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NEUROPHARMACOLOGY
Division of Biological Research, Alkermes, Inc., Cambridge, Massachusetts (H.C.S.-B., D.F.E., S.B., K.H., S.R., D.L., B.P., L.N., K.F., A.S.B., R.T.B.); and Department of Neurological Sciences, Rush Presbyterian Medical Center, Chicago, Illinois (E.-Y.C., T.K., J.H.K.)
Received February 3, 2003; accepted April 2, 2003.
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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) leading to a
massive Ca2+ influx that activates, among other
processes, the Ca2+-dependent phospholipases
A2. These phospholipases A2 cleave membrane
phospholipids to yield arachidonic acid, which is converted by cyclooxygenases
(COXs; Hurley et al., 2002)
into prostaglandin (PG)G2. PGG2 is subsequently reduced
to PGH2 with the production of a free radical intermediate that
rapidly converts to a reactive hydroxyl radical
(Kukreja et al., 1986). Two
isoforms of COX exist, the constitutive isoform COX-1 and an inducible isoform
COX-2. Although the products of COX-1 activity exert a cytoprotective role in
the periphery (Hawkey, 2001),
their function in the CNS remains unclear
(Zhang and Rivest, 2001;
Lin et al., 2002).
COX-2 expression is constitutive in some neurons
(Seibert et al., 1994) but is
induced by glutamate (Manev et al.,
2000) and proinflammatory stimuli
(Bazan et al., 1994) in
migratory immune cells, glia, and neurons
(Nogawa et al., 1997;
Luo et al., 1998;
Hurley et al., 2002). The
resulting increase in COX-2 activity may contribute to neurodegeneration
either by oxidative stress, or the neurotoxic actions of prostaglandins such
as PGA1 and PGE1
(Kukreja et al., 1986;
Bezzi et al., 1998). Increased
expression of COX-2 is associated with a number of acute and chronic
neurodegenerative states, including seizures, ischemia/stroke, Alzheimer's
disease (Hurley et al., 2002),
Parkinson's disease (Knott et al.,
2000), and amyotrophic lateral sclerosis
(Yasojima et al., 2001). The
involvement of COX-2 in acute and chronic neurodegenerative syndromes has
promoted the development of neuroprotective treatment strategies involving COX
inhibitors, such as the nonsteroidal anti-inflammatory drugs (NSAIDs).
Although epidemiological studies suggest that NSAIDS may be protective in
chronic neurodegenerative conditions
(McGeer et al., 1996), little
is known of their clinical efficacy in treating acute neurodegeneration. If
COX inhibition blocks neurodegenerative aspects of excitotoxicity, then
neurodegenerative disorders with an excitotoxic component may benefit from
this treatment. Moreover, the rapidity of onset of COX inhibition may play a
crucial role in protecting neurons impacted by acute neurological insults,
such as those associated with ischemia and/or trauma
(Dash et al., 2000; Strauss,
2000; Iadecola et al., 2001).
This need may be served by formulations of COX inhibitors that can be
delivered by the pulmonary route, allowing rapid entry of a drug into the
circulation.
In an attempt to determine whether inhibition of COX activity can suppress
acute neurodegeneration, we have used an animal model of excitotoxicity
(Beal et al., 1991) that allows
us to investigate the relative contributions of the COX-1 and COX-2 isoforms
to excitotoxic neurodegenerative processes. This was done by comparing the
neuroprotective efficacy of a currently prescribed, nonselective NSAID
(flurbiprofen) with specific, experimental COX-1 and COX-2 inhibitors (valeryl
salicylate and NS-398, respectively). In addition, we examined the
neuroprotective efficacy of an NSAID administered using a novel pulmonary
delivery system that optimizes the timeliness of delivery while decreasing the
neuroprotective dose of the drug.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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80 g; Taconic Farms, Germantown, NY) were used
in all studies. Rats were housed in pairs in polypropylene cages with free
access to food and water. The vivarium was maintained on a 12-h light/dark
cycle (lights on at 7:00 AM) with a room temperature of 22 ± 1°C
and relative humidity level of 50 ± 5%. All studies were approved by
Alkermes Institutional Animal Care and Use Committee and were conducted in
compliance with the National Institutes of Health Guide for the Care and Use
of Laboratory Animals. Preparation of Inhalable Flurbiprofen. Flurbiprofen, excipients (1 g/l), and ammonium bicarbonate (8 g/l) were mixed into a spray drying solution with ethanol/water [70:30 (v/v)] as the solvent. The solution was then is introduced into a NIRO spray dryer at 40 ml/min and atomized into droplets with a rotary atomizer at 20,000 rpm. The droplets contact the drying gas and the dry particles collected with a 6-inch cyclone. The final loading density of flurbiprofen in the particles is 20%.
