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Brain extraction of a calcium channel blocker.

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Brain Extraction of a Calcium
Channel Blocker
James C. Grotta, M D , L. Creed Pettigrew, M D , Alan H. Lockwood, M D , and Catherine Reich, BS
Dihydropyridine calcium channel blockers may be effective treatment for acute cerebral ischemia, but the uptake of
these drugs into the brain is unknown. A 0.2-ml bolus of ['*C]nicardipine hydrochloride and r3H}water was injected
into the common carotid arteries of 7 normal and 7 ischemic rats. T h e corrected first-pass extraction of nicardipine,
compared to water, was calculated to be 30.7% into the hemispheres and 42.3% into the hippocampi. T h e uptake was
greater into the ischemic hemispheres ( p < 0.001).These data suggest that dihydropyridines are available to binding
sites and calcium channels in neurons.
Grotta JC, Pettigrew LC, Lockwood AH, Reich C: Brain extraction of a calcium
channel blocker. Ann Neurol 21:171-175, 1987
Among their potential therapeutic applications, calcium channel blocking drugs may prevent the damaging flux of ionized calcium into ischemic neurons.
However, the availability in brain of biologically active
concentrations of the most potent and easily administered of these drugs has not been demonstrated. After
oral administration, verapamil has been found in the
cerebrospinal fluid (CSF) of schizophrenic patients IS},
but the concentration of {14C)nicardipine HC1 in rat
brain 30 minutes to 24 hours after an oral dose of 3
mg/kg was no higher than 0.16 ? 0.05 Fglgm, compared to levels of 1.37 t 0.44 pg/gm in plasma and
0.66 t 0.10 in heart @I. Such low levels in the brain
might be due to the rapid washout of the drug o r its
breakdown into inactive metabolites occurring within a
few minutes of drug administration, leaving essentially
no unchanged drug present 1 hour after a single dose
181; this suggests that measurement of first-pass extraction might reveal brain uptake not found at longer
intervals. This article describes measurement of the
first-pass uptake of nicardipine into the brains of normal and ischemic rats.
Production of Ischemia
Brain extraction of ['*C]nicardipine HC1-[2,6-dimethyl-4(3-nitrophenyl)-l,4-dihydropyridine-(4-'4C)-3,5-dicarboxylic
acid 3-[2-(N-benzyl-N-methylamino)]ethyl ester >-methyl
ester hydrochloride], molecular weight 5 15.99 (Syntex Research, Palo Alto, CA)-was
measured using the modified Oldendorf technique El5, 161 in 7 normal and 7 ischemic rats. Extraction of a nondiffusible internal standard
(['*C]inulin; New England Nuclear, Boston, MA) was measured in 5 normal rats.
Seven male Wistar rats (Hilltop Farms, Scottsdale, PA)
weighing 250 to 300 gm were subjected to transient forebrain ischemia according to a modification of the four-vessel
occlusion model [ 191.
Each animal undergoing the procedure was anesthetized
by an intraperitoneal injection of chloral hydrate (0.5 mg/gm
of body weight) and its vertebral arteries were cauterized.
The alar foramina of the C1 vertebra, beneath which the
vertebral arteries travel in their ascent through the posterior
cervical spine, were drilled to ensure access to the vessels at
the time of cautery {26}. A catheter was inserted into the tail
artery for determination of mean arterial blood pressure
(MABP), blood gas values, hematocrit, and glucose levels.
The common carotid arteries were carefully isolated and
tagged with loose ligatures. During surgery, all animals were
ventilated with room air pumped through a face mask connected to a Harvard animal respirator (Model #681; Harvard Bioscience, South Natick, MA). Rectal temperature
was monitored continuously and external heat was applied to
maintain core temperature at 37°C.
Each rat was given unlimited access to water and chow
following surgery. After 24 hours of recovery, four subdermal electrodes (two frontal and two parietal on the right and
left sides) were inserted, with the animal under ether anesthesia, to permit electroencephalographic (EEG) recording
f291. All animals were allowed to recover from the ether for
10 minutes before the common carotid arteries were located
and occluded with surgical clips. The cervical muscles were
ligated to reduce collateral circulation through vertebral artery branches that may not have been adequately cauterized.
