Novel -Hydroxybutyric Acid (GHB) Analogs Share Some, but Not All, of the Behavioral Effects of GHB and GABAB Receptor Agonists

-Hydroxybutyrate (GHB), a therapeutic for narcolepsy and a drug of abuse, has several mechanisms of action that involve GHB and GABAB receptors, metabolism to GABA, and modulation of dopaminergic signaling. The aim of these studies was to examine the role of GHB and GABAB receptors in the behavioral effects of GHB. Three approaches were used to synthesize GHB analogs that bind selectively to GHB receptors and are not metabolized to GABA-active compounds. Radioligand binding assays identified UMB86 (4-hydroxy-4-napthylbutanoic acid, sodium salt), UMB72 [4-(3-phenylpropyloxy)butyric acid, sodium salt], UMB73 (4-benzyloxybutyric acid, sodium salt), 2-hydroxyphenylacetic acid, 3-hydroxyphenylacetic acid (3-HPA), and 4-hydroxy-4-phenylbutyric acid as compounds that displace [H]NCS-382 [5-[H]-(2E)-(5-hydroxy5,7,8,9-tetrahydro-6H-benzo[a][7] annulen-6-ylidene) ethanoic acid] from GHB receptors at concentrations that do not markedly affect [H]GABA binding to GABAB receptors. In rats and pigeons, GHB discriminative stimulus effects were not mimicked or attenuated by UMB86, UMB72, or 3-HPA up to doses that decreased responding. In mice, GHB, GHB precursors ( -butyrolactone and 1,4-butanediol) and GABAB receptor agonists [SKF97541 [3-aminopropyl(methyl)phosphinic acid hydrochloride] and baclofen] dose-dependently produced hypolocomotion, catalepsy, ataxia, and loss of righting. The GABAB receptor antagonist CGP35348 (3-aminopropyl(diethoxymethyl)phosphinic acid) attenuated catalepsy and ataxia that was observed after GHB and GABAB receptor agonists SKF97541 and baclofen. UMB86, UMB72, UMB73, and 3-HPA, like GHB, produced hypolocomotion, ataxia, and loss of righting; however, catalepsy was never observed with these compounds, which is consistent with the cataleptic effects of GHB being mediated by GABAB receptors. Ataxia that was observed with UMB86, UMB72, UMB73, and 3-HPA was not antagonized by CGP35348, suggesting that ataxia induced by these analogs is not mediated by GABAB receptors and might involve GHB receptors. -Hydroxybutyrate (GHB) is a putative neurotransmitter and neuromodulator in brain (Maitre, 1997), a therapeutic for alcoholism (Poldrugo and Addolorato, 1999) and narcolepsy (Fuller and Hornfeldt, 2003), and a drug of abuse (Degenhardt et al., 2003; McDonough et al., 2004). GHB binds to GHB receptors (Benavides et al., 1982; Mehta et al., 2001) and GABAB receptors (Xie and Smart, 1992; Lingenhoehl et al., 1999), and it is metabolized to GABA, which can act at GABA receptors. GHB has also been hypothesized to modulate GABAA receptor function through GABAB receptor-mediated increases in neurosteroid concentrations in rat brain (Barbaccia et al., 2002). Furthermore, several studies have reported that GHB modulates dopaminergic transmission, although the mechanism of this modulation is not well understood (Feigenbaum and Howard, 1996). Effects of exogenously administered GHB include sedation, This work was supported by U.S. Public Health Service Grants DA14986 (to C.P.F.) and DA15692 (to W.K.). C.P.F. is the recipient of a Senior Scientist Award (DA17918). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.104.077578. ABBREVIATIONS: GHB, -hydroxybutyrate; UMB68, 4-hydroxy-4-methylpentanoic acid; [H]NCS-382, 5-[H]-(2E)-(5-hydroxy-5,7,8,9-tetrahydro-6H-benzo[a][7] annulen-6-ylidene) ethanoic acid; UMB72, 4-(3-phenylpropyloxy)butyric acid, sodium salt; UMB73, 4-benzyloxybutyric acid, sodium salt; UMB87, 4-(3-phenylpropyloxy)butyric acid, sodium salt; 2-HPA, 2-hydroxyphenylacetic acid; 3-HPA, 3-hydroxyphenylacetic acid; UMB86, 4-hydroxy-4-napthylbutanoic acid, sodium salt; GBL, -butyrolactone; 1,4-BDL, 1,4-butanediol; SKF97541, 3-aminopropyl(methyl)phosphinic acid hydrochloride; CGP35348, 3-aminopropyl(diethoxymethyl)phosphinic acid; %DR, drug-appropriate responding; CL, confidence limit. 