Drug Administration. All animals were fasted for 12 h before drug administration. Rats (n = 8) were administered either flurbiprofen (FLURBI; 2, 10, or 50 mg), N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS-398; 2 or 10 mg), valeryl salicylate (2, 10, or 50 mg) by the oral route, or an inhalable formulation of 20% flurbiprofen (2 mg) 10 to 15 min before injection of quinolinic acid (QA). Vehicles consisted of either blank powder for inhalable flurbiprofen, or 1% Tween in distilled water for the orally administered agents. All oral drugs were delivered by gavage in a total volume of 1 ml. The inhalable flurbiprofen formulation was administered using the following insufflation technique. Rats are anesthetized with 3% isoflurane/77% nitrous oxide/20% oxygen and a laryngoscope used to visualize the epiglottis. A blunt-tip insufflator (Penn Century, Philadelphia, PA) containing the premeasured dose is then inserted into the airway under visual guidance. A bolus of air (3 cc) from an attached syringe is used to deliver the powder from the chamber of the insufflator into the lungs. A second bolus of air is used to make certain that the entire dose is administered. A total of 10 mg of powder containing 2 mg of flurbiprofen was delivered to each rat.
Similar techniques were used for administering flurbiprofen for pharmacokinetic studies. Rats (n = 12) were administered 5 mg of powder containing 1 mg of flurbiprofen, or were orally administered 1 mg of flurbiprofen in 1% carboxymethylcellulose solution. Blood samples (500 µl in heparinized tubes) were obtained at 0, 2, 5, 15, 30, 60, 120, 240, and 360 min after administration. Only four samples were taken from each rat. The blood samples were centrifuged, plasma removed, and then rapidly frozen and stored at –80°C until assayed.
Plasma flurbiprofen levels were assayed using HPLC with UV detection.
Briefly, rat plasma samples (200 µl) were spiked with ketoprofen as an
internal standard and then extracted using Oasis HLB extraction cartridges
(Waters, Milford, MA). Samples were loaded onto a conditioned column, washed
with 1 ml of deionized water, and then eluted with 1 ml of methanol. The
eluate was dried and reconstituted with 1 ml of 0.1% trifluoroacetic
acid/acetonitrile [60:40 (v/v)]. Flurbiprofen and ketoprofen were separated
using a Luna C18 (5 µm; 150 x 3.0-mm i.d.; Phenomenex,
Torrance, CA) column with a guard column. The column temperature was 35°C
and samples were maintained at 25°C with a refrigerated autosampler. The
injection volume was 5 µl and the flow rate was 0.4 ml/min. Materials were
eluted from the column with a gradient consisting of 0.1% trifluoroacetic acid
(A) and 100% acetonitrile (B), using the following parameters. Initial
conditions: 50:50; 4 min, 50:50, isocratic; 6 min, 40:60, linear gradient; 10
min, 40:60, isocratic; 11 min, 20:80, linear gradient; and 13 min, 20:80,
isocratic. Eluates were detected by monitoring at 
= 254 nm.
