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ABSTRACT |
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Many mediators activate eosinophils via transduction pathways involving the enzyme phosphatidylinositol 3-kinase. The initial investigation of wortmannin, a specific inhibitor of PI3-kinase, was of its
effect on human and guinea pig eosinophil superoxide (O2
) release and degranulation in vitro. Subsequently, the effect on allergen- and Sephadex-induced bronchial inflammation and airway hyperresponsiveness (AHR) in vivo in guinea pigs was investigated. Wortmannin potently inhibited complement C5a-induced O2 generation and eosinophil peroxidase (EPO) release from human eosinophils, with 50% inhibition produced by a 1-10 nM concentration. Both aerosol allergen challenge of sensitized guinea pigs and intravenous injection of Sephadex beads in normal guinea pigs caused,
in 24 h, significant eosinophilia and increased EPO activity in bronchoalveolar lavage fluid (BALF) and AHR to intravenous acetylcholine and histamine. In the allergic model, intranasal pretreatment
with wortmannin had no effect on BALF eosinophilia, but dose dependently inhibited BALF EPO activity. At 1 mg/kg, the drug abolished the AHR to histamine, but not acetylcholine. In the Sephadex
model, the drug significantly inhibited all three parameters (eosinophilia, increased EPO activity, and AHR to both spasmogens). These
results show that wortmannin is a potent inhibitor of human eosinophil degranulation and that when administered intranasally can
prevent AHR in allergen-challenged guinea pigs, probably by inhibiting eosinophil degranulation, but not their accumulation in BALF.
This may be relevant to the possible clinical utility of wortmannin in
conditions involving eosinophilic inflammation and AHR.
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
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Keywords: airway hyperresponsiveness; eosinophils; guinea pigs; wortmannin
Lung inflammation and airway hyperresponsiveness (AHR) are important components of bronchial asthma (1, 2), and several reports have shown that eosinophil accumulation and subsequent activation in bronchial tissues play a critical role in the pathophysiology of the disease (3, 4).
Although the exact causal relationship between bronchial eosinophilic inflammation and AHR is not clearly understood, it is currently believed that the eosinophils infiltrating the asthmatic lung degranulate to release tissue-damaging granular proteins such as the major basic protein and eosinophil peroxidase (EPO), as well as release oxygen free radicals. These mediators, acting in concert, may then cause airway epithelial damage, resulting in the development of AHR (5, 6).
Wortmannin is a relatively specific inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase) (7). The use of this inhibitor has revealed the involvement of PI 3-kinase in the biochemical transduction of activation signals generated by many inflammatory mediators in eosinophils. For example, in isolated
human eosinophils, wortmannin potently blocks neuropeptide-induced chemotaxis (8) and interleukin 5 (IL-5)-induced 
2-integrin-dependent adhesion to intercellular adhesion molecule
type 1 (ICAM-1)-coated surfaces (9). It also inhibited C5a-
induced generation of leukotrienes (10), phosphoinositide metabolism, and production of reactive oxygen metabolites (11,
12). In guinea pigs, Palframan and coworkers (13) showed that
wortmannin inhibited IL-5-induced selective release of eosinophils from perfused bone marrow, as well as selective eosinophil chemokinesis in vitro. These observations may suggest a
potential effect for wortmannin in preventing the development of AHR in vivo, either by preventing the eosinophil infiltration of bronchial tissues or their activation on arrival. Despite the studies described above, none, to our knowledge,
have examined the effect of this drug in vivo in allergen-induced
eosinophilic lung inflammation and/or AHR in any animal
models. One reason may be that wortmannin, being an inhibitor of a widely distributed enzyme, may have an effect too unspecific to be useful.
In the present study, we set out first to confirm that wortmannin inhibits eosinophil responses in vitro and subsequently to study its effect on bronchial inflammation and AHR. To do this, we employed two guinea pig models, the allergen- and Sephadex-induced models of bronchial inflammation/AHR, and administered the drug by intranasal instillation to localize the effect to the lungs. The study also allowed us to assess the relative role of airway eosinophilia and eosinophil degranulation in the development of AHR in guinea pigs.
