Los grupos interactivos

The transporter hypothesis aims to explain resistance to AEDs as a result of changes in expression of transporters at the BBB, which may reduce AED accumulation in the brain. The majority of research surrounding this hypothesis has focused on the role of efflux transporters, in particular P-gp, whilst the role of influx transporters of the SLC family has been largely overlooked. Transporters of the SLC family are critical for uptake and homeostasis of many essential nutrients (Pardridge et al., 1977; Pardridge, 1986). In addition it is becoming increasing clear that a number of drugs are subject to transport by the SLC family and that SLC transporters may influence the

pharmacokinetics of substrate drugs (König et al., 2013).

The organic anion transporting polypeptides (OATPs) are a well characterised family of SLC transporters encoded by the SLCO gene family and have been shown to mediate the sodium-independent transport of a range of chemically diverse drugs (Tamai et al., 2000; Zhang et al., 2002). OATP1B1 and OATP1B3 are the best studied members of the OATP family due to their expression at the sinusoidal membrane of hepatocytes (Hagenbuch et al., 2003; Hagenbuch et al., 2004). Here they mediate the rate limiting step in the hepatic clearance of many substrate drugs such as statins by transporting them across the sinusoidal membrane into hepatocytes where they are metabolically cleared by the CYP450 system (Hirano et al., 2004; Hirano et al., 2006).

One isoform of this family, OATP1A2 is a 670 amino acid glycoprotein which shares 66-77% homology with its rodent orthologs (Hagenbuch et al., 2003). OATP1A2 is predominantly expressed at the BBB and although the precise location of its membrane expression is debated, the majority of evidence suggests that OATP1A2 is expressed on the apical membrane of brain endothelial cells (Urquhart et al., 2009). OATP1A2 would thus likely act to pump drugs into brain endothelial cells that comprise the blood-brain barrier from the blood, therefore a reduction in the expression of OATP1A2 might reasonably explain the lack of accumulation of substrate drugs in the brain.

OATP1A2 has been shown to transporter a number of therapeutic agents including imatinib (Eechoute et al., 2011a; Eechoute et al., 2011b), methotrexate (Badagnani et al., 2006), levofloxacin (Maeda et al., 2007) and antiretroviral protease inhibitors (Hartkoorn et al., 2010).

33 Monocarboxylic acid transporters (MCTs), encoded by the SLC16 gene family function endogenously to transport fuels and so are predominantly expressed in tissues with high energy demands (Halestrap, 2013). One MCT isoform, MCT1 is known to be

expressed at the basolateral and apical membranes of cerebral microvascular endothelial cells (Pierre et al., 2005). The MCT transporter system is critical for brain uptake of fuels such as lactate, pyruvate and in times of calorific restriction ketones and utilises a proton dependent transport mechanism (Pardridge et al., 1986).

MCT1 was the first MCT isoform to be cloned from Chinese hamster ovary (CHO) cells in 1994 (Garcia et al., 1994). MCT1 is a 494 amino acid protein, (Enerson et al., 2003) and is ubiquitously expressed (Halestrap et al., 1999). MCT1 transports lactate and pyruvate with high affinity and Km values of 3-5mM and 0.7mM respectively have

been reported depending on the expression system employed (Halestrap et al., 2004).

Both membrane expression and activity of MCT1 has been shown to be dependent on its association with an essential accessory protein basigin (Wilson et al., 2005). Although MCT1 preferentially associates with basigin, it can also associate with another accessory protein embigin in the absence of its primary partner (Poole et al., 1997; Kirk et al., 2000). Basigin and embigin are glycoproteins containing a single transmembrane span which acts as a chaperone, translocating MCT1 to the plasma membrane where the transporter and its accessory protein remain tightly bound. Furthermore dissociation from the accessory protein renders the transporter defunct (Manoharan et al., 2006).

MCT1 has been implicated in the transport of a number of drugs including the drug of abuse γ-hydroxybutyrate (GHB) and the gabapentin pro-drug XP13512 (Cundy et al., 2004; Wang et al., 2006; Morris et al., 2008). More interestingly, MCT1 has been implicated in the transport of VPA (Utoguchi et al., 2000; Fischer et al., 2008) and recently a deficiency of MCT1 on cerebrovascular endothelial cells has been shown in both animal models of temporal lobe epilepsy (TLE) and human tissue resected in the surgical treatment of TLE (Lauritzen et al., 2011; Lauritzen et al., 2012).

Many in vitro models have been used previously to investigate functional transport of drugs including surrogate transport cell lines such as Caco-2 cells, overexpressing cell

34 lines (where the transporter of interest is cloned and transfected into cell lines such as MDCK or HEK293), and Xenopus laevis oocytes.

The X. laevis oocyte method of transporter expression is a proven, robust tool in the identification of drug transport. cDNAs cloned from cells or other sources can, after in vitro transcription to cRNAs, be injected into individual X. laevis oocytes and

transporter function determined after just 3 days using a radiotracer uptake assay. The main advantages of the X. laevis system over somatic cell expression systems are that genetic information can be transferred easily and rapidly, transfection rates are high and the oocytes express very few endogenous transporter proteins to interfere with results (Xia et al., 2007; Markovich, 2008).

The aim of the study described in this chapter was to firstly develop X. laevis expression models for both OATP1A2 and MCT1 and to characterise their functional transport using well described control substrates, estrone-3-sulfate and L-lactic acid respectively. These models could then be used in radiotracer uptake assays to determine if OATP1A2 and MCT1 actively transport commonly prescribed antiepileptic drugs at therapeutically relevant concentrations.

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2.2

Materials and methods