Life Sciences 78 (2006) 3007 – 3012
www.elsevier.com/locate/lifescie
Comparison of rat dopamine D2 receptor occupancy for a series
of antipsychotic drugs measured using radiolabeled
or nonlabeled raclopride tracer
Vanessa N. Barth, Eyassu Chernet, Laura J. Martin, Anne B. Need, Karen S. Rash,
Michelle Morin, Lee A. Phebus ⁎
Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN, 46285, USA
Received 22 September 2005; accepted 27 November 2005
Abstract
Preclinical brain receptor occupancy measures have heretofore been conducted by quantifying the brain distribution of a radiolabeled tracer
ligand using either scintillation spectroscopy or tomographic imaging. For smaller animals like rodents, the majority of studies employ tissue
dissection and scintillation spectroscopy. These measurements can also be accomplished using liquid chromatography coupled to mass spectral
detection to measure the brain distribution of tracer molecules, obviating the need for radioligands. In order to validate mass spectroscopy-based
receptor occupancy methods, we examined dopamine D2 receptor dose–occupancy curves for a number of antipsychotic drugs in parallel
experiments using either mass spectroscopy or radioligand-based approaches. Oral dose–occupancy curves were generated for 8 antipsychotic
compounds in parallel experiments using either radiolabeled or unlabeled raclopride tracer. When curves generated by these two methods were
compared and ED50 values determined, remarkably similar data were obtained. Occupancy ED50 values were (mg/kg): chlorpromazine, 5.1 and
2.7; clozapine, 41 and 40; haloperidol, 0.2 and 0.3; olanzapine, 2.1 and 2.2; risperidone, 0.1 and 0.4; spiperone, 0.5 and 0.4; thioridazine 9.2 and
9.5; and ziprasidone 1.4 and 2.1 (unlabeled and radiolabeled raclopride tracer, respectively). The observation that in vivo application of both
techniques led to comparable data adds to the validation state of the mass spectroscopy-based approach to receptor occupancy assays.
© 2006 Elsevier Inc. All rights reserved.
Keywords: Rat; Dopamine; D2; Receptor occupancy; In vivo binding; Striatum; Liquid chromatography; Mass spectroscopy; Chlorpromazine; Clozapine;
Haloperidol; Olanzapine; Risperidone; Spiperone; Thioridazine; Ziprasidone; Raclopride
Introduction
Rat brain dopamine D2 receptors have often been the subject
of in vivo receptor binding studies examining the relationship
between percent occupancy and activity in preclinical models
associated with the therapeutic and side effect profile of antipsychotic drugs. These receptors are of particular interest since
their blockade is closely associated with clinical efficacy in
treating psychosis, where a consensus has developed regarding
therapeutic occupancy levels (Kapur, 2000) (Tauscher and
Kapur, 2001), which are most often measured by positron
emission tomography (PET) or single photon emission
computed tomography (SPECT). Both these imaging techni⁎ Corresponding author. Tel.: +1 317 276 0646; fax: +1 317 276 5546.
E-mail address: [email protected] (L.A. Phebus).
0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.lfs.2005.11.031
ques measure the concentration of a radiolabeled ligand, called
a tracer, in various brain areas over time. This ligand is injected
intravenously in very low doses to assess the receptor occupancy of another drug that has been given as a pretreatment. By
quantifying the tracer levels in various brain structures and
following the kinetics of its distribution, occupancy can be
calculated. While PET and SPECT can be applied to the smaller
animals typically used in preclinical pharmaceutical research,
the size of the brain, the need for anesthesia and the inherent
resolution of the imaging technologies put limitations on the
types of data that can be collected. Nonetheless, the advantage
to these imaging techniques include the ability to follow tracer
kinetics over time in the same animal and to generate data for
multiple doses within the same animal thus reducing measurement variability and decreasing animal use. More usually,
preclinical brain receptor occupancy measures in small animals
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V.N. Barth et al. / Life Sciences 78 (2006) 3007–3012
have been conducted by quantifying the brain distribution of a
radiolabeled tracer ligand at a single time point. In this case, the
animals are sacrificed, brain areas dissected and the radioligand
contained in those areas quantified using scintillation spectroscopy. In all of the above examples, the receptor occupancy
tracer is radiolabeled, which has been thought to be necessary in
order to measure the very small levels of tracer in brain tissue.
