Ojima I. (ed.) Fluorine in Medicinal Chemistry and Chemical Biology
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Study of Metabolism of Fluorine -
containing Drugs Using In Vivo
Magnetic Resonance Spectroscopy
Erika Schneider and Roger Lin
19.1 Introduction
Fluorinated compounds are widely used in medicine and treat a range of conditions includ-
ing those in psychiatry (e.g., mood disorders, anxiety disorders, psychotic disorders)
[1, 2] , cancer (e.g., antimetabolite agents) [3 – 10] , infection and infl ammation (e.g.,
anti - infl ammatory agents, antimalarial drugs, antiviral agents, antihistamines, steroids)
[3, 4, 13] , and anesthetics. Inclusion of a fl uorine atom can alter drug disposition as well
as modify its interaction with the pharmacological target [18] , making fl uorination of
compounds a common drug development technique.
In drug development, it is important to know that the compound has reached its
intended target as well as to understand the absorption, excretion, bioavailability,
metabolism, and distribution (e.g. , the pharmacodynamics and pharmacokinetics) of
the compound. Direct monitoring of human in vivo drug and metabolite concentrations
in the target organs or in adverse event - related organs, such as the brain, liver, or
heart, is often needed to understand the therapeutic impact, potential adverse events,
or other effects.
Magnetic resonance (MR) is routinely used in clinical practice as a powerful, nonin-
vasive diagnostic tool. While human in vivo MR is mainly an anatomical and morphologi-
cal imaging technique, it can also provide functional information using both imaging
and spectroscopic methods. MR spectroscopy has proved to be an important technique in
Fluorine in Medicinal Chemistry and Chemical Biology Edited by Iwao Ojima
© 2009 Blackwell Publishing, Ltd. ISBN: 978-1-405-16720-8
496 Fluorine in Medicinal Chemistry and Chemical Biology
drug design [13, 60, 61] and preclinical studies [14 – 16] . Extension of these laboratory
techniques into the clinic can be challenging, however
19
F MR spectroscopy has proved
to be highly specifi c for identifying fl uorinated drugs and their metabolites in vivo [1, 3 – 10,
17] . MR methods in vivo are very useful despite a relatively low detection sensitivity,
because they are noninvasive, because the effects of one or more doses can be measured,
and because they do not expose patients to ionizing radiation, thereby allowing repeat
measurements to be performed as often as needed and outcomes to be measured in longi-
tudinal studies. Competing methods of measuring the in vivo biochemistry of fl uorine
compounds use expensive radioactive labeling with detection by either positron emission
tomography (PET) or single - photon emission computerized tomography (SPECT). The
radioisotopes decay quickly, which limits the evaluation to only the initial kinetics, metab-
olism, and biodistribution of a single dose. However, fl uorodeoxyglucose (FDG) PET is
a factor of 10
6
– 10
9
more sensitive than
19
F MR methods, but has no ability to differentiate
metabolites from the parent compound [17] . Thus, when it is essential to differentiate
metabolic products that are not present in blood or urine, or to measure biodistribution
after administration of multiple doses, only biopsy or MR techniques are of use.
Since 1987 when Wolf and colleagues [6] published the fi rst use of
19
F MR to monitor
the human in vivo metabolism of a drug, a range of fl uorine - containing pharmaceuticals
and metabolites have been evaluated in patients. The most frequently examined organs
have been the brain and the liver, although heart, liver and extremity muscle, as well as
bone marrow, have also been evaluated. Fluorine MR spectroscopy is able to measure both
the relative and absolute concentration of administered fl uorinated compounds due in part
to the lack of naturally occurring MR - visible fl uorine metabolites. However, the accuracy
of
19
F MR assessments is critically dependent on the MR visibility of the compound, which
in turn is determined by the spin – lattice (T
1
) and spin – spin (T
2
) relaxation properties as
well as in vivo processes and interactions [19, 20] .
