Bile salts are endogenous surfactants which have been extensively studied as permeability enhancers to increase drug transport across various biological barriers such as the intestine, skin and buccal mucosa. However, only a few studies of the blood brain barrier (BBB) have been carried out. Previous animal studies have shown that 12-monoketocholate (MKC), a semisynthetic bile salt, enhanced brain uptake of quinine and increased the activity of morphine and pentobarbital in rat, which was speculated to be due to modulation of BBB permeability. Drug delivery to brain is largely prevented by the BBB with its densely packed lipid bilayers, tight junctions, efflux transporters and minimal endocytotic activity. The aim of this thesis was to study bile salts as permeability enhancers and to investigate the mechanism by which bile salts potentially enhance BBB permeability. MKC and three natural bile salts, cholate (C), deoxycholate (DC) and taurocholate (TC) were compared for their effects on the biophysical properties and transport characteristics of four different membrane models. From simple to complex, these were phospholipid monolayers, phospholipid bilayers (liposomes)), RBE4 cells (an immortalized rat brain capillary endothelial cell) and the whole animal (rat). The RBE4 cell line was used as a more complex bilayer model with some similarities to in vivo brain endothelium. In the phospholipid monolayer study using the Langmuir Blodgett trough, bile salts varied in their ability to penetrate into the monolayers in the order DC > TC > C> MKC. The penetration was dependent on the concentration of bile salt and on the surface pressure of the monolayer. Moreover, once bile salts inserted into the phospholipid monolayer, they increased membrane compressibility. In the phospholipid bilayer study, various techniques were used to study the effect of bile salts on their biophysical properties. Using electrophoresis and fluorescence polarisation spectroscopy, it was found that bile salts increased membrane surface negative charge and increased membrane fluidity. These effects subsequently modulated drug/membrane binding and membrane permeability. Capillary electrophoresis-frontal analysis showed that bile salts increased membrane binding of cationic compounds in a liposome/buffer system. Release studies using carboxyfluorescein as a model drug showed that, on initial exposure to bile salts, membrane permeability markedly increased but then stabilised. It is suggested that the initial insertion of the bile salt into the outer leaflet disrupts the membrane but then the membrane restabilises as the bile salt distributes approximately evenly between the inner and outer leaflets of the lipid bilayer. Although the lipid bilayer becomes relatively less permeable once the distribution of bile salt reaches equilibrium in both leaflets, the incorporation of bile salt in the bilayer still makes the membrane more permeable than a bilayer without bile salt as shown by a dithionite permeability study. In initial studies in RBE4 cells, cytotoxicity of bile salts was assessed using hemolysis, LDH and MTS assays. It was found that cytotoxicity of bile salts correlated with their lipophilicity with the exception of TC which was non-toxic. It is suggested that this is because TC is actively effluxed from RBE4 cells. In studies of drug transport via the transcellular pathway, rhodamine 123 (R123) was used as a model compound. It was found that C, DC and TC enhanced the uptake of R123 by increasing passive diffusion, probably as a result of their effect on cell membrane fluidity. DC showed a more significant effect on passive diffusion than other bile salts as it requires much lower concentration to increase R123 uptake significantly (ANOVA, α=0.05). Both uptake and efflux studies showed MKC inhibited efflux of R123 by P-glycoprotein (P-gp). Mechanistic modelling suggested that MKC decreased maximum efflux rate rather than the dissociation constant of P-gp-mediated efflux. In studies of drug transport via the paracellular pathway, it was shown that the tightness of the cell monolayers was increased by using astrocyte-conditioned medium. Relatively high concentrations of bile salts (2-5 mM) were required to increase permeabilities of sucrose and morphine-6-glucuronide (M6G). The former is a marker of paracellular transport and the latter is a very hydrophilic model drug. The effect of MKC on the pharmacodynamics and pharmacokinetics of M6G was studied in rat. The results show that MKC at a subcutaneous dose of 20 mg/kg significantly (ANOVA, α=0.05) enhanced analgesic activity of M6G in the hotplate test and increased M6G concentration in both brain and blood. Due to the decrease in plasma clearance of M6G in the presence of MKC, the blood concentrations of M6G in the treatment group (AUC = 8741±310 h•ng/ml) was much higher (no overlap of two 95% confidence intervals) than in the control group (AUC = 5561±142 h•ng/ml). As a result, the presence of MKC decreased the brain/plasma area under the curve (AUC) ratio. This may due to the active efflux transport of M6G in the in vivo BBB. In conclusion, the results of this research suggest bile salts increase membrane permeability by modulating some biophysical properties of membranes such as surface charge and fluidity. MKC inhibits P-gp efflux and may be useful to enhance the BBB permeability of P-gp substrates. In future, the effects of MKC on the BBB permeability of a P-gp substrate should be studied using microdialysis at a steady state blood level of the model drug.
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Effect of bile salts on drug delivery to the brain
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