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NEUROPHARMACOLOGY
Departments of Anesthesiology (T.L.Y., C.I.S., B.F., X.-Y.H.) and Chemistry and Biochemistry (D.S., E.A.D.), School of Medicine, University of California, San Diego, La Jolla, California; Department of Chemistry, University of Athens, Athens, Greece (G.K., C.G.K.); and Department of Pharmaceutical Chemistry, University of Thessaloniki, Thessaloniki, Greece (D.H.-L.)
Received for publication
June 28, 2005
Accepted
September 30, 2005.
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). Although early work suggested that this action resulted from a peripheral effect (Ferreira, 1972), it was subsequently found that inhibition of spinal COX also led to reversal of the facilitated state (Yaksh, 1982; Taiwo and Levine, 1988). These initial findings have been widely confirmed (Yamamoto and Nozaki-Taguchi, 1996; Turnbach and Randich, 2001) Consistent with this action, persistent small afferent input, as arises from tissue injury, was shown to evoke a significant spinal release of prostanoids in vivo in a manner that was blocked by spinally delivered COX inhibitors (Yaksh, 1982; Malmberg and Yaksh, 1992, 1995; Southall et al., 1998; Ebersberger et al., 1999; Samad et al., 2001; Yaksh et al., 2001). An important element of prostaglandin (PG) synthesis is phospholipase A2 (PLA2) because it is required to generate arachidonic acid, which is the substrate for COX-mediated prostanoid formation. In recent work, we have shown the presence of constitutive mRNA and protein in the spinal cord for group IVA calcium-dependent PLA2 (cPLA2) and group VIA calcium-independent PLA2 (iPLA2) and groups II and V secretory PLA2 forms (Lucas et al., 2005; Svensson et al., 2005b). Inhibition of group IV cPLA2 but not group VI iPLA2 isoforms using i.t.-delivered agents suggested a role for group IV cPLA2, but not group VI iPLA2 (Lucas et al., 2005), in inflammation-evoked hyperalgesia.
We have reported recently the discovery of a novel structural series of 2-oxoamides that inhibit group IVA cPLA2 in vitro and in vivo (Kokotos et al., 2002, 2004). In initial work, 2-oxoamides were observed to inhibit inflammation in the rat paw carrageenan-induced edema assay (Kokotos et al., 2004). In the present work, we have focused on the in vivo activity of four related analogs of this series, AX006, AX010, AX048, and AX057. These molecules were examined for their inhibitory effects on group IV cPLA2 and group VI iPLA2 as well as on COX activity in in vitro assays. Their actions were then characterized after systemic and i.t. delivery on thermal hyperalgesia induced by peripheral inflammation (intraplantar carrageenan). In addition, we have shown previously that spinal sensitization can be directly initiated in the absence of peripheral inflammation by spinal delivery of substance P (SP). Substance P, acting through the spinal neurokinin 1 receptor, will evoke the spinal release of PGE2 and subsequent thermal hyperalgesia. Both of these events are antagonized by spinal cyclooxygenase inhibition (Malmberg and Yaksh, 1992; Yaksh et al., 2001). Based on these observations, we examined the effects of the PLA2 inhibitors on the hyperalgesia and PGE2 release evoked by spinally delivered SP. We report here that one of these agents, after systemic delivery, displays significant antihyperalgesic effects in models of both centrally and peripherally initiated hyperalgesia and in an effective systemic dose blocks the spinally evoked release of spinal PGE.
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Materials and Methods |
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In Vivo Studies
Animals. Male Holtzman Sprague-Dawley rats (300-350 g; Harlan, Indianapolis, IN) were individually housed and maintained on a 12-h light/dark cycle with free access to food and water.
Intrathecal Catheter Implantation. For spinal drug injections, lumbar catheters were implanted in rats under isoflurane anesthesia according to a modification of the procedure described by Yaksh and Rudy (1976). A polyethylene catheter (PE-5, 0.014 in outside diameter; Spectranetics, Colorado Springs, CO) was inserted into the i.t. space and advanced to the rostral edge of the lumbar enlargement through an incision in the atlanto-occipital membrane. Five days after implantation, rats were entered into the study. In separate experiments to assess spinal prostaglandins release, rats were prepared with lumbar loop dialysis catheters with three lumens, as previously described (see Yaksh et al., 2001). In brief, the outer two lumens were connected to a length of dialysis tubing (10-kDa cut-off). The catheter was then implanted i.t. using the same technique as described above for the i.t. catheter. A 3-day interval was allowed to elapse prior to including the animal in a study. In all cases, the exclusion criteria were the presence of any neurological sequelae, 20% weight loss after implantation, or catheter occlusion.
