Abstract
Caffeine, an adenosine A1, A2A, and A2B receptor antagonist, is frequently used as an adjuvant analgesic in combination with nonsteroidal anti-inflammatory drugs or opioids. In this study, we have examined the effects of novel specific adenosine receptor antagonists in an acute animal model of nociception. Several A2B-selective compounds showed antinociceptive effects in the hot-plate test. In contrast, A1- and A2A-selective compounds did not alter pain thresholds, and an A3 adenosine receptor antagonist produced thermal hyperalgesia. Evaluation of psychostimulant effects of these compounds in the open field showed only small effects of some antagonists at high doses. Coadministration of low, subeffective doses of A2B-selective antagonists with a low dose of morphine enhanced the efficacy of morphine. Our results indicate that analgesic effects of caffeine are mediated, at least in part, by A2B adenosine receptors.
Caffeine has intrinsic antinociceptive properties and is frequently used as an adjuvant analgesic drug (Malec and Michalska, 1988; Sawynok and Yaksh, 1993). Although it is thought that caffeine analgesia is produced, at least in part, through adenosine receptor antagonism, it is unclear which receptor subtypes are involved. The adenosine receptor family comprises four subtypes: A1, A2A, A2B, and A3 (Fredholm et al., 2001). They are widely distributed in the CNS and peripheral tissues. The A1 receptors are found in high density in the brain (cortex, cerebellum, and hippocampus) and the dorsal horn of the spinal cord, and at lower levels in other brain regions and in peripheral tissues (Rivkees et al., 1995; Fredholm et al., 2001). The A2A receptors show a more restricted expression pattern in the CNS with high levels in the striatum, nucleus accumbens, and olfactory tubercle (Ongini and Fredholm, 1996). In the periphery, A2A receptors are highly expressed in spleen, thymus, leukocytes, and blood platelets (Ongini and Fredholm, 1996). A2B and A3 receptors are widely distributed, but have a low density in the CNS (Dixon et al., 1996). In the periphery, A2B receptors are highly expressed in the large intestine, on mast cells and hematopoietic cells (Feoktistov and Biaggioni, 1995). A3 receptors show a species-dependent distribution: in rats, testis and mast cells express A3 receptors in high density (Salvatore et al., 1993), whereas humans exhibit high A3 receptor expression in the lung and in cells of the immune system (Salvatore et al., 1993).
A1 receptors can couple to Gi, thus inhibiting the formation of cAMP, whereas stimulation of A2 receptors, which bind to Gs leads to an increase in adenylate cyclase activity (Fredholm et al., 2001). A1 receptors also activate phospholipase C and phospholipase D (Fredholm et al., 2001). A2 receptors are further subdivided into subtypes A2A and A2B, based on the recognition that stimulation of the adenylate cyclase by adenosine (through Gs or in the striatum through Golf) in rat brain was mediated via distinct high-affinity (localized in high density in striatal membranes) and low-affinity binding sites (present in low density throughout the brain) (Daly et al., 1983). A2B receptors can also stimulate phospholipase C via Gq activation (Feoktistov and Biaggioni, 1997). A3 adenosine receptors are coupled to Giα2-, Giα3-, and to a lesser extent to Gq/11 protein (Palmer and Stiles, 1995).
The role of adenosine receptors in nociception is complex and may involve different mechanisms in the central nervous system and in peripheral tissues. For example, spinal administration of adenosine receptor agonists produces antinociception in a variety of animal models of pain, presumably through the activation of spinal A1 and to a lesser extent through A2 receptors (Holmgren et al., 1986; Sawynok, 1998). Adenosine can produce analgesic or pronociceptive effects (Doak and Sawynok, 1995) through the activation of peripheral A1 and A2 receptors, respectively (Taiwo and Levine, 1990; Doak and Sawynok, 1995). It has been suggested that antagonism of peripheral adenosine A2 receptors accounts, at least in part, for caffeine analgesia (Taiwo and Levine, 1990; Karlsten et al., 1992).
A number of studies have shown interactions between adenosine receptors and the opioid system. Caffeine potentiated the analgesic effects of morphine, decreased the morphine-induced hyperactivity in mice, and inhibited the development of tolerance to morphine in rats (Malec and Michalska, 1988).
In this study, we examined the roles of specific adenosine receptor subtypes in analgesia and morphine synergism using several novel subtype-selective adenosine receptor antagonists. We demonstrate an analgesic effect of A2B receptor-selective compounds.
