Supplementary Materials and Methods Study 1: Screening panel for the mode of action of acamprosate Drugs



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Supplementary Materials and Methods

Study 1: Screening panel for the mode of action of acamprosate

Drugs

Calcium-bis (N-acetylhomotaurinate (Ca-AOTA, acamprosate) was manufactured by Laboratorio Chimico Internazionale (Labochim, Milano, Italy) (Batch # 183201). 2,3-Dioxo-6-nitro-1,2,3,4-tetrahydro-benzo(f)quinoxaline-7-sulfonamide disodium salt) (NBQX), ([R-(R*,S*)]-5-(6,8-dihydro-8-oxofuro[3,4-e]-1,3-benzodioxol-6-yl)-5,6,7,8-tetrahydro-6,6-dimethyl-1,3-dioxolo[4,5-g]isoquinolinium bromide) (()-bicuculline methobromide), (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl)phosphinic acid hydrochloride (CGP55845), 3,5-dihydroxyphenylglycine (DHPG), (S)-(+)-α-amino-4-carboxy-2-methylbenzeneacetic acid (LY367385), 2-methyl-6-(phenylethynyl)-pyridine(MPEP), R/S)-1-aminoindan-1,5-dicarboxylic acid (AIDA), (2S)-2-amino-3-(3,5-dioxo-1,2,4-oxadiazolidin-2-yl)propanoic acid (quisqualic acid), and all other compounds were obtained from Tocris (Ellisville, MI, USA).


Electrophysiology in Xenopus laevis oocytes expressing NMDA receptors

The two subunit types expressed for our purposes were hNR1A and hNR2B, a combination thought to be predominantly present in the forebrain. Xenopus laevis oocytes were harvested, defolliculated and injected with cRNA 1-3 days prior to recording. On the day of the assay, only oocytes displaying robust currents upon addition of glycine (3 to 10 M) and glutamate (30-100 M) were used (> 100 nA currents). Assay buffer for recordings consisted of the following components: 90 mM NaCl, 10 mM hemi-Na HEPES, 2 mM KCl, 1 mM MgCl2 and 1.9 mM CaCl2.


To determine NMDA receptor agonist activity, glycine and glutamate were substituted with Ca-AOTA. Multiple concentrations were tested up to a maximal concentration of 1 mM. Elicited currents were measured and compared to currents seen with saturating concentrations of glycine and glutamate. Results for acamprosate were expressed as a percent (%) of the maximal response seen with glycine or glutamate. To determine NMDA receptor antagonist activity, standard Schild-plot analyses were performed. It was evaluated whether or not acamprosate could shift the affinity of glycine and glutamate. Concentration response curves of glycine and glutamate were prepared with varying concentrations of acamprosate (100M, 500 M and 1 mM).
NMDA receptor dependent EPSPs in slice preparations from the nucleus accumbens

A concentric stimulation electrode was placed in close proximity (ca. 0.5 mm) to a recorded cell in the nucleus accumbens of acute brain slices prepared from 6-10 week old male Sprague-Dawley rats (Charles River, Hollister, CA, USA). Electrical stimulation (10-40V for 1-5 msec every 15 sec) was used to evoke presynaptic release of glutamate in presence of AMPA receptor antagonist NBQX, GABAA receptor antagonist () bicuculline methobromide, and GABAB receptor antagonist CGP55845. EPSPs were measured before and after addition of acamprosate; resulting change in EPSP amplitude is presented as % difference.


Cell lines expressing mGluR1/5 and Ca2+ flux assay

Human mGluR1 and 5 were transfected into tet-inducible T-RExTM-HEK 293 cells (Invitrogen, Carlsbad, CA, USA) using FuGENE® transfection reagents (Roche, Plesanton, CA, USA. Cells expressing mGluR1/5 were then seeded into black 96-well plates and allowed to grow for 2 days. Media was removed and cells were washed with Hank's balanced salt solution (HBSS). Cells were loaded with FLUO-4 (Invitrogen) in HBSS for 30 minutes at 37 °C. FLUO-4 binds Ca2+ and its fluorescence increases, producing a signal, detected by CCD camera in the fluorescent imaging plate reader instrument which was equipped with appropriate excitation and emission filters for this particular probe (excitation wavelengths: λ = 470-495 nm; emission wavelengths: λ = 515-575 nm).


Patch clamp electrophysiology to measure effects of mGluR1/5 on resting membrane potential and spike number

Transverse hippocampal brain slices (300 m) were prepared from 6-10 week old male Sprague Dawley rats (Charles River, Hollister, CA). Using whole cell patch clamp, resting membrane potential was measured in CA1 neurons of the hippocampal region. Effects of mGluR1/5 agonist (DHPG), mGluR1 antagonist (LY367385), mGluR5 antagonist (MPEP), and acamprosate on resting membrane potential were subsequently recorded. For spike number depolarizing current was applied intracellularly for 0.5 seconds to evoke action potentials in CA1 neurons; stimulation intensity was adjusted to activate 3-5 action potentials during stimulation period. The number of action potentials was measured before and after application of various combinations of compounds: DHPG, LY367385, MPEP, and acamprosate.


