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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Department of Pharmacology, East Carolina University, Greenville, North Carolina (P.C.M.O., A.N., S.J.M.); and Endacea Inc. (V.K.B., P.B., C.N.W.), Research Triangle Park, North Carolina
Received for publication
April 19, 2005
Accepted
July 12, 2005.
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Abstract |
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; Polosa, 2002; Rorke and Holgate, 2002). Adenosine levels are increased in the bronchoalveolar fluid of asthmatics and also in the plasma of patients with exercise-induced asthma (Driver et al., 1993; Vizi et al., 2002). There is also an association between allergen exposure and adenosine monophosphate (AMP) responsiveness in asthmatics (Currie et al., 2003). In asthma, the airway response to AMP seems to correlate more closely with disease activity than the response to other more conventional provocative agents, such as methacholine (de Meer et al., 2002).
Adenosine may contribute to the pathogenesis of airway responsiveness and airway inflammation associated with asthma by acting on specific cell surface ARs (Polosa, 2002; Rorke and Holgate, 2002; Livingston et al., 2004). Adenosine receptors belong to the superfamily of receptors known as G protein-coupled receptors. Four adenosine receptor subtypes, A1, A2A, A2B, and A3, are expressed in the lung, have been cloned in humans, and have been investigated as potential targets for drug development in asthma (Bjorck et al., 1992; Polosa, 2002; Rorke and Holgate, 2002). By acting through A1 ARs on a number of different human cell types, adenosine produces bronchoconstriction, inflammation, increased endothelial cell permeability and mucin production, a cardinal feature of airway remodeling, which increase airflow obstruction in asthma (Cronstein et al., 1990, 1992; Salmon et al., 1993; Marquardt, 1997; Wilson and Batra, 2002; McNamara et al., 2004).
Emerging scientific and clinical data support that the A1 AR is an important AR target in human asthma. Bamifylline is an A1 AR antagonist approved for the treatment of asthma in Europe (Abbrachio and Cattabeni, 1987; Catena et al., 1988; Morandini, 1988). Theophylline produces its anti-asthma effects in humans with an effective therapeutic plasma level that is less than that required to inhibit human phosphodiesterase enzymes and that would produce antagonism of ARs (Barnes, 2003). Further validation that the A1 AR is an important target in human asthma is supported by positive results in human asthmatics from early clinical trials with EPI 2010, a respiratory antisense oligonucleotide to the human A1 AR (Ball et al., 2003).
Previously, it was reported that the allergic rabbit model simulates the human condition of asthma (Metzger et al., 1989; Herd and Page, 1996). Both allergic rabbits and allergic humans behave similarly to airway hyperreactivity to adenosine, histamine, acetylcholine, and platelet-activating factor. After an inhalational challenge, adenosine increases airway reactivity in both allergic humans and allergic rabbits, but not in nonallergic, normal humans, and normal rabbits (Cushley et al., 1983; Ali et al., 1992a, 1994c). With the use of selective pharmacological probes for ARs, the A1 AR clearly mediates adenosine-induced acute bronchoconstrictor responses in the allergic rabbit model of asthma (Ali et al., 1994c; El-Hashim et al., 1996; Nyce and Metzger, 1997). Moreover, in small airways from allergic rabbits, the expression of the A1 AR is increased compared with that in small airways from normal rabbits (Ali et al., 1994b).
L-97-1 is a water-soluble small molecule A1 AR antagonist with high affinity and high selectivity for the human A1 AR. It is under development as an oral antiasthma treatment for humans. In an allergic rabbit model of asthma, after oral administration, the effect of L-97-1 on house dust mite (HDM) allergen induced early (bronchoconstrictor) and late (inflammatory) allergic responses (EARs and LARs, respectively) as well as histamine and adenosine-induced bronchial hyper-responsiveness were determined.
