0022-3565/99/2883-1327$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics
JPET 288:1327–1333, 1999
Vol. 288, No. 3
Printed in U.S.A.
Novel, Highly Lipophilic Antioxidants Readily Diffuse Across
the Blood-Brain Barrier and Access Intracellular Sites
GERI A. SAWADA, LAWRENCE R. WILLIAMS,1 BARRY S. LUTZKE, and THOMAS J. RAUB
Drug Absorption and Transport (G.A.S., T.J.R.), Central Nervous Systems Research (L.R.W.), and Discovery Technologies (B.S.L.), Pharmacia &
Upjohn, Inc., Kalamazoo, Michigan
Accepted for publication September 8, 1998
This paper is available online at http://www.jpet.org
Recently, novel 2,4-diamino-pyrrolo[2,3-d]pyrimidines
were identified as orally active antioxidants with protective
effects in both in vitro and in vivo models of oxidative injury
(Bundy et al., 1995). The first generations of antioxidants,
21-aminosteroids (Hall et al., 1994) and 2-methylaminochromans (Hall et al., 1991), have poor oral bioavailabilities and
low brain uptake (Raub et al., 1993). Poor blood-brain barrier
permeability is attributed to serum protein binding, slow
diffusion beyond the endothelial barrier, and rapid hepatic
clearance.
To aid the selection of lead compounds, transcellular permeability and cellular partitioning of pyrrolopyrimidine analogs were measured in a cell culture model (Sawada et al.,
1999). These data were expected to predict which compounds
were most likely to partition into target tissue. The assay
identified compounds that would most likely remain tissue or
cell associated after declining blood levels. The data show
that the prototype, PNU-87663, and one of its homologs,
PNU-89843, behave differently with regard to permeability
and partitioning (Sawada et al., 1999). These results predict
that PNU-89843 penetrates into brain more readily but that
retention would be more affected by declining blood levels.
The predicted uptake and penetration by PNU-87663 were
less obvious given their similarities to the 21-aminosteroid
Received for publication May 6, 1998.
1
Present address: Guilford Pharm., Inc., Baltimore, MD.
ties of PNU-87663, its distribution within brain and within cells
in culture was demonstrated using confocal scanning laser
microscopy. PNU-87663 rapidly partitioned into the cell membrane and equilibrates with cytoplasmic compartments via passive diffusion. Although partitioning of PNU-87663 favors intracytoplasmic lipid storage droplets, the compound was readily
exchangeable as shown by efflux of compound from cells to
buffer when protein was present. The results demonstrated that
pyrrolopyrimidines were well suited for quickly accessing target
cells within the central nervous system as well as in other target
tissues.
PNU-74006 and the 2-methylaminochroman PNU-78517F in
this assay (Raub et al., 1993), although the loss of brainassociated PNU-87663 was expected to lag significantly behind decreasing blood levels.
In this report, brain uptake of PNU-87663 and PNU-89843
was measured using two methods to test these predictions;
however, these techniques could not distinguish between accumulation within the endothelium of the blood-brain barrier
and permeation throughout brain parenchyma, or accumulation in specific brain regions other than the intended target.
Consequently, we exploited the unique fluorescent properties of PNU-87663 and used confocal scanning laser microscopy (CSLM) in conjunction with cryomethods to show that it
permeated beyond the blood-brain barrier, becoming localized in parenchymal cells. This approach also was used to
confirm that PNU-87663 passively diffuses through plasma
membrane of cultured cells and partitions into lipid-rich,
intracytoplasmic inclusions.
Materials and Methods
Reagents. PNU-87663 monomethanesulfonate salt (443.6 g/mol)
was synthesized by Medicinal Chemistry Research, Pharmacia &
Upjohn (Kalamazoo, MI). An egg lecithin-olive oil emulsion containing 4.1 mM PNU-87663 (free base) in the oil phase was kindly
provided by Cell Biology and Inflammation Research, Pharmacia &
Upjohn. PBS containing 1 mg/ml D-glucose (GIBCO BRL Life Sci-
ABBREVIATIONS: BUI, brain uptake index; CSLM, confocal scanning laser microscopy; SLM, scanning laser microscopy.
