FBAL (b) versus driving period TS of 1H spins (model solutions, 50 mM). Pulse sequence, see Fig. 17, with n = 0,1,...,30 (TS = 0,1,...,30 s), TR = 31 s, NEX = 4. Fit function, Eq. (23) (adapted with permission from Journal of Magnetic Resonance, B 108, Krems et al. [126], © 1995, Academic Press, Inc.).

pulses as was applied at the Larmor frequency of the 1H spins prior to the excitation of the 19F spins (continuous irradiation for the necessary long periods was not possible with the hardware of the whole-body scanner). The driving period TS was varied by increasing n from 1 to 30 in steps of 1. Cross-relaxation depends on the energy exchange between different spin systems. The characteristic time of this process (1/OlS) will be longer than the characteristic time of the energy exchange between spins and lattice (Tl), since the I-S spin coupling is weaker than the coupling of the spins to the lattice. Therefore, repetition times TR >> Ta of the involved nuclei were chosen to observe the temporal evolution of the NOE enhancement. The NOE signal enhancement is ~l(t) = [l(t) - Io]/ Io, Eq. (19), where l(t) is the 19F NMR signal observed with excitation of the proton spins and Io is the signal when the 19F spin system is in thermal equilibrium (otherwise the enhancement is ~). The existence of a large ]gF-{IH} NOE for 5-FU

and FBAL is demonstrated in Fig. 18 through 19F NMR spectra obtained without and with irradiation of the proton frequency. Signal amplifications are - 3 0 % for 5-FU and - 5 0 % for FBAL in this experiment. Jacobson et al. [152] observed negative NOE factors (signal decrease) in 19F-{1H} doubleresonance experiments with fluorinated compounds of larger molecular mass which were free or in interaction with macromolecules. This physical condition is called slow tumbling or spin diffusion limit; it implies the predominance of zero-quantum transitions over double-quantum trafisitions, W01s > W2~s,hence axs < 0, which corresponds to negative NOE factors. For spins in small, rapidly tumbling molecules (extreme-narrowing limit), W21Sis larger than W01S, resulting in a positive tr~s and positive NOE factors (signal increase). Fig. 19 shows the 19F-{ 1H} NOE enhancements of 5-FU and FBAL measured for various driving periods TS. The function Eq. (23), with T = 0 because of fully

Table 6 Maximum 19F_ {i H } NOE enhancement factors, ~/Lmax,and delays between 1H and 19F excitation pulses for maximum enhancement, t(~ Lma0, obtained from fits of truncated-driven and transient NOE data of 5-FU and FBAL (from Ref. [126]) Model solution

711 a....

T/l.max b

t(r/Lmax) (S) b

5-FU (50 mM) FBAL (50 raM)

0.31 --- 0.06 0.40 - 0.13

0.16 --- 0.06 0.20 -+ 0.04

2.8 -+ 0,5 1.7 -+ 0,7

Note. Deviations are the root-mean-square total errors of statistical estimate. a Asymptotic enhancement for TS ---* ~ of truncated-driven NOE. b Transient NOE.

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

I = 19F

-

29

AQ

S =IH

time

~ts

51200 I .................

1 000 000

315

I

500

I

300

51200

n_ _=_3_o__,

Fig. 20. Pulse sequence for frequency-selectiveproton excitation of 19F-{IH } truncated-drivenNOE experiment. The excitation bandwidth of the Gaussian-shaped RF pulse as is about 20 Hz. Duration of time intervals in/~s. relaxed 19F spins (TR = 31 s), was fit to the data by means of the Gauss-Newton algorithm. The evaluation of the results of several experiments gives maximum signal enhancements (for T S - - - , oo) of ~/I,max : 0.31 ___ 0.06 for 5-FU and 7/l,max ---~ 0.40 ___ 0.13 for FBAL (Table 6) [126]. For a 385 mM solution of 5-FU ~TI,max= 0.51 -+ 0.04 was observed. This corresponds to the theoretical maximum NOE factor of purely dipolar coupled 19F-1H spins in extreme narrowing. The signal amplification of 53% permits reduction of the measurement time by 24% without compromising fluorine S/N. Frequency-selective presaturation allows one to identify the protons that are involved in the 19F-1H cross-relaxation process. For this purpose, the proton spin system was irradiated in a truncated-driven NOE experiment (Fig. 20) with a train of n = 30 Gaussianshaped RF pulses a s with a narrow bandwidth of 2 0 H z (pulse width: 51.2ms). Fig, 21 shows the detected 19F-{lH} NOE enhancement as a function of the 1H excitation frequency. The NOE factor of 5-FU and F B A L is a maximum when the solvent water protons at 6 = 4.7 ppm are irradiated. The same effect was observed in the coupled 3 l p - I H spin system of phosphoruscontaining compounds in aqueous solution (e.g. phosphocreatine, nucleoside 5'-tfiphosphate) [153,154]. The NOE of this spin ensemble was attributed to the intermolecular dipole-dipole coupling between the phosphorus nuclei and protons of coordinated water molecules surrounding the phosphate group [ 155,156]. The NOE of 5-FU shows another maximum when aromatic protons are excited that resonate in the

chemical-shift range 8 ~ 7 - 8 p p m (Fig. 21). A detailed investigation of this spectral region reveals a doublet signal with J-splitting of 5 Hz centered at 8 = 7.5 ppm in the localized water-suppressed ] H N M R spectrum (STEAM) of the 5-FU solution (Fig. 22). This signal presumably originates from the C6bound pyrimidine ring proton close to the fluorine atom in the 5-FU molecule (Fig. la). The resolution of the small J-splitting is possible because of the localized shim in the stimulated I H echo experiment. Fig. 23 shows the corresponding J-splitting of - 5 Hz in the 19F NMR spectrum of 5-FU. In Fig. 22 the low-field range of the I H NMR spectrum is superimposed on the plot of 19F NOE enhancement versus 1H excitation frequency, thus

ql 0.35

_.,~

f'~

0.30-

o. o-

\

-FO

°"°I,",, 8

FBAL

7

! 1

I/ 6

,

,

5

4

6/ppm

3

2

Fig. 21. Truncated-driven 19F- {i H } NOE enhancement 7/i of 5-FU and FBAL versus ]H excitation frequency (model solutions, 50 raM). Pulse sequence, see Fig. 20, with TR = 20 s and NEX = 15. Water protonsresonate at 6 = 4.7 ppm (adapted with permission from Journal of Magnetic Resonance, B 108, Krems et al. [126], © 1995, AcademicPress, Inc.).

30

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

1"110.15

0.10

I I 19 F

j

0.05 -

0.00 ] 90

detected indirectly, e.g. by means of heteronuclear double resonance. For FBAL no 19F N M R signal amplification was observed when protons were excited that resonate outside the chemical-shift range of water.

8:5 8:0 7:5 7:0 615 610 5:5 5.0 6 /ppm

Fig. 22. Truncated-driven=9F-{1H} NOE enhancementr/1 (pulse sequence, see Fig. 20) and localized i H NMR signal of 5-FU model solution versus 1H excitation frequency (IH STEAM experiment with water-signal suppression, TR = 1500ms, TE = 50 ms, TM -----27 ms, NEX = 512, voxel size 2 x 2 × 2 cm3; RF antenna: volume resonator of 10cm diameter) (adapted with permission from Journal of Magnetic Resonance, B 108, Krems et al. [126], © 1995, Academic Press, Inc.).

showing simultaneously the effects of scalar and dipolar 19F-IH couplings. The observed NOE signal amplification attributed to the intramolecular dipoledipole interaction of t h e fluorine nucleus and the neighboring ring proton in 5-FU corresponds to expectation. Although the dipolar interaction is several orders of magnitude stronger than the scalar spin-spin coupling, its effect in liquids can only be 2.5 Signal intensity [a.u.]

:ot

4.5.1.2. Transient ~gF-{I H] nuclear O v e r h a u s e r effect. In the transient NOE experiment, a selective inversion pulse is applied to the S spins and the I-spin signal amplification is monitored for variable delays t between S- and I-spin excitation pulses. As distinguished from the truncated-driven NOE, where the S-spin magnetization is locked in a state with (Sz(t))= constant for a period TS, the coupled I - S spin system can evolve freely before the readout pulse. Hence both equations, Eqs. (21) and (22), must be considered. It turns out that the recovery of thermal equilibrium magnetization follows biexponential kinetics [39,146]. With insertion of the test functions 7~(t) = a(t). e x p ( - p t ) and 7 s ( t ) = ~/3(t).exp(- at), Eqs. (21) and (22) can be solved for the initial conditions 7i(0) = 70 and 7s(0) = 7 °. The result is [126]

7l(t) = 7°A + (t) + 70 "YSB(t) 3'1

(25)

7s(t) = 7°A - (t) + 7 ° "YIB(t) 3's

(26)

where A + (t) --= cosh(Xt) +

sinh(Xt) .exp( - pt)

A - (t) ~

sinh(Xt) -exp( - pt)

cosh(Xt) -

B( t ) ----- - -~-.sinh(Xt).exp( - pt ) A

1.0_

0.5-

P

0.0

P

loo

5b

6

Ps -t- Pl 2 _ PS -- PI 2

v/Hz Fig. 23. Fluorine-19 NMR spectrum of 5-FU model solution, with resolved doublet splitting owing to vicinal coupling (3Jxs = 5 Hz) of fluorine nucleus and C6-boundring proton (chemical shift scale: of 5-FU was set to 0 ppm).

In the case of complete inversion of S spin magnetization (n o = - 2) and thermal equilibrium of the I spins

31

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

1 = 19F

I-g]

AQ

S =Ill

time gs

500 I

t I

I

315

I

500

300

51200

Fig. 24. Pulse sequence of 19F-{ I H } transient NOE experiment. The delay between ' H (us) and 19F (cY1)excitation pulses is varied. Duration of time intervals in gs.