Cerebral ischemia was maintained for 30 minutes, during
which MABP and rectal temperature were monitored continuously. A Grass model 79D EEG machine (Grass Instrument Co, Quincy, MA), set at a paper speed of 30 mdsec,
sensitivity of 7.5 to 10 pV/mm, and appropriate filter set-
From the Stroke Research Laboratory, Department of Neurology,
University of Texas Health Science Center, 6431 Fannin, Houston,
TX 77030.
Received May 13, 1986, and in revised form June 23. Accepted for
publication June 24, 1986.
Address reprint requests to Dr Grotta.
tings, was used to record EEG data throughout the procedure. The loss of the righting reflex and an isoelectric EEG
indicate severe compromise of cortical blood flow [19, 20);
animals that did not meet both of these criteria were not
included. After 30 minutes, the clips were removed from the
carotid arteries and the cervical ligatures were released to
terminate the ischemic period.
Brain Extraction of Nicardipine
Seven normal animals and 7 animals studied 24 hours after
production of ischemia (vide supra) were anesthetized with
chloral hydrate and received a 0.2-ml bolus consisting of 1
pCi of [14C}nicardipine HCI, specific activity (SA) = 17.13
mCi/mmol, and 2 pCi of L3H}water, SA = 1 mCi/gm, via a
needle stick into a common carotid artery in the neck. Another 5 normal rats were treated identically except for the
substitution of 1 pCi of ['*C]inulin, SA = 2.7 mCi/gm, for
the radiolabeled nicardipine. Each animal was decapitated 5
seconds after injection and its brain was removed. The hemispheres were separated and one hemisphere was placed with
the frontal cortex forward in the barrel of a 5-ml syringe; 0.3
ml was then forced through a 20-gauge needle and dissolved
in 0.5 ml of dihydroxyacetone (Protosol; New England Nuclear, Boston, MA). Preliminary studies in our laboratory
have shown that using this 0.3-mi sample leads to similar
results, but with less quench than does dissolving the entire
hemisphere. The entire hippocampus was dissected from the
other hemisphere and dissolved in the same fashion. After
injection, the syringe that had contained radiolabeled compounds used to inject the animals was rinsed with 1 ml of
ethyl alcohol.
The brain tissue samples were placed in a 37°C bath and
left overnight. Each vial containing the dissolved tissue then
received 100 pl of 30% hydrogen peroxide to reduce color
quenching, was shaken by hand, and returned to the bath for
an additional 30 minutes. Ten ml of Econofluor (New England Nuclear, Boston, MA) was added to each vial containing tissue. The ethyl alcohol used to rinse the syringe was
diluted 1:50, and 1 ml of the resulting solution was mixed
with 10 ml of Aquasol (New England Nuclear, Boston,
MA). Tissue solutions that remained cloudy were cleared by
adding drops of Protosol. The samples were then kept in the
dark for 3 hours before being counted in a liquid scintillation
counter (Model #LS6800; Beckman Instruments, Irvine,
CA). Dilutions were calculated to result in counts of the
same magnitude and similar quench factors for all brain regions and infusate. This eliminated inaccuracies produced by
incorporating an automatic quench correction factor. Window settings of 0 to 260 keV for 3H and 350 to 670 keV for
'*C were defined by spectrum searches determined for brain
samples labeled with 1 pCi of i3Hfwater or ['*C)nicardipine
and processed according to protocol.
The first-pass extraction of ['*C]nicardipine by nonischemic brain was calculated according to the following formula
[ I s , 161:
Extraction = 0.84
['*C]nicardipine (brain)/L3H}water (brain)
['*C)nicardipine (infusate)/13H)water (infusate)
["C}inulin (brain)/['H)water (brain)
[14C]inulin (infusate)/{ 3H]water (infusate)
Oldendorf and Braun [l6] have determined from previous
studies in nonischemic animals that a correction factor (0.84)
will account for the incomplete extraction of E3H]water 1161.