In the past decade, the sensitivity of mass spectral detection
of ionizable molecules has increased greatly. The coupling of
modern single (LC/MS) or triple (LC/MS/MS) quad mass
spectral detectors with liquid chromatography has produced
analytical instruments capable of great sensitivity, selectivity
and quantitative accuracy. We have previously reported on the
use of LC/MS and LC/MS/MS to quantify brain tracer distribution in rat receptor occupancy experiments targeting the
dopamine D2, the serotonin 2A and the neurokinin NK1 receptors (Chernet et al., 2005). In these examples, the tracer
ligand was not radiolabeled, but injected in its ordinary “cold”
form. The use of mass spectroscopy to conduct in vivo receptor
occupancy experiments has a number of advantages, the most
obvious being avoidance of the environmental, regulatory and
purchase costs of working with radiopharmaceuticals. It also
confers other advantages including the ability to run these
experiments faster (1 versus 3 days), measure the test compound
in addition to the tracer, use multiple tracers in the same animal
and perhaps most importantly, the ability to search for new
tracers at rates orders of magnitude faster than is practical using
radioligands. Finding new tracers is the rate-limiting step in
generating new receptor occupancy assays for novel neuroscience targets. Since few ligands have the properties of a good
tracer, and the chemical features that confer this property are
poorly understood, one typically needs to examine many tracer
candidates in order to find one that works satisfactorily. This is
more easily accomplished using unlabeled tracers and LC/MS
or LC/MS/MS. In order to further validate the application of
mass spectral detection to in vivo receptor occupancy, we have
compared dose–occupancy curves for the rat brain dopamine
D2 receptor for a series of antipsychotic compounds using both
the radioligand and the LC/MS/MS-based methods.
Materials and methods
Test compounds
Haloperidol, clozapine, risperidone, spiperone, chlorpromazine hydrochloride, thioridazine hydrochloride and S-(−)raclopride tartrate were purchased from Sigma-Aldrich (St
Louis, MO, USA). Ziprasidone was isolated from commercially available material by chemists at Eli Lilly and
Company. Olanzapine was synthesized at Eli Lilly and
Company. Raclopride was used as a tracer in both the
radiolabel and mass spectroscopy-based experiments. Tritiated
raclopride with a specific activity of 75 Ci/mmol was
purchased from Perkin-Elmer (Boston, MA, USA) and diluted
with saline to 7.5 μCi in 0.2 mL, the volume injected. This
resulted in an intravenous dose of approximately 0.4 nmol/kg
(0.14 μg/kg). “Cold” S-(−)-raclopride tartrate was used as an
occupancy tracer in the LC/MS/MS-based experiments and
was administered at an intravenous dose of 6, 20 or 60 nmol/
kg (3, 10 or 30 μg/kg) dissolved in sterile saline.
Antipsychotic compounds for which dopamine D2 receptor
occupancy was assessed were administered orally in a 25%
(2-Hydroxypropyl)-β-cyclodextrin (Sigma-Aldrich) vehicle 1
h before tracer administration.
Animals
Adult male Sprague–Dawley rats (HSD, Indianapolis, IN,
USA) weighing 240–260 g were housed 6 to a cage in a
room using a 12-h on / off lighting schedule (lights on at 6
AM). Room temperature was maintained at 21 ± 3 °C.
Animals had ad libitum access to food and water and were
permitted at least 2 days after arrival at our site to adapt to
housing conditions before testing.