07o = 0) at the beginning of the cycle, Eq. (25) reduces

[126]. The time scale of the build-up of the 19F{1H } NOE is comparable to that of the 31p_ l H spin system of phosphorus-containing metabolites [153]. In contrast, t(r/l,max) -~- 0.2 S for the transient 13C{t H } NOE of fatty acids where the cross relaxation is mediated by the strong dipole-dipole interaction of 13C nuclei and directly bound protons [157]. Close proximity is favourable for cross relaxation, since the transition probabilities W21s and W0ts depend on ris 6, where ris is the spatial distance of the coupled nuclei. The slow build-up of the 19F-{1H} NOE (Figs. 19 and 25) indicates a weak dipolar interaction in agreement with the argument that water protons located at a larger distance from the 19F spin are involved (Fig. 21). The maximum transient NOE factor is lower than the truncated-driven NOE enhancement. This is a general result that also follows from an analysis of the solutions of Eqs. (21) and (22) [126]. Ratios of

to

hi(t) = 2.7s. OlS.sinh(ht).exp( _ Ot) 3'~ X

(27)

The transient 19F-{IH} NOE of 5-FU and FBAL in aqueous solution was observed with the pulse sequence displayed in Fig. 24. Fig. 25 shows the evolution of the fluorine signal enhancement r/l(t) as detected in a series of experiments where the delay t between the 1H inversion (C~s) and the 19F excitation pulse (al) was varied from 0 to 20 s in steps of 1 s. The data follow biexponential kinetics, as demonstrated by the fit of the theoretical function, Eq. (27). The evaluation of experiments with the 5-FU model solution gives a maximum NOE enhancement factor of ~ l,max= 0.16 -----0.06 for a pulse delay t(~ 1,max) = 2.8 -2_ 0.5 s; for FBAL, ~=. . . . ~ - 0.20 --- 0.04 at t(r/1.max ) = 1.7 4- 0.7 s was measured (Table 6) ql 0 . 2 5 [

TI I 0 . 2 5 -

b 0.20-

0.150.10 -

0.10-

0,05 -

St

0.00 ~

0

~

lb tls

1~

20

0

~

lb

1~

20

tls

Fig. 25. Transient ]9 F-{ I H} NOE of 5-FU (a) and FBAL (b) (model solutions, 50 mM). Pulse sequence, see Fig. 24, with t = 0,1,_,20 s, TR = 31 s and NEX = 5. Fit function, Eq. (27) (adapted with permission from Journal of Magnetic Resonance, B 108, Krems et al, [126], © 1995, Academic Press, Inc.).

32

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

Table 7 Rate constants for dipolar cross-relaxation, als, and dipolar longitudinal relaxation, Pc and Ps, obtained from fits of truncated-driven and transient 19F-{ IH } NOE data of fluorine-containingmodel solutions (5-FU and FBAL data from Ref. [ 126]) Compound

O'IS (s-I)

Pl

5-FU (50 raM) FBAL (50mM) 2,5-DFBP NaF

0.07 --- 0.03 0.19 --- 0.10 0.29 _+ 0.22 0.21 _ 0.03

0.18 +- 0.05 0.41 --- 0.17 1.06 + 0.53 0.50 -+ 0.06

(s-I)

PS (s-l) a

0.84 - 0.25 0.74 -+ 0.06

Note. Deviations are the root-mean-square total errors of statistical estimate. a Only from transient NOE data. m a x i m u m truncated-driven and transient N O E enhancements of about 2 were observed in experiments with 5-FU and F B A L (Table 6). The relaxation parameters that were derived from kinetic N O E data are given in Table 7. The PI values obtained from the fit of truncated-driven and transient NOE data are consistent with measured fluorine spinlattice relaxation rate constants (Section 4.3). As expected, the characteristic time of 19F-1H cross relaxation (1/Ois) is longer than the spin-lattice relaxation times of proton (--1/ps) and fluorine spins ( - 1 / p l ) ; the difference is particularly large in 5-FU. The 19F spin-lattice relaxation rates (PI) as well as the N O E build-up rates ( - o l s ) and signal enhancements o f F B A L are larger than those o f 5-FU (Tables 6 and 7; Figs. 19, 21, and 25). The kinetic NOE data discussed so far were measured with a long repetition time (TR = 31 s) to discover the " l i f e t i m e " of the nonthermal state and

111 .5

a

the dynamics o f the cross-relaxation process. The experiments show that for delays t > 15 s, the enhanced spin polarization vanishes, i.e. there is sufficient time for the perturbed spin system to return to thermal equilibrium (Fig. 25). For short TR, however, the pumped nuclear polarization can survive during the cycle and a steady-state NOE enhancement is established. Experimental results for 5-FU and F B A L are shown in Fig. 26. Using pulse sequences with successive (Fig. 24) or simultaneous excitation of proton and f u o r i n e spins (Fig. 27), N O E signal enhancements ~ were measured with different repetition times TR (TR = 1 - 5 s in steps of l s ) . Note that in this experiment Io has to be determined for each TR. For TR ----- 3 s, signal amplifications ~h ~ 0.25-0.35 are observed with the pulse sequence in Fig. 24 and t = 1 ms. The effect decreases for longer TR and eventually vanishes when TR >- 10 s. A theoretical

111.5.

b .4"

.4-

.~"

.2-

.2"

T-----

.1-

.0

.1 °

.0

0 TR I s

TR I s

Fig. 26. Steady-state 19F-{ IH} NOE of 5-FU (a) and FBAL (b) (model solutions, 50 mM). Pulse sequence, see Fig. 24, with t = 1 ms. Fit function, see Ref. [126] (adapted with permission from Journal of Magnetic Resonance, B 108, Krems et al. [126], © 1995, Academic Press, Inc.).

33

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

I

I = 19F

S =IH

time ItS

I

500

I

315

I

51200

Fig. 27. Alternative pulse sequence for steady-state 19F-{IH } NOE experiment. Duration of time intervals in #s.

model that yields the fit function ~7~(TR)in Fig. 26 is given in Ref. [126]. The combination of chemical-shift imaging with dynamic nuclear polarization is straightforward. The gradient-echo CSI technique with short TR suggests the use of the transient effect. The truncated-driven

NOE can be useful in combination with the ISIS localization method [144], which requires long pulse delays. This allows for the inclusion of the S-spin saturation period of several seconds into the I-spin localization sequence. Fig. 15 shows a 2D CSI pulse sequence that is

a

b

FBAL

5-FU

120

~

4b

I~ 6/ ~

-~,0

-~0

-120

120

~

4b

1~ -~0 4 / ppm

-~0

-120

ct

C

___L_ 12o

eb

4b

6

6 / ppm

-io

-~o

-12o

4 / ppm

Fig. 28. Transient 19F-{ tH} NOE induced by tH prepulse (C~s)in 19F 2D CSI experiment. Pulse sequence, see Fig. 15, with TR = 600 ms, N X N = 8 × 8, NEX = 16, FOV = 50 X 50 cm 2 (model solutions, 50 mM). CSI spectra from FBAL-containing voxel (a, b) and from 5-FUcontaining voxel (c, d) were obtained with (a, c) and without (b, d) NOE prepulse. Voxel bleeding is manifested by signal contamination from the other ~gF-containing species (chemical shift scale: 6 of 5-FU was set to 0 ppm).

34

P. Bachert/Progressin NuclearMagnetic ResonanceSpectroscopy 33 (1998) 1-56

extended by a rectangular RF pulse at the Larmor frequency of protons (Ors). The pulse C~s is applied before the slice-selective excitation pulse of 19F spins (oti) to induce the transient ~9F-{IH} NOE. The resulting signal amplification is appreciable, as demonstrated in experiments with model solutions. Localized 19F N M R spectra (TR = 600 ms) with steady-state NOE signal enhancements ~/I = 21 _ 1% for 5-FU and ~l = 25 ___ 2% for FBAL were obtained (Fig. 28). 4.5.2. Proton spin decoupling The large fluorine chemical-shift range favours high spectral resolution. However, in the case of in vivo 19F N M R spectra obtained after administration of 5-FU, the superposition of signals is critical for the resonances of total 5-FU nucleosides and nucleotides (5-FUranuc) at ~ ~ - 89 p p m as well as for the signal at ~ = - 113 p p m (Fig. 9). This peak is up to twice as broad as the 5-FU resonance (20% broadening in Fig. 9) and is unresolved at 1.5 T in 19FN M R spectra in vivo [31]. Although attributed to FBAL, it is presumably a superposition of the signals of FBAL and of its direct precursor in the catabolic pathway, t~-fluoro~-propanoic acid (FUPA) [31]. The scalar spin-spin coupling produces splittings of the resonances in multiplets. Scalar 19F-ill coupling constants of 5-FU and catabolites measured in urine of patients receiving chemotherapy are given in Table 8 [74]. The 19F spectrum of FBAL is a scalar coupled system with 19F-1H spin-spin coupling constants,

Table 8 Fluorine-19 chemical shifts and scalar 19F-tH spin-spin coupling constants of 5-FU and 5-FU catabolites in urine of patients receiving 5-FU chemotherapy (from Ref. [74]) Compound

6 (ppm)a'b

JLs (Hz)a

DHFU FBAL FUPA 5-FU

-126.27 -112.77 -111.07 -93.77

46.8, 14.2, 11.2c 50.7, 28.2, 19.1c'd Av > 20 5.0 ¢'e

-- 0.15 --- 0.05 - 0.05 - 0.10

a NMR parameter were measured at B0 = 11.7 T and 28°(2. b Chemical shift versus trifluoroacetic acid (t5 = 0) in phosphate buffered saline (pH = 7); deviations ( _+) are the range of measured values for pHi5-8. ¢pH = 7.84. d One geminal (2Jls) and two vicinal (3Jis) couplings. e One vicinal coupling.