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Materials and Methods |
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). Sensitized rabbits show higher titers of HDM-specific IgG (0.505-1.097 U) compared with nonsensitized rabbits (0.062-0.262 U). The rabbits were kept in community cages with 12-h periods of light and dark cycles and were maintained on a standard laboratory rabbit diet with access to water ad libitum. All animal care and experimentation was approved and carried out in accordance with the East Carolina University Institutional Animal Care and Use Committee and in accordance with the principles and guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Pulmonary Function Measurements. Four months after birth, the rabbits were screened for adenosine/histamine sensitivity by measuring lung dynamic compliance (Cdyn) changes in response to aerosol administration of serial dilutions of adenosine or histamine (0.17-20 mg/ml). Briefly, rabbits were anesthetized and relaxed with 1.5 ml of a mixture of ketamine hydrochloride (35 mg/kg) and acepromazine maleate (1.5 mg/kg) administered intramuscularly. After induction of anesthesia, the rabbits were laid supine on a soft molded animal board in a comfortable position. A salve was applied to the eyes, and they were closed. Each animal was then intubated with a 3.0-mm flexible cuffed Murphy 1 endotracheal tube (Webster Veterinary Supplies, Charlotte, NC) as described previously (Zavala and Rhodes, 1973). A polyethylene catheter of 
2.4 mm o.d. (BD Biosciences, Clay Adams, Parsippany, NJ) with an attached thin-walled latex balloon was passed into the esophagus and maintained at the same distance (16 cm) from the mouth throughout the experiment. The endotracheal tube was attached to a heated Fleish Pneumotach (size 00; DEM Medical, Richmond, VA) and flow (V) was measured using a Validyn differential pressure transducer (model DP-45-16-1927; Validyn Engineering, Northridge, CA) driven by a Gould carrier amplifier (model 11-4113; Gould Electronics, Cleveland, OH). The esophageal balloon was attached to one side of Validyn differential pressure transducer, and the other side was attached to the outflow of the endotracheal tube to obtain transpulmonary pressure (Ptp). Flow was integrated to give a continuous tidal volume. Measurements of total lung resistance (Rt) and dynamic compliance (Cdyn) were calculated at isovolumetric and zero flow points. Recording of flow, volume, and pressure were made amplified on an eight-channel Gould 2000W high-frequency amplifier. Cdyn was calculated using total volume and the difference in Ptp at zero flow. Rt was calculated as the ratio of Ptp and V at midtidal lung volumes. These calculations were made automatically with the Buxco automatic pulmonary mechanics respiratory analyzer (Biosystem XA system; Buxco Electronic Inc., Sharon, CT) running on a personal computer, as described previously (Giles et al., 1971). A period of 15 min is allowed after intubation to allow the animals to attain a steady baseline respiration before any procedure.
Measurement of Bronchial Hyper-Responsiveness (BHR). At 4 months, each of the sensitized rabbits were initially administered histamine or adenosine by aerosol to determine its baseline hyperresponsiveness. Aerosols of either normal saline, histamine, or adenosine were generated by a DeVilbiss nebulizer (DeVilbiss, Somerset, PA) for 2 min at each dose. The ultrasonic nebulizer produced aerosol droplets, of which 80% were <5 µm in size. Histamine or adenosine aerosol was administered in increasing concentrations (0.17-10 mg/ml) with measurements of pulmonary function for 15 min after 3 min of aerosol for each dose. Animals were not exposed to higher doses of histamine or adenosine after their PC30 [concentration (milligrams per milliliter) required to produce 30% reduction in Cdyn] was reached. An initial saline aerosol was used to establish baseline. Pulmonary function was summated every 10 breaths of even gradation. Data from spastic breathing were filtered out as artifact. The drug-induced response for each treatment was taken as the lowest consistent Cdyn value with 3 min of treatment. This point was typically achieved within the first 2 min after treatment. Allergic rabbits that do not attain a PC30 to histamine or adenosine above 20 and 10 mg/ml, respectively, were excluded from the study. Less than 1% of all sensitized rabbits did not attain this PC30 to histamine or adenosine.
Effect of L-97-1 on Allergen-Induced EAR and LAR. Sensitized rabbits exposed to allergen aerosol are susceptible to an allergic response characterized by a phasic bronchoconstriction (EAR) and airway inflammation (LAR). The following procedure was designed to investigate the effect of L-97-1 on HDM allergen-induced EAR and LAR in the allergic rabbit model. Rabbits that had not been used for any airway provoking procedure were anesthetized and intubated as described above. After a steady baseline respiration is attained, the animals were aerosolized with 2500 AU of HDM allergen for about 10 min or until the allergen was exhausted. Pulmonary function (Cdyn) was then measured at 15-min intervals during the next 6 h to determine the effect of HDM allergen on early (0-60 min) and late (120-360 min) allergic responses (EAR and LAR, respectively). The same procedure was repeated with L-97-1 (10 mg/kg) oral gavage administered 1 h before allergen challenge in the same animals after at least a 2-week washout period. Anesthesia was maintained with a mixture of ketamine/acepromazine (80:20) at a dose of 0.15 mg/kg given every 45 min to 1 h.