Behavioral Analysis
Thermal Hyperalgesia. Two approaches were employed to initiate a hyperalgesic state. An inflammation-evoked thermal hyperalgesia was induced by subcutaneous injection of 2 mg of carrageenan [Sigma-Aldrich, St. Louis, MO; 100 µl of 20% solution (w/v) in physiological saline] into the plantar surface of the left hind paw. The thermally evoked paw withdrawal response was assessed (Dirig et al., 1997). In brief, the device consists of a glass surface (maintained at 25°C) on which the rats are placed individually in Plexiglas cubicles (9 x 22 x 25 cm). The thermal nociceptive stimulus originates from a focused projection bulb positioned below the glass surface. The stimulus is delivered separately to either hind paw of each test subject with the aid of an angled mirror mounted on the stimulus source. A timer is actuated with the light source, and latency is defined as the time required for the paw to show a brisk withdrawal as detected by photodiode motion sensors that stop the timer and terminate the stimulus. Paw withdrawal latencies are assessed prior to any treatment (control) and at intervals after treatment. Left (injured) and right (uninjured) paw withdrawal latencies are assessed and plotted versus time. In addition, difference latency scores (uninjured - injured) are calculated, and the average withdrawal latencies over the postinjection observation intervals are calculated for comparison between treatment groups. In addition to the use of a peripheral inflammation, a thermal hyperalgesia is also initiated by the i.t. injection of SP (20 nmol/10 µl). The mean paw withdrawal latency of the left and right paws is assessed at each time point. The mean difference between the pre- and post-i.t. SP response latency scores is calculated for analysis.
Intrathecal Dialysis and PGE2 Assay. Spinal dialysis experiments to define the spinal release of PGE2 were conducted in unanesthetized rats 3 days after dialysis catheter implantation. A syringe pump (Harvard Apparatus Inc., Holliston, MA) was connected, and dialysis tubing was perfused with artificial cerebrospinal fluid at a rate of 10 µl/min. The artificial cerebrospinal fluid contained 151.1 mM Na+, 2.6 mM K+, 0.9 mM Mg2+, 1.3 mM Ca2+, 122.7 mM Cl-, 21.0 mM HCO3, 2.5 mM HPO4, and 3.5 mM dextrose and was bubbled with 95% O2/5% CO2 before each experiment to adjust the final pH to 7.2. The efflux (20 min/fraction) was collected in an automatic fraction collector (Eicom, Kyoto, Japan) at 4°C. Two baseline samples were collected following a 30-min washout and an additional three fractions after i.t. injection of NMDA (0.6 µg). The concentration of PGE2 in spinal dialysate was measured by enzyme-linked immunosorbent assay using a commercially available kit (Assay Designs 90001; Assay Designs, Ann Arbor, MI). The antibody is selective for PGE2 with less than 2.0% cross-reactivity to PGF1
, PGF2, 6-ketoPGF1, PGA2, or PGB2 but cross-reacts with PGE1 and PGE3.
Drug Delivery. Drugs were delivered systemically (i.p.) or spinally (i.t.). Intraperitoneal drugs were delivered uniformly in doses prepared in volumes of 0.5 ml/kg. Drugs injected i.t. were administered in a total volume of 10 µl followed by a 10-µl flush using vehicle.
Enzyme Assays. In vitro group IV cPLA2 and group VI iPLA2 assays were done as previously described (Kokotos et al., 2002). Briefly, 100 µM lipid substrate and 100,000 cpm radiolabeled analog were dried down under N2 and dissolved in assay buffer containing 400 µM Triton X-100 to yield a mixed micelle substrate solution. Inhibitors dissolved in DMSO were added to the reaction tubes and allowed to incubate with substrate for 5 min at 40°C. Pure enzyme was added to yield a final volume of 500 µl, and digestion was carried out at 40°C for 30 min. Reactions were quenched and extracted using the Dole method, and products were quantified by liquid scintillation counting (Dole, 1956). Percent inhibition was determined at a range of inhibitor mole fraction concentrations for XI(50) calculations.
