Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management, University of California, Berkeley, California 94720-3112
Received June 11, 2003; accepted August 7, 2003
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ABSTRACT |
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Key Words: cannabinoid receptor; CB1; dodecanesulfonyl fluoride; O-isopropyl dodecylfluorophosphonate; neuropathy target esterase; lysophospholipase; fatty acid amide hydrolase.
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INTRODUCTION |
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This study considers the toxicological and structural aspects of organophosphorus and organosulfur esters as cannabinoid CB1 receptor ligands acting at the proposed nucleophilic site. Octyl and dodecyl derivatives are emphasized not only as fluorophosphonates and sulfonyl fluorides but also as benzodioxaphosphorin oxides (BDPOs) (which are known to inhibit NTE and NTE-LysoPLA [Quistad et al., 2003; Wu and Casida, 1992
] and FAAH [Quistad et al., 2001
]). Comparisons are made with mice for inhibition in vitro and/or in vivo between CB1, NTE-LysoPLA, and AChE. The N-(2-hydroxyethyl) substituent, important in anandamide binding at CB1 and agonist action (Hillard and Campbell, 1997
; Khanolkar and Makriyannis, 1999
), is also examined here in the sulfonamide series along with several analogs for possible inhibition of 3H-CP 55,940 binding or pharmacological activity.
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MATERIALS AND METHODS |
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Chemicals.
The test chemicals used for this study are listed in Tables 13
. Sources for the chemicals were: 3H-CP 55,940 (120 Ci/mmol) from New England Nuclear (Boston, MA); MAFP (compound 3) from Cayman Chemical (Ann Arbor, MI); diisopropyl fluorophosphate (DFP, compound 6) from Sigma Chemical (St. Louis, MO). Compounds described before and available from previous or analogous syntheses in this laboratory were: alkylfluorophosphonates 1 and 2 (Segall et al., 2003a
); 4 (Wu and Casida, 1995
); 5, 7, and 18 (Quistad et al., 2002b
); BDPOs 813 (Quistad et al., 2001
; Wu and Casida, 1992
); and alkane- and alkenesulfonyl fluorides 1416 (Segall et al., 2003a
,b
).
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Animal studies.
Male Swiss-Webster mice (2228 g) from Harlan Laboratories (Indianapolis, IN) were used for studies in accordance with the Guiding Principles in the Use of Animals in Toxicology as adopted by the Society of Toxicology in 1989. Test compounds were administered ip as solutions in dimethyl sulfoxide (10100 µl) in comparison with dimethyl sulfoxide alone as a control. For possible cannabinoid or delayed toxic effects, mice were maintained and observed for up to 5 days.
Cannabinoid and other pharmacologic effects.
Mice were treated ip with compounds 2, 14, 18, and several dodecane- and octanesulfonamides as above for observation of depression of activity and recumbent posture at 15 min (Quistad et al., 2001). They were scored individually on a scale of 04 for activity depression (no deaths during 015 min): 0, no effect; 1, minimal depression; 2, moderate depression but sternal posture; 3, recumbent posture; 4, total immobility. These signs are consistent with the enhanced effects produced by anandamide administered to mice lacking FAAH, which adopt a flattened, rigid posture and remain completely motionless (Cravatt et al., 2001
). Results are given as the mean followed in parentheses by the range (a single number indicates the same rating for all mice), n = 36. Potential analgesic activity was determined with the mouse hot plate assay 15 min after ip administration of the test compound at 5 or 10 mg/kg following the procedure used earlier (Tomizawa et al., 2001
).
Inhibition of CB1 receptor in vitro and in vivo .
Mouse brain membranes were used for the CB1 receptor binding assay with 3H-CP 55,940 as the radioligand determining nonspecific binding with MAFP for the nucleophilic site or WIN 55,2122 mesylate for the agonist site (Quistad et al., 2002a). The assays involved brain membranes (150200 µg protein, pooled from 10 mice) in incubation buffer (50 mM Tris [pH 7.4] containing 1 mM EDTA, 1 mM MgCl2, and 3 mg/ml bovine serum albumin) (475 ul) to which was added in sequence the test inhibitor in Me2SO (5 µl) or MAFP or WIN 55,2122 (10 or 1 µM final concentration, respectively) in Me2SO (10 µl). After incubation at room temperature for 15 min, the radioligand (10 nM final concentration) was added in Me2SO (10 µl). Incubations were carried out for 90 min at 30°C before termination by the addition of ice-cold wash buffer (0.9% NaCl with 1 mg/ml bovine serum albumin) (1 ml) and vacuum filtration using Whatman GF/B glass-fiber filters that were presoaked in wash buffer for 2 h at 4°C. The filters were rinsed three times with 4 ml of ice-cold wash buffer prior to scintillation counting. In some cases, potential hydrolysis of candidate inhibitors by FAAH was examined and minimized by assaying with phenylmethanesulfonyl fluoride (PMSF) (50 µM, 15 min preincubation) or alone (Compton and Martin, 1997
). The concentration of compound inhibiting 50% of the 3H-CP 55,940 binding (IC50) was derived usually from threefold dose differentials giving 1585% inhibition. Mean IC50 and SE values reported represent at least three experiments.
Assays of mouse brain FAAH, NTE, NTE-LysoPLA, and AChE activities.
The methods have been described (Quistad et al., 2001, 2002b
, 2003
; Wu and Casida, 1996
). Hydrolysis assays used specific substrates. FAAH activity was assayed with 14C-oleamide. NTE activity is defined as paraoxon-resistant and mipafox-sensitive using phenyl valerate. This enzyme is referred to here as NTE-LysoPLA to recognize that NTE utilizes lysolecithin as a substrate (i.e., has LysoPLA activity) (Quistad et al., 2003
). AChE activity is determined using acetylthiocholine with Ellmans reagent.