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ABSTRACT
In an accompanying article, an in vitro assay for permeability
predicts that membrane-protective, antioxidant 2,4-diaminopyrrolo[2,3-d]pyrimidines should have improved blood-brain
barrier (BBB) permeation over previously described lipophilic
antioxidants. Using a first-pass extraction method and brain/
plasma quantification, we show here that two of the pyrrolopyrimidines, one of which is markedly less permeable, readily
partition into rat brain. The efficiency of extraction was dependent on serum protein binding, and in situ efflux confirms the in
vitro data showing that PNU-87663 is retained in brain longer
than PNU-89843. By exploiting inherent fluorescence proper-
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Sawada et al.
Vol. 288
BUI 5 k
F
test/ref ~ brain!
test/ref ~ injectate!
G
3 100
The constant k is the fractional BUI value for the reference tracer,
such as 0.84 for H2O (Oldendorf, 1981).
The injectate was prepared by adding radiotracers (0.5–1.0 mCi/ml
14
C compound and 1.5– 4 mCi/ml [3H]2O at .3:1 [3H]:[14C]) to PBS
containing 3% (w/v) BSA or 0.1 to 10% rat serum. After mixing for 10
min at 37°C, the solutions were centrifuged at 150,000g for 10 min to
remove insoluble compound, and the amount of radioactivity remaining in solution was measured.
Mouse brain, heart, lung, and plasma levels were measured by
reversed-phase HPLC at 5 and 60 min after tail vein injection of 23
mmol/kg (;10 mg/kg) of unlabeled PNU-87663 and PNU-89843 as
described by Hall et al. (1997).
Brain Efflux. The BUI was done as above, but termination of the
experiment by decapitation was increased from #10 sec to #2 min.
Under these conditions, brain-to-blood back flux occurs (Oldendorf
and Braun, 1976). The data were calculated as a percent of the dose
administered and analyzed as a first order process using nonlinear,
least-squares regression analysis (Yamaoka et al., 1981). Given the
relatively short times in all of these experiments, it was assumed
that metabolism of compound was negligible.
Scanning Laser Fluorescence Microscopy. All samples were
viewed with an Adherent Cell Analysis System (ACAS) 570 confocal
scanning laser microscope (Meridian Instruments, Okemos, MI) fitted with an Innova 5W argon ion laser (Coherent, Palo Alto, CA)
tuned to 310 nm (actual excitation wavelength, 352–358 nm). The
collimated first order beam was attenuated 90 to 99% with neutral
density filters and passed through a standard ultraviolet filter cube
[VG-1 excitation filter, 380-nm long-pass (LP) dichroic mirror,
390-nm LP barrier filter]. The data were collected by a photomultiplier tube fitted with standard INDO filters (445-nm LP dichroic,
485 6 45-nm band-pass), quantified, and transformed by system
software to a pseudocolor image. Corresponding numerical data were
saved on an optical disk for processing. The scanning parameters
were carefully chosen to detect changes in fluorescent signal over a
wide range of intensity while minimizing background and photobleaching effects.
Confocal Scanning Laser Microscopy. Cells grown in Lab-Tek
coverglass chamber slides (Nunc, Inc., Naperville, IL) were washed
twice with PBS and incubated for 60 min at 37°C with emulsion
diluted to 10 mM PNU-87663 with buffer. To ensure that the emulsion itself did not contribute to the cell or background fluorescence,
cells were incubated with an equivalent dilution of blank emulsion
without PNU-87663. For comparison, cells were also incubated with
50 mM PNU-87663 prepared by diluting a 4 mg/ml dimethyl sulfoxide stock solution into PBS containing either 0.5% (w/v) or 3% (w/v)
BSA. Occasionally, N18 cells were cultured for 24 h in the presence
of 10% (v/v) cholesterol-rich lipids before their use. Immediately after
rinsing, individual cells or small clusters of cells were scanned using
a 1003 ultraviolet, oil immersion objective lens and a 225-mm pinhole, to yield a section thickness of ;0.9 mm. Fluorescence data were
collected every 0.5 mm along the x- and y-axes for two-dimensional
reconstruction. To confirm intracellular localization of PNU-87663,
data also were collected every 0.5 mm along the z-axis for subsequent
three-dimensional image reconstruction.