Materials and Methods
Chemicals. The following compounds were tested in this study: 8-cyclopentyl-1,3-dipropylxanthine (DPCPX); 1-butyl-8-(3-noradamantyl)-3-(3-hydroxypropyl)xanthine (PSB-36); 3,7-dimethyl-1-propargylxanthine (DMPX); phosphoric acid mono-(3-{8-[2-(3-methoxyphenyl)vinyl]-7-methyl-2,6-dioxo-1-prop-2-ynyl-1,2,6,7-tetrahydro-purin-3-yl}propyl)ester (MSX-3); 8-(p-bromophenyl)-1-propargylxanthine (PSB-50); 4-(1-butylxanthin-8-yl)benzoic acid (PSB-53); 1-propyl-8-(p-sulfophenyl)xanthine (PSB-1115); 8-{4-[2-(4-benzylpiperazin-1-yl)-2-oxo-ethoxy]phenyl}-1-butylxanthine; enprofylline, 3-propylxanthine (PSB-55); (R)-8-ethyl-4-methyl-2-(2,3,5-trichlorophenyl)-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]-purin-5-one (PSB-10); 8-(p-sulfophenyl)theophylline (8-SPT); 8-(p-sulfophenylcaffeine) (8-SPC); morphine-HCl; codeine sulfate; acetylsalicylic acid; and caffeine. For structures and affinities of adenosine receptor antagonists and reference compounds, see Table 1. Methylcellulose sodium salt, caffeine, morphine-HCl, codeine sulfate, Δ9-tetrahydrocannabinol (THC), acetylsalicylic acid sodium salt, DMPX, DPCPX, and enprofylline were purchased from Sigma-Aldrich (Steinheim, Germany); carrageenan was from Carl Roth (Karlsruhe, Germany). PSB-36, MSX-3, PSB-50, PSB-53, PSB-1115, PSB-55, PSB-10, 8-SPT, and 8-SPC were synthesized in our laboratory. The adenosine receptor affinities of compounds used in this study were determined experimentally by published standard procedures or taken from the literature (Table 1). For the selective A2B antagonists PSB-1115 and PSB-53 effects on specific radioligand binding to 30 different receptors were determined by Cerep (Poitiers, France) at a high concentration of 3 μM.
Animals. Male NMRI mice (32-40 g; Charles River Deutschland GmbH, Sulzfeld, Germany) were housed in groups of five mice per cage at a temperature of 23-24°C and a 12-h light/dark cycle. Standard food pellets (Altromin 1324 R. Germany) and water were available ad libitum. Experiments were approved by a local ethics committee. All animals were acclimatized for 2 weeks before the initiation of the experiments. Animals were used only once and were euthanized after the test. The experiments were performed during the light phase between 7:00 AM and 4:00 PM.
Hot-Plate Test. Antinociceptive effects of the compounds were evaluated in the hot-plate test. Mice (10/group) were injected intraperitoneally with drug, or the control animals with solvent (1% methylcellulose) in a volume of 10 ml/kg. At the times indicated, they were placed individually on a hot-plate apparatus (Columbus Instruments, Columbus, OH), a 25 × 25-cm metal surface maintained at 52 ± 0.1°C and surrounded by a 40-cm high Plexiglas wall. The latency of hindpaw licking was determined using a timer integrated into the hot-plate system. A cut-off time of 60 s was used to prevent tissue damage. The paw-surface temperature was determined 50 min after drug injection using a contact thermometer. The level of analgesia was expressed as percentage of the maximum possible effect (MPE): MPE = [(latencydrug-treated - latencycontrol)/(cut-off time - latencycontrol)] × 100. If possible, we calculated ED50 values for analgesic drug efficacy. We used only those compounds where data from at least five doses were available. All calculations were performed using logarithmic dose-values and sigmoid curve fit. The ED50 values were calculated by applying the Hill's equation using the Prism software (GraphPad Software Inc., San Diego, CA).
Open-Field Test. To determine possible psychostimulant drug effects, we determined the locomotor activity in the open field in mice that were treated under the same conditions as for the analgesia testing. Briefly, animals (6-8/group) were injected i.p. with drug or methylcellulose (controls) and placed into the center of an open field apparatus (42 × 42 × 28 cm) after 30 min. Activity of the animals was tracked by an automatic monitoring system (ActiMot; TSE, Germany) for 10 min under normal lighting conditions. Horizontal motor activity was evaluated and expressed as distance traveled (in meters).
