Free Fatty Acids Cause pH-Dependent Changes in Drug-Lipid Membrane Interactions Around Physiological pH

Purpose. The influence of oleic acid (OA) and phosphatidylethanolamine (PhE) as membrane constituents on the partition behavior of (RS)-[³H]propranolol between unilamellar liposomes and buffer was studied as a function of pH. Methods. Partition studies were performed by means of equilibrium dialysis at 37°C over a broad pH range at a molar propranolol to lipid ratio in the membrane of 10^–6. Results. As compared to the standard phosphatidylcholine (PhC)-liposome/buffer partition system PhE and OA have an enhancing effect on the apparent partition coefficient (D) of (RS)-[³H]propranolol between pH 6 and 11. Data analysis with Henderson-Hasselbalch equations revealed that the neutral propranolol has a higher affinity to membranes containing net neutral charged PhE than to pure PhC-liposomes. Net negatively charged PhE seems to have no significant influence on the partitioning. Deprotonated OA caused an increase in the true partition coefficient (P) of the protonated propranolol. Neutral OA showed no influence on the partitioning. From the fit D vs pH curves and from zeta potential measurements of the liposomes the intrinsic pK_a values of the membranous lipids were calculated as 7.5 to 7.8 for OA and 9.7 to 9.8 for PhE. Conclusions. Since the pK_a of membranous OA is close to the physiological pH and D depends on the ionisation state of OA, small pH changes around the physiological pH may cause large differences in drug-lipid membrane interactions.     

Alpha-tocopherol influences the lipid membrane affinity of desipramine in a pH-dependent manner.

Phopholipidosis is a lipid storage disorder caused by cationic amphiphilic drugs (CADs) characterized by the lysosomal accumulation of phospholipids and drug. alpha-Tocopherol (alpha-Toc) has a reversible effect on phospholipidosis in rats and cell culture. We studied the influence of alpha-Toc on the partitioning of the CAD desipramine in a liposome/buffer system using equilibrium dialysis with the following lipid compositions: egg phosphatidylcholine (PhC) or wheat germ phosphatidylinositol (PhI) or a combination of PhC, PhI and cholesterol, containing between 1.5 and 20% (mol per mol total lipids) of alpha-Toc, alpha-tocopherol acetate (alpha-TocAc), 2,2,5,7,8-pentamethyl-6-chromanol (PMC) or cholesterol. alpha-Toc (1.5%) enhanced the partition coefficient of neutral desipramine by up to 1.1 log units while it had no influence on the partitioning of the ionized compound. In the PhC liposome system, at pH 7.4 logD increased with increasing alpha-Toc concentrations but was unchanged at pH 4.5. Similar effects were found with PMC while alpha-TocAc or cholesterol, between 1.5 and 20%, had no influence on the partitioning of desipramine. From these results we postulate that in vivo, alpha-Toc could mediate a redistribution of CADs from lysosomal membranes (pH approximately 4.5) to membranes and lipoproteins at physiological pH.     

Inhibition of multidrug resistance transporter-1 facilitates neuroprotective therapies after focal cerebral ischemia

The blood-brain barrier possesses active transporters carrying brain-permeable xenobiotics back into the blood against concentration gradients. We demonstrate that multidrug resistance transporter (Mdr)-1 is upregulated on capillary endothelium after focal cerebral ischemia; moreover, Mdr-1 deactivation by pharmacological inhibition or genetic knockout preferably enhances the accumulation and efficacy of two neuroprotectants known as Mdr-1 substrates in the ischemic brain. We predict that Mdr-1 inhibition may greatly facilitate neuroprotective therapies.     

Towards the Predictability of Drug-Lipid Membrane Interactions: The pH-Dependent Affinity of Propranolol to Phosphatidylinositol Containing Liposomes

Purpose. Prediction of the pH-dependent affinity of (RS)-[³H]propranolol to mixed phosphatidylcholine (PhC)/phosphatidylinositol(Phl) membranes from the partitioning in the single lipid liposome/buffer systems. Methods. Partition studies in liposome/buffer systems were performed by means of equilibrium dialysis at 37°C between pH 2 and 11 at a molar propranolol to lipid ratio of 10^–6 to 10^–5 in the membrane. Results. The Phl membrane more strongly attracts the protonated (RS)-[³H]propranolol than the neutral solute, i.e. the partition coefficient of the protonated base (P_i) is 17430 ± 1320, P of the neutral compound (P_n) is 3110 ± 1650. In the PhC-liposome system P_i is 580 ± 17, P_n 1860 ± 20. The partition coefficients show an exponential dependence on the molar Phl fraction in mixed liposomes. The partitioning in mixed PhC/Phl membranes is predictable from P_n and P_i in the single lipid liposome systems. Conclusions. The negative charge of biological lipid membranes causes strong electrostatic interactions with positively charged solutes. This strong attraction is not predictable from the octanol/buffer partition system, but it is important regarding drug accumulation in the tissue and drug attraction by certain lipids in the vicinity of membrane proteins.     