Two different experimental methods were used to examine the in
vivo distribution of PNU-87663. First, female mice (;25 g) were
injected via tail vein with 23 mmol/kg (;10 mg/kg) PNU-87663 in
propylene glycol and were sacrificed at 5 and 60 min (Hall et al.,
1997). Unperfused brain, lung, and heart were excised and prepared
for microscopy as described below. In a second experiment, male rats
were injected via the left common carotid artery with a 200-ml bolus
of either 50 mM PNU-87663 in buffered 3% BSA or undiluted PNU87663 emulsion. The rats were decapitated at 8 s, and the left
cerebral hemisphere was prepared for microscopy as described below.
Excised brains were bisected medially and fixed for 10 to 20 min at
25°C in 10% neutral buffered formalin. Tissues were embedded in
O.C.T. compound (Miles, Inc., Elkhart, IN), quickly frozen in isopentane cooled to 2160°C with liquid nitrogen and stored at 270°C.
Sections 6 to 8 mm thick were collected on poly-L-lysine-coated glass
microscope slides and were kept in an airtight container at 270°C.
Frozen sections were warmed quickly at 25°C and mounted with a
coverslip in 75% glycerol, and the slide was viewed as described
above. Using an oil immersion 1003 objective lens, an area 200 3
200 mm was scanned (scan strength was 15%) and optically sectioned
along the vertical axis (0.4-mm resolution) every 1.0 mm through the
tissue section. Only horizontal optical sections that were at least 1.5
mm into the tissue section were used to avoid surface artifacts.
Cell Uptake Experiments. N18 cells were rinsed and scanned in
PBS at either 4°C or 37°C to set software parameters to obtain a
faint cell image (below 255 fluorescence units). The buffer was replaced with 10 mM PNU-87663 emulsion, and the cells were scanned
at 120-s intervals over 30 min. Control cells were scanned in diluted
blank emulsion. Additionally, some of the cells scanned at 37°C were
pretreated for 30 min with sodium azide (1 mM) and 2-deoxyglucose
(5 mM) to inhibit active (energy-dependent) uptake and/or efflux.
Cellular ATP levels in these cells were measured according to the
method of Baccallao et al. (1994).
Cell Efflux Experiments. N18 cells were incubated for 60 min
with 10 mM PNU-87663 emulsion in buffer at 37°C and rinsed twice,
and a single 1-mm optical section was scanned every 5 min. After 30
min, the buffer solution was replaced with 3% BSA in PBS, and the
cells were scanned for an additional 60 min.
Results
First-pass extraction of [14C]PNU-87663 and [14C]PNU89843 (Fig. 1) by the brain was measured after a single bolus
injection into the common carotid artery of a rat. Both com-
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ences, Grand Island, NY) was prepared by diluting a 103 stock
solution with glass-distilled, deionized water; adding 10 mM HEPES,
and adjusting to pH 7.4 with 5 N NaOH. BSA, fraction V, and
cholesterol-rich lipids from adult bovine serum were obtained from
Sigma Chemical Co. (St. Louis, MO). [14C]PNU-87663 monomethanesulfonate salt (22.2 mCi/mg; 443.9 g/mol) and [14C]PNU-89843 z
HCl (30.8 mCi/mg; 322.2 g/mol) were obtained .98% pure by HPLC
from the Pharmacia & Upjohn Radiosynthesis Group. These were
reconstituted in dimethyl sulfoxide to give 0.5 mCi/ml, and the
solutions were stored at 220°C under argon. [14C(U)]Sucrose (671
mCi/mmol), [3H]2O (1 mCi/g), n-[1-14C]butanol (1.7 mCi/mmol), and
[14C]thiourea (0.75 mCi/mg) were purchased from NEN-DuPont
(Boston, MA).