A number of excellent review articles exist, including several that explain MR spec-
troscopy and detail the impact of MR spectroscopy in drug design and in preclinical
studies, and others that provide overviews of human in vivo metabolism and pharmacoki-
netics measured by
19
F MR in fl uoropyrimidine compounds as well as in psychotropic
compounds [1, 3 – 5, 7 – 10, 59, 60 – 63] . In keeping with other facets of drug development,
the number of
19
F MR clinical investigations is much smaller than the number of animal
model, specimen, cell line, or solution experiments.
In this chapter, assessment of fl uorine - containing drugs using human in vivo fl uorine
(
19
F) MR spectroscopy will be reviewed along with the technical challenges.
19.2 MR Properties of
19
F In Vivo
The fl uorine MR signal in vivo will depend upon both the fl uorine concentration and
the compound ’ s MR visibility [1, 3, 5, 7 – 9, 19, 20, 71] . Like protons,
19
F nuclei have spin
½ with 100% natural abundance; however,
19
F has sensitivity of 83% relative to protons
(see Table 19.1 ) [71] . Unlike protons, which are ubiquitous, there are no natural occur-
rences of MR - visible fl uorine at suffi ciently high concentration to be observed by MR in
biological systems and hence there is no background fl uorine signal. Physiological fl uorine
Study of Metabolism of Fluorine-containing Drugs 497
is immobilized in bones and teeth, and is thus invisible to clinical or research MR imaging
systems. The technology for obtaining
19
F MR spectra is identical to that for obtaining
proton spectra, but the MR system must be equipped with fl uorine Larmor frequency -
specifi c excitation and detection packages.
The frequency separation between resonances of different chemical species (chemical
shift) varies linearly with the magnetic fi eld strength [71] and is commonly indicated rela-
tive to a reference compound in parts per million (ppm). Since chemical shift is a normal-
ized quantity, it has no dependence upon magnetic fi eld strength. For example, water
protons resonate at 4.7 ppm relative to tetramethylsilane (TMS) and fat protons in CH
2
and CH
3
groups resonate at about 1.4 ppm at every magnetic fi eld strength. However, at
1.5 Tesla (T) the water and fat proton resonance frequencies are separated by about 220
Hertz (Hz), and at 3 T they are separated by ∼ 440 Hz.
The chemical shift range of fl uorine nuclei is over an order of magnitude larger than
that of protons (see Table 19.1 ) because the fl uorine nucleus is surrounded by nine elec-
trons. Due to this large range, the fl uorine chemical shift is much more sensitive to the
local environment than proton chemical shift. This means that fl uorine chemical shift
changes are often observed when fl uorinated small molecules bind to proteins or receptors
[14 – 16, 20] and it is important to investigate the compound under the experimental condi-
tions expected in vivo . In particular, the potentially large shift difference between the free
and bound species means that it is necessary to acquire
19
F MR spectra of the compound
in plasma or a cellular suspension to evaluate the potential impact of associative com-
plexes, protein binding, or lipophilic interactions [11, 14, 15, 23] .
Fluorine MR reference compounds are used for frequency and/or concentration stan-
dards and can also be used for monitoring chest motion due to breathing and thereby
reducing motion artifacts (respiratory compensation) [11] . A reference compound should
have only one spectral resonance line and it is usually selected on the basis of the chemical
shift (or chemical) similarity to the compound being evaluated (see Table 19.2 ). Since
fl uorine chemical shifts span a wide range, there is no one compound that will be suitable
for universal use as a reference compound. Many suitable reference compounds exist; the
reader is referred to the CRC Handbook of Basic Tables for Chemical Analysis [22] . Often
it is necessary to integrate the reference compound into the radiofrequency (RF) coil [5,
9, 11, 23 – 26, 28] ; in other cases, the reference can be the organ water content [13, 25,
68 – 70] or intrinsically contained in the
19
F MR spectrum. If the reference compound is
external to the patient, it is important to ensure that it is housed in an unbreakable vial
that will not react with either the solvent or the reference compound, and that will
not create a background signal. For respiration compensation the reference standard
Table 19.1 Nuclear spin properties of fl uorine compared with protons [21, 22, 71]
Nucleus Spin Gyromagnetic
ratio
(rad s
− 1
T
− 1
)
Larmor
frequency,
(MHz) at
3.0 T
Natural
abundance
(%)
Sensitivity
relative to
1
H
Chemical
shift
range
(ppm)
1
H ½ 26.7519 127.7 99.98 1.0 16
19
F ½ 25.181 120.1 100 0.83 400
498 Fluorine in Medicinal Chemistry and Chemical Biology
that performs best has a high concentration, whereas for quantitative analysis the best
concentration would have similar peak amplitude to the administered compound in vivo .