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Isolation of Human Blood Eosinophils
Granulocytes were isolated from citrate-anticoagulated (13 mM final concentration) blood (14) from consenting healthy adults according to a protocol approved by the Faculty of Medicine Ethics Committee (Kuwait University, Safat, Kuwait). Eosinophils were purified from the granulocyte fraction by the immunomagnetic method (15), using mouse anti-human CD16 monoclonal antibody to positively extract neutrophils. Eosinophils were 98% pure and greater than 98% viable.
Allergen- and Sephadex-induced Lung Inflammation
Male Dunkin Hartley guinea pigs (300-500 g) were sensitized by intraperitoneal injection of 10 µg of ovalbumin (OA) and 5 mg of Al(OH)3 in 1 ml of sterile saline on Days 1 and 7. Controls received Al(OH)3 alone. Twenty-one to 28 d later, all animals were pretreated with pyrilamine maleate (10 mg/kg, intraperitoneal; 1 h prior), and challenged with aerosolized OA (0.5% [w/v] in saline) or saline for 30 min.
For the Sephadex model, lung inflammation, was induced by a single intravenous injection, via the foot vein under local anesthetic (2% xylocaine solution) and light ether anesthesia, of a sterile suspension of Sephadex G-50 beads (24 mg/kg in saline) as previously described (16). Inflammation and hyperreactivity were assessed 24 h later.
Bronchoalveolar Lavage, Cell Count, and Isolation of Guinea Pig Bronchial Eosinophils
Animals were killed with an overdose of pentobarbitone sodium and bronchoalveolar lavage was performed as previously reported (17). The cells were separated and the supernatant was saved for determination of EPO activity. Total and differential counts were performed by using a standard hemocytometer and Wright-Giemsa-stained cytosmear.
Guinea pig eosinophils were isolated from normal animals and purified by centrifugation over a Percoll gradient, and eosinophils (92-
98% purity) were recovered as the pellet. The purified cells were then
washed twice, counted, and then used for in vitro superoxide anion
(O2) release experiments.
In Vitro Release of Superoxide Anions and Eosinophil Peroxidase
Superoxide anion generation from eosinophils was determined by the
superoxide dismutase-inhibitable reduction of ferricytochrome c (18).
Purified cells were incubated with wortmannin for 10 min, stimulated
with C5a (human) or platelet-activating factor (PAF, guinea pig), and
incubated at 37° C for 1 h, and the absorbance was read at 550 nm.
The amount of O2 generated was estimated as nanomoles of ferricytochrome c reduced per 106 cells per hour, using the extinction coefficient of 2.1 × 104/M/cm.
For EPO release, human eosinophils were incubated with cytochalasin B (5 µg/ml) for 5 min, and then stimulated with C5a for 30 min. Cells were removed and EPO was measured in the supernatant, as well as in Triton X-100-lysed cells.
EPO activity was measured by the o-phenylenediamine method as previously reported (19). For EPO released from human eosinophils in vitro, values were expressed as a percentage of total cell content, using the amount obtained in half the same number of cells, after lysis, as 50%. The amount of EPO in guinea pig bronchoalveolar lavage fluid (BALF) was determined from a standard curve constructed with horseradish peroxidase (Type 1, 0.1-15 ng/ml; Sigma, St. Louis, MO).
Treatment Groups and Drug Administration
For the assessment of AHR and BALF EPO activity, the data from saline/saline (SAL/SAL) and OA/SAL groups were combined into a control group (CONTR), as there were no significant differences between them with regard to these two parameters.
Wortmannin (dissolved in 10% dimethyl sulfoxide [DMSO, in saline]) or vehicle was administered in a volume of 200 µl by intranasal instillation to each animal 1 h before and 3 h after allergen challenge or Sephadex injection.