Cell Culture. N18-RE-105 (N18) cells, a mouse neuroblastoma
hybrid cell line (Malouf et al., 1984), were grown in Dulbecco’s
modified Eagle’s medium (GIBCO BRL Life Sciences) supplemented
with 44 mM sodium bicarbonate, 10 mM thymidine, 100 mM hypoxanthine, 1 mM aminopterin, and 5% fetal calf serum at 37°C in 5%
CO2. Cells were used 1 to 2 days after plating or at approximately
50% confluence.
Differentiated 3T3-L1 fibroblasts resembling adipocytes and undifferentiated 3T3-L1 cells, as a negative control, were allowed to
accumulate PNU-87663 before viewing with CSLM.
Brain Uptake. First-pass extraction of radiolabeled pyrrolopyrimidines in brain was measured using the brain uptake index (BUI)
(Oldendorf, 1970). A 200-ml bolus containing 14C compound and
reference compound or [3H]2O was injected rapidly into the left
common carotid artery of anesthetized 300- to 350-g male albino
Sprague-Dawley rats. The rats were decapitated within 5 to 10 s, and
the amounts of radioactivity within the left cerebral hemisphere
were measured. The BUI value is a corrected partitioning of the test
compound relative to the reference tracer of known extraction from
injectate to brain tissue:
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Distribution of Lipophilic Antioxidants
TABLE 2
Effect of serum on brain uptake of PNU-87663
First-pass extraction of [14C]PNU-87663 by rat brain as measured using brain
uptake index (BUI) method in the presence of different concentrations of rat serum
Seruma
PNU-87663b
mM
%
0.1
0.5
1
5
10
BUIc
0.0005
10
19
98
186
(4)d
(4)d
75 6 47 (3)
48 6 8 (4)
37 6 9 (4)
a
Added to Hanks’ balanced salt solution as %, v/v, and adjusted to pH 7.4.
Concentration that was soluble following centrifugation at 150,000g for 10 min.
Mean 6 S.D. with number of animals in parentheses.
d 14
[ C]PNU-87663 radioactivity was below limit of detection.
b
c
pounds are readily taken up by brain, but the extraction
efficiency of PNU-87663 is less and more variable (Table 1).
This was observed even after accounting for compound precipitation by ultracentrifuging the sample before injection.
When the insoluble fraction (0.54) was not considered, the
BUI value was 10 6 3 (n 5 4). PNU-89843 solubility in 3%
BSA was $90% at #280 mM and extraction was consistently
'90% (Table 1). The membrane-impermeant, hydrophilic
solutes sucrose and thiourea and the hydrophobic membrane-permeant butanol were measured against tritiated
water for comparison.
Uptake of PNU-87663 by brain was dependent on the
degree to which it was serum protein bound (Table 2). At
serum concentrations of ,1%, the levels of brain-associated
radioactivity were below detection attributed to low solubility (,10 mM) and low specific activity. Serum protein binding
had less of an effect on PNU-89843 uptake given its higher
extraction efficiency. The addition of 10% rat serum only
decreased PNU-89843 extraction from 89 6 6% in 3% BSA to
68 6 7% (n 5 4). Nevertheless, 5 min after a 10 mg/kg i.v.
TABLE 1
Brain uptake of solutes
First-pass extraction of radiolabeled compounds by rat brain was measured using
brain uptake index (BUI) method.
a
Female mice were injected via tail vein with 23 mmol/kg (;10 mg/kg) compound in
propylene glycol, and samples were collected at two time points after transcardiac
perfusion with normal saline.
BUIa
Compound
Sucrose
Thiourea
Butanol
PNU-87663
PNU-89843
TABLE 3
Concentrations of pyrrolopyrimidines in mouse tissues and plasma
after single intravenous dose
2.4 6 0.6 (15)
3.4 6 0.7 (5)
84 6 7
(3)
57 6 14 (10)
89 6 6 (11)
Mean 6 S.D. with number of animals in parentheses.