Statistical Analysis. Data were analyzed by one-way ANOVA followed by Dunnett's test or in case of the interaction studies by Bonferroni's test. The 0.05 level of probability was used as the criterion for significance. The group sizes are shown in the description of the methods above. All data were expressed as mean values ± S.E.M.
Results
Adenosine Receptor Affinity and Selectivity of Compounds. All noncommercially available compounds used in this study (Table 1) were synthesized in our laboratory. The affinities and selectivities for adenosine receptor subtypes of the test compounds were described previously or determined by published standard procedures in radioligand binding assays at native (A1 and A2A) or recombinant (A2B and A3) adenosine receptors. Ki values are summarized in Table 1. It should be noted that these binding data were mostly obtained with human and rat adenosine receptors. Mouse data were only available for some compounds at the A2B receptor (Brackett and Daly, 1994), although the Ki values are likely to be similar in the highly homologous human, rat, and mouse A1,A2A, and A2B receptors (Fredholm et al., 2001; Kim et al., 2002). Only the A3 adenosine receptor exhibits larger species differences, many antagonists exhibiting a considerably lower affinity for the rat than for the human A3 adenosine receptor subtype. Thus, it is likely that the A3 affinity at mouse A3 receptors may even be lower than at human A3 receptors for the compounds used in this study. The selectivity of the A2B adenosine receptor antagonists PSB-53 and PSB-1115 was further investigated in radioligand binding assays at a series of 30 different receptors, including adrenergic, angiotensin, benzodiazepine, bradykinin, cholecystokinin, dopamine, endothelin, GABA, N-methyl-d-aspartate, histamine, muscarinic, and nicotinic acetylcholine, neurokinin, opioid, serotonin, and vasopressin receptors, as well as neurotransmitter transporters for norepinephrine, dopamine, and serotonin. At a high concentration of 3 μM both compounds showed only low to negligible binding to all investigated drug targets, except for the adenosine receptors (data not shown). Therefore the compounds appear to be truly selective A2B adenosine receptor antagonists.
The compounds include 1) the potent A1-selective antagonists DPCPX and PSB-36, both compounds exhibiting high selectivity over all other adenosine receptor subtypes; 2) the water-soluble prodrug MSX-3 of the potent A2A-selective antagonist MSX-2; 3) the A3-antagonist PSB-10; 4) four recently developed A2B-selective adenosine receptor antagonists (PSB-1115, PSB-50, PSB-53, and PSB-55); 5) the moderately potent standard antagonists caffeine, DMPX, and enprofylline, which has moderate selectivity for A2B receptors (3-7-fold versus A1, 3-28-fold versus A2A, highly selective versus A3); and 6) two sulfophenylxanthine derivatives, structurally related to the A2B-selective antagonist PSB-1115, one of which is virtually inactive at adenosine receptors (8-SPC), the other one being a nonselective A1/A2B antagonist (Table 1). The A2B-selective antagonist PSB-1115 and the structurally related sulfophenyltheophylline (8-SPT) and sulfophenylcaffeine (8-SPC) would not penetrate into cells or into the brain (Daly, 2000) and can thus be used as pharmacological tools to distinguish extracellular from intracellular, and peripheral from central effects.
Hot-Plate Test. The antinociceptive activity of these compounds was evaluated in the hot-plate test in mice, an acute animal pain model. The results are summarized in Fig. 1, Table 2, and Table 3. A time-course analysis of several adenosine receptor antagonists was performed. As shown in Table 2, PSB-1115, PSB-55, and PSB-10 showed their maximum effect after 30 min. This time point was chosen for all further studies with independent groups of animals. Caffeine, a virtually nonselective A1/A2A/A2B adenosine receptor antagonist with a slight preference for the A2B receptor produced a robust dose-dependent analgesia in this test. In contrast, the A1-selective antagonists DPCPX and PSB-36 were not effective in doses up to 100 mg/kg. The A2 nonselective antagonist DMPX, which shows a slight preference for the A2B versus the A2A adenosine receptor (Table 1), exhibited antinociceptive activity at 30 mg/kg. When subtype-selective antagonists were used, we found no effect with the A2A-selective compound MSX-3, whereas all A2B-selective compounds (PSB-50, PSB-53, PSB-1115, PSB-55, and enprofylline) produced a dose-dependent effect in hot-plate analgesia. PSB-50 was effective at 75 mg/kg, but not at 100 mg/kg. However, we observed profound hypomotility/ataxia and sedation in animals treated with 100 mg/kg. Surprisingly, we found a dose-dependent decrease in hot-plate response latencies in mice treated with the A3 antagonist PSB-10. Thus, PSB-10 seemed to produce hyperalgesia. 8-SPT and 8-SPC, which are structurally similar to the A2B-selective compound PSB-1115, but were either nonselective (8-SPT), or virtually inactive at adenosine receptors (8-SPC), did not show any antinociceptive efficacy. The antinociceptive effects of codeine sulfate, morphine hydrochloride, and acetylsalicylic acid were also tested and are shown for comparison (Table 3). To determine whether the compounds had any effects on the body temperature of the animals that might interfere with the hot-plate result, we determined the paw-surface temperature using a contact thermometer 30 min after drug injection. The results are shown in Fig. 2. Neither PSB-1115, nor PSB-55, nor PSB-10, nor caffeine produced changes of the animals paw temperature. In contrast, injection of 4 mg/kg ethanol, which is known to produce hypothermia, led to a readily detectable, highly significant reduction in paw temperature.