0022-3565/05/3133-1314–1323$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 313, No. 3 Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics 77578/1199576 JPET 313:1314–1323, 2005 Printed in U.S.A. 1314 at A PE T Jornals on Sptem er 2, 2017 jpet.asjournals.org D ow nladed from seizures, catalepsy, ataxia, amnesia, coma, and death (Snead and Liu, 1993; Navarro et al., 1998; Mason and Kerns, 2002). The wide range of behavioral effects that can be observed following administration of GHB is likely due to its multiple mechanisms of action. Specific GHB receptors have been hypothesized (Benavides et al., 1982; Snead and Liu, 1984; Hechler et al., 1987; Castelli et al., 2000; Mehta et al., 2001) and recently cloned (Andriamampandry et al., 2003). However, behavioral studies suggest an important role for GABAB receptors (Colombo et al., 1998; Lobina et al., 1999; Carai et al., 2001, 2002a; Carter et al., 2003, 2004a,b; Ren and Mody, 2003), consistent with the finding that GHB binds to these receptors (Bernasconi et al., 1992; Xie and Smart, 1992; Mathivet et al., 1997; Lingenhoehl et al., 1999). The relative importance of GHB receptors in mediating behavioral effects of GHB is not clear, in part because GHB has direct and indirect effects at GABA receptors. Thus, GHB receptor-selective ligands are critical tools to examine the role of GHB receptors in the various effects of GHB and related compounds. We recently reported the synthesis of the GHB receptor-selective analog UMB68. UMB68 and GHB have similar affinities for [H]NCS-382-labeled GHB receptors; however, unlike GHB, UMB68 does not have affinity for GABAB receptors and does not have GHB-like discriminative stimulus effects in rats (Wu et al., 2003). In the present study, three approaches introduced aromatic substituents on the GHB molecule to prevent metabolism to compounds with affinity for GABA receptors. First, it has recently been shown (Macias et al., 2004) that the 4-hydroxyl group of GHB acts as a hydrogen bond acceptor when bound to GHB receptors, and not as a donor, as suggested previously (Bourguignon et al., 2000). Protecting the 4-hydroxyl of GHB as an aryl alkyl ether (UMB72, UMB73, and UMB87; Fig. 1) simultaneously converts the readily oxidizable alcohol function to the more inert ether function (Block and Beale, 2004), introduces an aromatic substituent, and allows for the retention of a hydrogen bond acceptor. Second, a phenolic hydroxyl cannot undergo metabolism to an amino group and also contains the required aromatic group. The presence of such a group in 2-hydroxyphenylacetic acid (2HPA) and 3-hydroxyphenylacetic acid (3-HPA) (Fig. 1) results in a conformationally restricted analog of GHB with a similar distance between the acid and the hydroxyl group. Third, 4-hydroxy-4-phenylbutyric acid has a sterically demanding phenyl ring in the 4-position and was previously shown to have affinity for [H]GHB-labeled receptors (Bourguignon et al., 2000). The same report showed that introducing 4-benzyl groups in the GHB molecule further increases the affinity for [H]GHB-labeled receptors. UMB86 (Fig. 1) has a napthyl group that occupies the same space as the phenyl group in the 4-benzyl analogs, whereas its steric bulk close to the alcohol would be expected to hinder enzymatic oxidation of the alcohol (Block and Beale, 2004). Of the compounds synthesized, first, two or three compounds from each of the three synthetic approaches were evaluated in the binding assays to determine whether compounds from different structural classes bound selectively to GHB receptors. [H]NCS-382 was used to examine the specific binding of GHB analogs to GHB receptors in rat cerebrocortical membranes (Mehta et al., 2001). The same analogs were also evaluated in different membrane preparations developed to study the specific binding of [H]GABA to GABAA or GABAB receptors. Second, a representative compound from each chemical approach was examined in drug discrimination experiments. Drug discrimination data in rats have shown that GHB precursors and compounds that act at GABAB receptors occasion substantial GHB-appropriate responding, whereas pharmacologically