Measurement of Lung Function
Guinea pigs were anesthetized with urethane (175 mg/kg, intraperitoneal) and paralyzed with pancuroniun bromide (50 µg/kg, intravenous). They were ventilated with room air (8 ml/kg, 1 Hz) using a model 683 ventilator (Harvard Apparatus, Natick, MA). Air flow and transpulmonary pressure were measured as previously reported (20). Lung resistance (RL) and dynamic compliance (Cdyn) were calculated by a digital lung function recording system (Mumed Systems, London, UK).
To assess airway hyperresponsiveness, acetylcholine (ACh) and histamine, both in the range of 1-128 µg/kg, were administered intravenously at 5-min intervals via a cannulated femoral vein. In experiments in which both spasmogens were tested, ACh was used first.
Drugs and Chemical Reagents
Recombinant human C5a, wortmannin, o-phenylenediamine, PAF, histamine dihydrochloride, ACh, urethane, Sephadex G-50, Percoll, and cytochalasin B were obtained from Sigma. Mouse monoclonal anti-human CD16 antibody (clone FcR gran1) was obtained from CLB (Amsterdam, The Netherlands). Immunomagnetic beads were supplied by Dynal (Oslo, Norway).
Statistics
Experimental data are presented as means ± SEM. For in vitro experiments, statistical significance was determined by one-sample t test (InStat; GraphPad Software, San Diego, CA). For the in vivo experiments, treatment groups were compared by one-way analysis of variance (ANOVA), and when significance was indicated, the Bonferroni test was applied. In all cases, significance was set at p < 0.05.
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RESULTS |
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Effect of Wortmannin on Human and Guinea Pig Eosinophil Responses in Vitro
As shown in Figure 1A, preincubation of human eosinophils with
wortmannin caused a concentration-dependent inhibition of O2
release induced by 108 M C5a. In the presence of cytochalasin B
(5 µg/ml), C5a induced EPO release from the eosinophils, which
was similarly inhibited by wortmannin. The concentration of the
drug producing 50% inhibition of response (IC50) (95% confidence interval, 95% CI) was 2.4 (1.2-4.5) nM for O2 release and
7.8 (3.6-11.8) nM for EPO release. In the same concentration range, the drug also inhibited PAF-induced O2 release from
guinea pig bronchial eosinophils, with an IC50 (95% CI) of 6.5 nM
(3.8-9.2 nM) (Figure 1B). The effect of wortmannin on EPO release from guinea pig eosinophils was not determined because of
the lack of an appropriate inducer for this response.
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Effect of Wortmannin on Allergen- and Sephadex-induced Cell Content of BALF
As shown in Table 1, the "saline-sensitized" and challenged control group that had been treated with intranasal saline 24 h before bronchial lavage (SAL/SAL+SAL) had a baseline total cell count of 45.83 (±6.42) × 106 cells, of which 13.9% were eosinophils. Intranasal instillation of such animals with the drug vehicle alone (SAL/SAL+VEH) had no significant effect on the total cell count, but significantly increased the number of eosinophils (6.25 [±0.80] versus 12.2 [±4.61] × 106 cells, p < 0.05) and percentage neutrophils (0.4 [±0.2] versus 1.3 [±0.3]%, p < 0.05). No further significant change was seen when such animals were treated with wortmannin at 1 mg/kg (SAL/SAL+ WTM). In OA-sensitized and challenged guinea pigs that had been pretreated with the drug vehicle (OA/OA+VEH), there was a highly significant increase in the number of cells recovered from BALF compared with the appropriate control group (OA/SAL+VEH) (110.53 [±11.30] versus 53.85 [±15.81] × 106 cells, p < 0.01). This increase was almost exclusively due to increase in eosinophil count (48.62 [±3.10] versus 12.78 [±0.49] × 106 cells, p < 0.001). Pretreatment of such animals with wortmannin (0.3-1 mg/kg, intranasal), produced no significant effect on BALF eosinophilia. The drug vehicle alone had no significant effect on any of these parameters in allergen-sensitized and challenged animals.