as reported by Malet-Martino et al. [71], of 2Jis = 5 0 . 6 H z (geminal), 3Jis ~- 28.3 Hz and 3jis ~18.8 Hz (vicinal). The multiplet is partially resolved in the 19F N M R spectrum of the model solution in Fig. 29a obtained at 1.5 T. For comparison, a theoretical spectrum of eight Lorentzian lines based on measured chemical shifts and the 19F-1H coupling constants of FBAL is also shown. The dispersion of the signal of the 19F nucleus among several resonances hampers the observation of this catabolite. Another consequence of the scalar coupling is J-modulation errors which must be taken into account, when long pulse delay times are used. In general, syystem splittings in I-spin spectra owing to scalar spin-spin couplings of I and S spins (as well as line-broadenings in the case of unresolved multiplets and long-range couplings) can be removed by selective irradiation with an additional RF field B2(t) at the Larmor frequency O~s of S spins during the detection of the N M R signal of I-spins. The method is called spin decoupling. The suppression of this interaction of 19F and IH spins leads to only one resonance for each distinguishable 19F atom. In the experiments discussed in Sections 4.5.1.1 and 4.5.1.2, decoupling effects were not observed because the proton spins were excited at times outside the 19F acquisition window. The effect is demonstrated in Fig. 29b, which shows the result of a 19F-{lH} decoupling experiment on the FBAL model solution. A single lowpower RF pulse t~s of width 10 ms was applied at the 1H Larmor frequency in the beginning of the 5 1 . 2 m s detection period of the 19F N M R signal (Fig. 30). Upon increasing the amplitude B2(1H) of the decoupling field, the multiplet collapsed and a reduction of the linewidth of the FBAL resonance by a factor of 6 occurred [126]. The 10 ms p u l s e suffices for decoupling of the signal because of the short fluorine T~ (Section 4.3). A significant change in the linewidth of the 19F N M R resonance of 5-FU was not seen in 19F-{IH} decoupling experiments at 1.5 T. This is expected, because the coupling constant is fairly small ( J i s - 5 Hz (Table 8, Fig. 23)). The difficulty with continuous wave decoupling is the restriction on a small range of the 1H excitation frequency and (in the case of long RF pulses and short TR) a high RF power deposition in the tissue. To

35

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

Signal intensity [a.u.] 3.0

a

b

2.5 2.0

J

1.5 1.0 0.5 0.0

150

1()o

-50

50

- 100

- 150

Av I Hz

I

I

I

0v

2 v

5v

I

15v

I

3O V

Fig. 29. Proton-decoupled 19FNMR spectroscopy.(a) Observed multiplet signal of FBAL (model solution) at 1.5 T and theoretical spectrum consisting of eight Lorentzian lines (chemical shift scale: 5 of FBAL was set to 0 ppm). (b) FBAL resonance versus field strength B2(t H) (in volts) of decoupler pulse c~s.Pulse sequence, see Fig. 30, with c~s resonant on the Larmor frequencyof water protons, TR = 2 s and NEX = 30 (adapted with permission from Journal of Magnetic Resonance, B 108, Krems et al. [126], © 1995, Academic Press, Inc.). circumvent these problems, composite pulse decoupling, e.g. the excitation of the proton spin system with a series of low-power RF pulses in W A L T Z - n cycles (n = 4 or 8), was applied to in vivo 13C and 31p N M R spectroscopy [10,148150,157]. A simple r-pulse cannot invert the S spins over the whole range A~ s of chemical shifts since it rotates the S-spin magnetization around the effective field Bef f = (B2, 0, A6os/'YS), i.e. its effect depends on the width of the S-spin spectrum. The W A L T Z - n cycles, on the other hand, consist of inversion pulse elements (~'/2)x(Tr)_x(37r/2)x = 153 and ( r / 2 ) _ x ( r C ) x ( 3 r d 2 ) _ x = 123, where x is the axis of rotation of the magnetization. A series of these pulses yields the W A L T Z - 4 cycle 17.3 123 i23 123 = 124:23 i2423 and correspondingly WALTZ-8 and W A L T Z - 1 6 [158,159].

These pulse schemes allow complete decoupling of the heteronucleus and the protons over the whole range of ~H frequencies even with RF coils with substantial B2-inhomogeneity. Composite pulse decoupling was first proposed by Levitt and Freeman [160]. Gonen et al. combined the W A L T Z - 4 cycle with 3D CSI and obtained localized broadband I Hdecoupled fluorine spectra [81]. The RF power of W A L T Z - 4 was --12 W; the decoupling period lasted 256 ms. Collapse of the FBAL multiplet and the resonance of FUPA at t5 = - 111 ppm were observed in 19F-{1H} N M R spectra obtained at 3 X 3 x 3 cm 3 spatial resolution from a 1 1 phantom containing urine from a patient undergoing 5-FU chemotherapy. The results of in vitro 19F-{1H} NOE and spin decoupling experiments indicate that double-

.o

I = 19F

]

S =Ill

time liS

I

500

300

10000

I

41200

I

Fig. 30. Pulse sequence for J9F-{IH } spin decoupling. Duration of time intervals in jzs.

36

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

resonance techniques can significantly enhance the NMR visibility of fluorinated compounds in the tissue.

5. Biomedical applications The methods discussed in Sections 3.2.2, 4.1, 4.4.3, 4.5.1.2 and 4.5.2 were applied in 19F NMR studies of patients undergoing 5-FU chemotherapy. Informed consent was obtained from each patient prior to his/her NMR examination. 5.1. Volume-selective fluorine NMR spectroscopy o f the human liver

Methods of spatial localization of 19F nuclei in tissue have been applied to studies of the human liver. While the catabolic pathway of 5-FU has been well characterized in normal liver tissue by 19F NMR in vivo, clinical studies of patients with hepatic tumors showed strong interindividual differences of 5-FU catabolism. The multivoxel rather than the single-voxel approach was employed to enable the simultaneous detection of 5-FU metabolism in different regions of the liver. Chemical-shift imaging was applied in one [125], two [112] and three dimensions [80,81,132]. In one of our own studies (A), three patients were examined with 2D CSI who were treated for advanced colorectal carcinoma (patients nos. 1 and 2, with and

without liver metastases, respectively) and advanced pancreatic carcinoma (patient no. 3, with liver metastases). Treatment schedules consisted of at least two cycles of 5-FU chemotherapy with 5-FU doses of 500 mg (patient no. 1), 600 mg (no. 2) and 2000 mg (no. 3) per day. For each cycle the cytotoxic compound was administered as 10 min intravenous infusion (nos. 1 and 2) or as a continuous infusion (no. 3) for five consecutive days according to the clinical protocol. Fluorine CSI was performed during the first treatment cycle on day 1 (nos. 1 and 2) and on day 5 (no.3). The 2D gradient-echo CSI pulse sequence employed is shown in Fig. 15. After selective excitation of fluorine spins in a transversal slice of width d = 4 cm with RF pulse cq (in the presence of gradient GS), two phase-encoding gradients (GP1, GP2) are applied. N × N = 8 × 8 phase-encoding steps and a FOV of 50 × 50 cm 2 yield an in-plane spatial resolution of 6.25 × 6.25 cm 2 (voxel size: 156 cm3). S/N was maximum for repetition time TR = 60 ms in experiments with model solutions (dwell time 50/zs, 14 cm diameter surface coil, B0 = 1.5 T). The 19F excitation frequency was 6 kHz lower than the Larmor frequency of the reference TFA in order to place the carrier frequency between 5-FU and FBAL resonances. FOV was 50 × 50 cm 2 in all examinations. Experiments with model solutions indicate that for smaller FOV the signal will drop below the detection limit.

Signal intensity

Signal intensity

.:/"' ' '"...•.•

q ....

:

/

:. |]

"

"-,...

.....................................

•/(t)

2b

40 t I min

.. ........

""

60

ao

•

•

-

'""",, =

,i

o

T(O

...........',., .... e

m

I(t)

2b

4b

..........

g •

6b

•

.

8b

~00

t / min

Fig. 31. Kinetic 19FNMR data frompatientsnos. 6 and 8 (studyC [33]). Squaresrepresentnormalizedsignal intensitiesl(t) of 5-FU from series of 19FNMR spectra obtained after infusion of 5-FU. The dotted curves represent5-FU signals l(t) accumulatedduring the period t (t = 0 at beginning of infusion). The intersectionof both curves yieldsthe measurementtime for optimum5-FU signal, typicallyof the order of 15 rain.

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

The delay between RF pulse oq and the beginning of the detection period of the FID due to the phaseencoding gradients is 1.7 ms (Fig. 15). Taking into account the damping time constants T ~ 5 - F U ~ 5-11 ms and T~FBAL~ 4 - 7 m s determined from resonance line widths of 5-FU and FBAL in the human liver in vivo [79], loss of transverse magnetization during the delay before signal acquisition will be 15-35%. The pre-acquisition delay also leads to J-modulation errors of the FBAL signal which is a coupled multiplet of eight resonances (Fig. 29a). The examinations were performed with a series of consecutive 2D CSI measurements. To maximize the chance of observing 5-FU at least in the initial CSI acquisition, the measurement time was chosen as follows. The NMR signal accumulated during the period TA, that is I(TA), is a function of the mean concentration of 5-FU in the tissue: ~(TA) =- 7~" 1 j0 I'TA c(t)dt, where c(t) is the concentration of the drug at time t after the beginning of the infusion. Maximum signal is obtained when TA is chosen de(t') I c= TA ~ O. This leads to the condition: such that -d?-

37

this process was repeated 200 times. The result is an averaging of the signal over the acquisition period TA. Imaging of the liver is susceptible to motional artefacts from both breathing and intestinal movements.

y

?( TA ) = c( TA ).

A simple approximation of the concentration-time profile is given by the biexponential function c(t) = al (e -"~t _ e - ~ t )

(28)

which has a maximum when a3 > a2. When applied to drug levels measured after bolus infusion of 5-FU, c(t) with a2 --- 0.1 min -1 and a 3 -----0.2 min -l gives a good model of the observed kinetics [33]. The analysis of c(t) yields a maximum at 6.9 min after the beginning of infusion and a measurement time TA -~ 12.6 min for optimum signal. This agrees with the result in Fig. 31 which shows an intersection of the curves of detected 5-FU signal intensity l(t) and of accumulated signal i(t) at t = T A ~ 1 5 min (data of patients nos. 6 and 8 of study C). Experimental parameters were therefore chosen according to TA = T R x N 2 x N E X = 60ms x 82 X 200 = 12.8min to obtain optimum S / N of 2D CSI data. Since the levels of 5-FU and FBAL change during TA, averaging for each sampling point ~.(m.,,) in k-space produces a signal weighting because of varying concentrations. Therefore the whole k-space lattice (8 x 8 phase-encoding gradient steps) was scanned in the short period of T R X N 2 = 3.8 s and

Fig. 32. Fluorine-19 and ~H NMR examination of patient no. 1 (study A [112]). (a) Transversal ~H FLASH image (obtained with surface coil of 18 cm diameter) of the liver region, the interpolated 16 x 16 CSI grid is superimposed. (b) ZgF NMR signal intensity map of FUPA + FBAL from 2D CSI showing catabolite distribution in the liver 48-60 rain after beginning of 5-FU infusion (TR = 60ms, N x N = 8 x 8, NEX = 200, FOV = 50 × 50cm2). Data processing: 2 K zero filling, digital exponential filter to impose 64 Hz line-broadening.