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RESULTS |
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Inhibitory Potency for CB1 Receptor, NTE-LysoPLA and AChE in Vivo Relative to Pharmacologic Response and Delayed Toxicity
Two very potent (2 and 14) and one moderately effective (18) CB1 inhibitors in vitro were examined for pharmacologic response at 15 min and CB1 inhibition in vivo 4 h after treatment (Table 2). The dodecyl compounds 2 and 14 both cause recumbent posture in mice at 400 mg/kg ip and inhibit CB1 strongly at 30 mg/kg, whereas octanesulfonyl fluoride (18) is much less active (requiring 100 mg/kg). Additional compounds giving less than 50% inhibition of CB1 receptor at 4 h with indicated dose (mg/kg) are: alkylfluorophosphonates 4 and 5 at 30, BDPOs 8 at 30, 9 and 11 at 10, and 13 at 100. After dosage of 14 at 30 mg/kg, mouse brain CB1 gave the following percentage inhibition as a function of time (n = 34): 87 ± 4 at 2 day and 17 ± 12 at 5 day. Thus, with 14 the half-time for CB1 activity recovery is estimated to be 34 days. Alkylfluorophosphonates 4 and 5 in this study and BDPOs 8 and 13 at 30 or 100 mg/kg are less active in vivo CB1 inhibitors than dodecyl compounds 2 and 14.
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Inhibitory Potency for CB1 Receptor in Vitro and Pharmacologic Responses for Dodecane- and Octanesulfonamides Modeled on Anandamide
A series of 23 C12 and C8 sulfonamide analogs of anandamide was synthesized as candidate agonists acting at CB1. The compounds were also tested for pharmacologic response in mice. Dodecanesulfonamides with cyclopropyl, 2-chloroethyl, isopropyl, and 2-fluoroethyl substituents on nitrogen (but not the 2-hydroxyethyl analog) showed moderate inhibition of CB1 binding (IC50 = 1032 µM), whereas the corresponding octanesulfonamides were inactive. Pharmacologic effects in mice at 15 min (hypomotility, but not death) were observed after a high ip dose (400 mg/kg) with most analogs, but C8 sulfonamides are generally more active than C12. The exception was a dodecanesulfonamide with bis(2-hydroxyethyl) substitutions at nitrogen. Stronger effects were observed for C8H17SO2-imidazole and C8H17SO2N- bound to 2-hydroxyethyl, 2-fluoroethyl, and 2-thiazolyl: recumbent posture or total immobility at 400 mg/kg and (although not tabulated) moderate reduction of mobility at 40 mg/kg. Overall, depressed mobility did not correlate to CB1 inhibition. Use of PMSF at 50 µM to inhibit possible FAAH hydrolysis of the sulfonamides (and thereby possibly enhance activity) had no effect on CB1 inhibitory potency (C12H25SO2-F, -NHCH2CH2Cl, and -NHCH2CH2F).
Six compounds were tested for possible analgesic activity in the mouse hot plate assay. No activity was observed at 5 mg/kg for C8H17SO2NHCH2CH2OH and at 10 mg/kg for C12H25SO2-NHCH2CH2OH, the C8 and C12 sulfonamides with the 2-fluoroethyl substituent, the C12 analog with bis(2-hydroxyethyl), and C12H25SO2N3 (17).
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DISCUSSION |
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Alkanesulfonyl fluorides can achieve essentially the same potency as their organophosphorus counterparts and, again, with specific chain length requirements. C12 is optimal in a C12C20 series (Deutsch et al., 1997a). The terminal unsaturation in 15 appears to reduce the potency versus the C12 compound 14, and arachidonylsulfonyl fluoride (16) with four cis double bonds is even less potent (Segall et al., 2003b
).
Selectivity for CB1 Receptor Relative to FAAH in Vitro
Since anandamide is both a ligand for CB1 and a substrate for FAAH, mutual inhibitors might be expected (Table 4). Long-chain (C18 and C20) alkylphosphonates have the highest selectivity for CB1 versus FAAH, while retaining high potency for CB1 (Martin et al., 2000
). In this investigation four series of C12 and C8 analogs (fluorophosphonates, BDPOs, phosphonic difluorides, and sulfonyl fluorides) were compared for selectivity between CB1 and FAAH. In each series, C8 versus C12 chain length has less effect on inhibition of FAAH activity than on CP 55,940 binding to CB1, where C12 is always preferred. Although C12 monofluorophosphonates were equally potent on CB1 and FAAH, all other C8 and C12 analogs were FAAH selective. Long-chain alkanesulfonyl fluorides are much more potent for FAAH inhibition compared to CB1, but compound 14 is somewhat more selective, favoring FAAH inhibition in vitro using rat brain by only 6-fold (Deutsch et al., 1997a
). Compound 18 is the most selective inhibitor of FAAH (Quistad et al., 2002b
). PMSF and palmitylsulfonyl fluoride are known to potentiate anandamide action at CB1 by inhibiting FAAH (Compton and Martin, 1997
; Gifford et al., 1999
).
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In conclusion, the most potent organophosphorus and organosulfur CB1 inhibitors examined here are also NTE-LysoPLA inhibitors and cause delayed toxicity in mice.
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ACKNOWLEDGMENTS |
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NOTES |
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2 To whom correspondence should be addressed at Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management, 115 Wellman Hall, University of California, Berkeley, CA 94720-3112. Fax: (510) 642-6497. E-mail: ectl{at}nature.berkeley.edu.
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