Inhibition of cyclooxygenase-1 and cyclooxygenase-2 was tested in vitro using the COX Activity Assay kit (catalog no. 760151) from Cayman Chemical (Ann Arbor, MI). Assays were performed in 96-well plates using 10 µl of supplied COX standard (catalog no. 760152) that contained COX-1 and COX-2 proteins. Activity was detected colorimetrically at 595 nm by the appearance of oxidized N,N,N',N'-tetramethylphenylenediamine, which has an absorption maximum of 611 nm (Kulmacz and Lands, 1983). Inhibitors dissolved in DMSO (study compounds) or ethanol (indomethacin) were added to 50 µM final concentration and allowed to incubate with the assay mixture including enzyme for 5 min. After addition of N,N,N',N'-tetramethylphenylenediamine and arachidonic acid, samples were mixed and allowed to incubate for 5 min at room temperature before reading absorbance at 595 nm to determine results. Results were calculated, and percent inhibition values were derived.
Drugs
PLA2 inhibitors employed in these studies were synthesized (see below). These agents were prepared for delivery in a vehicle of 5% Tween 80. Other agents used in these studies included the cannabinoid agonist anandamide and the CB1 antagonist SR141716A (supplied courtesy of Benjamin Cravatt, Scripps Institute, La Jolla, CA). Anandamide was prepared in 100% DMSO and SR141716A in ethanol Emulphor and saline (1:1:18). Control studies were run with the respective vehicles.
Drug Synthesis
AX006 and AX010 were prepared as previously described (Kokotos et al., 2002, 2004). The synthesis and the characterization of the novel agents AX048 and AX057 are described here in detail. Figure 1 summarizes the synthesis schema.
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Ethyl 4-[(2-hydroxyhexadecanoyl)amino]butanoate. Yield 72%; 1H NMR. 
6.68 (1H, t, J = 7 Hz, NH), 4.13 (3H, m, CH, COOCH2CH3), 3.34 (2H, m, CH2NH), 2.68 (1H, b, OH), 2.32 (2H, t, J = 7 Hz, CH2COO), 1.80-1.58 (4H, m, CH2CH2COO, CH2CH), 1.45-1.23 (27H, m, 12xCH2, COOCH2CH3), 0.85 (3H, t, J = 7 Hz, CH3); 13C NMR: 174.0, 173.8, 72.2, 60.6, 38.5, 34.9, 31.9, 31.7, 31.4, 29.7, 29.6, 29.5, 29.4, 29.3, 25.0, 24.6, 22.7, 14.1. Anal. calcd. for C22H43NO4 (385.58): C, 68.53; H, 11.24, N, 3.63. Found: C, 68.12; H, 11.32; N, 3.48.
Tert-Butyl 4-[(2-hydroxyhexadecanoyl)amino]butanoate. Yield 64%; 1H NMR. 6.49 (1H, t, J = 7 Hz, NH), 4.12 (1H, m, CH), 3.34 (2H, m, CH2NH), 2.73 (1H, b, OH), 2.27 (2H, t, J = 7 Hz, CH2COO), 1.82-1.49 (4H, m, CH2CH2COO, CH2CH), 1.45 [9H, s, C(CH3)3], 1.38-1.15 (24H, m, 12xCH2), 0.89 (3H, t, J = 7 Hz, CH3); 13C NMR: 173.9, 173.7, 80.1, 72.3, 38.3, 35.4, 31.9, 31.8, 31.4, 29.7, 29.6, 29.5, 29.4, 29.3, 28.7, 25.1, 24.5, 22.8, 14.1. Anal. calcd. for C24H47NO4 (413.63): C, 69.69; H, 11.45, N, 3.39. Found: C, 69.42; H, 11.61; N, 3.27.