Effects of tracer dose and survival interval on striatal and
cerebellar tissue levels of raclopride were examined in groups
of 3–4 rats that were briefly restrained and injected with
unlabeled raclopride in the lateral tail vein. To examine D2
dose–occupancy relationships for antipsychotic drugs, groups
of 3–4 rats were fasted overnight and pretreated orally with
test compound or its vehicle. One hour later, they were briefly
restrained and administered a low intravenous dose of either
radiolabeled or unlabeled raclopride tracer via the lateral tail
vein. Rats treated with the radiolabeled ligand were sacrificed
by cervical dislocation, whereas those treated with unlabeled
tracer were sacrificed by carbon dioxide asphyxiation. Both
sets of animals were sacrificed 15 min following intravenous
raclopride tracer. After sacrifice, brain striatum and cerebellum tissue samples were dissected and weighed.
Analysis of radiolabeled raclopride levels
Previously weighed brain tissue samples were placed in 20
mL scintillation vials containing 2 mL of Soluene-350, a tissue
solubilizing agent (Perkin-Elmer). Vials were allowed to agitate
slowly on an orbital platform for 24 h to ensure complete
tissue dissolution. Five milliliters of Ready Protein scintillation
cocktail (Beckman Coulter, Fullerton, CA, USA) was then
added and again tissue samples were allowed to slowly agitate
on an orbital platform for 24 h. Samples were then counted
using scintillation spectroscopy and results were expressed as
disintegration per minute per milligram of tissue (wet weight).
Analysis of unlabeled raclopride levels
Previously weighed brain tissue samples were placed in
conical 1.5 mL polypropylene centrifuge tubes to which 4
volumes (w/v) of acetonitrile containing 0.1% formic acid was
added. Samples were then homogenized using an ultrasonic
dismembrator probe (Fisher Scientific model 100, Pittsburgh,
PA, USA), vortexed and centrifuged for 16 min at 16,000 ×g
(Eppendorf model 5417R, Westbury, NY, USA). 100 μL of
supernatant was then added to 900 μL of water in 1.5 mL
autosampler vials and vortexed.
V.N. Barth et al. / Life Sciences 78 (2006) 3007–3012
Raclopride concentration measurements were made using a
liquid chromatograph (HPLC, Agilent Technologies model
1100, Wilmington, DE, USA) with triple quadrapole mass
spectral detection. The HPLC system employed a C18 column
(SB Zorbax, 4.6 × 75 mm, 3 μm particle size, Agilent) with an
aqueous mobile phase consisting of 50% acetonitrile with 0.1%
formic acid. Raclopride was quantified after elution from the
HPLC column using an API 3000 triple quad mass spectrometer
(Applied Biosystems, Foster City, CA, USA) in positive electrospray mode using MRM methods to monitor the transition
from parent to daughter ions with mass to charge ratios of 347.1
and 112.2, respectively. Chromatographic assays were calibrated using a standard curve generated by extracting a series of
brain tissue samples from non-treated animals to which known
quantities of raclopride had been added.
The efficiency of raclopride extraction was examined using
striatal and cerebellar tissue samples taken from non-treated rats.
Raclopride was added to each sample to generate a final concentration of 2.1 or 7 ng/g. Four tissue samples were used at each
concentration. In parallel, 4 water-based standards were generated at each concentration. All samples were extracted as
described above and the raclopride levels in the extracts were
assayed by LC/MS/MS. Since the water-based standards were
fully miscible with the acetonitrile used for the extraction, the
raclopride levels in these samples represented 100% extraction
efficiency. The raclopride levels seen in the tissue-based samples
were compared to these values to calculate the percent extraction
for striatal and cerebellar tissues at the two concentrations.