Role of drug efflux carriers in the healthy and diseased brain

The blood-brain barrier is a natural diffusion barrier, which expresses active carriers extruding drugs on their way to the brain back into the blood against concentration gradients. Whereas these so-called adenosine triphosphate-binding cassette (ABC) transporters prevent the brain entry of toxic compounds under physiological conditions, they complicate pharmacotherapies in neurological disease. Recent observations in animal models of ischemic stroke, drug-resistant epilepsy, and brain cancer showed that the prototype of ABC transporters, ABCB1, is upregulated on brain injury, deactivation of this carrier considerably enhancing the accumulation of neuroprotective, antiepileptic, and chemotherapeutic compounds. These studies provide the proof of concept that the efficacy of brain-targeting drugs may significantly be improved when drug efflux is blocked. Under clinical conditions, efforts currently are made to enhance drug accumulation by selecting new compounds that do not bind to efflux carriers or deactivating ABC transporters by targeted downregulation or pharmacological inhibition. We predict that strategies aiming at circumventing drug efflux may greatly facilitate progress in neurological therapies.     

Cholesterol-mediated activation of P-glycoprotein: distinct effects on basal and drug-induced ATPase activities

Cholesterol promotes basal and verapamil-induced ATPase activity of P-glycoprotein (P-gp). We investigated whether these effects are related to each other and to the impact of the sterol on bilayer fluidity and verapamil membrane affinity. P-gp was reconstituted in egg-phosphatidylcholine (PhC) liposomes with or without cholesterol, 1,2-dipalmitoyl-phosphatidylcholine (DPPC), alpha-tocopherol (alpha-Toc) or 2,2,5,7,8-pentamethyl-6-chromanol (PMC). Basal and verapamil-induced ATPase activities were studied with an enzymatic assay. Membrane fluidity was characterized with diphenyl-hexatriene anisotropy measurements and membrane affinity by equilibrium dialysis. DPPC (70% mol/mol) decreased the fluidity of PhC bilayers to the same level as 20% cholesterol. PMC (20%) and alpha-Toc (20%) decreased the fluidity to lesser extents. alpha-Toc and PMC, but not DPPC increased the verapamil membrane affinity. While 20% cholesterol strikingly enhanced the basal ATPase activity, none of the other constituents had a similar effect. In contrast, verapamil stimulation of P-gp ATPase activity was not only enabled by cholesterol but also by alpha-Toc and DPPC. PMC had no effect. In conclusion, cholesterol exerts distinct effects on basal and verapamil-induced ATPase activity. The influence on basal ATPase activity is sterol-specific while its effect on verapamil-induced ATPase activity is unspecific and not related to its influence on membrane fluidity and on verapamil membrane affinity.     

Immobilized Artificial Membrane (lAM)-HPLC for Partition Studies of Neutral and Ionized Acids and Bases in Comparison with the Liposomal Partition System

Purpose. To study the partitioning of model acids ((RS)-warfarin and salicylic acid), and bases (lidocaine, (RS)-propranolol and diazepam), with immobilized artificial membrane (lAM)-HPLC, as compared to partitioning in the standardized phosphatidylcholine liposome/buffer system. Methods. The pH-dependent apparent partition coefficients D were calculated from capacity factors (k_IAM) obtained by IAM-HPLC, using a 11-carboxylundecylphosphocholine column. For lipophilic compounds k_IAM, values were determined with organic modifiers and extrapolation to 100% water phase (k_IAMw) was optimized. Temperature dependence was explored (23 to 45° C), and Gibbs free energy (G), partial molar enthalpy (H) and change in entropy (S) were calculated. Equilibrium dialysis was used for the partitioning studies with the liposome/buffer system. Results. For extrapolation of k_IAMw, linear plots were obtained both with the respective dielectric constants and the mole fractions of the organic modifier. All tested compounds showed a similar pH-D diagram in both systems; however, significant differences were reproducibly found in the pH range of 5 to 8. In all cases, G and H were negative, whereas S values were negative for acids and positive for bases. Conclusions. In both partitioning systems, D values decreased significantly with the change from the neutral to the charged ionization state of the solute. The differences found under physiological conditions, i.e. around pH 7.4, were attributed to nonspecific interactions of the drug with the silica surface of the IAM column.     

P-Glycoprotein in cell cultures: a combined approach to study expression, localisation, and functionality in the confocal microscope.

