Introduction

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Adenosine, an endogenous purine nucleoside, has been proposed to modulate a variety of physiological responses by stimulating specific extracellular receptors (Collis and Hourani, 1993). Adenosine receptors have been classified as A₁, A_2A, A_2B, and A₃ receptors (Fredholm et al., 1994; Olah and Stiles, 1995). Under several adverse conditions, such as stress, trauma, ischemia, seizures, and pain, the local tissue concentrations of extracellular adenosine are increased after the release of adenosine itself, and/or that of AMP, which is metabolized extracellularly to adenosine. This increased adenosine can protect against excessive cellular damage or dysfunction in negative feedback manners (Ralevic and Burnstock, 1998). Indeed, exogenous adenosine and its agonists attenuate ischemic cerebral injuries (von Lubitz et al., 1988), seizures (Dunwiddie and Worth, 1982; Murray et al., 1985), and pain (Sawynok et al., 1986) in several animal models.

Adenosine is also released at inflamed sites (Cronstein, 1995; Cronstein et al., 1995) and exhibits anti-inflammatory effects (Cronstein, 1997; Rosengren and Firestein, 1997). Although adenosine and its agonists are effective in animal models of inflammation, their therapeutic application has been limited by systemic side effects, such as hypotension, bradycardia, and sedation (Williams, 1996). Moreover, adenosine usually disappears very rapidly in physiological conditions due to rapid uptake into the adjacent cells (erythrocytes, endothelial cells) and subsequent intracellular metabolism (Arch and Newsholme, 1978; Moser et al., 1989). Endogenous adenosine levels at inflamed sites are reported to increase further because of the increased need for energy supplied by ATP, which is metabolized to AMP and adenosine ultimately (Eigler et al., 1997). In addition, the activity of 5'-nucleotidase, which metabolizes AMP to adenosine, is reported to increase in inflammatory conditions (Johnson et al., 1999). It is therefore assumed that prevention of adenosine uptake into the cells and its subsequent metabolism can selectively enhance extracellular concentrations of adenosine at inflamed sites, resulting in an anti-inflammatory effect. In fact, adenosine kinase inhibitors have been shown to exhibit anti-inflammatory effects in vivo (Firestein et al., 1994; Cronstein et al., 1995; Kowaluk et al., 2000). However, there has been no report on anti-inflammatory effects of adenosine uptake inhibitors in vivo.

3-[1-(6,7-Diethoxy-2-morpholinoquinazolin-4-yl)piperidin-4-yl]-1,6-dimethyl-2,4(1H,3H)-quinazolinedione hydrochloride (KF24345) is a novel adenosine uptake inhibitor with a new chemical structure (Fig. 1). In the present study, we investigated the effects of KF24345 on lipopolysaccharide (LPS)-induced tumor necrosis factor- (TNF-) production and leukopenia in mice to determine whether the adenosine uptake inhibitor exerts anti-inflammatory effects in vivo. Moreover, we investigated the effects of adenosine receptor antagonists on the anti-inflammatory effects of KF24345 to determine the involvement of endogenous adenosine and adenosine receptors.

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Chemical structure of KF24345.

	Materials and Methods

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Animals. Male ddY mice (weighing 20-30 g), New Zealand White rabbits (weighing 2-3 kg), and golden Syrian hamsters (weighing 100-200 g) were purchased from Japan SLC Inc. (Hamamatsu, Japan). The animals were given access to food and water ad libitum and were maintained on 12-h light/dark cycle at 22-23°C. All experimental procedures were conformed to the Animal Care and Use Committee protocols filed at Kyowa Hakko Kogyo Co., Ltd. (Tokyo, Japan).

Chemicals. The sources of materials used in this study were as follows: dilazep (N,N'-bis[3-(3,4,5-trimethoxybenzoyloxy)-propyl]homopiperazine), dipyridamole, and adenosine from Sigma Chemical (St. Louis, MO); [2,8-³H]adenosine (41 Ci/mmol) from PerkinElmer Life Sciences (Boston, MA); LPS (Escherichia coli 055:B5) from Difco (Detroit, MI); ZM 241385 (4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo [2,3-a] [1,3,5]triazin-5-ylamino]ethyl)phenol) from Tocris Cookson (Bristol, UK); and 8-(p-sulfophenyl)theophylline (8-STP) from Sigma/RBI (Natick, MA). KF24345, 2-(aminocarbonyl)-N-(4-amino-2,6- dichlorophenyl)-4-[5,5-bis(4-fluorophenyl)pentyl]-1-piperazineacetamide (R75231; Beukers et al., 1994) and 8-(noradamantan-3-yl)-1,3-dipropylxanthine (KW-3902; Shimada et al., 1992; Mizumoto et al., 1993) were synthesized at Pharmaceutical Research Institute of Kyowa Hakko Kogyo. All other chemicals and solvents were used in their analytical pure form.