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Similarly, 24 h after intravenous administration of Sephadex into naive guinea pigs, there was a significant increase in the total cells recovered from BALF (115.70 [±15.13] × 106 cells in Sephadex-injected guinea pigs, compared with 52.62 [±6.80] × 106 cells in saline-injected guinea pigs, p < 0.01, both pretreated with drug vehicle) (Table 2). As seen in the allergen model, most of the increase was in the eosinophil count. In striking contrast to the allergen model, pretreatment with wortmannin (1 mg/kg) completely abolished the increase in total cell number, although no changes were apparent in the percentage composition of the different cells.
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Effect of Wortmannin on BALF EPO Activity of Allergen- and Sephadex-induced Lung Inflammation
The amount of EPO in the BALF supernatant of allergen-sensitized and challenged guinea pigs was significantly increased when compared with the controls, both pretreated with drug vehicle (8.8 ± 1.3 ng/ml [n = 8] versus 1.7 ± 0.5 ng/ml [n = 7]; p < 0.01) (Figure 2A). Pretreatment with wortmannin (0.3-1 mg/kg, intranasal) caused a dose-dependent decrease in the EPO content of BALF, with the 1 mg/kg dose producing a statistically significant inhibition of 45.5%, p < 0.05.
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Sephadex injection also caused a significant increase in BALF supernatant EPO after 24 h, when compared with the saline- injected group (7.3 ± 0.7 ng/ml [n = 6], versus 1.6 ± 0.3 ng/ml [n = 8]; p < 0.001), (Figure 2B). Pretreatment with wortmannin (1 mg/kg) significantly inhibited the increase by 41.1%, p < 0.05.
Effect of Wortmannin on Allergen- and Sephadex-induced AHR
Twenty-four hours after a single aerosol allergen challenge of sensitized guinea pigs, significant AHR to both ACh and histamine was seen (Figure 3A and 3B). This was characterized mainly as an increase in the slope of the dose-response curve of the spasmogens. For example, at 64 µg of ACh per kg, the increase in RL was 401.7 ± 108.0 cm H2O/L per s (n = 6) in vehicle-pretreated controls compared with 824.0 ± 122.5 cm H2O/L per s (n = 6) in sensitized and allergen-challenged animals, also pretreated with the drug vehicle, p < 0.05 (Figure 3A). For histamine (32 µg/kg), the corresponding values were 370.3 ± 104.0 cm H2O/L per s (n = 5), and 692.2 ± 132.3 cm H2O/L per s (n = 5), p < 0.05 (Figure 3B). For both spasmogens, there was little or no increase in the sensitivity. The pretreatment of allergen-sensitized and challenged animals with wortmannin (1 mg/kg, intranasal), a dose that had been established to be effective in inhibiting BALF EPO release, produced only a small, statistically nonsignificant attenuation of the allergen-induced AHR to ACh (Figure 3A). In contrast, AHR to histamine was almost completely abolished by the treatment. In four or five experiments, neither the vehicle (10% DMSO, 400 µl, intranasal) nor wortmannin (1 mg/kg, intranasal) had any significant effect on airway responses or the baseline RL to ACh and histamine in control animals (data not shown).
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Sephadex-injected guinea pigs also developed, within 24 h, a pronounced AHR to both ACh and histamine, reflected as a leftward shift in the dose-response curve of the two spasmogens (Figure 4A and 4B). For example, the increase in RL to ACh at 32 µg/kg was 93.2 ± 30.0 cm H2O/L per s (n = 5) in control animals compared with 551.7 ± 158.8 cm H2O/L per s (n = 5) in Sephadex-injected animals (both pretreated with the drug vehicle), p < 0.01. For histamine (16 µg/kg), the increase was from 133.6 ± 41.5 cm H2O/L per s (n = 5) in the controls to 690.0 ± 119.6 cm H2O/L per s (n = 5) in Sephadex-injected animals, p < 0.01. Pretreatment with wortmannin (1 mg/kg, intranasal) completely abolished the AHR to both spasmogens.