Subtracting the extraction rate for inulin, a nondiffusible substance, from that of nicardipine corrected for the amount of
nicardipine lingering in the cerebral circulation at the time of
decapitation. No correction for incomplete water extraction
or intravascular nicardipine was made in the ischemic brain,
as the distribution of inulin in brain with an abnormal bloodbrain barrier is unknown.
Statistical Analysis
Data are presented as the mean
the standard deviation.
Statistical significance ( p ) was determined using a two-tailed
Student's t test for independent samples.
Extraction of nicardipine or inulin for each hemisphere
or hippocampus was calculated by the following ratio
of counts:
14C (brain)/3H (brain)
I4C (infusate)/3H (infusate)
The results of these calculations are presented in the
Table. When intravascuiar nicardipine was corrected
Percent Extraction of 14C Tracer Compared t o {jH}Water
Mean t SD
Nonischemic ['4C)Inulin
Nonischemic [ '"C)Nicardipine
Ischemic { "CC)Nicardipine
42.0 t 14.Iy
55.7 -+ 22.5
49.7 t 23.4
5.3 t 1.2
"Significantly different than y at p < 0.001 (t
172 Annals of Neurology
No 2
Vol 2 1
7.58; df
February 1987
"CN,( brain)/'HH~O(broin)
Meon EN, hemisphere ==
Meon EN, hippocornpus
Fig 1. Percent extraction of {14C}nicardipine(Ni) and
{14C}inulin (IN) into nonischemic cerebral hemispheres and hippocampi, calculated by obtaining the I4C :jH ratio in the brain,
divided by infusate. Subtracting the mean inulin extractionfrom
the mean nicardipine extraction and multiplying times 0.84
gives EN,, the corrected nicardipine brain extraction (16).
EB lnuiin
Nicardipine in Normal brain
0Nicordipine in Ischemic brain
n = 5
n = 7
n = 7
Fi g 2. Percent extraction of inulin and nicardipine into normal
and ischemic cerebral hemispheres and hippocampi. Significantly
greater extraction of nicardipine was fotlnd in ischemic hemispheres compared to nonischemic hemispheres.
by subtracting inulin from nicardipine extraction, and
incomplete extraction of water was corrected by multiplying the result times 0.84, the mean first-pass extraction of nicardipine was calculated to be 30.7% into
nonischemic hemispheres and 42.3% into nonischemic hippocampi (Fig 1).
Increased extraction of nicardipine occurred in ischemic hemispheres but not in hippocampi where ischemic damage is most severe in this animal model (Fig
Calcium channel blockers are presently under evaluation as possible therapeutic agents for cerebral ischemia. Threatened but potentially viable tissue has been
identified in animal stroke models [201 and in human
stroke patients 11, 2, 61. One strategy for preserving function in these brain regions is to elevate the
cerebral blood flow (CBF) in regions where perfu-
sion is barely adequate to meet the metabolic needs
of ischemic neurons. Another approach is to block
the damaging and possibly irreversible disruption of
neuronal homeostasis caused by failure to maintain the
normal extracellular-intracellular gradient of calcium.
As a result of a failure of energy-dependent membrane
pumps and possibly also from the opening of membrane channels by the release of excitatory amino acids
[227, cerebral ischemia is associated with a rise in intracellular free calcium; activation of membrane phospholipases; and the production of cytotoxic leukotrienes, thromboxanes, and free radicals {12, 14, 18,
21, 23, 27, 281. Calcium channel blockers might preserve neuronal function by causing the relaxation of
vascular smooth muscle and increasing CBF, by preventing the accumulation of intraneuronal free calcium, or by both mechanisms.