The reference compound, its concentration, solvent, and temperature need to be reported
in publications to enable the results to be reproduced.
19.2.1 Signal
For all MR imaging and spectroscopy applications, the signal - to - noise ratio (SNR) is
directly proportional to the Larmor frequency and hence to the magnetic fi eld strength
[71] . This linear dependence motivates the use of high magnetic fi elds, because the greater
SNR improves sensitivity and measurement precision. MR SNR also has a square - root
gain achieved by increasing the number of acquisitions (or number of excitations, NEX).
To increase the SNR by a factor of 2, the NEX and hence the acquisition time must be
quadrupled.
SNR is particularly important in
19
F MR spectroscopy, because the maximum in vivo
concentration of fl uorinated drugs administered to humans is often only a few micromolar
[1, 11, 27 – 33] . In addition, patients can only tolerate about 60 – 90 min MR examinations,
inclusive of patient and coil positioning, proton imaging localization, and magnet shim-
ming as well as the fl uorine spectroscopic acquisition. Today, whole - body MR systems
for human examinations range from 0.2 T to 9.4 T, with most spectroscopy examinations
performed between 1.5 T and 4 T. Based on trends over the past two decades, the magnetic
fi eld strength will continue to increase in the future. This trend is part of the continuous
drive to obtain higher spatial resolution motivated in part by pharmaceutical development
[16] , but also to detect lower in vivo concentrations with MR spectroscopy [1, 5, 11,
38] .
Development of high - fi eld MR is limited by safety concerns including exposure to
magnetic fi elds [34] , exposure to time - varying gradient fi elds [58] , and RF power deposi-
tion [35, 36] . Power deposition, also called specifi c absorption rate (SAR), is related to
the potential increase in tissue temperature due to the RF exposure during acquisition. At
low magnetic fi eld strengths, power deposition increases with the square of the magnetic
fi eld [35, 36, 37] while at higher magnetic fi eld strengths the dependence on magnetic
fi eld strength is lower [36] .
There are other costs associated with utilizing high magnetic fi elds. In general, the
price of the MR system increases linearly with the magnetic fi eld strength (e.g. , a 7 T
Table 19.2 Examples of potential
19
F reference compounds (chemical shift measured
relative to trifl uoroacetic acid)
Compound Chemical shift (ppm)
Trifl uoroacetic acid (CF
3
CO
2
H) 0.0
Fluorobenzene (C
6
H
5
F)
− 34.0
2,5 - Difl uoro benzophenone (C
13
H
8
F
2
O)
− 41.0
Potassium fl uoride (KF)
− 44.0
Tricholorfl uoromethane (CFCl
3
)
− 78.5
Hexafl uorobenzene (C
6
F
6
)
− 87.3
Study of Metabolism of Fluorine-containing Drugs 499
whole - body MR system currently costs about twice that of a comparably confi gured 3 T
MR system). In addition, the T
1
relaxation time is lengthened, the sensitivity to magnetic
susceptibility is increased (e.g., the T
2
* can be shorter), and the impact of chemical shift
artifacts with spatial encoding is also increased.
The SNR and sensitivity gain at high magnetic fi elds (3 T and 4 T) has recently been
shown to outweigh the disadvantages [11, 38, 39] . However, optimization of the
19
F detec-
tion hardware and spectroscopic acquisition is important at any magnetic fi eld strength,
since SNR improvement by up to a factor of 2 can result [24, 71] .