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Correlation Analysis of AHR with BALF Eosinophilia and EPO Activity
As seen in Figure 5A and 5B, among all animals in which bronchial inflammation was induced with allergen, no significant correlation was found between BALF eosinophil count and AHR to both ACh and histamine as judged by the increase in RL to a 32-µg/kg concentration of both spasmogens. In contrast, BALF EPO activity positively correlated with AHR to both spasmogens (ACh: r = 0.4847, p = 0.0415; histamine: r = 0.4936, p < 0.0374). For the Sephadex model, a similar pattern of correlation was seen (Figure 5C and 5D).
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DISCUSSION |
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ABSTRACT
INTRODUCTION
METHODS
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Many inflammatory mediators attract and activate eosinophils
via transduction pathways involving the wortmannin-sensitive PI 3-kinase enzyme, and wortmannin has been shown to be a
potent inhibitor of a number of eosinophil responses in vitro
(8). Initially, we sought to confirm the direct effect of wortmannin on both human and guinea pig eosinophils in vitro.
The result obtained not only confirmed the high potency of
this drug in inhibiting O2 release from eosinophils of both
species, but also showed, for the first time, that the drug was
equally a potent inhibitor of human eosinophil degranulation.
In view of these observations, and given that eosinophils play
important roles in the pathophysiology of asthma, we set out
to determine whether the drug would be effective in inhibiting
bronchial eosinophilic inflammation and AHR
parameters that characterize clinical asthma. Because of the ubiquitous
nature of PI 3-kinase in the signal transduction pathway of many
cell types (7, 21), we chose to administer the drug by intranasal instillation in order to localize the effect to the lungs.
Drugs administered by this route in guinea pigs have been
shown to reach the bronchial tree easily (24). The results obtained have shown, for the first time, that intranasally administered wortmannin, in as low a dose as 1 mg/kg, could inhibit
both allergen- and Sephadex-induced AHR in vivo. In detail,
the results show some interesting points about this drug. First,
in allergen-induced lung inflammation, the drug completely
failed to prevent the increase in eosinophil influx into the
lung, but significantly attenuated the increase in BALF EPO
content (index of bronchial eosinophil degranulation) and AHR
to histamine, but surprisingly not to ACh. This might suggest
that it is the degranulation of eosinophils in the lungs, rather
than their mere accumulation, which is important for the development of allergen-induced AHR to histamine. Others (25) have reported a similar dissociation between AHR
and pulmonary eosinophilia in allergen-sensitized and challenged guinea pigs. In patients with asthma, however, BALF
eosinophil count has often been positively correlated with the
magnitude of AHR (29, 30).
Second, in contrast to the situation in allergic model, wortmannin almost completely abolished Sephadex-induced BALF eosinophil accumulation, in addition to significantly reducing BALF EPO levels and abolishing AHR. In both models, however, it is the EPO levels, not BALF eosinophil numbers, that correlated with the magnitude of AHR. This further supports the view that eosinophil degranulation plays an important role in the development of AHR in this species, as reported by others (31, 32). It has been hypothesized that the activation of eosinophils, and their subsequent release of cytotoxic cationic proteins, contribute substantially to the bronchial epithelial damage and the consequent AHR (5, 6, 33). In view of this hypothesis, one observation that is difficult to explain is why wortmannin was effective in inhibiting allergen-induced AHR to histamine, but not to ACh, in the same animals in which the drug inhibited EPO release. This may be related to the magnitude of the reflex component of their bronchoconstriction, which is greater for histamine than for ACh. A similar spasmogen-selective AHR has been reported in sensitized guinea pigs challenged with allergen (34). It is possible that in our study a higher dose of wortmannin or a longer duration of pretreatment was necessary to affect AHR to ACh. This would, perhaps, be consistent with the fact that the drug showed a trend toward reduction of AHR to ACh. There was also a positive correlation between BALF EPO levels and AHR to ACh in this model, even though the inhibition of EPO release by wortmannin was not accompanied by a significant inhibition of AHR to ACh. Furthermore, it should be realized that only at the highest dose of wortmannin tested (1 mg/kg) was a statistically significant inhibition of BALF EPO levels obtained, suggesting that this dose was the threshold of effectiveness. Unfortunately, the limitation posed by drug solubility, and the need to avoid vehicle effect that would likely occur with greater than 10% DMSO, did not allow us to test higher doses by this route of administration.