Open-Field Test. To evaluate possible effects of active compounds on locomotor activity, we used the open field test (Fig. 3). High dose of enprofylline (100 mg/kg) reduced locomotor activity, but neither with an antinociceptive dose of 100 mg/kg PSB-53, nor with enprofylline and PSB-50, at an analgesic dose of 75 mg/kg effects on the locomotor activity were seen. No significant locomotor effects were observed with the analgesic compounds DMPX and PSB-1115. The hyperalgesic compound PSB-10 also did not reduce locomotor activity at a high dose of 100 mg/kg.
Interaction with Morphine. Because caffeine is known to enhance the analgesic effect of morphine, we wanted to investigate possible interactions between A2B antagonists and morphine. We therefore evaluated hot-plate response latencies of animals treated with a low (noneffective) dose of A2B antagonists, a low dose of morphine, or a combination of both. As shown in Fig. 4, the efficacy of morphine was increased by each compound except PSB-50. We selected among the active compounds PSB-1115 (10 mg/kg) and tested whether it also affected the efficacy of THC (20 mg/kg, threshold dose of THC). However, we did not find any significant enhancement of THC analgesia (p > 0.36).
Discussion
Adenosine has dual activity on nociception. It acts centrally within the spinal cord to suppress nociceptive signaling (Sawynok and Yaksh, 1993), presumably through the activation of A1 and A2 adenosine receptors (DeLander and Hopkins, 1986). In the periphery adenosine has algogenic activity, which is probably mediated by A2 receptors (Taiwo and Levine, 1990; Karlsten et al., 1992; Sawynok and Yaksh, 1993). Caffeine, a virtually nonselective A1-, A2A-, and A2B-adenosine receptor antagonist, exhibits antinociceptive effects and shows adjuvant analgesic properties in combination with opioid and nonopioid analgesics (Sawynok and Yaksh, 1993; Sawynok, 1998).
In this study, we investigated the effects of systemic administration of adenosine receptor subtype-selective antagonists on pain sensation. Results obtained with subtype-selective antagonists may provide better insights into the (patho)physiological role of specific adenosine receptors in pain models than agonists, because agonists can target receptors that have no role in control by endogenous adenosine, whereas antagonists block physiological stimulation of the receptors.
A1-selective adenosine receptor antagonists were not hyperalgesic in the hot-plate test. Thus, although the activation of central A1 receptors seems to play an important role in spinal antinociception (Reeve and Dickenson, 1995; Nakamura et al., 1997), the pharmacological blockade of this receptor has no effect on pain responses in the applied animal models. This cannot be due to lacking CNS penetration of DPCPX, because it has been shown that the compound does penetrate into the brain in concentrations sufficient to block A1 receptors (Finlayson et al., 1997). In A1 knockout mice the analgesic effect of intrathecal adenosine analogs was lost, suggesting that this receptor subtype is responsible for the central analgesic effects of adenosine. These animals showed an increased pain sensation in the tail-flick test, but not in the von-Frey test (Johansson et al., 2001). It is likely that the role of A1 receptors in the central processing of nociceptive signals may not have become evident in our models. In a recent study, DPCPX had exhibited pronociceptive effects only at a low dose of 1 mg/kg, but not at 3 or 10 mg/kg (Bastia et al., 2002). This had been explained by the potentially low selectivity at higher concentrations. In fact, DPCPX is highly selective versus the other adenosine receptor subtypes in rodents. PSB-36, an even more selective A1 antagonist, also did not show any pronociceptive effects in our study.
