Advancing ABC transporter biology: from cannabinoid interactions to neurobehavioural function

Abstract

Chapter 1 reviews the scientific literature and justifies the 3 main aims of this thesis. ATP-binding cassette (ABC) transporters are membrane bound efflux proteins located at several important biological barriers throughout the body. P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), are two widely studied ABC transporters and are both expressed throughout the intestine, liver and at the blood brain barrier (BBB). These transporters move a range of substrate drugs across plasma membranes to reduce intracellular concentrations. Many central nervous system (CNS) therapeutics, including several antipsychotics and anticonvulsants, as well as a range of chemotherapeutics, antibiotics and endogenous hormones are substrates of these transporters. Both P-gp and BCRP mediate multidrug resistance in cancer, where their overexpression results in reduced intracellular accumulation of anti-cancer drugs, which reduces their cytotoxic actions. This phenomenon promotes treatment failure in cancer therapy, but is also being increasingly recognised in mediating drug resistance in the treatment of schizophrenia and epilepsy. Cannabis is the most widely used illicit drug in the world. In recent years, there has been a world-wide shift toward acceptance of the medicinal use of cannabis. Like 10 other countries, Australia has recently passed federal legislation allowing the manufacture and distribution of medical cannabis. The two main phytocannabinoids found in cannabis, Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), interact with ABC transporters and modulate their expression. THC is the most abundant cannabinoid component in street cannabis and gives rise to its psychoactive effects. Whereas CBD is much less abundant in the plant, is not psychoactive, and has antipsychotic, anticonvulsant, antioxidant and anti-inflammatory actions. For example, recent phase III clinical trials reported CBD to reduce seizures in treatment- resistant childhood epilepsy rates. Screening novel therapeutics against ABC transporters is becoming common practice in CNS drug development to ensure optimal brain disposition. P-gp and BCRP contribute to drug resistance as they are found on brain endothelial cells and strongly limit the brain disposition of substrate drugs. There is currently no definitive research addressing whether CBD is a substrate of ABC transporters. The phytocannabinoid THC is a substrate of both P-gp and BCRP, and these transporters limit brain THC concentrations and its consequent pharmacodynamic actions. Considering the structural similarity of THC and CBD, it is possible that CBD might also be an ABC transporter substrate. Chapter 2 of this thesis aimed to assess whether P-gp and Bcrp impacts the brain transport of CBD by comparing CBD tissue concentrations in wild-type (WT) mice versus mice devoid of ABC transporter genes. P-gp knockout, Bcrp knockout, combined P-gp/Bcrp knockout and WT mice were injected with CBD, before brain and plasma samples were collected at various time-points. CBD results were compared with the positive control risperidone and 9-hydroxy risperidone, antipsychotic drugs that are established ABC transporter substrates. Brain and plasma concentrations of CBD were not greater in P-gp, Bcrp or P-gp/Bcrp knockout mice than WT mice. In comparison, the brain/plasma concentration ratios of risperidone and 9-hydroxy risperidone were profoundly higher in P-gp knockout mice than WT mice. These results suggest that CBD is not a substrate of P-gp or Bcrp and may be free from the complication of these transporters reducing brain uptake. Such findings provide favourable evidence for the therapeutic development of CBD in the treatment of various CNS disorders. In Chapter 3 we continued to examine cannabinoid and ABC transporter interactions, but this time by examining whether THC reduces the neurobehavioural effects of antipsychotics via an ABC transporter-mediated mechanism. Cannabis use increases rates of psychotic relapse and treatment failure in schizophrenia patients. Clinical studies suggest that cannabis use reduces the efficacy of antipsychotic drugs, however there has been no direct demonstration of this in a controlled study. The present study demonstrates that exposure to the principal phytocannabinoid THC reverses the neurobehavioural effects of the antipsychotic drug risperidone in mice. THC exposure did not influence D2 and 5-HT2A receptor binding, the major targets of antipsychotic action, however lowered the brain concentrations of risperidone and its active metabolite, 9-hydroxy risperidone. As risperidone and its active metabolite are excellent substrates of P-gp, we hypothesized THC might increase P-gp expression at the BBB and thus enhance efflux of risperidone and its metabolite from brain tissue. Further, we demonstrated that THC exposure increased P-gp expression in various brain regions important to risperidone’s antipsychotic action. We then showed that THC exposure did not influence the neurobehavioural effects of clozapine. Clozapine shares a very similar antipsychotic mode of action to risperidone, but unlike risperidone is not a P-gp substrate. Our results imply that clozapine or non-P-gp substrate antipsychotic drugs may be better first-line treatments for schizophrenia patients with a history of cannabis use. Not all individuals exposed to a traumatic event develop PTSD, therefore the search for PTSD susceptibility genes is an area of intense research focus. P-gp via its localisation at the BBB regulates the brain uptake of corticosteroid stress hormones that are involved in PTSD pathophysiology. Moreover, P-gp is found on microglia, the brain’s immune cells, which are activated in response to stress and have an emerging role in psychiatric disorders. Chapter 4 aims to examine whether P-gp moderates the behavioural and microglial response to stress in an animal model of PTSD. P-gp knockout mice displayed an unusual, frantic anxiety response to intraperitoneal injection stress in the light-dark test. They also tended to display reduced conditioned fear responses compared to wild-type (WT) mice in a paradigm where a single electric foot-shock stressor was paired to a context. Foot-shock stress reduced social interaction and decreased microglia density in the amygdala which was not varied by P-gp genotype. Independently of stressor exposure, P-gp deficient mice displayed increased depression-like behaviour, social withdrawal, facilitated sensorimotor gating, reduced acoustic startle reactivity, and increased locomotor activity. In addition, P-gp deletion increased microglia density in the CA3 region of the hippocampus, and the microglia exhibited a reactive, hypo-ramified morphology. In conclusion, this research shows that germline P-gp deletion affected various behaviours of relevance to psychiatric conditions such as PTSD, schizophrenia and depression. We also report for the first time that P-gp deletion and foot-shock stress independently affected microglia in the hippocampus and amygdala respectively. Finally, Chapter 5 summarises the main findings of the thesis in regards to ABC transporter biology and examines future directions with implications for medicinal cannabinoids, drug treatment strategies and CNS disorders.