38

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

Because low sensitivity prevents rapid acquisition of 19F CSI spectra to remove or minimise these effects, 19F spectroscopic images, obtained in 12.8 min, will be compromised by physiological motion. However, this is less serious in the light of the large voxel size, and localization errors owing to voxel-bleeding effects, deviations of the slice-excitation profile from the desired rectangular shape, and chemicalshift artefacts. The effect of motion can be reduced with additional gradients, however, this will increase echo time and signal loss. No compensation was therefore made for motional artefacts in this study. The large frequency difference of 19F resonances leads to chemical-shift artefacts in the slice-selection direction (Section 4.4.2). The pulse sequence in Fig. 15 uses a gradient strength of 1.25 mT/m for selection of the slice of width d = 4 cm. For FBAL and 5-FU there is a resulting relative spatial shift Az = 23 mm of the excited slices at B0 = 1.5 T (Eq. (10)). This is comparable to spatial shifts due to physiological motion and limits the localization technique in studies of small lesions in the liver. The results of examinations of the liver in three patients undergoing 5-FU chemotherapy are displayed in Figs. 32-35. Fig. 32a shows a transversal 1H FLASH image obtained with the surface coil from the liver region of patient no. 1. A grid of 16 X 16 image elements (voxel size: 3.1 x 3.1 x 4 cm 3) is superimposed. The distribution of FBAL in the same region, detected by means of 2D 19F CSI during 4 8 - 6 0 m i n after administration of 350 mg 5-FU/(m 2 b.s.) is displayed as a signal intensity map in Fig. 32b. After interpolation of the acquired 8 X 8 matrix to 16 X 16 elements by zero-filling in k-space, nine voxels show appreciable in vivo 19F NMR signal of the catabolite. Measurements of enzyme activities in vitro [161] and 19F NMR studies on the isolated perfused mouse liver [137] and in vivo on mice [162] showed that conversion of 5-FU to FBAL primarily takes place in the liver (FBAL is synthesized in the hepatocytes of the liver parenchyma; as a charged molecule it hardly distributes into the extravascular space). This agrees with the tissue distribution of the catabolite demonstrated in Fig. 32b. The FBAL resonance in the in vivo t9F NMR spectrum from the marked voxel ( x ) in Fig. 32a has S / N ~- 6:1 (Fig. 33a). The sensitivity is sufficient

to observe the kinetics of the catabolite for a period of 60 min (Fig. 33b). The data follow classic pharmacokinetic behaviour as demonstrated by the fit of a double exponential function to the normalized peak integrals. Fig. 34 shows the results of the examination of patient no. 2. The contour plots of detected signal distributions of reference (TFA) and FBAL in a transversal slice are displayed in Fig. 34a and b, respectively, together with the IH FLASH localizer image. These maps demonstrate the quality of localization that can be achieved with this technique.

Signal intensity a

12o

8b

4b

6

40

-~o

-;2o

8 / ppm

I(0

b

=

0

10

2b

3b

4b

5b

60

t / rnin Fig. 33. Fluorine-19 2D CSI examination of the liver of patient no. 1 (study A [112]) following intravenous infusion of 5-FU. (a) Localized t9F NMR spectrum from the marked 3 × 3 x 4 cm 3 voxel ( x ) in Fig. 32a. The resonance of FUPA + FBAL is resolved with S/N--6:1 (chemical shift scale: 8 of 5-FU was set to 0 ppm). (b) In vivo 19F NMR signal-time profile l(t) of FUPA + FBAL detected in the same voxel ( X ). Fit function, Eq. (28). The horizontal bars represent the measurement time of 12.8 min, the vertical bars represent the errors of the fit process.

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

39

Fig. 34. Fluorine-19 2D CSI examination of patient no. 2 (study A). Contour plots of 19F NMR signals of (a) TFA (external reference, sample fixed in the center of the surface coil) and of (b) FUPA + FBAL, superimposed on the transversal I H image of the liver (measurement parameters and data processing, see Fig. 32).

In the examination of patient no. 3, the FBAL signal could be detected in the tissue during continuous infusion of 5-FU. The catabolite signal is clearly resolved (S/N ~-- 8:1) in the localized 19F NMR spectrum (Fig. 35) obtained in a measurement time of 60 min from the tumor region (voxel size 6.3 × 6.3 × 4 cm3). These results demonstrate volume-selective fluorine NMR in patients undergoing 5-FU chemotherapy. Short-TR, gradient-echo CSI in combination with a large surface coil yields localized 19F NMR spectra from selected regions within the human liver. The observation of the kinetics of the catabolite FBAL (Fig. 33b) shows that 2D CSI provides sufficient time resolution for monitoring the pharmacokinetics of 5-FU. The sensitivity is also sufficient for volume-selective detection of fluorine signals in vivo during continuous infusion of 5-FU where the steady-state level of the cytostatic agent is significantly lower than after bolus administration. 5.2. Monitoring 5-fluorouracil catabolism with 19F-{I H} double resonance

First applications of 1 9 F - { 1 H } NMR techniques in examinations of patients have been reported recently [81,1261. T h e 19F-{1H} NOE experiments discussed in

Sections 4.5.1.1 and 4.5.1.2 showed that progressive saturation of proton spins is more effective for the fluorine signal amplification than a simple ~H-inversion pulse. However, when applied in vivo, the recommended limits of the specific absorption rate (SAR) must be considered when short repetition times are used. This point gives preference to the simpler Signal intensity

FUPA+FBAL

TFA

120

80

4C)

(3 6 / ppm

-40

-BO

-120

Fig. 35. Fluorine-19 2D CSI examination of patient no. 3 (study A), undergoing continuous 5-FU infusion over a period of 24 h. Five CSI data sets were added; the resulting localized spectrum shows the signals of FUPA + FBAL and of the reference TFA (chemical shift scale: 6 of 5-FU was set to 0 ppm; measurement parameters and data processing, see Fig. 32).

40

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

transient NOE scheme where only two short RF pulses are applied during one cycle. The pulse sequence in Fig. 24 was therefore employed in the first patient study (B) [126]. The patient was examined during his regular treatment with 5-FU. After intravenous administration of the drug at a dose of 750 mg/(m 2 b.s.), a series of 19F NMR spectra was obtained alternating with and without I H excitation with the surface coil placed over the liver region. The time resolution was T A -- 4.2 min ( T R = 500 ms, N E X = 500). The kinetics of FBAL could be monitored for up to 60 min after administration of 5-FU (Fig. 36). In contrast to the catabolite signal, the 5-FU signal could not be evaluated because of poor S / N . The integral of the FBAL peak of each 19F-{IH} and 19F spectrum was divided by the reference signal giving the normalized signal intensities I(t) and Io(t), respectively (in a previous experiment no 19F- {1H } NOE signal enhancement was found for the reference TFA). The difficulty with the determination of the NOE factor in this study is the variation of the FBAL concentration during the observation period of

/(t) [a.u.] 140 •

.

13

2O

'° t 0

26

36

t / min

46

Fig. 36. In vivo ]9F-{IH} NOE. Kinetics of FUPA + FBAL observed in the liver of a patient undergoing 5-FU chemotherapy (intravenous infusion of 750 mg 5-FU/[m 2 b.s.] during 10 min). 19F NMR signals obtained with (11) and without ([-7) proton excitation. Transient NOE sequence, see Fig. 24, with t = 1 ms, TR -----500 ms and NEX = 500 (TA ----4.2 min). Fit function, Eq. (28), to guide the eye. Data processing: 2 K zero filling, 64 Hz line-broadening (adapted with permission from Journal of Magnetic Resonance, B 108, Krems et al. [126], © 1995, Academic Press, Inc.).

60 min. The signal-time curve appears to follow biexponential kinetics (Eq. (28)): the FBAL signal reaches a maximum at t - 3 5 min after the beginning of the 5-FU infusion and decreases afterwards. Accordingly, the function I ( t ) = al (e - ~:t _ e - ~3t) was fitted to the normalized signal intensities observed with irradiation of the I H spins. The intensities Io(t) measured without IH excitation were multiplied by a variable factor 1 + ~/* and then the function Io(t) = a l o ( e - a 2 ° t - e -aa°t) was fitted to the resulting set of data points. The factor 1 + ~7" was adjusted in order to give the minimum X 2 o f the leastsquares fit. Fig. 36 shows the time dependence of the FBAL signal measured with (1) and without (I-7) proton excitation together with the fitted curves for the NOE-enhanced and the conventional data. The resulting fit parameter yield an in vivo 19F-{IH} NOE enhancement of the FBAL resonance of ~*(t) --- ( a l / a l o ) - 1 : 0.11 _+ 0.01. This is significantly lower than the steady-state enhancement ~/*-35% that was observed in the experiment on the model solution. The low in vivo NOE factor is partly explained by the fact that the sequence parameters of the phantom measurements were employed (there was no time for an adjustment of the pulse sequence during the patient examination). Proton spin decoupling has also been applied in patient studies during 5-FU chemotherapy. Gonen et al. applied WALTZ-4 broadband I H-decoupling in combination with 3D CSI to 19F NMR spectroscopy of the human liver [81 ]. Their result was a significant reduction of the linewidth of the FBAL resonance accompanied by the resolution of a small resonance at the chemical-shift position of FUPA (about 2 ppm downfield from FBAL). The assignment of this catabolic intermediate agrees with results of 19F NMR studies at B0 = 2.4 T of 5-FU metabolism in liver and implanted tumors in mice [163] and, at 11.7 T, of plasma of patients receiving 5-FU chemotherapy [74]. Wolf et al. detected in their 19F NMR study, in the sum spectra of one patient, a small shoulder on the FBAL resonance that probably represents FUPA [31 ]. The signal gain owing to t h e 19F-{ 1H} NOE in the short-TR decoupling experiment ( T A ~ 45 min, T R = 350 ms) permits localized fluorine NMR spectroscopy of the human liver in vivo at 3 × 3 × 3 cm 3 spatial resolution [81 ].

