Effects of the Immunostimulant, Levamisole, on Opiate Withdrawal and Levels of Endogenous Opiate Alkaloids and Monoamine Neurotransmitters in Rat Brain Sydney Spector, Ph.D., Inam Munjal, Ph.D., and Dennis E. Schmidt, Ph.D. This report presents evidence that the immunostimulant receptors. These results raise the intriguing possibility that drug levamisole, (Ϫ)-(S)-2,3,5,6-tetrahydro-6- agents such as levamisole, which elevate the levels of the phenylimidazo[2,1-b] thiazole monohydrochloride, endogenous opiate alkaloids, might be useful for treating produced a significant elevation of endogeneous morphine narcotic withdrawal. The mechanism for the and codeine levels in brain regions and peripheral organs immunostimulatory properties of agents such as levamisole and attenuated the effects of naltrexone-induced withdrawal and muramyl dipeptide (MDP) have not been established. in morphine-addicted rats. Levamisole also significantly We suggest that the ability of MDP and levamisole to altered the metabolism of norepinephrine, dopamine, and increase endogenous opiate alkaloids may be related to their serotonin in specific brain regions. These results suggest immunostimulatory properties. that levamisole’s attenuation of opiate withdrawal may be [Neuropsychopharmacology 19:417–427, 1998] © 1998 related to its ability to increase endogeneous opiate alkaloid American College of Neuropsychopharmacology. Published levels and/or to alter central monoaminergic function. by Elsevier Science Inc. Levamisole does not have significant affinity for opiate KEY WORDS: Morphine; Codeine; Levamisole; Narcotic precursor compounds. Levamisole initially was studied addiction; Immunostimulation for its anthelminthic action, but more recently has been shown to possess immunomodulatory properties (Marx Our group (Oka et al. 1985; Donnerer et al. 1986, 1987) 1976; Del Giaco et al. 1979). Several studies have shown and others (Goldstein et al. 1985) have identified the that immunomodulatory drugs attenuate the severity presence of morphine and codeine in mammalian tis- of the naloxone precipitated withdrawal behavior of sues and have demonstrated that mammalian tissues morphine-addicted rats (Dougherty et al. 1987; Dafny et have the ability to synthesize the opiate alkaloids from al. 1983). We reported previously that the immunostim- ulant agent muramyl dipeptide (MDP) also modified the withdrawal behavior of morphine-addicted rats and From the Department of Psychiatry, Vanderbilt University School of Medicine, Nashville, Tennessee, USA. elevated brain levels of endogenous codeine and mor- Address correspondence to: Sydney Spector, Vanderbilt Univer- phine (Munjal et al. 1996). We now report that levami- sity School of Medicine, Department of Psychiatry, AA-2232, Medi- sole attenuated the severity of the withdrawal syndrome cal Center North, Nashville, TN 37232, USA. Received October 21, 1997; revised February 4, 1998; accepted precipitated by naltrexone in morphine-dependent rats February 25, 1998. and, concomitantly, increased the concentrations of the NEUROPSYCHOPHARMACOLOGY 1998–VOL. 19, NO. 5 © 1998 American College of Neuropsychopharmacology Published by Elsevier Science Inc. 0893-133X/98/$–see front matter 655 Avenue of the Americas, New York, NY 10010 PII S0893-133X(98)00034-7 418 S. Spector et al. NEUROPSYCHOPHARMACOLOGY 1998–VOL. 19, NO. 5 Table 1. Effect of Levamisole (35 mg/kg IP) on Opiate Withdrawal Syndrome Wet Dog Teeth Fecal Treatment n Shakes Chattering Boli Placebo ϩ naltrexone 6 0 0 8 Ϯ 2 Morphine ϩ naltrexone 6 8 Ϯ 1* 7 Ϯ 1* Diarrhea* Morphine ϩ levamisole ϩ naltrexone 6 3 Ϯ 1** 3 Ϯ 1** 5 Ϯ 2** Placebo ϩ levamisole ϩ naltrexone 6 0 0 7 Ϯ 2 n ϭ number of rats/group. Numbers represent mean Ϯ SEM of the times the rats exhibited the behavioral signs during the 10 min. observation period. *p Ͻ .05; placebo ϩ naltrexone vs. morphine ϩ naltrexone. **p Ͻ .05; morphine ϩ naltrexone vs. morphine ϩ levamisole ϩ naltrexone. opiate alkaloids in various brain areas and peripheral phine pellets (containing 75 mg morphine base Na- organs. Levamisole administration also resulted in a tional Institute of Drug Abuse [NIDA], Rockville, MD complex regionally dependent alteration of biogenic USA) were implanted subcutaneously daily for 3 days. amine metabolism in brain. Control rats received placebo pellets implanted using identical procedures. On the 4th day, 24 hours after the last pellet implantation, the two most recent pellets METHOD AND MATERIALS were removed from the rat under light halothane anaesthesia. Two hours and 15 min later, the rats re- Behavioral Assessment of Opiate Withdrawal ceived levamisole (35 mg/kg IP) and 20 min later were Male Sprague–Dawley rats (200–250 g) were used in given naltrexone (1 mg/kg IP). The number of wet dog these studies. Under light halothane anesthesia, mor- shakes, episodes of teeth chattering, and fecal boli were Figure 1. Time-dependent