unrelated compounds do not (Winter, 1981; Carter et al., 2003; Baker et al., 2004). Compounds were also examined in pigeons discriminating GHB because recent studies suggest that the discriminative stimulus effects of GHB in this species involve not only GABAB receptors, but also GABAA receptors (Koek et al., 2004). Third, mice were used to study the effects of the same compounds, with the addition of UMB73, on locomotion, catalepsy, ataxia, and righting, alone, and together with a GABAB receptor antagonist. These latter studies were designed to compare the effects of a broad range of doses, up to and including doses that produced loss of righting or at which other limitations became apparent, of GHB and GHB precursors [ -butyrolactone (GBL) and 1,4-butanediol (1,4-BDL)], conventional GABAB receptor agonists (SKF97541 and baclofen), and selective GHB analogs (UMB86, UMB72, UMB73, and 3-HPA) on several unconditioned behaviors that are known to be affected by GHB in mice (Carai et al., 2001; Cook et al., 2002; Itzhak and Ali, 2002). GHB precursors and GABAB receptor agonists were also examined in the observational assays in mice because they serve as important controls and have been studied in the same binding and discrimination procedures (Mehta et al., 2001; Carter et al., 2003). GBL and 1,4-BDL serve as positive controls for GHB because these compounds Fig. 1. Structures of GHB, UMB68, 4-hydroxy-4-phenylbutyric acid, UMB86, UMB73, UMB87, UMB72, 2-HPA, and 3-HPA. Novel GHB Analogs: Binding and Behavioral Effects 1315 at A PE T Jornals on Sptem er 2, 2017 jpet.asjournals.org D ow nladed from exert their GHB-like effects through conversion to GHB, and they do not displace [H]GABA or [H]NCS-382 from rat cerebral cortex (Carter et al., 2002, 2003). Similarly, the prototypical GABAB receptor agonists SKF97541 and baclofen serve as positive controls. Given that GHB can produce effects through multiple mechanisms of action, it is important to compare the effects of GHB to those produced by conventional GABAB receptor agonists, as well as to related compounds that are used recreationally. Materials and Methods Drugs. All chemicals used in the synthesis of the test compounds were purchased from Sigma-Aldrich (St. Louis, MO). All compounds showed H NMR (300 MHz, D2O) and mass spectra (m/z, Finnegan LCQ, negative ion mode) consistent with their assigned structures. Elemental analyses were performed by Atlantic Microlabs Inc. (Norcross, GA) and were within 0.4% of theory. UMB86 was prepared by adding sodium hydroxide to a methanol solution of -napthylbutyrolactone and stirring for 2 h. After removal of the solvent, UMB86 was recrystallized from methanol (m.p. 181– 182°C). UMB72 and UMB73 were prepared by the method of Sheehan et al. (1971) (monoalkylation of 1,4-BDL under basic conditions), followed by oxidation of the remaining hydroxyl group with pyridinium dichromate using standard conditions (Corey and Schmidt, 1978). The sodium salts of UMB72 and UMB73 were crystallized from methanol (m.p. 228–230°C). Application of the same alkylation procedure to the phenethyl analog (UMB87) was unsuccessful, presumably due to basic elimination of phenethyl bromide to give styrene. Thus, UMB87 was prepared using the mercury-assisted alkylation method of McKillop and Ford (1974), followed by pyridinium dichromate oxidation (m.p. 212–214°C). SKF97541 and CGP35348 were synthesized as described previously (Froestl et al., 1995). The radioligand [H]NCS-382 was synthesized as described previously (Mehta et al., 2001). [H]GABA was purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). GHB, GBL, 1,4-BDL, ( )baclofen, 3-HPA, and 2-HPA were purchased from Sigma-Aldrich. For in vivo studies, drug solutions were prepared as follows: GHB, SKF97541, CGP35348, and 3-HPA were dissolved in sterile water; baclofen was dissolved in physiological saline; GBL and 1,4-BDL were diluted in sterile water; and UMB72, UMB73, and UMB86 were suspended in a solution of physiological saline and 1% Tween 80 (Sigma-Aldrich). The pH of each drug solution was adjusted