Inhibition of Adenosine Uptake (in Vitro). Blood samples from male healthy volunteers were collected into tubes with 1/10 volume of 3.8% (w/v) sodium citrate. Blood samples from the animals were similarly collected from the abdominal vein under ether anesthesia. After the centrifugation (1000g, 10 min), erythrocytes were washed three times with a 2-fold volume of saline to remove the buffy coats. Washed erythrocytes were resuspended in a transport medium (bicarbonate-free Eagle's basal medium buffered with 10 mM HEPES, pH 7.4). Number of erythrocytes in the suspension was determined by a cell counter (Celltac MEK-6158; Nihon Kohden Corporation, Tokyo, Japan), and erythrocyte density was adjusted to 2.5 × 10⁹ cells/ml.

Inhibition of Adenosine Uptake (ex Vivo). In vivo effectiveness of adenosine uptake inhibitors was determined in mice according to the previous method (Baer et al., 1991). Each inhibitor was orally administered to mice at varying doses. Whole blood samples (450 µl) were collected with 50 µl of 3.8% (w/v) sodium citrate from the abdominal vein under ether anesthesia at various time intervals after the drug administration, and rapidly diluted 1.5-fold into the transport medium containing 0.38% (w/v) sodium citrate. Adenosine uptake was started by mixing 100 µl of 2 µM [2,8-³H]adenosine (100,000-150,000 dpm) with 100 µl of diluted whole blood. The following procedure was the same as described in "Inhibition of Adenosine Uptake (in Vitro)".

Effects of KF24345 on LPS-Induced TNF- Production in Mice. Mice were orally pretreated with KF24345 (10 mg/kg) or vehicle 1 h before an intravenous injection of LPS (1 mg/kg). Mice were anesthetized with ether and blood samples were collected from the abdominal vein 1 h after the LPS injection. KW-3902 (a selective adenosine A₁ receptor antagonist; 0.3 mg/kg) or ZM 241385 (a selective adenosine A₂ receptor antagonist; 3 mg/kg) was intravenously injected via the tail vein 10 min before the LPS injection. The intravenous injection of KW-3902 at a dose of 0.1 mg/kg prominently antagonizes 5'-N-ethylcarboxamidoadenosine-induced bradycardia without affecting hypotension in rats (unpublished observation). The intravenous injection of ZM 241385 at 1 mg/kg attenuates adenosine-induced hypotension by over 70% (Poucher et al., 1996), whereas its intravenous injection even at 10 mg/kg did not inhibit the hypotensive and bradycardiac effects of the A₃/A₁ receptor agonist N⁶-2-(4-amino-3-iodophenyl)ethyladenosine (Keddie et al., 1996). From these observations, we selected the doses of adenosine antagonists used in the present study so that they selectively antagonize each of adenosine receptors. Blood samples were centrifuged (1200g, 10 min, 4°C), and sera were obtained. Serum TNF- concentrations were measured by using the enzyme-linked immunosorbent assay kit from Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK) with a sensitivity of 10 pg/ml, and calculated from a standard curve generated with recombinant mouse TNF- (Amersham Biosciences UK, Ltd.).

Effects of KF24345 on LPS-Induced Leukopenia in Mice. Mice were orally administered with KF24345 (10 mg/kg) or vehicle 1 h before an intravenous injection of LPS (3 µg/kg). Mice were anesthetized with ether and blood samples anticoagulated with 2 mM EDTA were obtained from the abdominal vein 2 h after the LPS injection. 8-SPT (a nonselective adenosine receptor antagonist; 20 mg/kg) was intravenously injected via the tail vein 10 min before the LPS injection. The dose of 8-SPT used in this study is enough to inhibit the myocardial protective effect of adenosine (Toombs et al., 1992). Number of leukocytes was measured using the cell counter (Celltac MEK-6158; Nihon Kohden Corporation).

Statistical Analysis. Data are presented as means ± S.E. Statistical significance was calculated by the Student's t test when comparing two groups, or by one-way analysis of variance followed by the Dunnett's test for multiple groups. P < 0.05 was considered to be statistically significant.

	Results

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Inhibition of Adenosine Uptake (in Vitro). We determined the in vitro potencies of KF24345 as an adenosine uptake inhibitor in erythrocytes isolated from different species, and compared them with those of other adenosine uptake inhibitors.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Representative inhibition curves with various adenosine uptake inhibitors in isolated erythrocytes from human (A) and mouse (B). Erythrocytes were incubated with 1 µM [3H]adenosine for 10 s in the presence of various concentrations of adenosine uptake inhibitors. Values are means ± S.E. of five separate experiments. IC50 values are given in Table 1. , KF24345; , dilazep; , dipyridamole; , R75231.