PNU-87663
Brain (nmol/g)
Lung (nmol/g)
Heart (nmol/g)
Plasma (nmol/ml)
PNU-89843
5 min
60 min
5 min
60 min
17.7 6 1.1
19.4 6 2.1
31.2 6 5.2
8.0 6 0.3
6.7 6 3.6
4.0 6 0.5
2.7 6 0.8
1.1 6 0.5
12.1 6 1.3
4.0 6 0.1
6.3 6 1.3
8.0 6 0.6
1.6 6 0.2
0.8 6 0.7
1.2 6 0.1
1.3 6 0.1
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Fig. 1. Chemical structures of PNU-87663 and PNU-89843 and the
position of the [14C] isotope (arrow).
dose, brain levels of PNU-87663 were 1.5-fold greater than
for PNU-89843 relative to plasma levels (Table 3). This difference was even greater at 60 min after injection with loss of
PNU-87663 from brain lagging behind the plasma-proportionate loss from the other tissues tested (Table 3).
In vitro permeability measurements showed that PNU87663 has a buffer-cell partition coefficient greater than
PNU-89843 (Sawada et al., 1999). Consequently, PNU-89843
was released from membranes more rapidly, resulting in
faster permeability. These data predict, therefore, that the
retention time of PNU-89843 in target tissues will be shorter
than that for PNU-87663 when plasma levels decrease. To
test this, we measured the rates of efflux of the pyrrolopyrimidines from brain relative to loss of tritiated water (Fig. 2).
Loss of water and PNU-89843 followed first order kinetics
representing the back flux of radiotracer from brain to blood.
PNU-89843 efflux was only slightly slower (Student’s t test,
P , .01) than water. In contrast, the efflux of PNU-87663
was very slow. Rigorous analyses of these data were not
possible due to excessive variability and the lack of fit (R2 5
.45) by linear regression analysis of transformed data indicating that the slope was not significantly different from
zero. In light of these limitations, the T1/2 of PNU-87663
efflux was estimated to be ..8 min (Fig. 2).
The 352- to 355-nm line of the ACAS 570 argon laser
source was capable of exciting the pyrrolopyrimidinyl group
of PNU-87663 given its excitation (lex 5 332 nm) and emission (lem 5 410 nm) maxima in lipid vesicles to produce a
fluorescent image. Emitted light between 458 and 503 nm
was collected for image construction. The relatively high
fluorescence quantum yield of PNU-87663 made it impossible to keep all of the fluorescence within the linear detection
range, and areas that sequester the drug eventually saturate
the system. Further attenuation of either the excitation or
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Fig. 2. Efflux of [14C]PNU-87663 (E) and PNU-89843 (F), relative to
tritiated water (h, ■), from brain. Using the BUI, animals were decapitated at different times (10 –120 s) and the amount of radioactivity in the
brain measured as a fraction of the dose injected per wet weight of brain
tissue. Values are mean and S.D. for three to five animals.
emission signals results in a loss of signal at the cell membrane.
Partitioning of PNU-87663 into N18 cells was rapid, as
indicated by the initial increase in cellular fluorescence, and
reached equilibrium by 30 to 45 min (Fig. 3). Fluorescence
intensity in the plasma membrane was low and reaches
steady-state levels quickly within ;7 min (results not
shown). Moderate fluorescence throughout the cytoplasm
was punctuated by occasional foci of intense fluorescence
(Fig. 4A). Optical sections using CSLM confirm that these
compound-filled inclusions were intracellular. Fluorescence
accumulation in the inclusions was rapidly detected by 5 to
10 min, and distribution was uniform throughout the interior
of the inclusion at equilibrium.
PNU-87663 accumulation by cells treated with sodium
azide and 2-deoxyglucose, where cellular ATP levels are decreased 95%, was similar to untreated controls (Fig. 3). Com-
Fig. 4. Intracellular distribution of PNU-87663 fluorescence in cultured
cells. A 0.9-mm optical section through (A) a group of three N18 cells, (B)
one differentiated 3T3 cell or adipocyte, and (C) four undifferentiated 3T3
cells. Intensely fluorescent foci (arrows) are present throughout the cytoplasm. Cells were incubated with 10 mM PNU-87663 emulsion for 60
min, rinsed with buffer, and viewed with CSLM. Nu, nucleus.
