Primary biliary cirrhosis is characterized by the presence of autoantibodies to mitochondria with specific reactivity to proteins of 74 and 52 kilodaltons (kd). The 74-kd mitochondrial protein is the E2 component--dihydrolipoamide acetyltransferase--of the pyruvate dehydrogenase complex, and the 52-kd protein is the equivalent E2 component--dihydrolipoamide acyltransferase--of the branched-chain alpha-keto acid dehydrogenase complex. Current methods for the detection of antibodies to these proteins lack specificity or sensitivity, or they are time-consuming and not readily available. We therefore developed an enzyme-linked immunoassay to quantify specific antimitochondrial antibodies in patients with primary biliary cirrhosis. Recombinant polypeptides coding for both the 74-kd and the 52-kd mitochondrial autoantigens were used to analyze 217 coded serum samples, including samples from 93 patients with primary biliary cirrhosis and 124 controls, for reactivity by our immunoassay, immunoblotting, and immunofluorescence testing. Serum samples from 89 of the 93 patients with primary biliary cirrhosis reacted with either the pyruvate dehydrogenase-E2 or the branched-chain alpha-keto acid dehydrogenase protein. None of the 124 control samples from healthy volunteers (n = 86) or patients with primary sclerosing cholangitis (n = 38) had significant reactivity. Our results indicate that the use of recombinant, cloned autoantigens provides a simple, accurate, and rapid method of quantifying and monitoring the levels of specific mitochondrial autoantibodies in the serum of patients with primary biliary cirrhosis.

The renewed interest in inhalation delivery over recent years has led to an expansion in the understanding of lung pharmacokinetics. Historically optimisation of inhaled drugs focused largely on development of material properties, consistent with achieving a good lung deposition, alongside demonstrating appropriate in vivo efficacy with little understanding of the relationship to pharmacokinetics in the lung. Recent efforts have led to an increased understanding of lung concentrations and how to maximise exposure in order to achieve the desired pharmacological response at a dose consistent with development of an inhaled product. Although there is a prerequisite for excellent potency in inhalation delivery, it is essential that this be combined with pharmacokinetic properties that allow sufficient free concentration at the effect site in lung to exert the pharmacological response for an appropriate dosing interval. Increases in basicity, polarity and/or decreases in aqueous solubility can extend pharmacokinetic duration and assist in finding the right balance between lung and systemic exposure. Current evidence suggests there are similarities in lung retention in rat and dog and that animal lung concentration data can enable pharmacokinetic-pharmacodynamic relationships to be derived thus providing more confidence in the requirements for man. Although inhaled delivery is challenging from a pharmacokinetic point of view, direct evaluation of exposure in the target organ has enabled further understanding of the drivers for drug disposition and highlighted the need for further development of predictive lung pharmacokinetic tools in the future.

Multidrug resistance-associated protein (MRP) isoforms 1 through 6 mRNA are expressed in the human intestine and Caco-2 cells. In Caco-2 cells, the rank order for mRNA expression was MRP2 Ն MRP6 Ͼ MRP4 Ն MRP3 Ͼ MRP1 ϭ MRP5. The functional expression of MRP-like activity was quantified as the efflux of the fluorescent probe calcein from confluent, polarized monolayers of Caco-2 cells. Calcein efflux was sensitive to temperature, energy depletion, and the MRP an-Calcein efflux across the apical membrane of Caco-2 cells exceeded that across the basolateral by approximately 2-fold, correlating with the apical localization of MRP2 visualized by immunocytochemical staining. T84 cells do not express MRP2 and show a predominance of basolateral calcein efflux over apical efflux. MRP3 was localized by immunocytochemical staining to the basolateral membrane. MRP1 staining was not localized to either membrane domain and MRP5 staining was not detected. Thus, basolateral calcein efflux may reflect a function of MRP3 or MRP4 and 6 inferred by their basolateral localization in other tissues. Basolateral, but not apical, calcein efflux was sensitive to glutathione depletion with buthioninesulfoximine, indicating that whereas MRP2-mediated apical efflux is independent of glutathione, basolateral efflux is glutathione-dependent. Benzbromarone, probenecid, pravastatin, and diclofenac were able to inhibit both apical and basolateral calcein efflux. The apical calcein efflux in Caco-2 cells was selectively sensitive to indomethacin and propranolol, but not verapamil or erythromycin, whereas the converse was observed for basal efflux. The differential pharmacological sensitivity of apical (MRP2) and basolateral calcein efflux provides tools for dissecting MRP isoform functional roles.

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