41

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

5.3. Clinical studies. 19F NMR during fluoropyrimidine chemotherapy The aim of the application of 19F NMR in clinical studies is the noninvasive monitoring of 5-FU disposition in tumor tissue, in particular the production of cytotoxic 5-FUranuc versus the detoxification in the liver. Another goal is to observe the modulation of 5-FU metabolism by different drugs in combined chemotherapy. Levels of 5-FU measured ex vivo in plasma do not correlate well with patient reponse to treatment [164]. The clinical utility of in vivo 19F NMR spectroscopy would therefore be the differentiation of response and nonresponse during the early phase of treatment. The feasibility of monitoring 5-FU pharmacotherapy in individual patients by means of 19F NMR spectroscopy was demonstrated by the time-resolved detection of the unmetabolized drug and its catabolites FUPA + FBAL [31]. In subsequent studies, the signals of anabolites (5-FUranuc) and of the catabolic intermediates DHFU and FUPA could also be resolved (Table 4) [33,79,81,165]. A prolonged biological half-life of free 5-FU in tumors was observed in patients who responded to treatment [77,78]. Clinical studies showed metabolic changes associated with 5-FU modulation by interferon-c¢ [127,165], and changes in the intratumoral half-life of 5-FU upon its modulation by Leucovorin [166]. In the following, two clinical 19F NMR studies are discussed where the patients received 5-FU for the treatment of liver metastases [33,79]. The 19F NMR examinations were performed in a 1.5 T whole-body NMR scanner with a one-turn 15 cm diameter surface coil placed over the liver region. When these clinical studies were performed the fluorine spatial localization techniques discussed in Sections 4.4.3 and 5.1 were not available. The FOV of the coil included a significant portion of the right liver lobe, the tumor(s), and muscle and subcutaneous adipose tissue. Therefore, the discrimination between signals originating from healthy portions of the liver and those from tumor tissue was not possible. In study C [33], 19F NMR spectra were obtained from seven patients (nos. 1, 2, 4 - 8 ) who received 5-FU (Fluroblastin ® or Fluorouracil Roche ®) of a dose of 1000 mg/(m 2 b.s.) by infusion into the hepatic artery within 10 (in patient no. 1, 4.5) min. The

position of the intra-arterial catheter is illustrated in Fig. 37. Shimming was performed on the tissue water resonance, resulting in an average linewidth A~,I/2(IH,H20) = 62 _+ 25 Hz (mean + SD). 19F NMR spectra were recorded up to 210 min after the beginning of the 5-FU infusion. The measurement time for each spectrum was TA = 4.3 min (onepulse-acquire sequence, TR = 1 s, ]VEX = 256). The intra-arterial bolus injection of the cytostatic agent causes a first-pass effect in the liver and thus high concentrations of fluorinated compounds in the FOV of the surface coil. Three in vivo 19F NMR resonances were detected and assigned by calibrating their chemical shifts relative to the signal of the extemal reference TFA: 5-FU (6 = - 94.2 _+ 0.7ppm [mean +_ average error of Lorentzian line fits]), 5-FUranuc (-89.2 _+ 2.4 ppm [mean + SD], observed in one patient only), and a third peak (centered at 6 = - 112.8 ppm) that contains FBAL and probably a small contribution of FUPA signal [31 ], but cannot be resolved with the measurement technique employed. Fig. 9 shows one spectrum (40-45 min postinjection) of the examination of the patient (no. 8) with detectable levels of 5-FUranuc. The result of the evaluation of the complete series of spectra of the same patient is shown in Fig. 38 where normalized 19F NMR signal intensities of 5-FU and FUPA + FBAL are plotted as a function of time. Fig. 39 shows the signal-time profiles that were observed in study C, together with curves fitted i.a. port (subcutaneous)

Fig. 37. Schematic drawing of the position of the intra-arterial catheter for 5-FU infusion to patients in study C (adapted with permission from Radiology, 174, Semmler et al. [33], © 1990, Radiological Society of North America).

42

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

I(t) [a.u.] 10

mm

•

mmm

mm •

•

0

i

m~/'

~

" m , tb._ _

t

~

J

0

FUPA+FBAL

5-FU

i

~

t

t

~

1O0

I

200

t/rain Fig. 38. F l u o r i n e - 19 N M R s i g n a l - t i m e profile o f 5 - F U a n d F U P A + F B A L in the liver no. 8 ( s t u d y C [33]).

from pharmacokinetic modeling [124] (see Section 5.4). The errors of the integrated peak areas were less than 10% and nearly constant for both resonances during the observation period. 5-FU and FUPA + FBAL could be followed for at least 30 and 70 min, respectively. In all cases, the 5-FU concentrations reached their maximum at the end of the infusion. Patients nos. 1, 2, 6, 7 and 8 showed similar 5-FU and catabolite kinetics: 5-FU elimination was biphasic with a distribution phase of 10-20 min; a continuous increase of the FUPA + FBAL signal intensity was observed up to at least 50 min after the beginning of the 5-FU infusion. Thus synthesis and accumulation of the catabolites exceeds elimination during this period, in agreement with the results of animal experiments [21]. The elimination phase of the catabolites could be followed only in patient no. 8; in this examination the FUPA + FBAL signal began to decrease at about 110 min after the start of the 5-FU infusion. The sum of the signal-time curves of 5-FU and FUPA + FBAL can show a minimum after the steep initial increase, as for example the data of

observedafterintra-arterial

infusion o f 5 - F U in patient

patient no. 6 in Fig. 39. This course corresponds to activity-time profiles observed in the liver of animals in 18F-PET studies [116]. After bolus infusion of 5-[18F]U, the activity in the liver decreases because the drug distributes in the body, followed by a transient increase of activity when the recirculating drug is converted into 18F-labeled catabolites in the hepatocytes. Finally, during the elimination phase of the drug and its metabolites from the body, the 18F concentration in the liver parenchyma decreases again. In 18F-PET studies of patients, however, a continuous increase of activity in liver parenchyma up to 30 min after intravenous infusion of 5-[18F]U was observed, followed by a decline of activity [ 115]. This behaviour corresponds to most of the time courses of the sum signals of Fig. 39. Besides the long observation periods for 5-FU and catabolite kinetics that were possible in this study, the detection of 5-FUranuc is notable. The observation of the anabolites is desired, because a correlation of the 5-FUranuc level in the tumor tissue and 5-FU cytotoxicity towards tumors was found in animal studies

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56 ~oo-

Io.

po~'~ no. t.~

~o.

p o t ~ no. 2

poli,,nl no. 4

~o

poek~ no. 5 ~I£

43

= 16.4 ohm ualll

?I-o "e~4~.~l. 4.a ........................ .~...o ..... h~/z 0.I

o

m

~o

~o

;o

po~r~.o. 6

~

.o ° . ~ ° - " ~ ' o 0

0

k~/c < .0001 O h m unHs

o 0.1

(n~) ofmr ~ m ~

~ the ~U

oJl~ ....................................................................... o ~) ~ " " ~) " " ~

k,,,/,: do

,,;o

2;o

Fig. 39. Fluorine-19 NMR signal-time courses of 5-FU (O) and FUPA + FBAL (O) in the liver observed after intra-arterial infusion of 5-FU in patients nos. 1, 2, 4 - 8 (study C [33]). A nonlinear three-compartment model was fitted to the data (adapted with permission from Clinical Pharmacology and Therapeutics, 49, Port et al. [124], © 1991, Mosby Year Book, Inc.).

[76,162]. However, fluorine-containing metabolites must be in concentrations greater than 100 nmol/g (equivalent to 0.1 raM) to be detected in a measurement time that is tolerable for patients. As shown in several clinical studies, the tissue concentration of

5-FUranuc is below the 19F NMR-detectable level in most cases, particularly when the drug is administered on the intravenous route. This indicates efficient catabolism and excretion and a much lower distribution of 5-FU into the anabolic pathway.

44

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

The intracellular conversion of 5-FU into 5-FUranuc is a prerequisite for antitumor activity (Section 2.2). Patient no. 8, who showed high levels of cytotoxic anabolites and also a long 5-FU elimination half-life (tl/2 = 35 min, as estimated using a linear twocompartment model, see Section 5.4) in her 19F NMR examination, responded to 5-FU chemotherapy. The outcome of the treatment and the findings with NMR are consistent in this case. In another study [79], the 5-FUranuc signal was detected after intravenous infusion of the drug (dose: 650 mg 5-FU/[m 2 b.s.]) in a responsive patient. Findlay et al. observed 5-FUranuc in patients during continuous intravenous infusion at the low dose of 300 mg 5-FU/(m 2 b.s. × day) [165]. The 5-FUranuc resonance was unresolved at 1.5 T as expected from the small chemical-shift difference ( < 1 ppm [167]) of 5-FU nucleosides and nucleotides. In ex vivo 19F NMR spectra, however, obtained at B0 = 11.7 T and 4°C sample temperature of excised liver metastases and unaffected liver tissue of patients receiving a treatment dose of 1000 mg 5-FU/(m 2 b.s.), the resonances of total 5-FU nucleosides (e.g. FUrd, FdUrd) and of total 5-FU nucleotides (e.g. FUMP, FUDP, FUTP, FdUMP) as well as the resonances of the catabolites DHFU, FUPA and FBAL could be resolved [168]. The fraction of 5-FU nucleotides of the total NMR-detectable fluorinated metabolites was estimated at 5 % - 1 0 % ( < 50nmol/g). However, quantification was difficult because of the instability of the nucleotides in the excised tissue. In 19F NMR spectroscopy studies of the Walker carcinosarcoma in the rat, FUTP was the predominant compound of 5-FUranuc [76]. A detailed analysis of 5-FU anabolites was possible with extracts from cells and tissue. In the first place, high-performance liquid chromatography (HPLC) using 3 H - o r lnC-labeled 5-FU was employed for this purpose [169,170]. In contrast, extracts of liver tissue of rats receiving 180 mg 5-FU/(kg b.w.) were measured by means of ]9F-{IH} NMR at B0 = 7 T [167]. In ]H-decoupled spectra, resonances of ribosyl derivatives of fluoronucleotides (FUDP, FUTP, FUMP, with chemical shifts in the range 6 = - 8 9 . 1 0 to - 8 9 . 2 0 ppm relative to TFA) and of FUDP-sugars (6 = -88.97 to -89.04 ppm) could be resolved. The catabolic intermediate 5,6-dihydro-5-fluorouracil (DHFU) was not observed in study C, in accordance with Hull et al. [163], who did not detect