The dihydropyridine calcium blockers, including
nicardipine, nitrendipine, nifedipine, and nimodipine,
are the most potent regulators of voltage-sensitive calcium channels [13} and block calcium uptake by membrane fractions from cerebral arteries 131. This results
in the relaxation of smooth muscle in the walls of cerebral arteries and arterioles [ l l ] and increased CBF in
experimental cerebral ischemia [9, 251. The effect of
the dihydropyridines on neuronal calcium channels is
still being studied. Dihydropyridine binding sites are
present in cerebral tissue C4, 131, but macromolar
concentrations of these drugs may be necessary to
suppress calcium entry into neurons [lo], as depolarization-induced calcium flux into synaptosomes is
relatively insensitive to the dihydropyridines 131.
Recently, however, a second type of dihydropyridinesensitive calcium channel has been identified in neutonal soma, suggesting that the more easily achieved
brain levels of these drugs might play a role in modifying calcium entry into neurons [13].
Previous data from our laboratory suggest that the
dihydropyridines, specifically nicardipine, have a direct
effect on blocking neuronal calcium influx during the
reperfusion period and do not support the contention
that the drug works by increasing CBF during the ischemic period [7]. Cellular function as measured by
somatosensory evoked-potential amplitude was preserved once treatment was begun after the ischemic
period when CBF had returned to normal {7}. We
chose to study nicardipine because of its potency and
solubility in both lipid and water {S], and subsequently
nicardipine has been selected for therapeutic trials in
cerebral ischemia following atherothrombotic stroke
and subarachnoid hemorrhage.
The present study demonstrates that nicardipine is
extracted into the brain when measurements are performed before metabolism of the drug has occurred,
and suggests that biologically active amounts of the
drug may be available to dihydropyridine-sensitive cal-
Grotta et al: Brain Extraction of a Calcium Channel Blocker
cium channels in neurons. The uptake demonstrated in
this study, however, must be considered a rough estimate of nicardipine availability. More than 90% of
circulating nicardipine is reversibly bound to plasma
proteins (data on file, Syntex Research). The uptake
into the brain demonstrated in the present experiment
occurred within the first 5 seconds, and we do not
know what percentage of nicardipine became proteinbound during that interval or how protein binding affected the uptake of recirculating nicardipine. Also,
oral or intravenous administration and first-pass liver
metabolism would probably alter the quantity of circulating unchanged nicardipine and uptake kinetics. Finally, the animal model used in this study causes global
cerebral ischemia followed by reperfusion. The results
might be different in a model that produces focal necrosis or no reflow.
The method of measuring nicardipine uptake into
brain used in this study has been validated Il5, 161.
There was minimal mixing of the labeled bolus with
unlabeled plasma [ 171 and the correction for incomplete extraction of the diffusible reference (water) and
inclusion of a nondiffusible reference (inulin) [24] increased accuracy {l5, 167. However, as the reliability
of double-tracer counting is compromised by overlap between the 3H and ‘*C windows and relatively
inefficient counting of low energy 3H, by increasing
the activity of injected 3H and giving careful attention
to window settings, we minimized these sources of
error. As described in Methods, the thorough solubilization of brain protein is also necessary and ratios of
brain substance and organic solvent must be found that
minimize the need for quench correction. Chemoluminescence and bioluminescence must also be eliminated by the appropriate choice of scintillation fluids
and delayed counting after storage in the dark for 3
Increased extraction of nicardipine in ischemic
hemispheres might be due to a damaged blood-brain
barrier, but if so it is hard to explain our failure to find
increased extraction in ischemic hippocampi where
neuronal damage is most marked. It is possible that a
subnormal CBF 24 hours after ischemia limits the delivery of nicardipine to the hippocampi. In addition,
many hippocampal regions demonstrate no ischemic
histological changes { 191, and increased extraction into
discrete hippocampal regions where damage is most
marked (i.e., CA-1) might be missed by measurements
combining all hippocampal regions. Nevertheless, the
data show that nicardipine uptake does occur in normal and ischemic brain and lend support to the
rationale behind the use of nicardipine in clinical
stroke trials.
Supported by a grant from The Cullen Trust for Health Care.
174 Annals of Neurology
Vol 2 1 No 2
February 1987
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Grotta et al: Brain Extraction of a Calcium Channel Blocker
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