Inhibition of Adenosine Uptake (ex Vivo). We measured adenosine uptake in blood samples obtained from mice after the oral administration of KF24345, and the effect of KF24345 was compared with those of other adenosine uptake inhibitors.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Time course of adenosine uptake inhibition in sampled blood cells after oral administration of KF24345 (A), dilazep (B), dipyridamole (C), and R75231 (D) in mice. Blood was sampled at various time intervals and incubated with 1 µM [3H]adenosine for 10 s. Adenosine uptake inhibition was expressed as percentage of inhibition compared with the vehicle-treated group, and values represent means ± S.E. of five animals. *, P < 0.05, **, P < 0.01, and ***, P < 0.001, differ significantly from the vehicle-treated group.

Effects of KF24345 on LPS-Induced TNF- Production in Mice. KF24345 (10 mg/kg p.o.) significantly suppressed the elevation of serum TNF- concentrations in the control mice treated with LPS (Fig. 4A). ZM 241385 (3 mg/kg i.v.), but not KW-3902 (0.3 mg/kg i.v.), completely reversed the suppressing effect of KF24345 (Fig. 4A). Neither KW-3902 nor ZM 241385 alone affected LPS-induced TNF- production (Fig. 4B).

View larger version (26K): [in this window] [in a new window]   Fig. 4.   A, suppression by KF24345 of LPS-induced TNF- production and the effects of selective adenosine receptor antagonists on the suppression by KF24345. B, effects of selective adenosine receptor antagonists on LPS-induced TNF- production. Mice were pretreated with KF24345 (10 mg/kg p.o.) 1 h before intravenous injection of LPS (1 mg/kg). KW-3902 (a selective adenosine A1 receptor antagonist; 0.3 mg/kg) or ZM 241385 (a selective adenosine A2 receptor antagonist; 3 mg/kg) was intravenously administered 10 min before the LPS injection. Blood samples were collected 1 h after the LPS injection, and serum TNF- concentrations were measured by enzyme-linked immunosorbent assay. Values are means ± S.E. of six animals. **, P < 0.01 and ***, P < 0.001 differ significantly from the LPS-treated group; , P < 0.001, significantly different from the KF24345 + LPS-treated group.

Effects of KF24345 on LPS-Induced Leukopenia in Mice. LPS caused a significant reduction in leukocyte counts, and pretreatment with KF24345 (10 mg/kg p.o.) significantly inhibited the drop in leukocyte counts (Fig. 5A). 8-SPT (20 mg/kg i.v.) almost completely reversed the inhibitory effect of KF24345 on LPS-induced leukopenia (Fig. 5A). 8-SPT alone did not affect LPS-induced leukopenia (Fig. 5B).

View larger version (18K): [in this window] [in a new window]   Fig. 5.   A, inhibition by KF24345 of LPS-induced leukopenia and the effect of 8-SPT (a nonselective adenosine receptor antagonist) on the inhibition by KF24345. B, effect of 8-SPT on LPS-induced leukopenia. Mice were pretreated with KF24345 (10 mg/kg p.o.) 1 h before intravenous injection of LPS (3 µg/kg). 8-SPT (20 mg/kg) was intravenously administered 10 min before the LPS injection. Blood samples were collected 2 h after the LPS injection, and total leukocyte counts were obtained. Values are means ± S.E. of 5 to 10 animals. *, P < 0.05, significantly different from the LPS-treated group; , P < 0.05, significantly different from the KF24345 + LPS-treated group.

	Discussion

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In this study, we demonstrated that a newly synthesized KF24345 is an orally effective and long-lasting adenosine uptake inhibitor in mice. We also found that KF24345 suppressed LPS-induced TNF- production and leukopenia in mice. The suppressing effects of KF24345 were completely reversed by adenosine receptor antagonists, suggesting that the effects of KF24345 were mediated by endogenous adenosine and adenosine receptors. These are the first demonstrations that an adenosine uptake inhibitor exhibits anti-inflammatory effects via endogenous adenosine in vivo.