3009
Table 1
Effect of raclopride dose on Levels ± SEM of unlabeled raclopride in the rat
striatum and cerebellum 15 min after various intravenous doses and the striatal /
cerebellar concentration ratio
6 nmol/kg (N = 4) 20 nmol/kg (N = 3) 60 nmol/kg (N = 3)
Striatum, ng/g
4.9 ± 0.19
Cerebellum, ng/g 1.0 ± 0.06
Ratio
4.9
14.6 ± 0.48
3.6 ± 0.23
4.1
20.5 ± 1.04
10.2 ± 1.06
2.0
Sigmoidal dose–occupancy curves with variable slopes were
calculated using a 4-parameter (top, bottom, slope and ED50)
logistic fit. Relative ED50 values, the occupancy seen at a point on
the curve half way between the calculated curve top and bottom,
are reported. Rather than constrain the top and bottom of the curves
to 100% and 0% respectively, arguably a rational approach for this
set of experiments, a better fit was obtained by letting the software
determine all 4 parameters wherever possible. In the case of
clozapine, thioridazine and the radiolabeled curve for chlorpromazine, the top of the curve was constrained to 100%. This was
necessary for these less potent compounds since the data points did
not adequately define the top of the curve. For the unlabeled tracerderived risperidone curve, the bottom of the curve was constrained
to 0% since, in this case, the bottom of the curve was not completely defined. In all other cases, the 4 parameters of the fit were
determined by the software. Differences in raclopride extraction
efficiency were compared using analysis of variance (JMP
software, version 5.1, SAS Institute, Cary NC, USA).
Results
Striatal protein content determination
In order to estimate the percent of the total D2 receptors
occupied by the tracer dose used in the mass-spectroscopy-based
studies, we measured the protein content of 5 striatal tissue
samples using the Coomassie Plus Protein Assay Reagent (Pierce
Chemical Co., Rockford, IL, USA). This allowed us to determine
the percent of striatal wet weight composed of protein.
Receptor occupancy calculation
Receptor occupancy calculations were made for each animal
employing the widely used ratio method (Farde et al., 1988; Kapur
et al., 1999; Wadenberg et al., 2000) and the following equation:
100*f1−½ðRatiot −1Þ=ðRatioc −1Þg ¼ % Occupancy
The ‘Ratiot’ represents the ratio of raclopride concentrations
measured in the striatum to those measure in the cerebellum in
individual animals pretreated with antipsychotic compounds or
vehicle. The ‘Ratioc’ represents the average ratio of raclopride
levels measured in the striatum to that measured in the
cerebellum for the vehicle-pretreated group.
Statistical analyses
Prism (GraphPad Software Inc., version 4.0, San Diego, CA)
software was employed for calculations, curve fitting and graphics.
Raclopride was nearly quantitatively extracted from striatal
and cerebellar tissue samples with no significant differences
between either tested concentrations or between brain areas
(P = 0.22, f = 1.696). The percent extraction (mean ± SEM, N = 4)
was 89 ± 1.9 and 97 ± 3.3 at the 2.1 ng/g concentration and 107 ±
11.7 and 108 ± 5.8 at the 7 ng/g concentration for the cerebellum
and striatum, respectively. If both concentrations are combined,
the average percent recovery was 98 ± 6.5 and 103 ± 3.6 for the
cerebellum and striatum, respectively.
The 5 striatal samples assayed for protein content showed
that an average of 9.23% of the tissue wet weight was protein.
The standard error for this value was 0.4%.
Unlabeled raclopride tartrate was administered intravenously at doses of 6, 20 and 60 nmol/kg and 15 min later,
raclopride levels were assessed in the striatum and cerebellum
(Table 1). As the dose increased, so did the raclopride levels in
both brain structures. The striatal to cerebellar concentration
Table 2
Levels ± SEM of unlabeled raclopride in the rat striatum and cerebellum at
various time points after an intravenous dose of 6 nmol/kg and the striatal /
cerebellar concentration ratio
Striatum, ng/g
Cerebellum, ng/g
Ratio
N = 4.