to between 5 and 9 with lactic acid or sodium hydroxide (SigmaAldrich), as necessary. Subjects. All animals were housed individually on a 12-/12-h light/dark cycle (experiments conducted during the light period) with free access to water in the home cage. Adult male Sprague-Dawley rats (Harlan, Indianapolis, IN) were used for binding studies and drug discrimination experiments. Rats used in the binding studies had free access to food and weighed between 250 and 300 g. Rats discriminating 200 mg/kg GHB i.p. (n 9; Carter et al., 2003; Wu et al., 2003) were maintained at 340 to 360 g by providing rodent chow (rodent sterilizable diet, Harlan Teklad, Madison, WI) in the home cage after daily experimental sessions. Adult white Carneau pigeons (Columbia livia; Palmetto, Sumter, SC) discriminating 100 mg/kg GHB i.m. (n 6; Koek et al., 2004) were maintained at 80 to 90% of their free-feeding weight, ranging from 590 to 620 g, by providing mixed grain in the home cage after daily sessions. Eighty male C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME) were used to examine directly observable effects; all mice had free access to food (rodent sterilizable diet; Harlan Teklad), weighed between 17 to 30 g, and were experimentally naive before testing. On arrival, mice were allowed at least 5 days to habituate to the experimental room, then were handled for at least 1 day prior to the start of testing. All animals were maintained and experiments were conducted in accordance with the Institutional Animal Care and Use Committee, The University of Texas Health Science Center (San Antonio) and with the 1996 Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources on Life Sciences, National Research Council, National Academy of Sciences). Binding. Membranes were prepared as described previously (Mehta et al., 2001). Briefly, rats were decapitated, and the cerebral cortex and cerebellum were dissected. Tissue was stored at 80°C until it was thawed and homogenized in ice-cold 0.32 M sucrose, pH 7.4 (20 ml/g tissue), and centrifuged at 1000g for 10 min at 4°C. The supernatant was then centrifuged at 140,000g for 30 min at 4°C to obtain the mitochondrial plus microsomal (P2 P3) fraction. This fraction was dispersed in ice-cold double-distilled deionized water and homogenized with a Brinkman Polytron at a setting of 6 for two 10-s bursts, 10 s apart. The suspension was centrifuged at 140,000g for 30 min at 4°C. The pellet was then resuspended in ice-cold Tris buffer (50 mM, pH 7.4) and centrifuged at 140,000g for 30 min at 4°C. This step was repeated twice. After the final centrifugation step, the pellet was suspended in a small volume of ice-cold Tris buffer (50 mM, pH 7.4) and stored at 80°C. On the day of the assay, the tissue was thawed and washed twice with buffer as before (140,000 g, 30 min, 4°C) and then resuspended in the buffer for use in the assay. [H]NCS-382 binding was measured using a centrifugation assay as described previously (Mehta et al., 2001). Briefly, aliquots (0.3– 0.4 mg of protein) of membrane preparation in Tris buffer (50 mM, pH 7.4) were incubated with [H]NCS-382 (16 nM) in triplicate at 4°C for 20 min in a 1-ml total volume. Nonspecific binding was determined using NCS-382 (500 M). The binding reaction was stopped by centrifugation (50,000g, 10 min, 4°C). The supernatant was decanted, and the vials were rapidly rinsed twice with 4 ml of ice-cold Tris buffer (50 mM, pH 7.4) without disturbing the pelleted tissue. Pellets were solubilized with 0.3 ml of Soluene-350 (Canberra Industries, Meriden, CT) for 4 to 6 h. Scintillation liquid (3 ml) was added to the solubilized material in the biovials. Radioactivity was quantified by liquid scintillation spectrometry. For determination of IC50 values, [ H]NCS-382 (16 nM) binding was carried out in the absence and presence of concentrations of unlabeled compounds. [H]GABA (10 nM) binding to GABAA receptors in cerebral cortex was performed in a similar manner, using a 10-min incubation period at 4°C, and GABA (100 M) to define nonspecific binding. Binding