Oxidation of 2-Hydroxy-Amides
To a solution of a 2-hydroxy-amide (1.00 mmol) in a mixture of toluene-EtOAc (15 ml), a solution of NaBr (0.11 g, 1.05 mmol) in water (1.3 ml) was added, followed by 4-acetamido-2,2,6,6-tetramethylpiperidine-1-yloxy free radical (2 mg, 0.01 mmol). To the resulting biphasic system, which was cooled at -5°C, an aqueous solution of 0.35 M NaOCl (3.1 ml, 1.10 mmol) containing NaHCO3 (0.25 g, 3 mmol) was added drop wise while stirring vigorously at -5°C over a period of 1 h. After the mixture had been stirred for a further 15 min at 0°C, EtOAc (15 ml) and H2O (5 ml) were added. The aqueous layer was separated and washed with EtOAc (10 ml). The combined organic layers were washed consecutively with 5% aqueous citric acid (15 ml) containing KI (0.04 g), 10% aqueous Na2S2O3 (6 ml), and brine and dried over Na2SO4. The solvents were evaporated under reduced pressure, and the residue was purified by column chromatography [EtOAc-petroleum ether 1:9 (b.p., 40-60°C)].
AX048. Yield 86%; white solid; mp 63-64°C; 1H NMR. 7.16 (1H, m, NH), 4.12 (2H, q, J = 7 Hz, COOCH2CH3), 3.33 (2H, m, CH2NH), 2.89 (2H, t, J = 7 Hz, CH2COCO), 2.34 (2H, t, J = 7 Hz, CH2COO), 1.87 (2H, m, CH2CH2COO), 1.57 (2H, m, CH2CH2COCO), 1.40-1.15 (25H, m, 11xCH2, COOCH2CH3), 0.85 (3H, t, J = 7 Hz, CH3); 13C NMR: 199.0, 172.7, 160.2, 60.4, 38.5, 36.5, 31.7, 31.4, 29.5, 29.4, 29.3, 29.2, 28.9, 24.2, 23.0, 22.5, 14.0, 13.9; MS (FAB) m/z (%) 384 (100) [M++ H]. Anal. calcd. for C22H41NO4 (383.57): C, 68.89; H, 10.77, N, 3.65. Found: C, 68.71; H, 10.88; N,3.54.
AX057. Yield 95%; white solid; mp 61-62°C; 1H NMR. 7.11 (1H, m, NH), 3.33 (2H, m, CH2NH), 2.91 (2H, t, J = 7 Hz, CH2CO), 2.28 (2H, t, J = 7 Hz, CH2COO), 1.84 (2H, m, CH2CH2COO), 1.60 (2H, m, CH2CH2COCO), 1.45 [9H, s, C(CH3)3], 1.38-1.23 (22H, m, 11xCH2), 0.89 (3H, t, J = 7 Hz, CH3); 13C NMR: 198.6, 171.6, 159.7, 80.0, 38.1, 36.1, 32.2, 31.3, 29.0, 28.9, 28.8, 28.7, 28.4, 27.4, 23.8, 22.5, 22.0, 13.5; MS (FAB) m/z (%) 412 (17) [M++ H], 356 (100). Anal. calcd. for C24H45NO4 (411.62): C, 70.03; H, 11.02, N, 3.40. Found: C, 69.89; H, 11.32; N, 3.47.
Statistics
Escape latency data are presented as the mean ± S.E.M. For carrageenan and i.t. SP analysis of thermal escape, latencies were carried out over time and compared with one-way ANOVA. For carrageenan, difference scores between control and injured paws over time were calculated for each group. Comparison of drug with vehicle treatment was performed using an unpaired Student's t test. For dose-response analyses, least-squares linear regression was performed, and the drug dose required to produce a 50% reduction in the hyperalgesia otherwise observed in the vehicle-treated control animals was estimated. For release studies, release was expressed as percentage of baseline, and the area under the release curve following i.t. SP was calculated. Group comparisons were carried out using nonparametric statistics for repeated measures over time and (Friedman analysis) across treatment groups with post hoc analyses being undertaken with Dunns Multiple Comparison analysis. Analyses were performed using Prism statistical software (GraphPad Prism version 4.02 for Macintosh; GraphPad Software Inc., San Diego, CA).