Surgery. Immediately after drug administration, rats were
anesthetized with ketamine (25 mg/kg), xylazine (1.3 mg/kg), and acepromazine
(0.25 mg/kg intramuscularly) and positioned in a stereotaxic instrument (Kopf,
Tujunga, CA). A midline incision was made in the scalp and a hole drilled
through the skull for injection of QA (225 nmol in phosphate-buffered saline)
at the following coordinates: 1.2 mm anterior, 2.6 mm lateral to bregma, and
5.5 mm ventral to the surface of the brain
(Emerich et al., 1996). QA was
infused into the striatum using a 28-gauge blunt-tip syringe (Hewlett Packard,
Palo Alto CA) in a volume of 1 µl over 5 min. The injection cannula was
left in place for an additional 2 min to allow the QA to diffuse from the
needle tip, after which the cannula was removed, the bone window waxed over,
and the overlying skin sutured closed. A similar procedure was followed on the
contralateral side, with the exception that only vehicle was injected. The
rats were then injected with lactated Ringer's solution (10 ml, subcutaneous)
to prevent dehydration and allowed to recover on a heating pad. Surgery was
timed so that QA was injected exactly 10 min after administration of COX
inhibitors.
Behavioral Testing. Four tests were used to measure unilateral motor
impairment. Placement and akinesia tests
(Schallert and Tillerson,
2000) were performed 27 days after QA lesions. The placement test
requires holding a rat parallel to the edge of a tabletop in such a way as to
allow it to place its forelimb atop the table in response to stimulation of
its whiskers by contact with the table edge. For each trial, the subjects were
held with their limbs hanging unsupported and then placed with their bodies
parallel to and within the distance of their whiskers (approximately 4 cm)
from the edge of the table. Each rat was tested in 10 consecutive trials per
forelimb, and the total number of times the rat placed its forelimb on top of
the table was recorded. In the akinesia test, the rat was supported on one
forelimb and allowed to move independently. The number of "steps"
taken with each weighted forelimb was recorded over 30 s. Rats were tested in
the bracing task 34 days after surgery. Subjects were individually placed on a
smooth stainless steel surface and gently pushed laterally a distance of 90 cm
at a rate of approximately 20 cm/s. The number of braces made with the
forelimb opposing the direction of movement was recorded. Each trial involved
moving the rat twice on each side.
Apomorphine-induced rotations were recorded 4-weeks after surgery to
further assess the extent of damage to the striatum
(Carman et al., 1991). Rats
were administered apomorphine (1 mg/kg s.c.) and then placed into a
cylindrical acrylic container. Each 360 degree rotation made by the rat was
counted over a 30-min trial period. Partial rotations and reversals were not
recorded. Rats were tested in this paradigm once a week for 2 weeks, with the
data for the third trial presented under Results. Apomorphine-treated
rats with QA-induced lesions of the striatum typically rotate 150 times/30 min
(Nakao et al., 1998).
Histology. At the conclusion of behavioral testing, all animals were sacrificed for histological analysis. Rats were anesthetized with ketamine, xylazine, and acepromazine solution and then transcardially perfused with heparinized phosphate-buffered saline (5000 U/l, 20 ml, pH 7.4, 0–4°C), followed by 4% Zamboni's fixative (500 ml, 0–4°C). The brains were then removed, placed in 30% phosphate-buffered sucrose (pH 7.4), and stored (48 h, 0–4°C). Before sectioning, the brains were rapidly frozen in methylbutane (–60°C), mounted on a freezing microtome, and 40-µm-thick sections cut and stored in a solution of 30% sucrose/30% ethylene glycol in phosphate-buffered saline at –20°C until processed for assessment of lesion size and DARPP-32 immunohistochemistry.
Immunohistochemistry. Sections were processed for the histochemical
visualization of DARPP-32-like or NeuN-like immunoreactivity using
biotin-labeled antibodies (Hsu et al.,
1981). Endogenous peroxidases were eliminated with a 20-min
incubation in 0.1 M sodium periodate in Tris-buffered saline. Background
staining was suppressed with a 1-h incubation in Tris-buffered saline (pH 7.4)
containing 3% normal goat serum, 2% bovine serum albumin, and 0.05% Triton
X-100. The sections were then incubated in the primary antibodies, either
DARPP-32 (1:25; Cell Signaling Technology Inc., Beverly, MA), or NeuN (1:100;
Chemicon International, Temecula, CA) for 48 h at room temperature. After
several washes, sections were sequentially incubated in the biotinylated IgG
secondary antibody (1:200; Vector Laboratories, Burlingame, CA) for 60 min and
the avidin-biotin (ABC Elite) substrate (1:500) for 75 min.