affinity for GABAB receptors was measured in tissue from cerebellum, an area where GHB binding to GABAB receptors has been shown previously (Mathivet et al., 1997; Wu et al., 2003). For [H]GABA (10 nM) binding to GABAB receptors in rat cerebellum, all of the assay tubes contained 40 M isoguvacine HCl (MP Biomedicals, Irvine, CA) to displace [H]GABA binding to GABAA receptors. These assay tubes also contained calcium chloride (2.5 mM), and the incubation was carried out at 25°C for 10 min. GABA (100 M) was used to define nonspecific binding. All other assay conditions for [H]GABA binding were the same as those used for the [H]NCS-382 binding assays. Drug Discrimination. Discrimination experiments using rats were conducted in commercially available operant chambers (model no. ENV-008CT; MED Associates, St. Albans, VT) located within sound-attenuating, ventilated enclosures (model no. ENV-022M; MED Associates), described in detail elsewhere (Carter et al., 2003). Data were collected using MED-PC IV software and interface (MED Associates). Rats were previously trained to discriminate 200 mg/kg GHB i.p. from saline. Discrimination training and experimental sessions were carried out as described previously (Carter et al., 2003). Immediately prior to each daily session, rats received 200 mg/kg GHB or saline i.p. and were placed into the operant chamber. A 15-min pretreatment period, during which the chamber was dark and responses (i.e., lever presses) had no programmed consequence, was followed by a 15-min response period, during which the stimulus lights above both levers were transilluminated, and 10 consecutive responses (fixed ratio 10; FR10) on the correct lever resulted in the delivery of a food pellet (45 mg; Research Diets; New Brunswick, NJ). 1316 Carter et al. at A PE T Jornals on Sptem er 2, 2017 jpet.asjournals.org D ow nladed from A response on the incorrect lever reset the FR requirement on the correct lever. The response period ended after 15 min or the delivery of 100 food pellets, whichever occurred first. Experimental sessions were conducted 5 to 7 days a week, and the order of training sessions was generally double alternation (e.g., saline, saline, drug, drug). All rats had satisfied the following testing criteria before this study: at least 90% of the total responses on the correct lever and fewer than 10 responses on the incorrect lever before delivery of the first food pellet for five consecutive sessions or six of seven sessions (Carter et al., 2003). Subsequently, rats were required to satisfy these criteria for at least one saline and one drug training session in two of the three sessions before a test (including the day immediately before the test). Test sessions were identical to training sessions, except that 10 consecutive responses on either lever resulted in the delivery of food. Test compounds (UMB72, UMB86, and 3-HPA) were studied alone (given immediately prior to the 15-min pretreatment period in the operant chamber) and 10 min before the training dose of GHB, up to a dose that markedly decreased responding in more than half of the subjects (UMB72 and UMB86) or at which other limitations were apparent (e.g., solubility; 3-HPA). The order of treatment with different doses was unsystematic. Discrimination experiments using pigeons were conducted in sound-attenuating, ventilated operant chambers (BRS/LVE, Laurel, MD) equipped with two translucent response keys that could be transilluminated by red lights. Data were collected using MED-PC IV software and interface (MED Associates) to monitor and control inputs and outputs to the operant chambers and to record the data. The procedure has been described in detail elsewhere (Koek et al., 2004). Briefly, before each daily session, subjects received either 100 mg/kg GHB or saline (i.m.) and were immediately placed into the chamber. Sessions started with a pretreatment period of 15 min, during which the lights were off and key pecks had no programmed consequence. Subsequently, the left and right keys were transilluminated red, and 20 