5 min
15 min
30 min
60 min
6.1 ± 0.61
2.6 ± 0.24
2.3
4.9 ± 0.35
1.1 ± 0.09
4.5
3.4 ± 0.23
0.44 ± 0.05
7.7
1.3 ± 0.15
0.17 ± 0.01
7.6
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V.N. Barth et al. / Life Sciences 78 (2006) 3007–3012
100
100
80
80
60
60
Spiperone
Haloperidol
40
40
20
20
Chlorpromazine
0
0.001
0.01
0.1
1
10
100
Thioridazine
0
0.001
100
100
80
80
60
0.01
0.1
1
100
60
Risperidone
Ziprasidone
40
40
20
20
Olanzapine
Clozapine
0
0
0.001
10
0.01
0.1
1
10
100
0.01
0.1
1
10
100
Fig. 1. Dopamine D2 receptor dose–occupancy curves for orally administered antipsychotic drugs were measured using raclopride as a tracer. Curves were generated
for each of eight drugs using either radiolabeled (solid symbols) or unlabeled (open symbols) raclopride tracer. The X axis represents the oral dose in milligrams per
kilogram administered 1 h before raclopride tracer. The Y axis represents the percent dopamine D2 receptor occupancy. For clarity, two drugs are shown per panel.
ratio, decreased with increasing dose. When a 6 nmol/kg
intravenous dose of raclopride tartrate was given at various
intervals before sacrifice, raclopride tracer levels decreased in
both brain structures over time (Table 2). The concentration
ratio increased with time reaching a maximum at 30 min and
maintaining this approximate level at 60 min after raclopride
administration.
Striatal dopamine D2 dose–occupancy curves were generated for 8 antipsychotic drugs administered orally 1 h before
raclopride tracer. Two independent curves were generated for
each antipsychotic compound; one using radiolabeled raclopride as the tracer and scintillation spectroscopy, the other using
unlabeled raclopride and LC/MS/MS to analyze tracer levels.
For these experiments, the post-tracer survival interval was
15 min. Radiolabeled raclopride tracer was administered at
doses of 7.5 μCi/rat (0.4 nmol/kg). Unlabeled raclopride tartrate
was injected at a dose of 6 nmol/kg (approximately 3 μg/kg). The
resultant curves are shown in Fig. 1. Calculated relative ED50
values for each curve are listed in Table 3.
Table 3
Relative ED50 values (mg/kg) ± standard error derived from dose–occupancy
curves for a series of antipsychotic drugs measured using an LC/MS/MS or
radioligand-based method
Chlorpromazine
Clozapine
Haloperidol
Olanzapine
Risperidone
Spiperone
Thioridazine
Ziprasidone
LC/MS/MS method
Radioligand method
5.1 ± 1.4
41 ± 1.0
0.2 ± 1.6
2.1 ± 1.3
0.1 ± 1.2
0.5 ± 1.2
9.2 ± 1.3
1.4 ± 1.6
2.7 ± 1.9
40 ± 1.1
0.3 ± 1.1
2.2 ± 1.1
0.4 ± 1.1
0.4 ± 1.1
9.5 ± 3.2
2.1 ± 1.1
Linear regression analysis demonstrated P b 0.0001, r 2 of 0.995 and a slope
of 0.97.
Discussion
We have previously reported on the use of liquid chromatography coupled to mass spectral detection to analyze tracer
distribution in brain receptor occupancy experiments targeting
dopamine D2, serotonin 2A and neurokinin 1 receptors (Chernet
et al., 2005). In order to further validate the use of LC/MS/MS in
preclinical receptor occupancy experiments, a rigorous comparison of results obtained using the well characterized and
accepted dopamine D2 receptor occupancy assay, based on
radiolabeled raclopride as a tracer, and an LC/MS/MS-based
unlabeled raclopride assay was conducted.
For this comparison, we decreased the dose of nonlabeled
raclopride tracer by a third and increased the post tracer survival
time from 10 to 15 min, as compared to our previous publication.
The dose of radiolabeled raclopride was one frequently used in
the literature, but the 15 min survival interval was shorter than
the more typical 30 min. The choice of this survival interval was
based on the intention to minimize the tracer dose of unlabeled
raclopride in the mass spectroscopy-based experiments. We
chose the raclopride dose to be as low as we felt could be reliably
measured using our chromatographic equipment and protocol.
