Endocannabinoid Modulation of Scratching Response in an Acute Allergenic Model: A New Prospective Neural Therapeutic Target for Pruritus

Abstract

Pruritus is the general clinical term for itch, characterized by a generally unpleasant sensation attributed to an area at or just below the skin. The sensation is closely related to the reflex desire and action to scratch the afflicted area. Pruritus can greatly reduce the quality of life of those inflicted and is a common symptom attributed to discomfort from dermatological conditions (Stante et al., 2005). Although there are no collective data on the prevalence of patients seeking treatment for pruritus, the numbers on skin diseases alone estimates an incidence of one in three Americans afflicted at any given moment, costing a yearly $28.3 billion in medical treatment (Bickers et al., 2006). Because of the great variety of pathophysiological conditions (e.g., anemia, parasites, liver/kidney disease, thyroid disorders, HIV) underlying pruritus symptomology, common treatments are not always effective. Recent studies show that pruritus shares much of its neural substrates and signaling characteristics with that of another nocifensive modality, pain. Many of the same receptor systems shown to modulate analgesic actions also are being examined for their effects on itch (e.g., serotonin and opioid), often with antinociception-enhancing perception of itch (for review, see Paus et al., 2006; Schmelz and Handwerker, 2006).

Δ9-Tetrahydrocannabinol (THC), the primary psychoactive component in marijuana, and other cannabinoid receptor agonists produce analgesic effects through the activation of cannabinoid receptors in the periphery, spine, and brain (for review, see Pacher et al., 2006). However, given the occurrence of motor suppression, abuse potential, physical dependence, and psychomimetic effects of THC and other direct-acting cannabinoid agonists, recent efforts in cannabinoid research are focused on examining drugs that prevent the catabolism of endogenous cannabinoids. The endocannabinoid system consists of CB1 and CB2 receptors and several phospholipid-derived endogenous ligands that bind to these receptors. The most characterized endogenous cannabinoid, anandamide, is rapidly degraded predominantly by the enzyme fatty acid amide hydrolase (FAAH). Inactivation or inhibition of FAAH greatly increases anandamide levels in brain, spinal cord, liver, and other tissues (Cravatt et al., 2001; Boger et al., 2005; Fegley et al., 2005). FAAH also degrades several other fatty acid amides with known physiological functions, including oleamide (sleep), N-palmitoyl ethanolamine (PEA; anti-inflammatory), and oleoylethanolamide (satiety) (Cravatt et al., 2001). FAAH(-/-) mice and wild-type mice treated with FAAH inhibitors possess cannabinoid receptor-mediated hypoalgesic and anti-inflammatory phenotypes, without any signs of significant sedation or muscle relaxation (Lichtman et al.,2004a,b).

Although minimal study has been performed in the area of cannabinoids and pruritus, cannabinoid receptor agonists tend to decrease scratching behavior, whereas cannabinoid receptor antagonists, such as rimonabant, elicit a dose-dependent increase in scratching behavior in mice (Darmani and Pandya, 2000). It is interesting that several mixed CB1/CB2 receptor agonists (i.e., THC, WIN55212-2, CP55940, and HU-210) potently reversed rimonabant-induced scratching in an order similar to their potency to elicit known cannabinoid-mediated behavioral effects (Janoyan et al., 2002). In addition, each of these cannabinoids also reduced scratching elicited by the 5-hydroxytryptamine2A/C receptor agonist, 2,5-dimethoxy-4-iodoamphetamine (Darmani, 2001). In limited case reports, dronabinol (oral THC) relieved intractable pruritus resulting from liver cholestasis (Neff et al., 2002), a common clinical cause of severe and prolonged pruritus. Finally, transdermal application of the potent cannabinoid receptor agonist, HU-210, significantly reduced the perception of itch elicited by histamine iontophoresis in a small group of healthy volunteers (Dvorak et al., 2003). Taken together, the above evidence suggests that cannabinoids may provide a novel therapeutic target for both peripheral and central inhibition of pruritus. At present, there are no published studies of which we are aware that have examined the role of FAAH in an animal model of pruritus.