a signal of DHFU in vivo in liver or experimental tumors of the mouse. DHFU was found by in vitro 19F NMR at 11.7 T in plasma and urine samples of patients receiving 5-FU [74]. A possible explanation for the missing signal of DHFU is rapid conversion of DHFU to FUPA. However, in a single case, reported in Ref. [79], a fluorine signal was detected in vivo in the human liver that was assigned to DHFU on the basis of its chemical shift (Fig. 40). In this patient, who received 1000 mg 5-FU/ (m 2 b.s.) with intra-arterial infusion, a delayed increase of FUPA + FBAL (at 18 min after the beginning of the 5-FU infusion) and enhanced 5-FU levels were observed (in the 50 min sum spectrum the signal intensity of 5-FU was higher than that of FUPA ÷ FBAL). This indicates a partial block of the catabolic pathway after the DHFU-producing step resulting in an accumulation of 5-FU and DHFU and a slow production rate of subsequent catabolites. In study D [79], 16 colon cancer patients (14/16 with liver metastases) were examined by means of 19F NMR spectroscopy of the liver during their initial treatment cycle. 5-FU (Fluroblastin ®) was administered with 10 rain intravenous infusion in a dose of 650mg/(m 2 b.s.). Spectra were sampled during 17 min intervals (one-pulse-acquire sequence, dwell time = 50/zs, TR = l s , N E X = 1024) with the same equipment as in study C. Observation periods ranged from 55 to 141 min (mean 89 min). Fluorine S / N was significantly lower than in study C, where the patients received 1000 mg 5-FU/(m 2 b.s.) with intra-arterial infusion. In particular, the Signal intensity [a.u.]

5-FU FUPA+FBAL

-40

I

I -60

I

I -80

I

I -100

I

I -120

I

8 / ppm

Fig. 40. Fluorine-19NMR spectrum(sumof spectraobtainedduring 0-58 min after the beginning of 5-FU infusion) of the liver of a patient in studyD showingresonancesassignedto DHFU, FUPA + FBAL, and 5-FU (adapted with permission from Magnetic Resonance Imaging, 12. Schlemmeret al. [79], © 1994, Elsevier Science Ltd.).

45

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

signal o f the cytostatic drug was often difficult to detect, as seen in F i g . 41 in a selection of s i g n a l time curves o f 5 - F U and F U P A + F B A L o b s e r v e d in five patients [123]. In most e x a m i n a t i o n s of study

D, 5 - F U was o b s e r v e d only in the first two spectra (0-34min). The signal o f F U P A + FBAL (unresolved) could be d e t e c t e d until the end o f the e x a m i n a t i o n in all cases.

b) _>, c-

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/."

m

•g~

60 90 time (min)

"

8

8

30

.... e- .....

:":

ii ...... Q... o "'""

0 0

30

60 90 time (min)

120

0

30

60 90 time (rain)

120

Fig. 41. Fluorine-19 NMR signal-time courses of 5-FU (0) and FUPA + FBAL (©) in the liver observed after intravenous infusion of 5-FU (study D [79]). Patient no. 7 (c) was a responder, patients nos. 8 (a, b) and 9 (e) were nonresponders. A determination of response/nonresponse was not possible for the other two patients (d, f). A nonlinear two-compartment model of 5-FU catabolism in the liver was fitted to the data (adapted with permission from European Journal of Clinical Pharmacology, 47, Port et al. [ 123], © 1994, Springer-Verlag).

46

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

The most useful spectral parameter in study D was 150, i.e. the normalized peak integral of a fluorine resonance in the sum of spectra obtained during 5 0 m in after the beginning of the infusion. Since 5-FU was not observed later than 50 min, I~o FU Can be interpreted as a measure of the fraction of the administered dose that is offered to the target organ ("bioavailability"). In patients with large volumes of metastases ( > 20 cm 3) a correlation was found between/550 FU /SO5"FU

a

I O.OS

I-0.03

0

...."/

100

200

300

400

volume of metastases I cm 3

ll~t~FU

I

b

0.05

F-0.04

~

[oo, 0

and the volume of liver metastases in the FOV of the surface coil. In this group of patients (6/16) responders showed high /550FU values. Fig. 42a gives a plot of 155oFU versus the volume of metastases in the examined liver region of 16 patients (17 examinations). The data for responsive ([3) and nonresponsive patients (11) in Fig. 42b are found within the 90% confidence interval (Pearson productmoment correlation). Another observation is a prolonged visibility of 5-FU in responders compared with patients who did not respond to therapy. This is demonstrated, for example, in the data of patients no. 7 (Fig. 41c) and no. 8 (Fig. 41a, b). Figs. 43 (no. 7) and 44 (no. 8) show transversal ~H N M R images of the liver region of these patients. Patient no. 7, with a large metastasis and maximum intensity of 5-FU in the second spectrum ( 1 7 - 3 4 m i n ) , responded to treatment. Patient no. 8, with multiple metastases in the right liver lobe close to the surface coil, showed much lower drug levels at the same administered dose and was identified as a nonresponder. These findings indicate an accumulation and retention of 5-FU in the tumor tissue in the case of response, confirming the results of Wolf et al. They observed that a prolonged half-life of free 5-FU in

100

200

300

400

volume of metastases I cm ~ Fig. 42. (a) In vivo 19F NMR sum signal of 5-FU (15o Fv) versus volume of liver metastases within the FOV of the surfacecoil (study D). (b) 155oFv values in (a) from patients with large volumes of metastases ( > 20cm3), identified as responder ([3) and nonresponder (11). The 90% confidence interval (Pearson productmoment correlation) is indicated showing the correlation between 155otru and the volume of metastases (adapted with permission from Magnetic Resonance Imaging, 12, Schlemmer et al. [79], © 1994, Elsevier Science, Ltd.).

Fig. 43. Transversal ]H NMR image of patient no. 7 (study D) (adapted with permission from Magnetic Resonance Imaging, 12, Schlemmer et al. [79], © 1994, Elsevier Science, Ltd.).

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

Fig. 44. Transversal ~H NMR image of patient no. 8 (study D) (adapted with permission from Magnetic Resonance Imaging, 12, Schlemmer et al. [79], © 1994, Elsevier Science, Ltd.).

malignant tumors following administration of a bolus dose of 5-FU is predictive of a therapeutic response [77,78,125,166]. Half-lives of 5-FU of 5 - 2 0 m i n during the terminal phase (tl/2,~), when most of the drug is eliminated, were measured in plasma of humans and animals [ 118,171 ]. The prolongation of the half-life of intratumoral 5-FU to 20 min or greater is called "trapping" [77]. The effect was observed both in human tumors [77,78,172] and in tumors in animals [172,173]. Direct proof of tumor trapping requires fluorine spatial localization. In study A, however, no evidence of 5-FU trapping was seen in the two patients with liver metastases who were examined with 19F 2D CSI (Section 5.1). Whether voxel bleeding, partial volume effects and physiological motion counteract a sufficiently precise localization of 5-FU for the detection of this effect in liver tumors requires further investigations. FUPA+FBAL did not depend on the volume of 50 metastases. This corresponds to expectation, since the production of FBAL occurs primarily in the normal liver tissue [137,162] (Section 5.1). The relatively high signal intensity of FUPA ÷ FBAL (Fig. 41 a-f), which also allowed the volume-selective detection of this resonance after intravenous drug

47

infusion (Fig. 33), agrees with results of biochemical studies demonstrating that most of the administered 5-FU is processed via the catabolic route (Section 2.2). Information on quantitative 5-FU turnover can be obtained from measurements of absolute concentrations of FBAL (CFBAL)in the liver. In vivo CFBALwas /FUPA + FBAL estimated from -50 [79] assuming that the signal contribution of FUPA is small according to 19F-{IH} NMR measurements [81]. It was also assumed that the FOV of the coil comprises two compartments, one without FBAL (subcutaneous adipose tissue, muscle, bone) and a second with a homogeneous distribution of FBAL (liver). For the density of liver tissue in vivo, the density of blood (1.06 g/ml) was taken. With correction for full T~ relaxation of fluorine spins and for signal loss owing to B l-inhomogeneity, an average FBAL concentration of 0.92 +_ 0.26 mmol/(kg liver tissue) (mean +_ SD) for the first 50 min after the beginning of drug infusion was estimated. From the peak intensities in the signaltime curves, a mean maximum FBAL concentration of 1.31 _+ 0.33 mmol/(kg liver tissue) from the patient examinations of study D was derived. Li et al. report CFBAL ~--- 1.0 + 0.2 mM at 60 + 10 min after 5-FU administration [ 132]. Spectral parameter and absolute concentrations of the catabolites were not related to clinical response to treatment. The steady-state FBAL level measured by means of in vivo 19F NMR spectroscopy was largely independent of the administered 5-FU dose. This indicates a saturation of catabolism in the human liver for drug doses greater than 1 g infused for 10 min [79]. 19F NMR studies on human plasma and urine also showed this effect for 5-FU doses > 90 #mol/kg [74] which is of the order of the doses that the patients of study D received. However, restricted transport of the charged compound FBAL out of the liver cells is also discussed in this context [74,162]. The tumor toxicity of 5-FU depends on drug uptake and the extent of anabolism in the tumor tissue. 5-FU bioavailability is reduced by the catabolism in the liver and the excretion via the kidneys. Clinical studies demonstrated that these processes can be monitored in patients during treatment by means of 19F NMR spectroscopy. A possible (and important) application of fluorine NMR could therefore be the guidance of dose escalation schemes for the