The selective A2A antagonist MSX-3 was also ineffective, although A2A knockout mice displayed a hypoalgesic phenotype (Ledent et al., 1997). Disparate results from knockout and pharmacological studies are not uncommon. They may be due to pharmacokinetic effects, or to developmental effects of the gene knockout, or both. In this case, pharmacological blockade of the A2A receptors may not be sufficient to produce antinociception, or the central and peripheral effects of A2A receptor inhibition counterbalance each other. Recently, antinociceptive effects of the A2A-selective antagonist SCH-58261 were described (Bastia et al., 2002), which is structurally different from the styrylxanthine derivative MSX-3. However, the A2A antagonist was only effective in the hot-plate test after intrathecal and not after systemic application.
Here, we report for the first time that A2B receptor antagonists are potent analgesic agents. Because the virtually nonselective A2 receptor antagonist DMPX (only slightly selective for A2B versus A2A receptors) was active in our models but not the A2A-selective antagonist MSX-3, we feel that the DMPX effects may be due to the inhibition of A2B receptors. A variable response was observed with compound PSB-55, which was effective only at the highest dose tested (100 mg/kg), although this compound has the highest affinity to the A2B receptors of all compounds tested (Hayallah et al., 2002). PSB-55 has a relatively high molecular weight and rather low water solubility. We therefore feel that the probably unfavorable pharmacokinetic properties, of this compound may be responsible for the variable in vivo effects.
To determine possible locomotor effects of adenosine receptor antagonists, which may complicate the interpretation of hot-plate results, we studied locomotor activity after drug treatment in the open-field. We found that a high dose of the analgesic compound enprofylline (100 mg/kg) reduced locomotor activity. However, no locomotor effects were observed with PSB-53 at 100 mg/kg where strong analgesia was observed, nor with enprofylline and PSB-50 at an analgesic dose of 75 mg/kg. Also, we did not observe any locomotor effects with the antinociceptive compounds DMPX and PSB-1115. In addition, PSB-10 also did not alter locomotor activity at 100 mg/kg, although animals showed significantly decreased hot-plate response latencies. Thus, the hot-plate analgesia observed after A2B antagonist administration cannot easily be accounted for by their locomotor effects.
We found that the peripherally acting A2B antagonist PSB-1115, which probably cannot penetrate the blood-brain barrier due to its polar sulfonate group (Baumgold et al., 1992), is a potent analgesic compound. Therefore, A2B analgesia must be produced by a peripheral effect. Sulfonates such as PSB-1115 will also not penetrate cell membranes and therefore cannot inhibit intracellular enzymes (Daly, 2000). Thus, the observed effects clearly have to be due to an extracellular mechanism and are believed to be mediated by a blockade of peripheral A2B adenosine receptors. This is confirmed by the lacking affinity of PSB-1115 and another A2B-selective antagonist, PSB-53, for a series of 30 receptors, which are known drug targets.
The A3 adenosine receptor antagonist PSB-10 significantly increased pain sensation in the hot-plate test. It should be noted that PSB-10 is a very potent and selective A3 antagonist in humans (Ozola et al., 2003), but it may be less potent at mouse A3 receptors. Highly potent and selective rodent A3 antagonists are currently not available (Müller, 2003).
Caffeine has a virtually nonselective A1, A2A, and A2B antagonist activity on adenosine receptors, with a slight preference for A2B receptors. Caffeine analgesia in our models was similar to that described in the literature (Malec and Michalska, 1988), although the nonselective adenosine receptor blocker 8-SPT remained ineffective in our pain tests. The main differences between caffeine and 8-SPT with regard to their actions at adenosine receptors are 1) the high polarity of 8-SPT, which cannot penetrate the blood-brain barrier in contrast to caffeine; and 2) the (small) selectivity of caffeine for A2B versus A1 adenosine receptors. Other activities of caffeine (alteration of catecholamine or acetylcholine release and turnover, inhibition of phosphodiesterases, influence on intracellular calcium concentrations, interaction with GABAA receptors, or an as yet unknown mechanism) may contribute to its antinociceptive effects as well (Sawynok and Yaksh, 1993; Daly, 2000). Also, selectivity for A2B versus A1 receptors may play a role because A1 antagonism may counteract the antinociceptive effect mediated by A2B antagonists. In vivo, in the presence of high adenosine concentrations released by noxious stimuli, the A2B selectivity of caffeine may even be higher than in vitro, because A2B adenosine receptors exhibit low affinity for adenosine (EC50 value approx. micromolar range), whereas A1 adenosine receptors are high-affinity receptors (EC50 value adenosine approx. nanomolar range) (Fredholm et al., 2001).