19.2.2 Relaxation
Fluorine spins exchange energy through interactions with the surrounding proton and fl uo-
rine spins. Interactions through chemical bonds (spin – spin coupling) and over short dis-
tances through space (dipole – dipole coupling) are powerful relaxation mechanisms [1,
13 – 16, 71] that are tissue specifi c. The T
2
relaxation time depends upon the rate of energy
exchange between neighboring nuclei (spin – spin interaction). The T
1
relaxation time
depends upon the time required for the spins to align with the magnetic fi eld, or equiva-
lently, the time required for the spins to transfer their energy to the surrounding nuclei. A
critical aspect of both the spin – spin or transverse (T
2
) and spin – lattice or longitudinal (T
1
)
relaxation processes is the local mobility of the structure containing the fl uorine nucleus.
The T
2
relaxation times of small, rapidly tumbling molecules is generally long compared
with T
2
relaxation times for large, slowly reorienting molecules such as proteins. As a
result, the resonance lines in the MR spectra of small molecules, or of gas - phase com-
pounds, are much narrower than those from macromolecules or from small molecules that
are bound to a receptor or proteins [13 – 16, 20, 21, 23, 40, 71] . Conversely, the T
1
relaxation
times of small, rapidly tumbling molecules should be proportional to the T
2
value and
moderately long, whereas the T
1
relaxation times for large, slowly reorienting molecules
such as proteins tend to be longer.
A second important mechanism for fl uorine spin – lattice and spin – spin relaxation is
produced by the chemical shielding anisotropy (CSA) [13, 14, 21, 71] . The magnetic fi eld
experienced by a nuclear spin depends on both the electronic structure of the molecule
and how easily the electrons can move in the molecular orientations. In addition, the CSA
depends on how the molecule is oriented in the magnetic fi eld. Like spin – spin and
dipole – dipole interactions, the CSA of small, rapidly tumbling molecules will be an aver-
aged value (the chemical shift). However, these tumbling motions cause fl uctuations of
the local magnetic fi eld that lead to relaxation. Also slower reorientation, or an environ-
ment that restricts the molecular motion, will result in broader lines due to CSA.
Broad resonance lines, accompanied by T
1
relaxation times much longer than the T
2
relaxation times, are indications that the fl uorine nucleus is in a restricted - mobility envi-
ronment [14, 20, 21, 71] . The specifi c MR characteristics of a compound in its in vivo
state must be evaluated and the acquisition and analysis adjusted to account for the values
of these characteristics prior to performing human
19
F spectroscopy studies. Effective T
2
values (T
2
* ) that are shorter than 2 ms have spectra that are diffi cult to measure using
whole - body MR systems. In general, acquisition of spectra that contain short T
2
* - value
species require the use of short ( < 0.5 ms) excitation RF pulses and careful attention
500 Fluorine in Medicinal Chemistry and Chemical Biology
to minimizing the lost time between the end of the excitation pulse and the fi rst useful
detection time point. Even when signal decay is sampled appropriately, short T
2
* values
can limit the ability to perform quantifi cation due to broader lines and reduced signal - to -
noise levels.
The differences in local fl uorine environment are refl ected in the T
1
[20] and T
2
*
values reported in Table 19.3 . In theory, the trifl uoromethyl groups found in dexfenfl ura-
mine, fl uoxetine, fl uvoxamine, and nifl umic acid undergo less restrictive internal rotation
and should also be less sensitive to their local environment than monofl uorinated aromatic
rings such as in tecastemizole or 5 - FU. However, these compounds all have quite short
in vivo T
2
* values except for nifl umic acid [12] . Because these T
2
* values are signifi cantly
shorter than those found in solution, it is fair to anticipate that all the compounds, except
potentially nifl umic acid, interact with their environment. However, as demonstrated with
fl uoxetine and FBAL (Table 19.3 ), reported T
1
values may vary considerably. This illus-
trates the diffi culty of making a robust in vivo T
1
measurement under conditions of low
SNR and broad resonance lines [8, 11, 12, 20, 24, 28, 31, 32, 35, 41 – 43, 50, 56, 68] . In
addition, the variable bioavailability, due to the small number of patients generally evalu-
ated, compounds the measurement diffi culty.