DARPP-32-immunostained sections were then reacted in a chromogen solution
containing Tris-buffered saline, 0.05% 3,3' diaminobenzidine, and 0.005%
H2O2. All sections were mounted on chrome alum-treated
slides, air-dried, and coverslipped with Permount. Control sections were
treated in an identical manner except for the substitution of the primary
antibody solvent or an irrelevant IgG matched to the protein concentration of
the primary antibody.
Volumetric Measurements. Lesion volume was quantified using a point
counting procedure, (Cavalieri procedure;
Gundersen and Jensen, 1987)
and StereoInvestigator software (MicroBrightField, Colchester, VT).
Stereological measures were made using a microscope (BX-60; Olympus, Tokyo,
Japan), integrated with a computer-controlled three-dimensional motorized
stage (Ludl Electronic Products, Hawthorne, NY), and a high-sensitivity
charge-coupled device videocamera system (Hitachi, Tokyo, Japan). Every sixth
section throughout the rostrocaudal extent of the NeuN-stained lesion was
analyzed. The StereoInvestigator software internally calculates the total
volume for each case. Because of the shrinkage of the lesioned striatum over
time, we normalized the volume of the lesioned striatum
(VN) to the intact side in all animals by applying the
formula VN = VLesion ·
(VIntact striatum/VLesion
striatum).
Neuronal Counts. Counts of DARPP-32-immunopositive neurons were
performed using the MicroBrightfield stereological software and stereological
principles (Gundersen et al.,
1988). The total number of neurons was estimated by the optical
fractionator method using a 100x plan-apo oil immersion objective with a
1.4 numerical aperture. For each tissue section analyzed, section thickness
was assessed empirically (approximately 12.5 µm), and upper and lower
"guard zones" with a 4- to 5-µm thickness were established
before each series of measurements. The striatum was outlined under a low
magnification (4x) and approximately 5% of the outlined region was
analyzed using a systematic random sampling design. The total number of
neurons (N) for each case was calculated using the following formula:
NTotal = (N1 +
N2 + NN) · 12. The coefficients
of error were calculated according to the procedure of Gunderson and
colleagues as estimates of precision. Values of <0.10 were accepted
(West et al., 1996).
Data Analysis. The ED50 estimates of drug potency in the placement, akinesia, and bracing tests were derived using nonlinear regression fitting of a sigmoidal dose-response curve to the data (GraphPad Prism; GraphPad Software Inc., San Diego CA). The ED50 estimates of drug potency were derived using nonlinear regression fitting of a sigmoidal dose-response curve to the data, with the minimum constrained to the value obtained for the mean motor performance of the lesioned side from the vehicle-treated group, or zero for lesion size. The maximum value of the curve was constrained to the mean level of performance by the unlesioned limb, or the size of the lesion from the vehicle-treated group. The significance of the differences between populations was assessed using multiway ANOVA followed by Bonferroni's post hoc comparison matrix, except for the placement test, on which Kruskal-Wallis tests were performed as the nature of scoring the placement task skews the distribution.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Orally administered flurbiprofen dose dependently spared rats from QA-induced decrements in the performance of a number of motor assessments (Fig. 1; Table 1). Specifically, the number of placements by the impaired limb of rats treated with 50 mg of flurbiprofen was not significantly different from the unlesioned side (Fig. 1A), constituting a 74-fold higher level of performance than observed in vehicle-treated rats. Performance in this test was also improved relative to vehicle (p < 0.05) after 10 mg of flurbiprofen. Flurbiprofen dose dependently protected limb function in the bracing and akinesia tests at doses of 10 and 50 mg, but not 2 mg (Fig. 1, B and C). Similarly, the 10 (p < 0.01) and 50 mg (p < 0.01) doses of flurbiprofen resulted in 53 and 69% fewer apomorphine-induced rotations compared with vehicle-treated rats (Fig. 1D). Stereological assessment of the striatum revealed that pretreatment with 2, 10, and 50 mg of flurbiprofen before QA administration resulted in 50, 50, and 70% decreases in lesion volume compared with vehicle-treated rats (p < 0.01), with no significant differences between the three doses (Table 1; Figs. 2 and 3A). Furthermore, the number of DARPP-32-immunoreactive neurons was 290, 340, and 355% higher in the striata of rats receiving 2 (p < 0.05), 10 (p < 0.01), and 50 mg (p < 0.01) of flurbiprofen compared with vehicle-treated animals (Fig. 3B).