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Characterization of PLA2 Inhibitory Activity: Enzymatic Assay. The inhibitory effects of AX006, AX010, AX048, and AX057 on pure group IVA PLA2 and group VIA PLA2 were examined, and the results are presented in Table 1 as XI(50). The XI(50) is the mole fraction of inhibitor in the total substrate interface required to inhibit the enzyme by 50%. The reason that XI(50) is used instead of the more common IC50 or KI is that PLA2 is active only on phospholipid surfaces such as cell membranes, phospholipid vesicles, or phospholipid micelles, where its substrate phospholipids reside. Almost all inhibitors of PLA2s partition at least to some degree into the phospholipid surface because they usually have a hydrophobic portion that complements the hydrophobic active site of the PLA2. When these inhibitors partition into the surface, an important physical effect called surface dilution comes into play. In this case, the affinity of the PLA2 for the inhibitor depends not on the three-dimensional (bulk) concentration of the inhibitor in molar units but on the two-dimensional (surface) concentration of the inhibitor in mole fraction units. As indicated (Figs. 2 and 3; Table 1), AX048 and AX057 were potent against group IVA PLA2 and group VIA PLA2, AX006 was potent against group IVA PLA2 alone, and AX010 was less effective against both.
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Characterization of COX Inhibitory Activity. Incubation with indomethacin produced a near-complete inhibition of the COX activity in the assay. In contrast, incubation with the AX compounds at concentrations that had significant effects upon PLA2 (50 µM) had no inhibitory effects upon COX activity (Fig. 4).
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Dose Dependence. The effects of i.p. AX048 were observed to be dose-dependent over the range of 0.2 to 3 mg/kg (slope; P < 0.0004) (Fig. 6). The ED50 was defined as the dose that reduced the hyperalgesia observed in a vehicle-treated animal by 50%. On this basis, the estimated i.p. ED50 value for i.p. AX048 was 1.2 mg/kg (95% confidence interval, -0.5572 to 0.7713).
Time Course of Action. To determine the time course of the drug action, i.p. delivery of AX048 (3 mg/kg) was undertaken at -15, -30, and -180 min (Fig. 7). As indicated, peak effects were noted at 30 min, and minimal effects were observed at 15 min. The effects persisted through for 180 min but were no different from the control by 360 min.
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Intrathecal Delivery and Carrageenan-Induced Thermal Hyperalgesia
Control. In animals receiving i.t. injections of vehicle, the intraplantar injection of carrageenan resulted in a significant unilateral thermal hyperalgesia as compared with the uninjected paw (Fig. 8).
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Intrathecal Substance P-Induced Thermal Hyperalgesia
Control. Baseline thermal escape latencies were on the order of 10 to 12 s. In systemic vehicle-treated animals, the i.t. injection of SP (20 nmol/10 µl) evoked a significant reduction in thermal escape latency as early as 15 min after injection, which persisted through the 45-min test interval, returning to baseline by 60 min (Fig. 9).
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Side Effect Profile. After delivery of the highest systemic dose (3 mg/kg) or i.t. dose (20 µg) of any of the compounds, there were no changes in any assessed reflex end points including eye blink, pinnae, placing, or stepping. The animals showed no change in righting response, symmetric ambulation, or spontaneous activity.
Spinal Prostaglandin Release
Control. Overall baseline dialysate concentrations after the initial washout and prior to drug treatment were determined to be 555 ± 75 pg/100 µl perfusate. Intrathecal injection of SP (20 µg) but not vehicle (saline, not shown) resulted in a statistically significant increase in PGE2 concentrations in spinal dialysate as compared with the vehicle-treated control (Fig. 10).
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Effects of CB1 Inhibition. To determine whether the effects of the active agent AX048 might be acting directly or indirectly through a central cannabinoid CB-1 receptor, rats were pretreated with i.t. vehicle or i.t. SR141716, a CB1 receptor antagonist, followed after 15 min by i.t. AX048 (30 µg) or i.t. anandamide (100 µg). Intrathecal SR141716 had no effect when delivered alone (data not shown). As shown in Table 2, in vehicle-pretreated animals, i.t. anandamide resulted in a significant increase in the thermal escape latency of the uninjured paw and that of the injured paw. Both effects were prevented by pretreatment with i.t. SR141716. Intrathecal AX048 significantly reversed the respective hyperalgesia but had no effect upon the thermal escape latency of the uninjured paw. The antihyperalgesic effects of i.t. AX048 were not altered by i.t. SR141716. These observations suggest that i.t. anandamide, but not i.t. AX048, were interacting with a spinal CB1 receptor to alter thermal escape latency.