Madin Darby canine kidney (MDCK) cells transfected with the multidrug resistance mdr1 gene, MDR1-MDCK (Pastan et al., 1988, Proc. Natl. Acad. Sci. USA 85 4486-4470), were used in a combined approach to study expression, localisation and functionality of the P-glycoprotein (P-gp) membrane transporter in the same cell culture preparations. Cells were characterised with regard to their growth curve, transepithelial electrical resistance (TEER), and cytoarchitecture. Efflux of the P-gp substrate rhodamine123 (rho123) was monitored with confocal laser scanning microscopy (CLSM). The transfected cells grew in multilayers. After reaching confluence they exhibited a complete tight junction (TJ) network. P-gp was strongly expressed at the uppermost apical surface of the multilayer already after 4 days in culture. The lower cell layers were not clearly polarised. P-gp-mediated transport could be followed by efflux of the fluorescent rho123 from the cells into the apical extracellular space. Verapamil, a P-gp inhibitor, significantly decreased efflux. For MDCK parent cells the rho123 assay was negative up to about day 20, and only at later times (day 25) low P-gp activity was detected. These results clearly show that despite the fact that the transfected cells form irregular layers, they provide a good model for screening of P-gp substrates and inhibitors.     

Transfer of lipophilic markers from PLGA and polystyrene nanoparticles to caco-2 monolayers mimics particle uptake

Purpose. The objective of this study was to evaluate nanoparticle uptake by the Caco-2 monolayer model in vitro. Special emphasis was placed on the localization and the quantification of the uptake of fluorescently labeled polystyrene and poly(lactic-co-glycolic acid) (PLGA) nanoparticles. Methods. Intracellular fluorescence was localized by fluorescence and confocal laser scanning microscopy. Particle uptake was quantified either directly, by counting internalized nanoparticles after separation from the Caco-2 monolayers, or indirectly, by extraction of the lipophilic fluorescence marker. In vitro release studies of lipophilic markers from nanoparticles were performed in standard buffer systems and buffer systems supplemented with liposomes. Results. Instead of uptake of polystyrene and PLGA nanoparticles by Caco-2 monolayers an efficient transfer of lipophilic fluorescence markers from nanoparticles into Caco-2 cells with subsequent staining of intracellular lipophilic compartments was observed. Whereas in standard buffer no release of fluorescent marker from polystyrene and PLGA nanoparticles was observed, the release studies using liposome dispersions as receiver revealed an efficient transfer of fluorescent marker into the liposome dispersion. Conclusions. The results suggest that the deceptive particle uptake is caused by a collision-induced process facilitating the transfer of lipophilic fluorescent marker by formation of a complex between the nanoparticles and the biomembranes. Diffusion of the marker within this complex into lipophilic compartments of the cell strongly affects quantitative evaluation of particle uptake.     

Differences in the Intracellular Distribution of Acid-Sensitive Doxorubicin-Protein Conjugates in Comparison to Free and Liposomal Formulated Doxorubicin as Shown by Confocal Microscopy

Purpose. To investigate differences in the cellular uptake and intracellular distribution of protein-bound doxorubicin in comparison to free doxorubicin and a liposomal formulation (CAELYX®) Methods. LXFL 529 lung carcinoma cells were incubated with an acid-sensitive transferrin and albumin conjugate of doxorubicin, a stable albumin doxorubicin conjugate, and free and liposomal doxorubicin for up to 24 h. The uptake of doxorubicin was detected with confocal laser scanning microscopy (CLSM). To investigate the intracellular localization of the anticancer drug, lysosomes, Golgi apparatus, and mitochondria were also stained by various organelle-specific fluorescent markers. In vitro efficacy of the doxorubicin derivatives was examined with the BrdU incorporation assay. Results. The acid-sensitive albumin and transferrin doxorubicin conjugates showed enhanced cytotoxicity in comparison to liposomal doxorubicin, whereas the stable albumin-doxorubicin conjugate showed only marginal activity. Of all compounds tested, doxorubicin showed the highest cytotoxicity. CLSM studies with specific markers for lysosomes, mitochondria, and the Golgi apparatus demonstrated that protein-bound doxorubicin or liberated doxorubicin was accumulated in the mitochondria and Golgi compartments, but not in the lysosomes after 24 h. Free doxorubicin showed a time-dependent intracellular shift from the nucleus to the mitochondria and Golgi apparatus. Fluorescence resulting from incubation with CAELYX was primarily detected in the nucleus. Conclusions. Our results indicate that other organelles in addition to the cell nucleus are important sites of accumulation and interaction for protein-bound doxorubicin or intracellularly released doxorubicin as well as for free doxorubicin.