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After establishing the neuroprotective efficacy of flurbiprofen in this model, we attempted to enhance its pharmacokinetics by formulating flurbiprofen into inhalable microparticles. Comparison of the plasma pharmacokinetics of flurbiprofen (1 mg) administered through oral and pulmonary routes of delivery indicated that inhaled flurbiprofen yielded maximal plasma levels of 79 ± 9.0 to 84 ± 7.3 µg/ml (statistically indistinguishable) by 2 to 5 min after inhalation (Fig. 4). Detectable levels of inhaled flurbiprofen (9.5 ± 1.5 µg/ml) were found in the plasma as long as 6 h after administration, with the area under the curve equal to 9310 µg ml–1 min. In contrast, 1 mg of flurbiprofen administered orally reached a maximum plasma level of 3.6 ± 0.6 µg/ml by 5 min after dosing. Plasma levels six h after oral administration were maintained at approximately 3 µg/ml, with the are under the curve equal to 1260 µg ml–1 min.
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Administration of 2 mg of flurbiprofen via the pulmonary route fully protected limb performance as assessed in the placement task (Fig. 5A). Similar results were observed in the akinesia test (Fig. 5C) and apomorphine-induced rotations (Fig. 5D), where performance levels were not significantly different from unlesioned animals. Interestingly, the bracing task did not reveal any lesion effect in the animals administered inhalable flurbiprofen or blank powder (Fig. 5B). Consistent with the behavioral data, the lesion volumes of rats insufflated with flurbiprofen (Figs. 6C and 7A) were 92% smaller than those in vehicle-treated animals (Figs. 6, A and B, and 7A), and there was a 74% increase in survival of DARPP-32-immunoreactive neurons (Figs. 6, D–F, and 7B).
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To determine which isoform of COX was responsible for the neuroprotective efficacy of flurbiprofen, the effects of selective COX inhibitors were investigated. The COX-1-selective agent valeryl salicylate exerted no notable neuroprotective effects. Even at the highest dose tested (50 mg), limb performance was not significantly improved in either the placement or akinesia tasks (Fig. 8, A and C), or in the number of apomorphine-induced rotations (Fig. 8D). Only in the bracing test did the 50-mg dose of VS improve motor function to levels observed on the unlesioned side (p < 0.05).
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In contrast, rats receiving either 2 or 10 mg of the COX-2-selective inhibitor NS-398 orally before the QA lesion showed significant sparing of limb performance in the placement task compared with vehicle-treated animals (Fig. 9A; Table 1). The 10-mg dose of NS-398 fully protected limb function on the side contralateral to the QA injection. Similar results were observed in the performance of the bracing (Fig. 9B) and akinesia tests (Fig. 9C), where rats pretreated with 10 mg of NS-398 demonstrated contralateral limb performance that was not significantly different from that of the ipsilateral limb. NS-398 also dose dependently reduced the number of apomorphine-induced rotations relative to vehicle-treated rats (Fig. 9D). Stereological analysis indicated that 2 mg of NS-398 resulted in a lesion volume 90% smaller than vehicle (p < 0.01), whereas no lesions were apparent after pretreatment with 10 mg of NS-398 (Figs. 10, A–C, and 11A). Similarly, the 2- and 10-mg doses of NS-398 significantly increased DARPP-32 immunoreactivity compared with vehicle treatment (p < 0.01; Figs. 10, D–F, and 11B).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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;
Kelley et al., 1999;
Manev et al., 2000). Given the
close association of COX-2 with neurodegenerative states, we set about
comparing the relative neuroprotective efficacy of the currently prescribed,
nonselective COX-1/COX-2 inhibitor flurbiprofen, as well as selective
inhibitors of COX-1 and COX-2 in an animal model of acute, excitotoxic
neurodegeneration.
The mixed COX-1/COX-2 inhibitor flurbiprofen was found to be a highly
efficacious neuroprotectant from both histological and behavioral standpoints.