48

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

optimization of 5-FU bioavailability in the individual patient. 5.4. Pharmacokinetic modeling: 5-fluorouracil and ot-fluoro-13-alanine in vivo

Further information on 5-FU disposition in patients was obtained by kinetic modeling of in vivo 19F NMR spectroscopy data. Complete pharmacokinetic models, including volume and clearance parameters, were applied to describe 5-FU catabolism in the human liver (Fig. 3) as reflected in signal-time courses of 5-FU and the catabolites FUPA + FBAL. The models depicted in Figs. 6 and 7 were employed to fit the data observed in studies D and C, respectively [123,124]. Plasma pharmacokinetics of 5-FU in patients was studied extensively [118,119,122,174]. Following bolus infusion of the drug, total body clearance ranges from 0.5 to 2 l/min. During the first 24 h after injection, 50%-100% of the administered dose appears in urine as 5-FU or catabolites (mainly FBAL) [74,120,175,176]. The kinetics of 5-FU and its metabolites in tissues and plasma was described by mathematical models using systems of coupled differential equations. However, only those pharmacokinetic parameters that do not refer to volumes, such as half-lives (t~/2) or rate constants, can be estimated in a straightforward way [77,78,166,177]. Using 19F NMR and compartmental modeling, E1Tahtawy and Wolf [177] determined transfer rates of the pharmacokinetics of 5-FU and its cytotoxic anabolites in tumors in vivo. Time courses of 5-FU and 5-FUranuc signal intensities were measured noninvasively in tumor-bearing rats at 4.7 T. A twocompartment model with distribution volumes for 5-FU (V1) and 5-FUranuc (V2) was applied to estimate the apparent first-order rate constant of 5-FUranuc formation in tumor tissue, k12----CLld2/V1 (CL~ 2 = distribution clearance in one direction). Following methotrexate pretreatment, a significant change of k~2 was observed demonstrating the modulation of 5-FU anabolism by this drug. A first attempt to fit a complete pharmacokinetic model, i.e. a model that also yields information on volume and clearance parameters, to in vivo NMR data was made by Port et al. [124].

Since the 5-FU and FUPA + FBAL data obtained in studies C and D represent a mixture of signals from normal liver, tumor, muscle and subcutaneous tissue, a kinetic model had to be formulated without knowing exactly the organ or tissue that the model had to refer to. Therefore, a highly simplified picture of 5-FU catabolism in intact liver was chosen. The approximation was justified by the detection of differences in the kinetic data depending on the tumor properties. The nonlinear three-compartment model that was used to describe the signal-time curves obtained in study C was introduced in Section 3.2.2 (Fig. 7). In this model, 5-FU is infused (infusion rate R0) into a central compartment (volume V1) from which saturable elimination occurs (maximal velocity Vmax and Michaelis-Menten constant kM for both 5-FU elimination and catabolite formation); simultaneously the drug distributes between the central and a peripheral compartment (V2), the distribution clearance (CLd) being equal in both directions. FUPA and FBAL are assumed to be produced instantaneously by the 5-FU elimination step; a common distribution volume (V3) and a common elimination clearance from the liver (CLe) were assigned to both catabolites. The model corresponds to Eqs. (6)-(8), where Cl(t), c2(t) and c3(t) are the concentrations in the three compartments (5-FU central and peripheral and cataholites). The central compartment contains the liver, the target organ of the NMR examination. The major problem with the kinetic analysis of in vivo NMR data is the lack of accurate data on absolute concentrations of the detected compounds in the tissue. Quantification is limited by factors like low S/N, B l-inhomogeneity (causing different flip angles in the VOI under investigation), inhomogeneous distribution of the detected compounds in the tissue, variations of concentrations during the measurement, different relaxation times of the compounds of interest, and violation of the condition TR >- 5 × TI (full relaxation of all spins). In the case of 5-FU and FUPA + FBAL, with relatively high 19F SIN (in study C), two distinct resonances and comparable spin-lattice relaxation times (ratio of Tt times less than 2, Section 4.3), it was assumed that the normalized NMR signal intensities I(t) are related to the tissue concentrations by a common proportionality factor: Ik(t) × e = ck(t),

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

where e is the (unknown) molar tissue concentration of each compound corresponding to one peak area unit and k = 1,2,3. At least for 5-FU and FBAL this relation is a good approximation regarding measurements of model solutions (Fig. 10). It is further assumed that the spatial distribution in the VOI is the same for the parent drug and the catabolites and does not change during the measurement. Finally, the signal contribution of FUPA is expected to be small according to in vivo NMR measurements [81]. The substitution of Ck(t) in Eqs. (6)-(8) transforms the concentration-time model into a signal-time model [124]: d/1 (t)

[ dt -- Ro - CLd'e'[Ii(t) - 12(0] _

Vmax .I ] 1 (kM/e) + I i ( t ) l(t) • Vl'e

d12(t) 1 = CLd.e.[Ii(t) - 1 2 ( t ) ] ' d V2"e d/3(t )

dt

(29)

(30)

Vmax ]l(t ) _ CLe.e.i3(t)l ' 1 I (kMle) + 11(t) V3"e

[

(31) Only the parameter Vm~ remains unchanged upon transition to the signai-time model; it can be identified on the basis of information on dose and detected concentration changes alone. The fit of the kinetic model to the observed profiles l(t) was performed with the extended least-squares modeling program MKMODEL (Biosoft, Cambridge, UK). The results are estimates of the parameters V~e, V2e, V3e, CLde, CLee, Vm~x and kM/e. The unknown factor e is eliminated by calculating ratios of the parameters corresponding to volumes or clearances and multiplying one of the volume or clearance parameters with kM/e. Since the steady-state distribution volume Vs = V~ + V2 had the smallest variation in a reference study [178] and is expected to be less sensitive to local tissue properties than any of the other parameters, the relative kinetic parameters V1/Vs, V2/Vs, V3/Vs, CLd/Vs, CLdVs, kM X Vs were chosen. The ratios Va/Vs and CLdVs give a measure of the catabolite kinetics. The nonlinear three-compartment model (Fig. 7) yielded satisfactory fits to all pairs of 5-FU and

49

FUPA + FBAL signal-time curves observed in study C (Fig. 39). The corresponding parameter values, for example for the data of patient no. 8, are VffVs = 0.49, V3/Vs = 0.27, CLd/Vs = 0.032 min -1, CLdVs = 0.0033 min-l, kM × Vs = 520/xmol, Vmax= 101/~mol/min. The coefficients of variation of parameter estimates ranged from 2%-30% in patient no. 8 to 59%-143% in patient no. 4. Partly, the fit values fell in the range of data on 5-FU plasma kinetics of the reference study, but there were also deviations. For example, the rather low Vmax and high V3/Vs values may be interpreted as an indication of a slow 5-FU catabolism in the tumors examined (or of significant catabolism occurring outside the liver, contrary to the model assumptions). The results show that ratios for volume and clearance parameters can be estimated from in vivo NMR data. All parameters that do not refer to volumes, including rate constants and maximal velocity of metabolic conversion (Vm~x) of a nonlinear model, can be estimated in absolute terms. Only kM has to be multiplied by a volume parameter to obtain an identifiable quantity (kM/e has the same dimension as the measured quantity l(t); it indicates the signal intensity that is observed when kM = Ck). The knowledge of only one distribution volume of the model could enable the estimation of absolute tissue concentrations, which is still one of the major problems of in vivo NMR spectroscopy [100]. The kinetics of 5-FU alone can be equally well described with the linear two-compartment model (Fig. 5), or with the linear one-compartment model (Fig. 4) in the single patient (no. 5) with monoexponential 5-FU elimination. The 5-FU elimination tl/2 was estimated using these linear models. However, when the catabolite formation was included, satisfactory fits of the FUPA + FBAL signal-time courses are possible only for the data of the patients nos. 4 and 5. Interindividual comparison of the fit results confirmed the exceptional kinetics in these two patients. Patient no. 4 had the lowest values of V1/Vs (0.13), CLd/V S (0.011 min-l), Vmax(70 #mol/min) and V3/Vs (0.08) and by far the longest 5-FU elimination tl/z (139min). Patient no. 5, with no indication of biphasic 5-FU elimination, had the shortest tl/2 (9 min) and the maximum V3/Vs (2.5). The extremely low CLd/Vs value of patient no. 4

50

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

(0.011 min -I versus --0.03 min-l in patients nos. 1, 2, 6-8), paralleled by an extremely long tl/2, appears to indicate a case of tumor trapping of 5-FU [77] (Section 5.3). In contrast to patients nos. 1, 2, 6-8, quantification of tumor volumes was not possible in patients nos. 4 and 5, who had a disseminated tumor and diffuse liver infiltration, respectively. It seems that the tumors, rather than normal liver tissue, determined the outcome of the measurements in these two cases. The five patients with a similar pattern of 5-FU and catabolite kinetics (nos. 1, 2, 6-8) (Fig. 39) were those in whom single metastases were under investigation. Obviously, analyses of this kind could greatly facilitate quantitative comparisons between drug kinetics or treatments. In the case of the signal-time profiles obtained in clinical study D, a pharmacokinetic model could not be fitted individually. The reason was data sparsity because of low concentrations of the compounds and long measurement times. Even a clearancevolume ratio will not be capable of being calculated when the drug is detected for one measurement interval only (Fig. 41a, b, d, e), or when a metabolite is followed while the signal of the parent drug cannot be identified in any of the acquired spectra (Fig. 41f). In this situation the "population approach" [179] could be a useful tool. Population models of pharmacokinetics were applied successfully to drugs of a variety of classes [180,181] taking advantage when the number of data points is less than the number of parameter of the kinetic model [182,183]. For the description of the data observed in study D by a population model [123], the nonlinear twocompartment model (Fig. 6) was chosen as its "structural" part [184]. The kinetic model corresponds to Eqs. (6) and (8) with the distribution clearance CLd - O. The model is highly simplified in that it ignores the known biphasic [ 118] or even triphasic [ 120] disposition of 5-FU, the variation of V~ over time, the distribution of the catabolites outside the liver [74] (implying a possible reflux of catabolites from plasma into liver), and the consecutive formation and elimination of FUPA and FBAL [185]. As in the previous study (where also the implicit assumptions of the following analysis are discussed), translation into a signal-time model is achieved by

assuming the relations ll(t) X e = Cl(t) and 13(t) × e = C3(t) between NMR signal intensity and drug and catabolite concentrations in the distribution volumes V~ and V3. The common proportionality factor was defined as e = ~//V1. Insertion into Eqs. (6) and (8) gives (CLd =---0): d/l (t)