Third, the drug clearly distinguished between allergen- and Sephadex-induced eosinophil accumulation by significantly inhibiting this response when induced by the latter, but not the former. This would suggest an important difference in the mediators or mechanisms mediating eosinophil chemotaxis in the two models. Unlike in allergen-induced eosinophil accumulation, for which several eosinophil chemotactic mediators such PAF, IL-5, and eotaxin are known to be involved (35), the mediators involved in Sephadex-induced lung eosinophil accumulation in guinea pigs are less well defined although IL-5 (38) and complement activation products (39) have been proposed. Differential mediator involvement may also contribute to the differences in the ability of wortmannin to inhibit AHR to ACh in the two models.
Because the inhibition of AHR by wortmannin in both models was invariably accompanied by significant inhibition of EPO release, and given that in both models the magnitude of AHR positively correlated with BALF EPO levels, but not eosinophil count, it could be concluded that wortmannin inhibited AHR by virtue of its ability to inhibit eosinophil degranulation. However, because the drug could abolish AHR even when it reduced the elevated BALF EPO levels by only about 50%, it is possible that other eosinophil-independent mechanisms may contribute to its ability to prevent AHR. Wortmannin-sensitive PI 3-kinase enzyme appears to play signal transduction roles in many cell types other than eosinophils (7, 21). Although the intranasal route of administration adopted in this study may have ensured that systemic effects were minimized, many other cell types within the lungs may have been targets as well. For example, the drug may have inhibited the activity of resident T cells, macrophages, or epithelial cells, preventing their release of inflammatory cytokines and neurokinins that may contribute to the development of AHR (40).
In view of the high potency with which wortmannin inhibited human eosinophil degranulation in vitro, is it likely that the drug will exhibit such activity in vivo in humans. If that is the case, it would be reasonable to expect that it would prevent the development of allergic as well as nonallergic AHR in humans, as has been seen in guinea pigs in this study. This possibility would be consistent with the close relationship that is known to exist between bronchial eosinophil degranulation and AHR in individuals with asthma (3, 30).
In summary, wortmannin has a direct inhibitory effect on guinea pig and human eosinophils in vitro. When given by intranasal instillation to guinea pigs, it significantly inhibited allergen-induced AHR in a spasmogen-selective manner, but without affecting BALF eosinophilia. In the Sephadex model, it inhibited all parameters assessed. The ability to prevent AHR appears to be due, at least in part, to the inhibition of degranulation of bronchial eosinophils rather than the prevention of their accumulation in the lungs. These results are relevant to the possible clinical application of this class of drug in the treatment of airway diseases characterized by eosinophilic inflammation and AHR.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Charles I. Ezeamuzie, Ph.D., Department of Pharmacology, Faculty of Medicine, Kuwait University, P.O. Box 24923, Safat 13110, Kuwait. E-mail: ezeamuzie{at}hsc.kuniv.edu.kw
(Received in original form January 24, 2001 and accepted in revised form July 23, 2001).
Acknowledgments:
Supported by grant MR 039 from Research Administration, Kuwait University.
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References |
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