Effect of L-97-1 on Allergen-Induced BHR to Histamine. To determine the effect of L-97-1 on BHR, the following protocol was used. Allergic rabbits that had not been aerosolized with allergen, histamine, or adenosine for at least 2 weeks (n = 5) were aerosolized with histamine as described above to determine their baseline PC30 to histamine and BHR to histamine. Twenty-four hours later, the rabbits were given an aerosol challenge of 2500 AU of HDM allergen. Then, BHR measurement with histamine aerosolized challenge was repeated at 24 h after allergen challenge. The same protocol was employed to test the effectiveness of L-97-1 (10 mg/kg) administered as an oral gavage 1 h before HDM allergen challenge on BHR to histamine.
Effect of L-97-1 on BHR to Adenosine. To determine the effect of L-97-1 on BHR to adenosine, the following protocol was used. Allergic rabbits that had not been aerosolized with allergen, histamine, or adenosine for at least 2 weeks (n = 5) were aerosolized with adenosine as described above to determine their baseline PC30 to adenosine. The next day, 1 h after oral administration of L-97-1 (10 mg/kg), measurements of BHR to adenosine were again taken at 15 min, 6 h, and 24 h in the same rabbits.
Plasma Levels of L-97-1. An ear artery sample of blood was collected in tubes containing EDTA at 0 min, 15 min, 30 min, 1 h, 2 h, 3 h, 6 h, 8 h, and 24 h after L-97-1 administration. The samples were centrifuged at 5000g for 5 min, and plasma was collected and frozen at -20°C until used. Serum levels of L-97-1 were measured by electrospray liquid chromatography/tandem mass spectrometry method validated at Prevalere Life Sciences, Inc. (Whitesboro, NY).
Effect of L-97-1 on Contractile Responses in in Vitro Muscle Tension Studies in Small Airways of Allergic Rabbits. Allergic rabbits were euthanized with sodium pentobarbital (100 mg/kg i.v) in accordance with the guidelines of the Animal Use and Care Committee of the Brody School of Medicine (East Carolina University). Lungs were removed and immediately placed in oxygenated, ice-cold Krebs-Henseleit buffer, pH 7.4. Secondary (5 mm) and tertiary (2-4 mm) airways were dissected out of the lung tissue. During the dissection, tissue was immersed in ice-cold oxygenated buffer. Bronchioles were cut into small rings and mounted in 10-ml organ baths with stainless steel hooks and surgical thread (000) with a resting tension of 500 mg. Organ baths contained oxygenated and heated (37°C) Krebs-Henseleit buffer. Bronchiole rings were equilibrated with the organ bath environment for 2 h, with a complete change of buffer every 15 min. Contractions of each bronchiole ring were expressed as a percentage of the force measured when rings were treated with 50 mM KCl. Isometric tension was measured by force displacement transducers (BIOPAC Systems Inc., Santa Barbara, CA) connected to BIOPAC MP100 data acquisition and analysis hardware from BIOPAC Systems Inc. After bronchioles were stimulated with KCl and the buffer in each organ bath was exchanged three times in rapid succession, tissues were given a 30 min recovery period before challenge with the increasing concentrations of the nonselective AR agonist 2-chloroadenosine (2-CADO) (5 x 10-5 and 10-4 M). After washing out the agonist from the organ baths, the bronchioles were given a 30-min period to return to baseline tension before a 30-min treatment with L-97-1 at a concentration of 10-6 or 10-5 M. Bronchioles were again challenged with the same concentrations of 2-CADO. This protocol was repeated on the bronchioles using a single dose of histamine (5 x 10-6 M) before and after treatment with L-97-1. Each experimental condition was performed in bronchioles from three different rabbits in replicates of 8.
Radioligand Binding Assays. To determine the affinity for L-97-1 for the human A1 AR, A2A AR, and A2B ARs, the following protein sources were used: membranes from human pulmonary artery endothelial cells (Cambrex Bio Science Walkersville, Inc., Walkersville, MD) expressing the human A1 AR and membranes from human embryonic kidney 293 cells expressing the recombinant human A2A and human A2B ARs (Receptor Biology, Inc., Beltsville, MD).