consecutive responses (FR20) on the injectionappropriate key resulted in the key lights being extinguished for 4 s, during which time a white light illuminated the hopper where food (Purina Pigeon Checkers; Purina, St. Louis, MO) was available. Responses on the incorrect key reset the FR requirement on the correct key. The response period ended after 30 food presentations or 15 min, whichever occurred first. Experimental sessions were conducted 5 to 7 days a week, and the order of training sessions was generally double alternation (e.g., saline, saline, drug, drug). All pigeons had satisfied the following testing criteria before this study: at least 90% of the total responses on the correct key and fewer than 20 responses on the incorrect key before the first food presentation for at least seven of nine consecutive sessions (Koek et al., 2004). Thereafter, tests were conducted when these criteria were satisfied during two consecutive (drug and saline) training sessions. Test sessions were the same as training sessions, except that food was available after completion of 20 consecutive responses on either key. Test compounds were studied under the same temporal parameters, and doses were selected by the same criteria as were used in the rat studies. Behavioral Effects in Mice. Locomotor activity was assessed using four 301515-cm customized acrylic boxes (Instrumentation Services, University of Texas Health Science Center, San Antonio) that were separately enclosed in commercially available sound-attenuating chambers (model no. ENV-022M; MED Associates). Four infrared light beams were spaced 6 cm apart and located 2 cm above the floor of each box. Occlusions of the infrared light beams were counted using commercially available computer software (Multi-Varimex version 1.00, Columbus Instruments, Columbus, OH). The floor of the boxes consisted of a parallel grid of 2.3-mm stainless steel rods mounted 6.4 mm apart or of perforated 16-gauge stainless steel with 6.4-mm round holes (9.5-mm staggered centers). Floor types were counterbalanced between animals but were always the same for individual animals. Between tests, the floor and inside of the boxes were wiped with a damp sponge, and the litter paper beneath the floor was changed. Catalepsy was measured using a 1-cm-diameter horizontal bar supported 4 cm above the floor by two 88-cm square pieces of Plexiglas. Ataxia was studied by means of an inverted screen apparatus (Instrumentation Services, University of Texas Health Science Center), consisting of four 1313-cm wire screens (no. 4 mesh) located 23 cm above the floor of four Plexiglas containers. The four screens were connected to a rod and handle that could be rotated 180° to simultaneously invert the four screens. Mice were tested one to three times per week with at least 48 h between tests. Individual mice were tested on average nine times (range 1–16). Mice were not habituated to the locomotor activity boxes; however, the number of times that an animal had been tested did not significantly affect the mean number of beam breaks during the second half of the 30-min session under control conditions (i.e., after saline administration; F12,71 1.10, p 0.37), indicating that repeated testing of the animals did not confound the measurement of locomotion. Doses used in this study were based on preliminary dose finding data. The order in which the drugs and doses were tested was randomized within groups of 20 mice (with the exception of the largest dose of each compound studied, which was studied last due to the potential toxicity of those doses). Two groups of 20 mice were used to study the test compounds (UMB72, UMB73, UMB86, 3-HPA) alone, and two groups of 20 mice were used for the antagonism studies. On test days, one subgroup of mice (n 4) received 10 ml/kg saline, and four subgroups of mice (n 4 each) received a dose of a particular drug; saline and drug conditions were randomly assigned to individual subgroups, and no mouse received the same dose of drug twice. All drugs were administered i.p. in a volume of 0.1 to