Study 2: Testing different salt formulations of acamprosate in the ADE model

Drugs

For the synthesis of Na-AOTA (sodium 3-(acetylamino)propanesulfonate) a modified procedure according to Durlach, US 4,355,043 (Novel derivatives of 3-aminopropanesulfonic acid having a reinforced activity on membrane) was used. Briefly, 13.92 g (100.0 mmol) of 3-aminopropylsulfonic acid (homotaurine) (Toronto Research Chemicals, Cat # H656200) and 8.41 g (100.0 mmol) of sodium bicarbonate (NaHCO3) (EMD Chemicals, Cat # SX0320-1) were dissolved in 240 mL of a mixture of acetonitrile (MeCN) and deionized Millipore water (1:1 v:v) and 21.3 g (135.6 mmol) 2,5-dioxoazolidinyl acetate (acetic acid N-hydroxysuccinimide ester) were added to the solution. The reaction mixture was stirred overnight at room temperature and extracted three times with ethyl acetate (EtOAc) to remove excess of the acetylation agent. The solvents were removed through lyophilization. To remove N-hydroxysuccinimide, the residue was repeatedly (three to four times) dissolved in a minimal amount of hot methanol (MeOH), filtered hot, and the target compound was precipitated through addition of excess acetonitrile with vigorous stirring. After filtration, the precipitate was washed twice with diethyl ether (Et2O) to remove residual solvents. The residue was dried at 70 – 85 °C in high vacuum for two days to yield 17 g (84 %) of the target compound as a fine colorless powder. LC/UV (220 nm): 96.5 % by AUC. 1H NMR (400 MHz, D2O):  3.27 (t, J = 6.8 Hz, 2H), 2.94-2.88 (m, 2H), 1.97 (s, 3H), 1.96-1.87 (m, 2H) ppm. 13C NMR (125 MHz, D2O): 174.28, 48.79, 38.36, 24.34, 22.28 ppm. MS (ESI) m/z 182.2 (M+H)+, 204.1 (M+Na)+, 180.1 (M–H). The analytical data was consistent the proposed structure.


Animals

One hundred and eighteen 2-month-old male Wistar rats (from our own breeding colony at the CIMH, Mannheim, Germany) were used for the ADE experiments. All animals were housed individually in standard rat cages (Ehret, Emmendingen, Germany) under a 12 h artificial light-dark cycle (lights on at 7:00 a.m.). Room temperature was kept constant (temperature: 22±1 °C, humidity: 55±5 %). Standard laboratory rat food (Ssniff, Soest, Germany) and tap water were provided ad libitum throughout the experimental period. Body weights were measured weekly. All experimental procedures are approved by the Committee on Animal Care and Use, and will be carried out in accordance with the local Animal Welfare Act and the European Communities Council Directive of 24 November 1986 (86/609/EEC).


Long-term voluntary alcohol consumption in the home cage with repeated deprivation phases

After two weeks of habituation to the animal room, rats were given ad libitum access to tap water and to 5 %, 10 %, and 20 % ethanol solutions (v/v) as well. Spillage and evaporation were minimized by the use of special bottle caps. Drinking of alcohol and water was monitored by weighing bottles. For this purpose an electronic scale as accurate as 0.01 g was used. When placing bottle on the scale, the weigh is recorded automatically by a personal computer connected to it. From these data, water consumption (mL per kg of body weight per day; mL/kg/day) and alcohol consumption (calculated in g of pure alcohol per kg of body weight per day; g/kg/day) was calculated. The first two-week deprivation period was introduced after eight weeks of continuous alcohol availability. After the deprivation period, rats were given access to alcohol again and 4 more deprivation periods were introduced in a random manner. The long-term voluntary alcohol drinking procedure including all deprivation phases lasted in total 28 weeks.


Pharmacological studies

In order to study the effects of drug treatment on the expression on ADE, rats were divided into groups (for number of animals per group see figure legend 3) in such way that the mean baseline total alcohol intake was approximately the same in each group (i.e., 2.9 g/kg/day). Baseline drinking was monitored for at least three days. After the last day of baseline measurement, the alcohol bottles were removed from the cages leaving the animals with free access to food and water for several days (see above). Thereafter, each animal was subjected to a total of 5 intraperitoneal (i.p.) injections (starting at 7 p.m. with 12 hr intervals) of either vehicle or Ca-AOTA (200mg/kg), Na-AOTA (200 mg/kg), calcium chloride (CaCl2×2H2O; 73.4 mg/kg), or calcium gluconate (Ca-gluconate, 215 mg/kg). Note that all calcium salts contain equivalent amounts of Ca2+ ions (0.499 mmol/kg). The alcohol bottles were reintroduced after the second injection (at ~ 9 a.m.) and the occurrence of an ADE was determined. Total ethanol (g/kg of body weight/day) and water intake (ml/kg of body weight/day) were measured daily at ~ 9 a.m. for a subsequent week.


Home cage locomotor activity measurements by the E-motion system


In order to test for any sedative effects resulting from the drug treatment, home cage locomotor activity was monitored by use of an infrared sensor connected to a recording and data storing system (Mouse-E-Motion by Infra-e-motion, Henstedt-Ulzburg, Germany). A Mouse-E-Motion device was placed above each cage (30 cm from the bottom) so that the rat could be detected at any position inside the cage. The device was sampling every second whether the rat was moving or not. The sensor could detect body movement of the rat of at least 1.5 cm from one sample point to the successive one. The data measured by each Mouse-E-Motion device were downloaded into a personal computer and processed with Microsoft Excel. Monitoring of locomotor activity started four days before drug treatment procedure and was continued for five more post-treatment days. The percentage of each rat’s locomotor activity during and after treatment days was calculated by using the “before treatment” activity data as a reference.
Statistical analysis

For data analysis the statistical package Statistica was used (StatSoft, Tulsa, OK, USA). Data obtained from ADE measurements (total alcohol intake, water intake) and locomotor activity was analyzed using a two-way ANOVA with repeated measures (factors were: treatment group and time). Data analysis regarding the effects of treatment on the change in the rat body weight was performed using either a one-way ANOVA or independent two-tailed t-test (factor was: treatment group). Whenever significant differences were found, Tucky post-hoc tests were performed. The chosen level of significance was p < 0.05.


Study 3: Testing different salt forms of acamprosate in alcohol-seeking rats in the cue-induced reinstatement model



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