effects of levamisole (35 mg/kg, IP) on morphine content in brain regions at various times. Val- ues represent the mean pmoles/mg tissue ϩ SEM of six rats per time point. Significance of the individual time points were determine by Bonferroni’s post hoc testing. NEUROPSYCHOPHARMACOLOGY 1998–VOL. 19, NO. 5 Effects of Levamisole on Opiate Withdrawal 419 counted for 10 min following the administration of nal- were rapidly homogenized without prior thawing in trexone by observing the rats in a clear Plexiglass cage ice-cold 10 mM HCl (polytron homogenized, setting 9 to which the animals had been previously adapted for 30 s, to yield 100 mg tissue/ml). A 100 ␮l portion of prior to drug administration. These behavioral with- the brain homogenate was removed for analysis of drawal indicators were chosen, because they represent monoamine metabolites. Concentrated HCl was added readily identified and distinctive withdrawal character- in a ratio of 6 ml of acid to 10 ml of remaining tissue ho- istics that can be accurately quantified with a minimum mogenate, the samples were vortexed, and the glucu- of subjective interpretation. ronide and sulphate conjugates of morphine and co- deine were hydrolyzed by heating in a water bath for 30 min at 95 to 100ЊC. The samples were centrifuged for 30 min at 10,000 ϫ g at 4ЊC and filtered through a small Extraction of Morphine and Codeine plug of glass wool contained in a small glass funnel to The method described by Donnerer et al. (1987) was remove particulate material. The pH of the samples used for the extraction and quantitation of codeine and were adjusted to 8.5 to 9.0, and the hydrolyzed free al- morphine from tissues. Groups of six morphine-naive kaloids were extracted using 5 volumes of 1:9 mixture rats were pretreated with either saline or levamisole (35 of n-butanol in chloroform. The organic phase was col- mg/kg, IP). At the indicated times, the groups were lected, the compounds of interest were back extracted rapidly rendered unconscious with 50% CO2 and sacri- into 2 to 2.5 ml of 10 mM HCl, and the resulting aque- ficed by decapitation. Brains and peripheral organs ous acid phase was evaporated to dryness in a Savant were removed and washed free of blood with ice-cold evaporation centrifuge. normal saline. The peripheral organs were frozen, and The dried sample was reconstituted in 2 ml of phos- the brains were further dissected on wet ice and the re- phate-buffered saline (pH 7.4), and the pH was ad- sulting brain regions immediately frozen on dry ice. For justed to 8.5 to 9.0 with 1N NaOH. The sample was then analysis, the brain regions or whole peripheral organs passed through C-18 Sep Pak cartridge, which had been Figure 2. Time-dependent effects of levamisole (35 mg/kg, IP) on codeine content in brain regions at various times. Values represent the mean pmoles/mg tissue ϩ SEM of six rats per time point. Significance of the individual time points were determine by Bonferroni’s post hoc testing. 420 S. Spector et al. NEUROPSYCHOPHARMACOLOGY 1998–VOL. 19, NO. 5 pretreated with 5 ml methanol wash. Following appli- Radioimmunoassay for Morphine and Codeine cation of the samples, the column was washed twice The dried samples were resuspended in 100 ␮l of phos- with 10 ml of distilled water, and the eluate was dis- phate buffered saline (pH 7.4), 50 ␮l of the morphine carded. The alkaloids were then eluted with 7 ml of a mix- antibody, l125 morphine (Abuserum Kit, Roche Pharma- ture of H O (95.5%), 1-propanol (3%), pyridine (0.8%), 2 ceuticals) were added, and the samples were incubated and glacial acetic acid (0.3%). The eluate was evapo- for 90 min at 4ЊC. Following equilibration, 250 ␮l of sat- rated to dryness in a Savant evaporation centrifuge. urated ammonium sulfate was added, and after 45 min, samples were filtered through GF/B glass filters. Filters were counted in a gamma counter, and concentrations HPLC Separation of Morphine and Codeine of the alkaloids were estimated using the Riacalc pro- gram (LKB Wallace). The samples were resuspended in 250 ␮l of the high- performance liquid chromatography (HPLC) mobile phase (water, 98.4%; 1-propranol, 0.5%; pyridine, 0.8%; glacial acetic acid, 0.3%; pH 5.2). The samples were fil- Analysis of Monoamine tered through 6.2-␮m Z-bind filter (2-spin, Gelman Sci- Neurotransmitter Metabolites ϫ ences) and injected onto a 25 cm 4 mm Lichrosorb The monoamine neurotransmitters and their metabo- RP-18 10 ␮m column (Alltech) at a flow rate 1.5 ml/ lites were determined in the dissected brain regions us- min, and 1 min fractions were collected using Foxy frac- ing the method of Schmidt et al. (1990). The HCl homo- tion collector (ISCO). The retention times were approxi- genate from above was centrifuged at 10,000 ϫ g for 10 mately 8 min for morphine and 19 min for codeine. The min, and the clear supernatant was decanted into a eluated fractions were then evaporated to dryness.