the BBB has been demonstrated both in epileptogenic brain tissue of human pharmacoresistant patients and in animal models of pharmacoresistant epilepsy (Tishler et al., 1995; Sisodiya et al., 2002; Aronica et al., 2003; Potschka et al., 2004; Volk et al., 2004a, b; Volk and Löscher, 2005; Hoffmann et al., 2006). In pharmacoresistant epileptic tissue, P-gp is also over-expressed in astrocytic foot processes and neurons limiting the AED concentration at the target even further (Aronica et al., 2003; Volk et al., 2004a). Over-expression of the multidrug transporter P-gp was demonstrated in dogs after increased seizure activity, status epilepticus and cluster seizures (Pekcec et al., 2009b). This is an interesting finding as seizure frequency and severity are, as aforementioned, clinical risk factors for pharmacoresistant epilepsy.
More than 50 SNPs and insertion/deletion polymorphisms have been reported for ABCB1, with some of them resulting in a change of P-gp expression and/or function (Löscher and Potschka, 2005). An aforementioned study has also described a polymorphism in the promoter region of ABCB1 in Border collies associated with poor controlled epilepsy (Alves et al., 2011). This polymorphism could also have resulted in a change of function or over-expression of P-gp. However, no functional study was performed. The authors also found three other SNPs in the coding region of ABCB1, which were not associated with drug response or epilepsy. Apart from genetic influences, drug-induced mechanisms will interfere with multidrug transporter expression at the BBB. The BBB efflux multidrug transporter expression adapts continuously to ensure protection and detoxification of the CNS from xenobiotic substances. Efflux transporter expression is regulated by pregnane X receptor (PXR), which reacts to xenobiotic (foreign toxic) compound exposure (Masuyama et al., 2005; Miller, 2010; Shukla et al., 2011). This results in a clearing of these compounds from the brain and/or body. In addition to the up-regulation of multidrug transporter expression, PXR co-regulates drug metabolizing CytP450 enzymes (Potschka, 2012). The binding domain of PXR and P-gp have many similarities and will interact with similar compounds, resulting in a dynamic process of regulating the efflux of xenobiotic compounds. AED have been reported to cause an up-regulation of P-gp expression via PXR activation. However, a clear interaction of PXR with standard AEDs remains contentious (Potschka, 2012). It appears that the main driving force for P-gp over-expression is seizure activity (Potschka, 2010). Over-expression is transient and includes brain regions involved in seizure initiation and propagation (Kwan et al., 2002). A high seizure frequency therefore results in an accumulation of efflux transporters at the BBB.
Each epileptic seizure results in a glutamate release, which activates an intracellular signalling cascade in BBB endothelial cells (Bankstahl et al., 2008; Bauer et al., 2008). The glutamate binds to endothelial N-methyl-Daspartate (NMDA) receptors, which starts arachidonic acid signalling. Cyclooxygenase-2 (COX-2) processes the arachidonic acid and produces prostaglandin E2 (PGE2). PGE2 binds on the prostaglandin E receptor (EP1) resulting in P-gp expression (Pekcec et al., 2009a).
Should P-gp over-expression be one of the major reasons for drug refractoriness, blocking or reducing the expression of P-gp could reverse the lack of drug response. Several case reports in human medicine and studies in animal models of refractory epilepsy have shown an improved seizure control when P-gp inhibitors were used (Brandt et al., 2006; Potschka, 2012). However, a recent study performed in dogs using verapamil as a P-gp inhibitor did not show a significant reduction of the seizure frequency. Verapamil is not a very specific P-gp inhibitor, which could explain the negative results. Furthermore, verapamil dosage was limited due to its cardiovascular side effects. The other problem with using a P-gp inhibitor is the lack of specificity for the BBB, such that the rest of the excretory body system will be limited in function, therefore the long-term safety of this treatment approach needs to be questioned. A more promising route might be the use of COX-2 inhibitors as they have been shown to decrease P-gp in rodent epilepsy studies that also resulted in a reversal of the drug refractoriness (Potschka, 2012).
Conclusion
In conclusion, our understanding of pharmacoresistance has grown significantly over the last several years. Focusing on seizure freedom and quality of life will change the approach in veterinary medicine. In conjunction with a better understanding of mechanisms involved in drug refractoriness a new era of treatment development will have to evolve, hopefully improving animals’ welfare.
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