COX-1 activity apparently has little involvement in excitotoxic
neurodegeneration because the COX-1 selective inhibitor VS showed little or no
neuroprotective efficacy over the dose range tested, which should be
sufficient to completely inhibit COX-1 activity
(Bhattacharyya et al., 1995).
Moreover, COX-1 does not seem to produce any neuroprotective agents in the
short term (Teismann and Ferger,
2001; Zhang and Rivest,
2001; Lin et al.,
2002) because inhibition of COX-1 by VS neither increased lesion
size nor impaired motor performance to a greater extent than vehicle
treatment. In contrast, the selective COX-2 inhibitor NS-398 was a very potent
and efficacious suppressor of excitotoxic neurodegeneration in vivo. This was
manifested not only by a significant reduction in lesion area and preservation
of DARPP-32-immunoreactivity neurons, but also functionally, as indicated by
the preservation of rat performance in four different motor assessments.
Histological and neurobehavioral indices of neuroprotection were not precisely
correlated, with evidence of motor dysfunction present despite histological
evidence of relatively small lesions. This may reflect the influence of
environmental factors upon behavioral performance at any given time, as well
as the inability of the histological markers to distinguish between fully
healthy versus functionally impaired neurons
(Carman et al., 1991).
Nonetheless, these data support a significant role for COX-2 in acute
neurodegeneration involving excitotoxic processes
(Hewett et al., 2000).
The potent and significant neuroprotection offered by both NS-398 and
flurbiprofen is consistent with the involvement of COX-2 with inflammation and
neurodegeneration. Both mixed and COX-2-selective inhibitors suppressed
neuronal damage in an 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated
mouse model of Parkinson's disease
(Teismann and Ferger 2001),
with the COX-2 inhibitor being more potent. Similarly, NS-398 blocks LPS- and
NMDA-induced neuron death in vitro (Hewett
et al., 2000; Araki et al.,
2001). Although COX-2 inhibitors were acutely neuroprotective in
the above-mentioned studies, selective inhibition of COX-2 may present
liabilities. Selective COX-2 inhibition increases chemotactic eicosanoid
(leukotriene B4) formation via 5-lipoxygenase, possibly as a
compensatory response to the anti-inflammatory effects of COX-2 inhibition.
COX-2 selective inhibitors also carry the burden of unwanted cardiovascular
effects, resulting from the unbalanced inhibition of cyclooxygenases
(Cheng et al., 2002).
Furthermore, the efficacy of selective COX-2 inhibitors in treating chronic
neurodegeneration has yet to be established in clinical trials, despite the
apparent effectiveness of the nonselective COX inhibitors
(Rogers et al., 1993;
McGeer et al., 1996;
Stewart et al., 1997;
Veld et al., 2000;
Zandi et al., 2002). Together,
these observations support the consideration of nonselective COX inhibitors
for the treatment of neurodegenerative syndromes.
The promising therapeutic potential of NSAIDs for treating various
neurodegenerative diseases raises the question of how to optimize the delivery
method of these drugs to effectively protect neurons, particularly against
acute CNS insults. For example, enhancing the rapidity of onset of an NSAID
may make a critical difference in preserving neurons after acute trauma or
ischemic attacks. Given that drug administration via pulmonary pathways often
yields pharmacokinetics comparable with those of intravenous delivery
(Vanbever et al., 1999), we
created a formulation of flurbiprofen that could be administered by
inhalation. Pharmacokinetic studies indicated that the
Cmax for inhaled flurbiprofen (1 mg) was 24 times higher
than an equivalent oral dose. Moreover, the increase in plasma levels after
pulmonary administration of flurbiprofen was too rapid to accurately resolve.
Although the Tmax for oral flurbiprofen was also on the
order of 5 min, the oral formulation used in this study (flurbiprofen in
aqueous Tween) is a departure from the typical tablet or capsule, which would
require more time to dissolve and be systemically absorbed
(Tmax: 0.7–2 h;
Davies, 1995). Therefore, it
seems that not only does the pulmonary delivery route allow rapid entry of
flurbiprofen into the circulation, it achieves higher plasma concentrations
than an equivalent, orally administered dose
(Davies, 1995).