Table 1 shows that, at least at the 15-min time point, the lowest
dose tested resulted in the highest ratio between total and
nonspecific binding. Using this dose but looking at longer posttracer survival intervals (Table 2), the ratio increased to a
maximum at 30 min and remained essentially unchanged at the
60-min survival period. Concern about the relatively low
raclopride levels in the cerebellum at time points longer than
15 min led to the choice of the 15 min survival period. It would
have been possible to increase the raclopride dose and thereby
increase raclopride levels at later time points, perhaps obtaining
a higher concentration ratio, but concern to keep the unlabeled
raclopride dose as low as possible (see below) discouraged this
V.N. Barth et al. / Life Sciences 78 (2006) 3007–3012
option. This compromise resulted in our using a shorter posttracer survival interval than that used by others. It is possible that
the occupancy values we measured were influenced by the
uptake phase of the raclopride. This uptake can also be
influenced by pretreatment with the test compound and the
level of occupancy it generates. Despite these complications and
the possibility of inaccuracy in our measurements due to these
considerations, the limited number of direct comparisons
available between our data and those obtained by researchers
using longer post-tracer survival periods demonstrate ED50
values that are quite similar (Chernet et al., 2005). Tracers with
slower kinetics than raclopride could be even more problematic,
but ongoing increases in the sensitivity of mass spectral
detection may soon obviate these concerns.
Most, if not all, widely used antipsychotic drugs bind to and
block dopamine D2 receptors, a property that is thought to be
important to their efficacy toward the positive symptoms of
schizophrenia. Even though this receptor has been long known
and studied, it is still the subject of intense research because of
its therapeutic potential and the differing medicinal profiles of
drugs that block it. Receptor occupancy measurements can be
used to better understand the preclinical effects of these agents
in the context of a therapeutically relevant dose (Kapur et al.,
2003). Sigmoidal dopamine D2 dose–occupancy curves were
fit to the individual occupancy data points and relative ED50
values were calculated. The dose–occupancy curves obtained
for the 8 antipsychotic drugs tested using the traditional
radioligand and LC/MS/MS-based methods were highly similar
(Fig. 1). The ED50 values calculated for individual compounds,
measured by either the radiolabeled or unlabeled tracer
methods, were also comparable (Table 3). Of the eight antipsychotic drugs tested, only risperidone administration generated ED50 values that differed by more than 2 fold. The reason
for the relatively larger difference in the risperidone curves
(4 fold) is unknown, but we occasionally see this level of variability in other experiments. In order to get a better understanding of the risperidone variability, additional curves
would need to be produced. This work took the approach of
generating one curve by each method for 8 different antipsychotic compounds rather than many examples of an individual
drug. This allowed us to survey compounds of this class with a
wide range of in vivo potencies and thereby further reinforce the
comparability of the two tracer measurement methods.
There are a number of advantages to using LC/MS/MS to
conduct preclinical occupancy experiments. One is that the use
of unlabeled ligands as tracers obviates the need for radioligands and their associated environmental, purchase and bureaucratic costs. Unlike scintillation spectroscopy, which does
not differentiate between radiolabeled tracer and its metabolites,
the use of mass spectral detection permits absolute identification
and quantification of the tracer. It also permits simultaneous
measurement of the pretreatment, blocking drug in the tissue
extract. This allows exposure–occupancy curves to be generated removing the influence of dose–linearity deviations. LC/
MS/MS-based occupancy experiments can be conducted in a
shorter time frame since there is no wait for the tissue to
dissolve and for the auto-fluorescence to subside. Most
3011
importantly, the ability to use unlabeled tracers in preclinical
occupancy experiments greatly accelerates the search for new
tracers for novel targets. The properties that allow a ligand to
serve as an in vivo binding tracer are the subject of great interest
since the discovery of suitable tracers is rate limiting in novel
assay development (Fowler et al., 2003). While generalizations
have been made regarding lipophilicity and other physicochemical properties, the structural features that confer suitable
tracer-like properties are poorly understood (Wong and Pomper,
2003) (Waterhouse, 2003). Using LC/MS/MS-based occupancy
experiments, it is possible to test unlabeled tracer candidates
allowing unprecedented screening rates. The structure–activity
relationships that can be built from the increased data produced
can help medicinal as well as computational chemists better
understand the unique properties of tracer molecules and further
speed the discovery of new in vivo tracers.