Fig. 3. Accumulation of PNU-87663 by cultured N18 cells. Fluorescence
intensity associated with total cell increases immediately upon addition
of PNU-87663 in the presence (Œ) or absence (■) of 1 mM sodium azide
and 5 mM 2-deoxyglucose at 37°C. Accumulation of PNU-87663 at 4°C (F)
was markedly slower.
parison of drug uptake at 4°C, where active processes were
blocked, and 37°C (Fig. 3) showed that the rate of uptake at
4°C was significantly slower. This was most likely due to a
reduction in the diffusion coefficient at low temperature because detectable intracellular levels of PNU-87663 fluorescence appeared rapidly within 5 min at both temperatures
(results not shown).
Because the cytoplasmic inclusions in which compound
1999
Fig. 5. PNU-87663 efflux kinetics from cultured N18 cells. Cells were
incubated for 60 min in 10 mM PNU-87663 emulsion, rinsed, and scanned
by SLM in buffer. On the addition of 3% BSA (arrow), total cell-associated
PNU-87663 fluorescence decreased markedly, but not completely, over 60
min.
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measured above rapidly equilibrated with brain parenchyma
or was retained by the endothelium. PNU-87663 gained immediate access to the neuronal cells under these conditions
(Fig. 6D). There were no qualitative differences in PNU87663 distribution between bolus injections of 50 mM PNU87663 in 3% BSA and undiluted PNU-87663 emulsion, implying that precipitation was not a limiting factor in
partitioning under the conditions used.
Discussion
In the accompanying paper, PNU-87663 was shown to be
less permeable than PNU-89843 using an in vitro cell model
(Sawada et al., 1999). Because of a greater cell distribution
coefficient and weaker binding to albumin, PNU-87663 is
expected to remain in tissues for a longer period of time.
Similar data with two different chemical classes of lipophilic
antioxidants indicate that highly lipophilic compounds like
PNU-87663 may have difficulty permeating through lipid
membranes and, thus, through the blood-brain barrier (Raub
et al., 1993; Sawada et al., 1994). These predictions were
tested in this report with an in situ brain uptake assay
combined with microscopy and showed that although PNU89843 was more rapidly accumulated by brain during first
pass extraction, PNU-87663 equilibrated within neural tissue and intracellular sites and had a longer retention time.
The data confirm that pyrrolopyrimidines were capable of
quickly accessing target cells within the central nervous system as well as in other target tissues.
Brain uptake of the pyrrolopyrimidines was significantly
greater than the other lipophilic antioxidants or the 21-aminosteroid, PNU-74006 and the 2-methylaminochroman, PNU78517 that had BUI values of 5.7 6 0.8% and 7.5 6 1.5%,
respectively, which are only slightly greater than the impermeant sucrose (Raub et al., 1993). This result was not entirely predicted from the in vitro permeability model where
the apparent permeability coefficients for transcellular flux
of PNU-74006, PNU-78517, PNU-87663, and PNU-89843 are
slow at ;1, 1 to 3, 2.4, and 8.2 3 1026 cm/s, respectively.
From these data, PNU-87663 was expected to have an in vivo
performance similar to the earlier generation antioxidants. It
was concluded, however, that the in vitro assay markedly
underestimates absorption of highly lipophilic solutes
(Sawada et al., 1999).
The brain efflux experiment confirmed predictions that the
significantly greater permeability of PNU-89843 will result
in a shorter retention time. These data show that PNU-89843
was lost at .5-fold the rate of PNU-87663, which was consistent with the HPLC results showing that the mouse brainto-plasma ratio for PNU-87663 was 6-fold greater than for
PNU-89843 at 60 min. Fluorescence imaging of PNU-87663
after 60 min showed a marked reduction in its overall distribution, and this, too, was consistent with the 62% decrease in
the amount of brain-associated PNU-87663.
The initial fraction of the dose per gram tissue that was
extracted during first pass through the brain vasculature for
water and PNU-89843 was 10%. This inferred that 100% of
the compound entering the brain was extracted because it is
reported that ;10% of a bolus dose injected into the common
carotid artery accessed the brain via the internal carotid
artery (Oldendorf, 1981). In contrast, only ;3% of the PNU87663 dose was extracted or equivalent to a BUI value of
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accumulates were suspected of being lipid storage droplets,
PNU-87663 fluorescence distribution was examined in
3T3-L1 fibroblasts. The cytoplasm of the differentiated cells
or adipocytes was filled with large (5–10 mm in diameter)
triglyceride droplets that accumulate PNU-87663 (Fig. 4B).