Human Pulmonary Artery Endothelial Cells (PAECs): Culture and Membrane Preparation. Human PAECs were obtained from Cambrex Bio Science Walkersville, Inc., and grown in a multilayer tissue culture vessel for membrane preparation in an atmosphere of 95% O2 and 5% CO2. The cells were grown and maintained in medium recommended by the manufacturer, EMB-2 (Cambrex Bio Science Walkersville, Inc.) which contains 2.0% fetal bovine serum. The cells were washed three times with phosphate-buffered saline and then suspended in lysis buffer (10 mM Tris HCl, pH 7.4, containing 5 mM EDTA, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml benzamidine, and 2 µg/ml pepstatin). The cells were homogenized by sonication. The homogenate was centrifuged at 1000g at 4°C for 10 min. The supernatant was centrifuged at 30,000g for 45 min. The pellet was reconstituted in reconstitution buffer (50 mM Tris HCl, pH 7.4, 5 mM EDTA, 10 mM MgCl2, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml benzamidine, and 2 µg/ml pepstatin). The protein content was determined by Bradford reagent using bovine serum albumin as standard. The aliquots were stored at -80°C until used.
Determination of the Affinity of L-97-1 for Human A1 and Recombinant Human A2A, and A2B Adenosine Receptors in Inhibition, Competition Radioligand Binding Assays. Radioligand competition binding experiments were performed with membranes from human PAECs or human embryonic kidney-transfected cells in a total volume of 0.2 ml in incubation buffer at room temperature with the selective adenosine receptor antagonist radioligands and under the conditions as determined or recommended by the supplier of the membranes that are presented in Table 1. After incubation, the samples were filtered rapidly under vacuum through polyethyleneimine-treated filters and washed four times with 3 ml of ice-cold buffer using a cell harvester (Skatron, Lier, Norway). The filters were dried and counted for radioactivity with the use of a liquid scintillation counter (PerkinElmer Life and Analytical Sciences, Boston, MA). Two to four experiments were performed and assayed in duplicate.
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The affinity of L-97-1 for the recombinant human A3 AR was determined in another laboratory (Dr. Gary L. Stiles, Duke University Medical Center, Durham, NC) in Chinese hamster ovary cells expressing the recombinant human A3 AR with the use of N6-(4-amino-3-[125I]iodobenzyl)adenosine-5'-N-methylcarboxamide, as the competing radioligand in competition radioligand binding assays (n = 2).
To further validate the selectivity of L-97-1 for the human A1 AR, the affinity of L-97-1 for a number of other receptors was determined by NovaScreen (Hanover, MD) with the use of radioligand competition binding assay protocols similar to that described above and selective radioligands for the following receptors. L-97-1 (10 µM) was tested in competition radioligand binding assays for the rat adrenergic, 
1 and 2, peripheral benzodiazepine, nonselective dopamine, glutamate [-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate, NMDA agonist, and glycine NMDA sites), strychnine-sensitive glycine, H1 histamine, H2 histamine, and H3 histamine, nonselective central muscarinic, nonselective peripheral muscarinic, nonselective serotonin, and nonselective opiate receptors.
Assay for Inhibition of Human Phosphodiesterase Enzymes. Assays for inhibition of human phosphodiesterase enzymes II, III, IV, and V, were determined by Cerep (Celle l'Evescault, France). Test compounds, L-97-1 (100 µM) and theophylline (100 µM), were tested in duplicate. In each experiment, the respective reference compound was tested at a minimum of seven concentrations in duplicate to obtain an inhibition curve to validate the experiment. Radioactivity was determined with a scintillation counter (TopCount; PerkinElmer Life and Analytical Sciences). General procedures and experimental conditions are given in Tables 2 and 3, respectively.
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Chemicals. [3H]DPCPX and [3H] CGS 21680 were purchased from PerkinElmer Life and Analytical Sciences. (R)-N6-Phenylisopropyladenosine, 2-chloro-N6-cyclopentyladnenosine, 5'-N-ethylcarboxamidoadenosine, DPCPX, and all other common reagents of analytical grade were purchased from Sigma-Aldrich (St. Louis, MO). L-97-1 was custom synthesized by ChemSyn Laboratories (Lenexa, KS) and provided by Constance N. Wilson (Endacea, Inc., Research Triangle Park, NC).