There are drawbacks that accompany the use of LC/MS/MS
to conduct preclinical receptor occupancy experiments. The
high cost of the LC/MS/MS instrumentation itself can be a
factor. Another concern is the necessarily higher dose of tracer
that is used when compared to traditional radioligand-based
methods. For example, the dose of radiolabeled raclopride
employed in these experiments was 0.4 nmol/kg whereas that
used in the LC/MS/MS-based experiments was 6 nmol/kg,
approximately 15 fold higher. Using the equation described by
Hume et al. relating fractional occupancy to dose and ED50, and
using their intravenous raclopride ED50 value of 17.1 nmol/kg
(Hume et al., 1998), the radiolabeled tracer would be expected
to occupy about 2.3%, and the 15-fold higher dose of unlabeled
tracer perhaps 26% of striatal D2 receptors. Our quantitative
LC/MS/MS data allow a direct estimate of the D2 occupancy of
the higher tracer dose. There are a number of Bmax estimates in
the literature for rat striatal dopamine D2 receptors. An average
of 7 reported values (Hennies et al., 1984; Boyson et al., 1986;
MacRae et al., 1987; Noisin and Thomas, 1988; Dewar et al.,
1989; Bardo and Hammer, 1991) resulted in a mean value of
515 (with an SEM of 57) fmol/mg protein. Our data, obtained
from rat striatum, suggest that dissected tissue contains 9.23%
protein by weight. Therefore, we estimate that the dissected rat
striatum contains D2 binding sites at a density of 47.5 fmol/mg
or 47.5 pmol/g. This Bmax is equivalent to 16.45 ng/g of
raclopride bound. Our data averaged from Tables 1 and 2 show
that 15 min after a 6 nmol/kg iv raclopride dose, 3.85 ng/g,
or 11 pmol/g, of raclopride is specifically bound. Assuming that
each receptor binds one raclopride molecule, this suggests that,
at the tracer dose and survival period used in our comparisons,
raclopride bound approximately 23% of the total striatal D2
sites. Using the data from Table 2, this level of occupancy is
quite stable with values of 21%, 23% and 21% at the 5, 15 and
30 min time points, respectively. By 60 min after tracer
injection, only 6.8% of striatal D2 receptors were still labeled,
as assessed using our methods. The mass spectroscopy-derived
values matches quite well with the value calculated based on the
reported raclopride ED50 and the doses we administered. Interestingly, the two higher doses of raclopride tracer described in
Table 1 generated a specific raclopride binding of approximately
11 ng/g, equal to 31 pmol/g, a value close to the 23.5 pmol/g
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for striatal wet weight Bmax reported by Kohler et al. (1985). If
this value is a more accurate estimate of the striatal Bmax of
raclopride, the estimated occupancies of our tracer doses would
be doubled. It is suggested in the literature that, in order to
avoid errors in occupancy estimates based on tracer kinetics,
one should label no more than 1% to 5% of the total receptors
(Hume et al., 1998; Laruelle et al., 2003). One of the major
conclusions drawn from this set of experiments is that, despite
this different tracer dose, the D2 occupancy estimates obtained
for the antipsychotic drugs tested using the two different tracer
analysis methods were remarkably similar. Frequent advances
in the sensitivity of mass spectral detection are now routine,
and with these advances, the need for higher tracer doses may
disappear, along with this concern.
Conclusion
Dopamine D2 receptor occupancy was assessed for a series
of 8 antipsychotic drugs in the rat striatum using the traditional
method employing a radiolabeled raclopride tracer and a
version of the assay using LC/MS/MS to measure unlabeled
raclopride tracer. While there were differences in the dose–
occupancy curves for some compounds, most notably risperidone, in general the two methods generated very comparable
data. The use of LC/MS/MS for preclinical receptor occupancy
experiments conveys a number of advantages, allowing the
search for novel tracers to be conducted without the need for
radiolabeled molecules.
Acknowledgment
This work was funded by Eli Lilly and Company.
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