The pharmacodynamic characteristics of the inhalable flurbiprofen
formulation are as dramatic as its pharmacokinetics. Inhalation of 2 mg of
flurbiprofen afforded almost complete neuroprotection, as indicated by >90%
retention of motor function relative to the unlesioned side, and >90%
reduction of lesion volume compared with vehicle-treated control. Moreover, 2
mg of inhaled flurbiprofen was 2 to 6 times more effective than the same dose
administered orally, which was only slightly more efficacious from a
neurobehavioral standpoint than vehicle. Indeed, oral administration of 50 mg
of flurbiprofen was necessary to provide almost complete neuroprotection
(66–86% preservation of behavioral and histological indices). Together,
the pharmacokinetic and pharmacodynamic observations indicate that the
pulmonary route of drug administration is capable of rapidly delivering an
agent into the circulation and hence, the brain, whereas achieving higher
plasma levels than oral administration. Moreover, acute, as opposed to
chronic, administration of a COX-2 inhibitor in close temporal association
with the onset of a neurological insult may prove to be the most effective way
to minimize neuronal damage (Gilroy et
al., 1999; Dash et al.,
2000).
The underlying mechanisms responsible for the profound neuropreservation
observed after the pulmonary administration of such small amounts of
flurbiprofen remain unclear. After an acute insult to the brain, COX-2
expression increases in two phases. Initially, glutamate receptor activation
rapidly increases neuronal COX-2 expression
(Hewett et al., 2000;
Manev et al., 2000) and the
production of reactive oxygen species. In addition to suppressing necrosis,
reducing free-radical damage to the mitochondria by COX-2 inhibition would
reduce the probability of neuronal apoptosis manifested many days after the
initial insult (Luetjens et al.,
2000). Subsequent to this initial insult, cellular inflammatory
processes would increase the amount of active COX-2 available at the lesion
site (Luo et al., 1998),
expanding the neurodegeneration beyond the initial area impacted
(Barone and Feuerstein, 1999).
Based on our observations, rapid suppression of the initial, glutamate
receptor-stimulated activation of COX-2 expression by inhaled flurbiprofen
seems sufficient to reduce the immediate neuronal damage that would trigger
subsequent neurodegeneration by inflammatory mechanisms. Therefore, an
inhalable, rapidly acting preparation of flurbiprofen holds promise as a
neuroprotectant in cases where the time to achieve effective concentrations in
the target organ is a critical factor, as in acute CNS insults.
In summary, the current investigation compares the efficacy of three
different classes of COX inhibitors dispensed using two different
administration modalities to an animal model of excitotoxic neurodegeneration.
We demonstrate significant histological preservation and functional protection
with both nonselective and COX-2-selective inhibitors, with even more robust
effects achieved by using pulmonary over oral routes of administration. Thus,
an inhalable formulation of a NSAID may have a significant impact on the
severity of acute neurological insults, such as stroke and trauma
(Hurley et al., 2002), where
time is critical in establishing a therapeutically effective dose and where
COX-2 activity in the CNS is the target.
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Footnotes |
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ABBREVIATIONS: COX, cyclooxygenase; PG, prostaglandin; CNS, central nervous system; NSAID, nonsteroidal anti-inflammatory drug; FLURBI, flurbiprofen; QA, quinolinic acid; NeuN, neuron-specific nuclear protein; DARPP-32, dopamine and adenosine 3',5'-monophosphate-regulated phosphoprotein, 32 kDa; HPLC, high-pressure liquid chromatography; ANOVA, analysis of variance; VS, valeryl salicylate.
1 Current address: Ceregene, Inc., 9381 Judicial Dr., San Diego, CA
92121. 
Address correspondence to: Anthony S. Basile, Biological Research, Alkermes, Inc., 88 Sidney St., Cambridge, MA 02139. E-mail: anthony.basile{at}alkermes.com
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, contralateral to brain side
receiving lesion) is compared with that of the unimpaired limb (
,
ipsilateral to lesion) in A, B, and C. Rotation scores are compared with
vehicle group in D. * and **, performance is significantly different from
corresponding unimpaired limb (p < 0.05 and 0.01, respectively); a
and aa, vehicle control group (p < 0.05 and 0.01, respectively)
two-way ANOVA + Tukey's test (A–C), one-way ANOVA + Dunnett's test
(D).