Statistical Analysis. To assess the effect of L-97-1 on the changes in pulmonary function after adenosine, histamine, and allergen challenge, the area under the curve in square centimeters is digitized by computer-assisted plenometry for each rabbit during a 6-h period after adenosine and allergen challenge and at 24 h after adenosine, histamine, and allergen challenge. The early and late phase responses are determined at 0 to 1 h and 1 to 6 h, respectively, as previously established in this allergic rabbit model. The percentage change in Cdyn is calculated for each time point for allergen challenge (every 15 min for 6 h). Statistical significance in time series between the control and drug-treated groups were determined by two-way multiple ANOVA (MANOVA). Comparisons between control and test values at the same time point were determined by post hoc comparison of two means. Airway hyper-responsiveness for adenosine and histamine is calculated by determining the concentration of adenosine or histamine (milligrams per milliliter) required to reduce the Cdyn by 30% from baseline (PC30). Significance for histamine responses is determined by comparing these values using ANOVA with post hoc least square difference determination between values and for adenosine responses using Kruskal-Wallis test. In the in vitro muscle tension studies statistical significance of the results was determined using the Student's t test for paired data. Results are expressed as mean ± S.E.M. A value of p < 0.05 is considered significant. Radioligand binding data were analyzed by nonlinear regression using a sigmoidal dose-response curve with variable slope (Prism version 3.0; GraphPad Software Inc., San Diego, CA).
Results from assays for inhibition of human phosphodiesterase enzymes are expressed as a percentage of inhibition of control values obtained in the presence of the test compounds. IC50 values (concentration causing a half-maximal inhibition of control values) and Hill coefficients (nH) were determined for the reference compounds by nonlinear regression analysis of their inhibition curves. These parameters were obtained by Hill equation curve fitting. The IC50 values obtained for the reference compounds have passed the required inspections. They are within accepted limits of historic averages obtained ± 0.5 log units.
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Results |
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Effect of L-97-1 on BHR to Adenosine. Figure 3 shows the effect of L-97-1 (10 mg/kg oral) administration on bronchial hyper-responsiveness to adenosine in allergic rabbits. PC30 adenosine increased significantly from baseline 4.14 ± 0.83 to 38.33 ± 1.67 mg/ml (p < 0.05; n = 5), 15 min after the single administration of L-97-1 (10 mg/kg administered as oral gavage 1 h before adenosine challenge). The PC30 after 6 h (11.67 ± 4.41 mg/ml) and 24 h (5.83 ± 2.2 mg/ml) of single administration of L-97-1 (10 mg/kg oral) was higher compared with baseline reaching statistical significance at 6 h compared with the baseline (p < 0.05; n = 5).
Plasma Levels of L-97-1. Table 4 shows the levels of L-97-1 in a 24-h period after a single oral administration of L-97-1 (10 mg/kg). The effect of L-97-1 on LAR and adenosine-induced bronchial hyper-responsiveness at 6 h after administration of L-97-1 (10 mg/kg) correlates with a plasma level of 13 ng/ml. L-97-1 blocked bronchial hyper-responsiveness significantly increasing the PC30 histamine at 24 h after allergen challenge. This lasting effect of L-97-1 at 24 h suggests that a plasma level of 3 ng/ml is an effective plasma concentration.
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Effect of L-97-1 on Contractile Responses in in Vitro Muscle Tension Studies in Small Airways of Allergic Rabbits. Figure 4 shows that in in vitro muscle tension pharmacology studies in small airways from allergic rabbits, L-97-1 (10-5 and 10-6 M) selectively blocks the contractile responses of 2-CADO (5 x 10-5 and 10-4 M) (p 
0.006) in a concentration-dependent manner without blocking those of histamine (5 x 10-6 M).
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100 µM). Moreover no binding was demonstrated for L-97-1 ( 1 mM) to the recombinant human A3 AR (data not shown). There was no binding of L-97-1 to rat adrenergic, 1 and 2, peripheral benzodiazepine, nonselective dopamine, glutamate (-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate, NMDA agonist, and glycine NMDA sites), strychnine-sensitive glycine, H1 histamine, H2 histamine, and H3 histamine, nonselective central muscarinic, nonselective peripheral muscarinic, nonselective serotonin, and nonselective opiate receptors (data not shown).