In summary, our results indicate an important role of adenosine A2B and A3 receptors in pain signaling. A2B receptors have previously been proposed to mediate pronociceptive effects at peripheral sites (Sawynok et al., 1997). Because the compounds in these studies are likely to produce their antinociceptive effects also peripherally, they support the role of A2B receptors in peripheral pain signaling. However, our study revealed a pronociceptive effect of an A3 receptor antagonist, which was unexpected, because previous studies have indicated that those receptors may also be involved in peripheral pain signaling (Sawynok et al., 1997).
Caffeine is frequently used as an adjuvant analgesic in the medical practice for the treatment of various types of pain such as headache, postpartum pain, postoperative pain, and dental surgery pain (Sawynok and Yaksh, 1993) in combination with nonsteroidal anti-inflammatory drugs (Cass and Frederik, 1962; Sawynok and Yaksh, 1993) or in combination with morphine (Malec and Michalska, 1988). There is a large body of evidence pointing to an interaction between the opioid and adenosine systems in the modulation of pain signaling. Morphine enhances the release of adenosine from the spinal cord and cortex (Phillis et al., 1980; Cahill et al., 1996), presumably through a synergistic activation of μ and δ opioid receptors (Cahill et al., 1996). Pharmacological studies have indicated that the A1 and A2A receptors may be downstream mediators of morphine analgesia (Keil and DeLander, 1994; Sawynok, 1998), although studies in A2A knockout mice failed to reveal any differences in μ receptor-dependent analgesia (Bailey et al., 2002). The results from this study indicate that the analgesic effects of centrally released adenosine may be counterbalanced by pronociceptive A2B receptor-mediated peripheral effects of adenosine. The lack of synergistic effects between A2B-selective compounds and THC indicates that the synergism is specific for the opioid system and not due to a general increase in antinociceptive drug efficacy. Specific A2B receptor antagonists might therefore be valuable adjuvant drugs for opioid analgesia with minimal side effects.
Footnotes
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O.M.A.-S. was on leave from the Faculty of Pharmacy (Al-Azhar University) with a scholarship of the Egyptian government. A.M.H. was on leave from the Faculty of Pharmacy (Assiut University) with a scholarship of the Egyptian government. A.Z. was supported by Deutsche Forschungsgemeinschaft (FOR425), the State of North-Rhine-Westfalia (Innovationsprogramm Forschung), and the BONFOR Program. C.E.M. was supported by the Deutsche Forschungsgemeinschaft (FOR425).
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DOI: 10.1124/jpet.103.056036.
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ABBREVIATIONS: CNS, central nervous system; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; PSB-36, 1-butyl-8-(3-noradamantyl)-3-(3-hydroxypropyl)xanthine; DMPX, 3,7-dimethyl-1-propargylxanthine; MSX-3, phosphoric acid mono-(3-{8-[2-(3-methoxyphenyl)vinyl]-7-methyl-2,6-dioxo-1-prop-2-ynyl-1,2,6,7-tetrahydropurin-3-yl}propyl)ester; PSB-50, 8-(p-bromophenyl)-1-propargylxanthine; PSB-53, 4-(1-butylxanthin-8-yl)-benzoic acid; PSB-1115, 1-propyl-8-(p-sulfophenyl)xanthine; PSB-55, 8-{4-[2-(4-benzylpiperazin-1-yl)-2-oxo-ethoxy]phenyl}-1-butylxanthine; enprofylline, 3-propylxanthine; PSB-10, (R)-8-ethyl-4-methyl-2-(2,3,5-trichlorophenyl)-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]purin-5-one; 8-SPT, 8-(p-sulfophenyl)theophylline; 8-SPC, 8-(p-sulfophenylcaffeine); THC, Δ9-tetrahydrocannabinol; MPE, maximal possible effect; ANOVA, analysis of variance; 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine.
- Received July 1, 2003.
- Accepted August 21, 2003.
- The American Society for Pharmacology and Experimental Therapeutics