Spin – spin coupling, dipole – dipole coupling, and CSA also have the potential to split
and broaden the
19
F spectral lines, both which reduce the fl uorine signal amplitude and
sensitivity. These mechanisms can also probe the chemical structure of complex molecules
such as proteins [13, 14, 15, 72] . If proton excitation and polarization transfer and/or
decoupling sequences can be implemented [1, 5, 13 – 16, 56] , increases in the
19
F SNR can
be realized by removal of the two couplings. Spin – spin polarization transfer techniques
such as those used for nuclear Overhauser effect (NOE) spectroscopy should be used
judiciously and the acquisition parameters should be tuned in vivo , because either signal
decreases or increase can result [2, 5, 13, 16, 56] . These dual - frequency (fl uorine – proton)
methods can increase SNR and may provide additional information about structure and
physiology as they have in liquid and solid - state samples in the laboratory setting [13 – 15,
71, 72] , but are RF energy intensive [35, 36] and SAR constraints may be encountered
even at 1.5 T [24] .
19.2.3 Spatial Localization
Proton MR spectroscopy has limited spatial resolution compared to morphological imaging
due to low in vivo metabolite concentrations. In the clinic, morphological MR imaging
has moderate spatial resolution, with typical two - dimensional (2D) acquisitions having
3 – 5 mm slice thickness with in - plane resolution on the order of 0.3 – 0.5 mm × 0.3 – 0.5 mm.
In clinical research, MR imaging is often a higher spatial resolution technique, with three -
dimensional (3D) acquisitions having 0.5 – 1.5 mm slice thickness with in - plane resolution
on the order of 0.25 – 0.5 mm × 0.25 – 0.5 mm. This relatively high spatial resolution can be
achieved because the tissue proton concentration is approximately 55 M [13] . However,
since proton metabolite concentrations are 1 – 10 mM, voxel volumes of 1 – 10 cm
3
are
required for proton MR spectroscopy at 1.5 T [1] . Even at these high concentrations, the
MR spectroscopic spatial resolution is poor compared with PET which can detect much
lower concentrations ∼ 10
− 12
M of the radiolabeled compound [17] . In general, PET has
Study of Metabolism of Fluorine-containing Drugs 501
Table 19.3 Examples of human in vivo
19
F relaxation times. While T
2
relaxation times are relatively consistent, in vivo T
1
values (e.g., for
fl uoxetine and FBAL ) vary considerably, indicating the diffi culty of implementing a robust T
1
measurement method with low signal levels
and broad spectral linewidths
Compound Organ Field strength (T) Group T
1
(ms) T
2
* (ms) Reference
Dexfenfl uramine (Redux) Brain 1.5 CF
3
–
2.89 ± 0.55
31
Dexfenfl uramine (Redux) Brain 1.5 CF
3
– 2.7 – 3.5 32
Fluoxetine (Prozac ® ) Brain 1.5 CF
3
190 – 41
Fluoxetine (Prozac ® ) Brain 1.5 CF
3
650 – 28
Fluoxetine (Prozac ® ) Brain 1.5 CF
3
– 2.6
(2.2 – 3.3)
42
Fluvoxamine (Luvox ® , Faverin ® ,
Fevarin ® , Dumyrox ® )
Brain 1.5 CF
3
140 – 230 2.7 – 3.5 30
Nifl umic acid Liver 1.59 CF
3
– 33 12
Tecastemizole (Soltara
™
) Liver/heart 4.0 4 - Fluorophenyl 300 3.1 11
Haloperidol decanoate Muscle 1.5 3 - Fluorophenyl 365 – 25
Fluorouracil (5 - FU) Liver 1.5 5 - Fluoropyrimidine
1600 ± 200
5 – 11 50, 56
α - Fluoro - β - alanine (FBAL)
Liver 1.5 CHF
1730 ± 130
4 – 7 50, 56
FBAL Liver 1.5 CHF
380 ± 80
– 24
FBAL Liver 3.0 CHF
400 ± 140
– 68
Capecitabine Liver 3.0 5 - Fluoropyrimidine
250 ± 70
– 68