A similar distribution was observed in lipid-fed N18 cells
(results not shown). In contrast, PNU-87663 fluorescence in
undifferentiated 3T3-L1 fibroblasts was similar to that of the
N18 cells (Fig. 4C).
The kinetics of efflux of PNU-87663 from a 0.9-mm-thick
optical section through a single N18 cell that has been preincubated with PNU-87663 emulsion for 60 min at 37°C are
shown in Fig. 5. Quantitative and qualitative distribution of
drug remained unchanged during 30 min in buffer. The addition of 3% BSA caused an immediate and rapid decline in
total cellular fluorescence, reaching a new steady state by
;60 min.
To confirm that PNU-87663 penetrated into brain parenchyma and did not accumulate in the endothelium of the
blood-brain barrier, PNU-87663 fluorescence was examined
in brain tissue using CSLM. PNU-87663 readily diffused into
the cerebral parenchyma of mice within 5 min after tail vein
injection (Fig. 6A). Fluorescence was distributed heterogeneously throughout the tissue between cerebral blood vessels
and did not appear to be associated with a particular cell
population. Similar distributions were observed for the other
tissues sampled (data not shown). One hour after tail vein
injection, the apparent amount of PNU-87663 within the
brain was markedly decreased (Fig. 6B). The amount of fluorescence, imaged under identical parameters, in brain sections from untreated mice was negligible (Fig. 6C).
Brain uptake of PNU-87663 within 5 min after tail vein
injection, when plasma concentrations were ;8 mM (Table 3),
was extensive and showed that it diffused across the bloodbrain barrier. We examined whether PNU-87663 that is accumulated during first-pass extraction of a 50 mM solution as
Distribution of Lipophilic Antioxidants
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Sawada et al.
Vol. 288
38 6 7, and this variability in PNU-87663 extraction was
realized by the large standard deviation in the BUI value
obtained in 3% BSA (see Table 1). The reason for this difference is unclear. Although this technique assumes no mixing
between bolus and blood, in practice this is not true
(Pardridge et al., 1985). Therefore, the decrease in BUI value
with increase in serum concentration suggested that mixing
resulted in variable serum protein concentrations in the bolus injectate, thus affecting extraction efficiency. This effect
was not observed with PNU-89843 given its higher apparent
extraction efficiency in the presence of serum proteins.
The BUI value and directly measured mouse brain levels
for PNU-87663 indicated that drug has partitioned into the
brain, but further evidence is needed to prove that compound
accesses underlying neuronal cells by diffusing through the
vascular endothelium comprising the blood-brain barrier.
Penetration of PNU-87663 into brain parenchyma was confirmed by combining cryopreservation methods and CSLM.
Even though PNU-87663 spectral properties were marginal,
because excitation was barely within the absorption range,
the relatively high fluorescence quantum yield compensates.
Detection of PNU-89843 fluorescence was precluded by an
insufficient fluorescence quantum yield and a lower partition
coefficient, which contributes to diffusion artifact.
Because extraction from blood may continue after the animals were euthanized and during immersion fixation before
freezing, we repeated the 5-min experiment in mice but removed the brain and immediately froze it without fixation.
Ultralow temperatures prevent diffusion artifacts involved in
localization of lipid-like compounds (Raub et al., 1992). Under these conditions, there was no difference in the distribution of PNU-87663, suggesting that continued diffusion is
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Fig. 6. Distribution of PNU-87663 fluorescence around blood vessels in cerebral cortex after intravenous injection. All pseudocolor images are blended
with the phase-contrast image. The fluorescence intensity scale is shown. A, five minutes after tail vein injection of a mouse. The lumen (*) of a blood
vessel sectioned longitudinally containing red blood cells (small arrows) that are adjacent to endothelium (large arrow) with PNU-87663-associated
fluorescence. B, sixty minutes after dosing a mouse. C, background fluorescence of cerebral cortex from a control mouse that did not receive
PNU-87663. D, rat cerebral cortex within 8 s of an intracarotid bolus injection dose of undiluted emulsion containing 4.1 mM PNU-87663 in the oil
phase. PNU-87663 fluorescence is located on the neural side of the blood-brain barrier. Brains were removed and immersion fixed before freezing.