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). In contrast, in those studies, an irreversible group VIA iPLA2 inhibitor (bromoenol lactone), given i.t., failed to show any antihyperalgesic effects at doses that did not produce motor dysfunction and, at a higher dose, failed to block evoked spinal PGE2 release. Yeo et al. (2004) reported that i.c.v. injection of AACOCF3 or bromoenol lactone produced antihyperalgesia as measured using facial carrageenan in mice (Yeo et al., 2004). Burke et al. (2001) reported that BMS-229724, a group IVA cPLA2 inhibitor, was orally active in inhibiting edema and neutrophil infiltration. The present data thus continue to leave the issue open regarding the relative contribution of group IVA cPLA2 and group VIA iPLA2.
Role of Spinal PLA2 Isoforms in Cascade. Western blotting and reverse transcription-polymerase chain reaction have shown that group IVA cPLA2, group VIA iPLA2, and secretory PLA2 (groups IIA and V) are constitutively expressed in the spinal cord (Sapirstein and Bonventre, 2000; Lucas et al., 2005; Svensson et al., 2005b). The role of these respective isoforms has been difficult to assess given the lack of potent and selective inhibitors. Based on our earlier work noted above, we have had a particular interest in group IVA cPLA2. In the present work, AX048 displayed a dose-dependent suppression of both centrally and peripherally evoked thermal hyperalgesia. Importantly, the comparable antihyperalgesic action of AX048 after i.t. delivery with 20 µg versus the dose of 3 mg/kg, given i.p., emphasizes an important spinal action. The effects of systemic delivery showed an onset of approximately 30 min and a duration of action that exceeded 180 min. Importantly, this dosing was shown to have a significant effect upon i.t. SP-evoked spinal PGE2 release, a downstream biomarker believed to be essentially dependent upon PLA2 activity (Svensson and Yaksh, 2002).
Although the primary target of these molecules examined in the present study is PLA2, we note that other possibilities may also be relevant including a direct effect upon cyclooxygenase or the endocannabinoid system, both of which may lead to a change in pain behavior in a hyperalgesic state (see Rice et al., 2002; Svensson and Yaksh, 2002). The present studies, however, showed no effects at the highest concentrations on either COX-1 or COX-2 activity. Recent work suggested that agents interacting with the COX cascade may exert effects though an endocannabinoid pathway (Seidel et al., 2003). We, however, do not think that an effect through the cannabinoid-1 receptor is likely. The effect upon the centrally mediated hyperalgesia excludes a peripheral cannabinoid-2 receptor action. Moreover, after i.t. delivery, anandamide elevated the thermal escape latency of the normal paw, an effect not mimicked by the AX048. Finally, SR141716A, a potent CB1 antagonist (Shire et al., 1999), given i.t. at a dose that reversed the i.t. effect of anandamide, failed to alter the effects of AX048. These data suggest an effect of spinal AX048 that is independent of an action upon either endogenous cannabinoid release or upon the receptor itself. These experiments provide supportive evidence consistent with the assertion that AX048 was indeed acting though a PLA2 enzyme. We recognize that these are complex systems, and other potential targets might be considered in future studies and include a variety of upstream enzymes (such as mitogen-activated protein kinase) (Svensson et al., 2005a) as well as downstream effects (such as inhibition of prostaglandin synthases or receptors) (Guay et al., 2004; Reinold et al., 2005).
Factors Governing Central Bioavailability and Activity. These compounds are constructed based on a 2-oxoamide with a hydrocarbon tail and four-carbon tether. An important consideration in the functionality of these agents is their high cLog P values, in the range of 6 to 8. It is widely considered that agents with log P values greater than 5 may not be "druggable" (Lipinski et al., 2001). It is important to note that in the present systems, the target of drug action is within the cytosol. This requires that the molecule have a lipophilicity that allows it to readily cross the cell membrane to interact with PLA2. In the present work, we found that three of the molecules, AX048, AX057, and AX006, possessed appropriate enzyme inhibitory activity in a cell-free in vitro assay. Yet, only AX048 was observed to show in vivo activity.