We first studied the in vitro activity of KF24345 as an adenosine uptake inhibitor. KF24345 inhibited adenosine uptake into washed erythrocytes in concentration-dependent manners in all the species examined. KF24345 was slightly less potent than dilazep, but essentially as potent as R75231. The IC₅₀ values of dilazep in this study were similar to those previously reported (Baer et al., 1990). In the ex vivo studies in mice, examining the adenosine uptake into sampled blood cells, KF24345 was the most potent inhibitor with long duration of action among all the inhibitors tested, although its in vitro potency was not remarkably different from those of other inhibitors. Plasma concentrations of KF24345 2 h after its oral administration at 1, 3, and 10 mg/kg were 77, 292, and 1277 nM, respectively (H. Ohtsuka, unpublished observation). The plasma concentrations of KF24345 were 383 and 252 nM, respectively, at 8 and 12 h after the oral administration at a dose of 10 mg/kg. In another experiment, in vitro inhibitory activities of adenosine uptake by KF24345 at various concentrations scarcely changed even after the washout of the drug with saline (unpublished observation). Thus, it is considered that the strong binding of KF24345 to nucleoside transporters on erythrocytes as well as the long-lasting plasma level contributed to its long duration of action in vivo. The present results suggest that KF24345 is a useful drug for examining the in vivo effects of adenosine uptake inhibition in mice.

At inflamed sites, production and release of adenosine are increased due to enhanced cellular ATP catabolism in response to oxidants and other toxins. In addition, neutrophils are shown to release adenosine at inflamed sites (Newby et al., 1983). A previous report demonstrates that not only exogenous but also endogenous adenosine attenuates TNF- production from human mononuclear cells in vitro (Eigler et al., 1997). However, the effects of extracellular adenosine are extremely short-lived because it is rapidly taken up from the extracellular fluid into cells and metabolized (Dawicki et al., 1985; Moser et al., 1989). In contrast, if the adenosine uptake inhibitor could continuously increase endogenous adenosine at inflamed sites, it would be able to retard the inflammatory response. In this study, indeed, KF24345 reduced TNF- production and diminished leukocyte accumulation, which supposedly result from leukocyte accumulation at inflamed sites, suggesting an anti-inflammatory property of this drug. One possible mechanism by which KF24345 diminished leukopenia is considered to be inhibited expression of adhesion molecules in the stimulated vascular endothelium and/or neutrophils. Indeed, adenosine has been reported to inhibit expression of E-selectin and vascular cell adhesion molecule 1 in the activated vascular endothelial cells (Bouma et al., 1996). These are the first demonstrations that the adenosine uptake inhibitor is anti-inflammatory in vivo.

Adenosine has been described to inhibit various functions of inflammatory cells through activation of adenosine receptors on the cell surface. Adenosine suppresses neutrophil functions, such as oxygen radical production, adhesion to vascular endothelium and phagocytosis, and macrophage functions, including TNF- production, in vitro (Cronstein et al., 1985; Salmon and Cronstein, 1990; Cronstein et al., 1992; Parmely et al., 1993; Bouma et al., 1994). Furthermore, previous reports have shown that adenosine and its agonists exert anti-inflammatory effects in vivo (Schrier et al., 1990; Le Vraux et al., 1993). In the present study, the inhibition by KF24345 of LPS-induced leukopenia was completely reversed by 8-SPT, a nonselective adenosine receptor antagonist. In addition, the suppression of LPS-induced TNF- production by KF24345 was completely blocked by ZM 241385, a selective adenosine A₂ receptor antagonist. Moreover, the suppressing effect of KF24345 on LPS-induced TNF- production was also reversed by 8-SPT (unpublished observation). These results suggest that the anti-inflammatory effect of KF24345 is mediated by endogenous adenosine and adenosine receptor pathway, especially adenosine A₂ receptor stimulation.

Although the anti-inflammatory effects of KF24345 were blocked by adenosine receptor antagonists, systemic blood pressure in mice was shown to be unaffected from 1 to 8 h after the oral administration in mice (K. Yao, unpublished observation), suggesting that systemic adenosine levels were unchanged during this period. Thus, we could expect that KF24345 would diminish inflammation with minimal systemic side effects, as was previously reported in an animal model of cardiac infarction (Belardinelli et al., 1989). On the other hand, adenosine kinase inhibitors are reported to exhibit anti-epileptic, neuroprotective, and analgesic effects as well as anti-inflammatory effect (Kowaluk et al., 1998). Thus, when used as an anti-inflammatory drug, adenosine kinase inhibitors may elicit side effects, especially those in the central nervous system, after systemic administration. Collectively, KF24345 may be a potent and new anti-inflammatory agent with minimal systemic side effects and also may render effective therapies for some inflammatory diseases.

In conclusion, we have shown that KF24345, an orally effective adenosine uptake inhibitor, suppresses LPS-induced TNF- production and leukopenia in mice. Endogenously increased adenosine, which inhibits leukocyte accumulation and suppresses cytokine production, could contribute to the anti-inflammatory effects of KF24345 at inflamed sites. These data suggest that adenosine uptake inhibitors could be effective in the treatment or prevention of various inflammatory diseases.