Bio-behavioural evaluation of efavirenz and Δ9-tetrahydrocannabinol alone and in combination in rats with respect to psychosis and anxiety

Abstract

The highly active antiretroviral therapy, efavirenz (EFV), has been noted to occur as a constituent of a potent new designer drug cocktail called “Nyaope” or “Whoonga”, which is reported to have mind-altering and addictive-like properties. As an anti-retroviral medicine for treatment against human immunodeficiency virus type-1 (HIV-1) induced acquired immunodeficiency syndrome (AIDS), EFV has been noted to induce a number of neuropsychiatric side effects viz. dizziness, hallucinations, sleep disturbances, anxiety, depression, impaired concentration, aggression, paranoia and acute psychosis. These neuropsychiatric effects clearly suggest the involvement of the central nervous system, which could provide insight on the reported addictive and abuse profile of EFV. Allegedly EFV is mixed with common household items (e.g. vinegar, detergents, baby powder, and rat poison) and other illicit substances (e.g. marijuana (containing Δ9- tetrahydrocannabinol (Δ9-THC), methamphetamine and/or heroin). This multi-drug cocktail is then either smoked or injected to produce a somnolent, euphoric sensation. The abuse profile and addictive properties of “Nyaope” increases the financial burden on the health support systems and agencies, encourages criminal activity and promotes resistance to antiretroviral therapies. Few preclinical studies have sought to elaborate on the addictive profile of EFV. One study noted that the pre-dominate behavioural profile of EFV is similar to that of the hallucinogen, lysergic acid diethylamide (LSD), with EFV having weak partial agonist activity at the serotonin 5-HT2A receptor subtype, while another study reported that EFV induces depression and anxiogenic behaviour. More recently, the addictive properties of EFV has been established in a preclinical study, which reported that sub-acute and sub-chronic exposure to EFV (5 mg/kg) produces significant rewarding effects in rodents together with associated monoamine alterations in reward pathways of the brain. However, the deeper underlying mechanisms of this response remain unknown. The primary aim of this study was to investigate the effects of a rewarding dose of EFV (5 mg/kg), Δ9-THC (0.75 mg/kg) and the combination of EFV + Δ9-THC on social interactive behaviour, sensorimotor domains as well as anxiety-like behaviour in rodents. The study (NWU-00278-17-A5) used 72 male, adolescent Sprague-Dawley rats (150g – 180g) divided into four equal groups (n = 18 per group). The animals were bred and housed under identical conditions in the Vivarium of the North-West University (NWU). The rats received alternate day drug-vehicle exposure to intraperitoneal injections of vehicle (pharmaceutical grade olive oil), EFV (5 mg/kg), Δ9-THC (0.75 mg/kg) or the combination of EFV + Δ9-THC (5 mg/kg and 0.75 mg/kg) for 17 days. The rats were subjected to well-validated behavioural tests to assess social interaction, anxiety and sensorimotor gating using the social interaction test (SIT), elevated plus maze (EPM) and previ pulse inhibition (PPI) of startle test, respectively. The SIT was specifically used to measure alterations in self-directed and social interactive behaviour. The study secondary aimed to simultaneously investigate whether alterations in the abovementioned behaviours could be attributed to neuroendocrine and immune-inflammatory alterations. Consequently, alterations in hippocampal oxytocin (OT) levels as well as alterations in plasma pro- and anti-inflammatory cytokines, viz. tumor necrosis factor alpha (TNF-𝛼) and interleukin-10 (IL-10) respectively, were measured using enzyme-linked immunosorbent assay (ELISA) kits. Moreover, plasma tryptophan and its kynurenine metabolites (kynurenine, kynurenic acid (KYNA) and quinolinic acid (QA)) were determined using high-performance liquid chromatography (HPLC). One-way and two-way analysis of variance (ANOVA) and Bonferroni post hoc testing with multiple comparisons were used for statistical analysis, with p < 0.05 deemed statistically significant. The results indicate that EFV induces deficits in social interaction, anxiogenic and psychotogenic behaviour when compared to the vehicle control. Δ9-THC similarly induced alterations in social interaction and psychotogenic behaviour, but in contrast with EFV induced anxiolytic behaviour when compared to the vehicle control. The combination of EFV + Δ9-THC promoted social behaviour and profound psychotogenic-like behaviour when compared to the vehicle control. Only the combination of EFV + Δ9-THC was found to increase hippocampal OT concentrations compared to the control group, which paralleled the effects of this combination to increase social interaction behaviour compared to the control group. Exposure to EFV and Δ9-THC alone induced a pro-inflammatory state by increasing plasma pro-inflammatory (TNF-𝛼) and decreasing antiinflammatory (IL-10) cytokines levels, respectively. Moreover, EFV, Δ9-THC and the combination of EFV + Δ9-THC induced significant disturbances in tryptophan metabolism as indicated by increased plasma levels of neurodegenerative QA and decreased plasma levels of KYNA, resulting in a reduced neuroprotective ratio (KYNA : Kynurenine). This animal study provided insight on the bio-behavioural profile evoked by the use of EFV alone or in combination with Δ9-THC, which may resemble that of “Nyaope”-cocktail users. Furthermore, the study has provided insight into possible neuroendocrine and peripheral immune-inflammatory mechanisms through which EFV induces its psychological behavioural profile.