The overall objective of the present study was to elucidate the therapeutic potential of targeting the endocannabinoid system to treat pruritus. A model using the mast cell degranulator, compound 48/80, that directly activates signaling events characteristic of an acute allergenic response was adapted from Sugimoto et al. (1998). Local application of compound 48/80 elicits short-term and predictable levels of scratching behavior in the affected area. Clinical treatments of allergenic pruritus (e.g., antihistamines and glucocorticoids) effectively suppress scratching compound 48/80-induced scratching. The prototypical cannabinoid receptor agonist, THC, was evaluated in the compound 48/80 pruritus model for both scratching attenuation and levels of behavioral locomotor inactivity. FAAH(-/-) mice and wild-type mice treated with FAAH inhibitors were also assessed in the compound 48/80-induced scratching model. In addition, both pharmacological and transgenic tools were employed to elucidate cannabinoid receptor mechanisms and whether blockade of neuronal FAAH was critical in reducing scratching responses.

Materials and Methods

Subjects. Tests utilizing transgenic animals (i.e., FAAH, CB1, and CB2 knockout animals) and their controls were performed on mice from the Center Transgenic Colony at Virginia Commonwealth University (Richmond, VA) backcrossed onto either a C57BL/6J (13 generations) or DBA/1 (six generations) background. All other experiments utilized adult male C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME), with the exception of one intravenous study employing male ICR mice (Harlan, Indianapolis, IN). Mice were housed four to six per cage in a temperature (20–22°C)-controlled environment, with food and water available ad libitum while in their home cages. Mice were kept on a 12-h light/dark cycle, with all experiments performed during the animals’ light period. All experiments were performed with the approval of the Institutional Animal Care and Use Committee at Virginia Commonwealth University in accordance with the Guide for Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996).

Drugs. THC and rimonabant were obtained from the National Institute on Drug Abuse (Bethesda, MD), and OL-135 was synthesized as described previously (Boger et al., 2005). URB597 was purchased from Cayman Chemical (Ann Arbor, MI), and compound 48/80, diphenhydramine, dexamethasone, and loratadine were purchased from Sigma-Aldrich (St. Louis, MO). Compound 48/80, diphenhydramine, dexamethasone, and loratadine were dissolved in 0.9% saline, whereas all other drugs were dissolved in vehicle mixture of ethanol/alkamuls-620 (Sanofi-Aventis, Bridgewater, NJ)/saline in a ratio of 1:1:18. Drugs were diluted to an injection volume of 10 μl/g body mass and injected via intraperitoneal injection unless otherwise specified. Compound 48/80 was administered as a fixed quantity diluted per 200-μl subcutaneous injection.

All pretreatments and genotypic comparisons were made against a compound 48/80 challenge dose of 30 μg. The CB1/CB2 mixed receptor agonist THC, the first-generation antihistamine diphenhydramine, the nonsedating antihistamine loratadine, and the glucocorticoid dexamethasone, were each administered 30 min before compound 48/80 injection. The reversible FAAH inhibitor OL-135 and the irreversible FAAH inhibitor URB597 were both administered 1 h before compound 48/80, based on previous reports showing anandamide levels peaked at that time (Lichtman et al., 2004a; Fegley et al., 2005). In one study, URB597 was administered intravenously 10 min after application of compound 48/80 to examine reversal of scratching. In all studies utilizing rimonabant, the selective CB1 receptor antagonist was administered 15 min before compound 48/80.