[R

d/3(t)

[

dt=

dt=

Vmax ii(t).ll(t)l

o - (k~t'V1/'y) +

1 "-~

(32)

Vmax CLe~l.,Y.I3(t)] (kM'V1/'y) +11(0 "11(0- --~3 "

V1 3"V3

(33)

Only a brief summary will be given for the population model employed; detailed information can be found in Ref. [123]. The fit parameters Vmax,3', kM × VI, V3/V~ and CLJV3 were allowed to vary randomly between individuals according to

Ppi = Pp.exp(r/pi)

(34)

where Ppi is the value of parameter Pp in patient no. i, Pp is the population mean of this parameter, and ~/is a random variable which is normally distributed with mean zero. Inter-occasion random variation was allowed for 3' and Vmax, i.e. Ppij = Ppi'exp(rlpij) at examination no. j. Finally, the 5-FU and FUPA + FBAL signal intensities detected in spectrum no. k were assumed to vary according to llij(tk) = Ilij(tk).(1 q- rlli~k) and ^ I3ij(tk) = Iaij(tk).(1 q- 173ijk), respectively; llij(tk) and I3ij(tk) are the model predictions. Normalized signal intensities Ii(t) and 13(0 in the 17 min 19F NMR spectra were treated as having been measured at the mid-points of the corresponding time intervals. The model fits were performed using the program NONMEM [ 186]. The resulting parameter estimates were: 1Jmax 121/~mol/min (coefficient of variation: ± 23%), kM × V~ = 2590/zmol ( - 85%), V3/V~ = 0.065 ( ± 48%), and CLJV3 = 0.056 min -1 ( ± 28%) [123]. The estimate of Vmaxis close to the value of 2.02 ~zmol kg -1 min -a which was derived from 5-FU kinetics in plasma [122]. The estimated CLe/V3 results in a half-life of 12 min for catabolite elimination from the liver. A model with linear conversion of 5-FU into the catabolites turned out to be inferior to the nonlinear model. =

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (I 998) 1-56

Among the fit parameters of the model, only y cannot be interpreted in a purely pharmacokinetic sense ("hybrid" parameter). In Fig. 41 the model predictions which are based on the mean population parameter are shown as solid lines whereas the predictions which take into account inter-occasion random variation of 3' are represented by dotted lines (the NMR sensitivity is a function of the distance of the surface coil with respect to the liver which can change from one examination to the other and thus influences 3"; moreover, the observed weak dependence of 3' on body weight probably reflects a body-weight dependence of V0. The results of two examinations of patient no. 8 (transversal NMR image, Fig. 44) are shown to demonstrate the effect of random variation of 3' (Fig. 41a, b). No additional random effect on 3' is needed to explain the data of patient no. 7 (transversal NMR image, Fig. 43) with relatively low body weight (Fig. 41c). Fig. 41f shows a case where the model predictions are based on catabolite signals only. The large residual variation of the NMR data is attributed to physiological motion (respiration, pulsating blood flow, etc.) of the patient causing changes of local tissue susceptibility and of coil loading. In this section, modeling tools for pharmacokinetic analysis of relative concentration data have been discussed. The differences between the two approaches, which both allow fitting of a complete pharmacokinetic model to in vivo NMR data, are summarized in Table 9. The "population approach" is of particular interest, because it enables one to fit a complete kinetic model to sparse, relative concentration data as are usually obtained in clinical NMR studies. All ambiguities that result from the lack of absolute concentration data can be isolated using the parameter 3', which relates the amount of drug in one compartment

51

to the corresponding NMR signal intensity. The simplifications of the analysis were unavoidable given the sparsity and the large residual variation of the data which precluded the extraction of further information by means of a more complex structural model.

6. Conclusions and perspectives In vivo 19F NMR spectroscopy provides a highly specific tool for identifying fluorine-containing drugs and their metabolites in the tissue. The potential of the method was explored in vitro and in experimental studies in animals. Following the first demonstration of 19F NMR in humans in 1987 [31], various fluorinecontaining pharmaceuticals and metabolites could be monitored noninvasively in patients. The examined organs were brain and liver. Tumors have also been studied in patients undergoing chemotherapy with fluoropyrimidines. The biodistribution as well as absolute concentrations of fluorinated compounds in the tissue can be measured noninvasively by means of 19F NMR. The assessment of individual drug levels is important, since the efficiency of a cytostatic agent for killing tumor cells depends not only on the sensitivity of the cells, but also on the drug amount that can be deposited in the environment of the cells. Information on dose rate and effective dose in the tissue as provided by 19F NMR is needed to optimize therapy in the individual patient. Pharmacokinetic data acquired after drug administration allow for monitoring of the course of cancer treatment. 19F NMR could help to identify responsive versus nonresponsive patients at an early stage of treatment and to assess induced resistance of tumor cells against the cytostatic drug during therapy. In

Table 9 Characterization of two approachesof kinetic modelingof in vivo 19F NMR data Ref. [124]

Ref. [123]

All fitting parameters,except •max, are "contaminated"by the scaling factor e of NMR signal intensity and concentration. After fitting, ratios and products are calculated: VJVs, V2/V s, V31Vs, CLd/Vs, CLJVs and kM × Vs. Individual analysis Nonlinear three-compartmentmodel

The fitting parametersinclude one "hybrid"parameter(7), one absolute parameter(Vmax),and three relative parameters(kM × V~, VJVt and CLJV3) Sparse data, population analysis Nonlinear two-compartmentmodel

52

P. Bachert/Progress in Nuclear Magnetic Resonance Spectroscopy 33 (1998) 1-56

many studies on 5-FU disposition in vivo the kinetics of the drug and of FBAL could be monitored with sufficiently high temporal resolution. Kinetic modeling of these data is possible and yields additional information on drug disposition in the tissue. Data interpretation is facilitated by the absence of 19F background signals from endogenous compounds. 19F NMR emerges as a complementary method to radiotracer techniques. For example, the NMRvisibility of FBAL makes it possible to avoid the cumbersome synthesis of lSF-labeled FBAL needed for PET studies of the biodistribution of this compound. Volume-localized 19F NMR spectroscopy is a comparatively simple method which does not require excessive hardware modifications for implementation on conventional whole-body scanners. The possibility of detecting local 5-FU accumulation and regions of FBAL production suggests the combination of 19F CSI and 5-[18F]U-PET studies in humans. The methods now available of volume-selective high-resolution 19F NMR in whole-body scanners have to be applied in broader clinical studies. The kinetic modeling techniques discussed in Section 5.4 may then become useful in predicting individual response to cancer pharmacotherapy once volumeselective in vivo NMR spectroscopy data on drug kinetics and metabolism in malignant tumor tissue are available. Focusing signal accumulation to deftnite regions within the examined organ permits the observation of 5-FU trapping in tumors in patients. Notwithstanding the advantages of 19F NMR, particularly the sensitivity and chemical specifity, limitations remain. Methodological improvements of 19F NMR in whole-body scanners have been possible with antenna systems adapted to the anatomical conditions and by introducing volume-selection and 19F_ {1H } double resonance techniques. Despite these advances in the measurement techniques, the low S/N of 19F-labeled drugs in the tissue precludes, at present, the use of in vivo 19F NMR for mapping dynamic processes at a spatial resolution better than 3 × 3 × 3 cm 3. Furthermore, in studies on the model system 5-FU, the 5-FUranuc resonance band is unresolved in vivo and many metabolic intermediates could not be detected until now in patients during treatment. 19F-labeled groups that are attached to macromolecular structures (nucleic acids, proteins,

phospholipid aggregates) in the course of metabolic transformation are invisible. An improvement of volume-selective 19F NMR of the human liver is expected when phased-array coils are employed. Fast fluorine imaging techniques with high spatial resolution should be studied in conjunction with these coils. Echo planar imaging will be difficult because of the rapid decay of the fluorine signal. Larger enhancements of sensitivity than with NOE could be obtained by 19F-1H cross-polarisation techniques or polarisation transfer of scalar coupled nuclei. Finally, compounds that contain more than one fluorine atom - - an example is the novel anticancer agent gemcitabine (2',2'-difluoro-2'-deoxycytidine) - - are good candidates for drug monitoring in vivo by means of 19F NMR. NMR offers a variety of nuclei that are present in pharmaceuticals. For example, the spin-l/2 nucleus 195pt (natural abundance 33.8%) is bound in the anticancer agent carboplatin. In the future, studies of pharmacokinetics in humans could be extended beyond the nuclei 7Li, 13C and 19F that were already employed for this purpose, to other nuclei in administered drugs.

Acknowledgements The author wishes to thank Prof. Ulrich Haeberlen (Max-Planck-Institut fiir Medizinische Forschung, Heidelberg) and Prof. Gerhard van Kaick (Head of the Research Program Radiological Diagnostics and Therapy, Deutsches Krebsforschungszentrum, Heidelberg) and his coworkers Markus Becker, Dr. Michael Bock, Friedrich Hanisch, Dr. Boris Krems, Dr. Riidiger Port, Dr. Heinz-Peter Schlemmer, Petra Wollensack and Hans-Joachim Zabel.

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