We suspect that the dissociation between in vitro and in vivo activity that these agents display may well depend on the complex issue of distribution that these molecules face. At present, we believe that AX048 may be acting as a prodrug. The most common prodrug moiety in marketed drugs is the esterification of an acid group with a simple alkyl alcohol. A variety of ester prodrugs, in particular ethyl esters, are summarized in a recent review (Beaumont et al., 2003). A number of ethyl ester angiotensin-converting enzyme inhibitors, for example enalapril, exhibit greater oral activity than would be expected purely from the increased lipophilicity due to the conversion to ethyl ester. Furthermore, there is evidence that this ethyl ester is actively absorbed by a carrier mechanism (Swan and Tukker, 1997). These data could explain why only ethyl ester (AX048) of the four agents is active in vivo, whereas the other three agents are inactive at a dose of 3 mg/kg. Nevertheless, the observation that AX048 was able to produce an antihyperalgesic effect indicates that this molecule has properties that allow penetration of cellular membranes. Further work will be required to define the critical physical chemistry that defines the ability of AX048 to gain access to the central nervous system and inhibit intracellular PLA2.
Multiple Effects of PLA2 Inhibition. In the face of peripheral inflammation and tissue injury, an exaggerated processing of nociceptive stimuli ensues. This facilitation reflects in part an afferent-evoked cascade leading to enhanced nociceptive processing at the spinal level. An important component of this cascade is associated with the actions of spinally released prostanoids. Support for this thesis arises from the observation that the spinal delivery of prostaglandins will induce hyperalgesia and that these lipidic acids are released into the spinal extracellular space after tissue injury (see references in Introduction). In addition, i.t. COX inhibitors reduce prostaglandins release and the facilitated state induced by peripheral injury or by the direct activation of these circuits with i.t. SP and/or glutamate (see Svensson and Yaksh, 2002). This cascade suggests the relevance of pursuing the upstream PLA2 linkages which precede those mediated by cyclooxygenase. We note, however, that there is substantial evidence that other products of PLA2 activity are important in nociceptive processing. Arachidonic acid generated by PLA2 isoforms can directly augment NMDA ionophore function (Richards et al., 2003). The NMDA receptor is believed to play an important role in pre- and postsynaptic facilitation at the spinal level (L'Hirondel et al., 1999; Richards et al., 2003). Arachidonic acid formed by the action of PLA2s also provides the essential substrate necessary for the cyclooxygenase-independent synthesis of isoprostanes. Spinal isoprostanes initiate facilitated transmitter release and neuronal discharge, and their spinal delivery will lead to hyperalgesia (Evans et al., 2000). Platelet-activating factor, an alkyl-phospholipid, arises from the membrane lipid hydrolysis by PLA2. Platelet-activating factor produces a prominent allodynia after spinal delivery (Morita et al., 2004). This lipid mediator is present in the spinal cord and is released from stimulated microglia cells (Jaranowska et al., 1995). PLA2 activity forms lysophosphates. These products have been implicated in facilitated states of pain processing (Inoue et al., 2004; Seung Lee et al., 2005). In short, we hypothesize that a more pronounced effect on spinal nociceptive processing might arise by blocking these linkages upstream to COX,. Finally, the present studies showing the development of systemically bioavailable PLA2-selective agents may be relevant to therapeutic targets other than pain. A variety of neuron inflammatory processes may also be mediated through their activation of neuraxial PLA2 isoforms.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: COX, cyclooxygenase; PG, prostaglandin; PLA2, phospholipase A2; cPLA2, calcium-dependent PLA2; iPLA2, calcium-independent PLA2; AX006, 4-[(2-oxohexadecanoyl)amino]butanoic acid; AX010, methyl 4-[(2-oxohexadecanoyl)amino]butanoate; AX048, ethyl 4-[(2-oxohexadecanoyl)amino]butanoate; AX057, tert-butyl 4-[(2-oxohexadecanoyl)amino]butanoate; SP, substance P; NMDA, N-methyl-D-aspartate; DMSO, dimethyl sulfoxide; SR141716A, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride; ANOVA, analysis of variance; AACOCF3, arachidonyl trifluoromethylketone; BMS-229724, 4-[4-[2-[2-[bis(4-chlorophenyl)methoxy]ethylsulfonyl]ethoxy]phenyl]-1,1,1-trifluoro-2-butanone.
Address correspondence to: Dr. Tony L. Yaksh, Department of Anesthesiology University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0818. E-mail: tyaksh{at}ucsd.edu
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