Behavioral Evaluation of Scratching Response. Animals were pretreated with test drugs at times described for individual experiments. All animals were placed into white (for contrast) acrylic chambers (20 × 20 × 20 cm), with a clear acrylic front panel and a mirrored back panel, for 30 min to acclimate to the observational environment. The chambers were enclosed in sound-attenuating cabinets, designed and custom-built at Virginia Commonwealth University, that contained an indirect filtered light-emitting diode light source and fans for air circulation and white noise. At the 30-min time point, animals were briefly removed from the chambers, which were wiped clean with water. Subjects were then given an injection of compound 48/80 under the scruff at the most dorsal point of the back just beneath the head and were returned to the chambers for 30 min. Behavior was recorded through the clear front panel using a series of Fire-i digital cameras (Unibrain, Inc., San Ramon, CA), and the videos were processed and saved using ANY-maze video tracking software (Stoelting Co., Wood Dale, IL). Chambers were fully sanitized at the end of each testing day using ammonia-based cleansers and soap, then left to air dry for at least 2 days to dissipate any odors.

The videos were subsequently placed in randomized order in a separate ANY-maze protocol for a trained observer to score using a keyboard-based behavioral tracking system, blinded to treatment group. Either ANY-maze or ODLog2 (Macropod Software, Armidale, NSW, Australia) software was used to track key presses assigned to specific behavioral endpoints for both time pressed and number of occurrences. The scratching response was tracked as hind leg scratching of the injection site and surrounding areas, excluding behaviors focused at areas within the ears or flicking and pulling at ear tags. Behavioral locomotor inactivity was also monitored, scored as complete lack of voluntary movement for any continuous period longer than 5 s.

Data Analysis. All data are reported as mean ± S.E.M. and represent the total number of seconds a specific behavior was scored from a total observational period of 1800 s. Experiments with only two treatment groups were analyzed for statistical significance using Student’s t test. Experiments with more than two groups were analyzed using one- or two-way analysis of variance (ANOVA), with specified appropriate post hoc test. Resulting p values of less than 0.05 were considered significant. ED50 values (with 95% confidence intervals) and the potency ratios derived from the ED50 values were calculated using the least-squares linear regression method. Significant differences in potency were determined by the 95% confidence intervals of the potency ratio, not including a ratio of 1.

Results

Pruritic Response to Compound 48/80. In an initial experiment, we evaluated the compound 48/80 model as a reliable model of pruritus by examining a similar dose range as used by Sugimoto et al. (1998) in eliciting scratching. Injections of compound 48/80 in caudal regions of the back induced increased grooming behavior (data not shown) and intense scratching with the hind paws directed at the rostral area of the back. As shown in Fig. 1, compound 48/80 dose-dependently increased time mice spent scratching the area of injection more than 100-fold above that of saline control injections [F(5,30) = 55.3, p < 0.001]. Based on these results, 30 μg of compound 48/80 was used in all subsequent experiments because this was the lowest tested dose that significantly differed from saline at p < 0.01 (Dunnett’s post hoc test).

Fig. 1.

Dose-response of time spent hind leg scratching induced by subcutaneous compound 48/80 (1, 3, 10, 30, 100 μg). Totals represent time scratching within 30 min postinjection. , p < 0.05; , p < 0.01 versus …

To validate further the allergenic pruritus model, a pair of drugs representing the most common classes of clinically effective treatments for allergenic itch was tested against compound 48/80 (30 μg). The glucocorticoid compound, dexamethasone (2 mg/kg i.p.), significantly reduced scratching compared with saline pretreatment [t(14) = 6.1, p < 0.001]. In an initial study, we attempted to examine the dose-response relationship of diphenhydramine, an over-the-counter and commonly used H1 receptor antagonist. However, a significant reduction in scratching only occurred at doses that also elicited profound hypothermia and almost complete immobility (data not shown). The nonsedating second generation H1 receptor antagonist, loratadine (10 mg/kg i.p.), significantly reduced scratching behavior compared with vehicle controls [t(10) = 4.8, p < 0.001]. No significant effects of behavioral locomotor inactivity were found for either dexamethasone or loratadine. The results of these tests are summarized in Table 1.