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Phosphodiesterase Inhibition Assays. The effects of L-97-1 (100 µM) and theophylline (100 µM) on the human phosphodiesterase II, III, IV, and V, enzymes are summarized in Table 6 where the IC50 values for the reference compounds are also indicated. As opposed to theophylline (100 µM), which inhibits human PDE enzymes II, III, and IV at 19, 28, and 21% inhibition, respectively, L-97-1 (100 µM) does not inhibit human PDE enzymes II, III, IV, or V.
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Discussion |
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The endogenous nucleoside, adenosine is reported to produce acute bronchoconstriction through indirect effects by inducing the release of preformed and newly formed mediators from mast cells and possibly direct effects on airway smooth muscle (ASM) and adrenergic nerve endings (Polosa, 2002; Livingston et al., 2004). Previously, it is reported that A1 ARs on ASM are a direct target for adenosine in humans and allergic rabbits (Ali et al., 1994b; Nyce and Metzger, 1997; Mundell et al., 2001). Moreover, in the allergic rabbit model of asthma, administration of a respiratory antisense oligodeoxynucleotide specific for the A1 AR, EPI 2010, reduced the density of A1 ARs on ASM and attenuated adenosine-induced acute bronchoconstriction (Nyce and Metzger, 1997). In the present study, PC30 for adenosine is increased significantly at 15 min and 6 h after oral administration of L-97-1. In previous studies, it was reported that BHR to adenosine in allergic rabbit model of asthma is mainly due to up-regulation of A1 ARs on ASM (Ali et al., 1994b; Nyce and Metzger, 1997). Thus, in the present study, L-97-1 may block the BHR to adenosine in allergic rabbits due to the antagonistic action by this A1 AR antagonist on A1 ARs on ASM.
Airway hyper-responsiveness to allergen is considered to be a hallmark of allergic asthma. The rabbit model of allergic asthma has been previously used to test antiasthma drugs that are in current use for treatment of human asthma (Ali et al., 1992a,c, 1994a). Both theophylline and beclomethasone administered as inhalational treatments inhibit EAR and LAR responses in this rabbit model of allergic asthma (Ali et al., 1992c, 1994a). Both allergic rabbits and allergic humans share many common features of asthma. These features include airway hyperreactivity to adenosine, histamine, acetylcholine, platelet-activating factor, development of inflammation and permeability changes in the airways, release of mediators, including neutrophil and eosinophil chemotactic factors, production of antigen-specific IgE antibodies, and mast cell dependence (Larsen et al., 1984; Metzger et al., 1989; Herd and Page, 1996; Gozzard et al., 1997; Gascoigne et al., 2003). Moreover, adenosine levels are increased in the bronchoalveolar fluid of both humans and rabbits with allergic asthma (Ali et al., 1992b; Driver et al., 1993).
We therefore investigated the effect of L-97-1, an A1 AR antagonist in development as an oral treatment for asthma in humans, on HDM allergen-induced EAR and LAR as well as histamine-induced increases in BHR 24 h after allergen challenge, in the allergic rabbit model of asthma. L-97-1 (10 mg/kg) administered 1 h before HDM allergen administration significantly increased Cdyn at all time points after 30 min up to 6 h, thus preventing the decline in Cdyn and blocking both EAR and LAR responses after allergen challenge in rabbits with allergic asthma. The increase in Cdyn during the EAR response can be explained by the direct blocking of A1 ARs on airway smooth muscle as some of the previous studies have suggested (Ali et al., 1994b; Nyce and Metzger, 1997). Previously, it was reported that EPI 2010 reduces EAR response in allergic rabbits and decreases the expression of A1 ARs in ASM in allergic rabbits (Nyce and Metzger, 1997). Moreover, theophylline inhibits EAR and LAR responses in the allergic rabbit model at a dose that is lower than that which would produce plasma levels to inhibit PDE enzymes (Ali et al., 1992c). The inhibitory activity of L-97-1 on A1 ARs cannot be explained by the inhibition of PDE class of enzymes, since the plasma levels of L-97-1 detected after oral administration of this compound are too low to cause any inhibition of PDE enzymes. Together, the results of these studies suggest that L-97-1 may block allergen-induced EAR responses by blocking activation of A1 ARs by adenosine present in bronchoalveolar lavage fluid of allergen-challenged rabbits. This effect of L-97-1 on EAR response in allergic rabbits may also be, in part, a function of its antagonistic effect on the release of preformed or newly formed mediators from mast cells or adrenergic nerve terminals. A1 ARs may be up-regulated in human mast cells which are immunologically sensitized by IgE (Peachell et al., 1988). The effect of L-97-1 on the release of preformed or newly formed mediators, i.e., histamine and leukotrienes, from immunologically sensitized mast cells, are studies for future investigations.