Cryosections of 6 to 8 mm were viewed by CSLM, giving a 0.9-mm-thick optical section.
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1333
posed to confer added potency in vivo (Andrus et al., 1997;
Hall et al., 1997). The in situ results reported here confirmed
predictions, based on an in vitro cell culture model (Sawada
et al., 1999), that brain-associated PNU-87663 was retained
longer than PNU-89843.
Acknowledgments
We gratefully acknowledge the support of the following Pharmacia
& Upjohn personnel: S. E. Buxser and D. E. Decker for supplying the
N18 cells and the PNU-87663 emulsion, M. L. Swanson and J. E.
Bleasdale for supplying the 3T3-L1 fibroblasts, D. E. Epps for valuable advice and the suggestion that this work might be possible, V. E.
Groppi for providing access to the ACAS 570 and for performing
preliminary experiments showing that this approach works, and
Y. H. Gu for technical assistance.
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unlikely. It also was unlikely that diffusion continues after
cryopreservation and during storage at 280°C because images obtained immediately after the experiment and after
storage of the tissue for 2 months were indistinguishable.
The sections were kept at 220°C until immediately before
microscopy to minimize compound diffusion after thawing.
Because no gross changes in distribution were observed
while viewing the sections at 25°C mounted in an aqueous
medium, post-thaw diffusion also appeared to be minimal.
Thus, we are confident that the PNU-87663 distribution was
not influenced by postprocessing diffusion. The absence of
PNU-87663 fluorescence within the blood vessel lumen or red
blood cells was not surprising despite the fact that plasma
concentrations were 8 mM at 5 min after injection. This was
a very small volume of blood within the optical section, and
individual molecules of PNU-87663 were not sufficiently concentrated to emit a visible fluorescence like that seen within
cells or tissues where the compound has been concentrated
into small volumes by virtue of its partitioning into membranes (Sawada et al., 1999). Using depth-dependent fatty
acid and aqueous fluorescence quenching in artificial lipid
bilayers, a majority of membrane-associated pyrrolopyrimidines were located at the aqueous-lipid bilayer interface at
equilibrium (Hinzmann et al., 1992). At these plasma concentrations, most of the PNU-87663 will be serum protein
bound, keeping concentrations in circulating cells low.
PNU-87663 was not selectively accumulated within the
endothelium at either very short time intervals or after an
apparent equilibrium was reached within 5 min. This suggested that the neuronal compartment was acting as a sink.
It is for this reason that the in vitro permeability model
potentially underestimated tissue uptake of highly membrane-interactive molecules because the receiver compartment in this model was aqueous and, thus, disfavors accumulation of poorly water-soluble lipophiles (Sawada et al.,
1994, 1999).
In accord with rapid tissue penetration, CSLM shows that
PNU-87663 accessed intracellular sites and did not accumulate within the plasma membrane. PNU-87663 diffused
through the plasma membrane via a passive process because
metabolic inhibitors and low temperature had no effect on
intracellular accumulation. This indicates, for example, that
the apparent concentration of PNU-87663 within intracellular foci was not a result of endocytosis or an energy-dependent transmembrane transporter. Movement of such waterinsoluble compounds between membranes most likely
involved interaction with undefined cytoplasmic proteins as
modeled using serum proteins (Raub et al., 1993; Sawada et
al., 1994, 1999). PNU-87663 appeared to accumulate preferentially in neutral lipid storage droplets in cultured cells;
however, this must be confirmed with direct quantification
because the apparent amounts represented by fluorescence
intensity are likely influenced by an increase in quantum
yield in response to the lipid environment.
In conclusion, both PNU-87663 and PNU-89843 readily
cross the blood-brain barrier and diffuse into the brain parenchyma gaining access to intracellular sites within neurons. The improved accessibility of these antioxidants over
previously described lipophilic antioxidants has been pro-
Distribution of Lipophilic Antioxidants