The bronchoconstrictor effect of adenosine in the asthmatic lung is mediated through its specific cell surface receptors: 1) the effects of adenosine are not reproduced by inosine, the deaminated metabolite of adenosine, or a closely related purine nucleoside, guanosine; however, adenosine mono- and diphosphates, i.e., AMP and ADP (which are rapidly converted to adenosine in the lung under physiological conditions), are equipotent with adenosine as bronchoconstrictor agents (Mann et al., 1983); 2) theophylline and bamifylline preferentially produce their antiasthma effects in humans at concentrations that do not inhibit phosphodiesterase enzymes (Foutillan et al., 1983; Ginesu et al., 1987; Catena et al., 1988; Clarke et al., 1989; Spoto et al., 1995); and 3) dipyridamole, an uptake blocker of adenosine into the cells, enhances adenosine-induced bronchoconstriction in asthmatic patients (Cushley et al., 1986).
In humans bamifylline produces its antiasthma effects by selectively blocking A1 ARs. Bamifylline binds to the human A1 AR and human A2A AR with higher affinity for the human A1 AR (1.93 µM) than the human A2a AR (12.9 µM) and does not bind to human A2B or human A3 ARs (100 µM; Constance N. Wilson, personal communication). The therapeutic plasma concentration of bamifylline is 500 times less than that needed to inhibit human II, III, IV, and V PDE enzymes (Ginesu et al., 1987; Mann et al., 1983; Constance N. Wilson, personal communication). Moreover, in humans theophylline produces its antiasthma effects most likely by blocking ARs. Theophylline binds nonselectively to all the human AR subtypes (Klotz et al., 1998). The effective therapeutic plasma concentrations for theophylline in humans is 10 to 100 times below that needed to inhibit human PDE enzymes in vitro (Clarke et al., 1989). Compared with bamifylline and theophylline, L-97-1 binds selectively to the human A1 AR. Moreover, L-97-1 produces its antiasthma effects in allergic rabbits at concentrations that are 1000 to 10,000 times less than that needed to inhibit human PDE enzymes.
Together, these data suggest that L-97-1 is a potent, selective antagonist of A1 ARs and its efficacy in inhibiting the allergen-induced EAR, LAR responses and attenuating BHR to histamine after allergen challenge and adenosine-induced BHR in allergic asthmatic rabbits is most likely related to its antagonism of A1 ARs on ASM and perhaps on mast cells, as well as other cell types, such as inflammatory cell types that play an important role in the LAR response. Activation of A1 ARs on human neutrophils and macrophages has been shown to produce proinflammatory effects (Cronstein et al., 1990, 1992; Salmon et al., 1993). The effect of L-97-1 on airway inflammation in the allergic rabbit model of asthma is an area for future investigation.
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: AR, adenosine receptor; L-97-1, 3-[2-(4-aminophenyl)-ethyl]-8-benzyl-7-{2-ethyl-(2-hydroxy-ethyl)-amino]-ethyl}-1-propyl-3,7-dihydro-purine-2,6-dione; HDM, house dust mite; EAR, early allergic response; LAR, late allergic response; AU, allergen unit; Cdyn, dynamic compliance; BHR, bronchial hyper-responsiveness; 2-CADO, 2-chloroadenosine; PAEC, pulmonary artery endothelial cell; NMDA, N-methyl-D-aspartate; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; CGS 21680, 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosine; ANOVA, analysis of variance; MANOVA, multiple analysis of variance; PDE, phosphodiesterase; ASM, airway smooth muscle.
1 Current address: Department of Physiology and Pharmacology, School of Medicine, West Virginia University, Morgantown, WV. 
Address correspondence to: Dr. S. Jamal Mustafa, Department of Physiology and Pharmacology, School of Medicine, West Virginia University, Morgantown, WV 26505. E-mail: smustafa{at}hsc.wvu.edu
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References |
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Abbrachio MP and Cattabeni F (1987) Selective activity of bamifylline on adenosine A1-receptors in rat brain. Pharmacol Res 19: 537-545.
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