Use of the intestinal bile acid transporter for the uptake of cholic acid conjugates with HIV-1 protease inhibitory activity.

PURPOSE: To investigate the ability of the human intestinal bile acid transporter to transport cholic acid conjugates with potential HIV-1 protease inhibitory activity. METHODS: Cholic acid was conjugated at the 24 position of the sterol nucleus with various amino acids and amino acid analogs. The CaCo-2 cell line was used as a model to investigate the interaction of these bile acid conjugates with the human intestinal bile acid transporter. Interaction between the carrier and the conjugates was quantified by inhibition of taurocholic acid transport and confirmed by transport of radiolabelled conjugates in this cell line. RESULTS: The highest interaction with the transporter, as quantified by inhibition of taurocholic acid transport, occurred when a single negative charge was present around the 24 to 29 region of the sterol nucleus. A second negative charge or a positive charge significantly reduced the interaction. Transport of radiolabelled cholyl-L-Lys-epsilon-tBOC ester and cholyl-D-Asp-beta-benzyl ester was inhibited by taurocholic acid. Of all tested compounds, only cholyl-D-Asp-beta-benzyl ester showed modest HIV-1 protease inhibitory activity with an IC50 of 125 microM. CONCLUSIONS: Cholic acid-amino acid conjugates with appropriate stereochemistry are recognized and transported by the human bile acid transporter and show modest HIV-1 protease inhibitory activity. Transport of these conjugates by the bile acid carrier is influenced by charge and hydrophobicity around the 24 position of the sterol nucleus.

In vitro gene delivery by degraded polyamidoamine dendrimers.

Transfection of cultured cells has been reported using complexes between DNA and spherical cationic polyamidoamine polymers (Starburst dendrimers) that consist of primary amines on the surface and tertiary amines in the interior. The transfection activity of the dendrimers is dramatically enhanced (> 50-fold) by heat treatment in a variety of solvolytic solvents, e.g., water or butanol. Such treatment induces significant degradation of the dendrimer at the amide linkage, resulting in a heterodisperse population of compounds with molecular weights ranging from the very low (< 1500 Da) to several tens of kilodaltons. The compound facilitating transfection is the high molecular weight component of the degraded product and is denoted as a "fractured" dendrimer. Transfection activity is related both to the initial size of the dendrimer and its degree of degradation. Fractured dendrimers exhibit an increased apparent volume change as measured by an increase in the reduced viscosity upon protonation of the terminal amines as pH is reduced from 10.5 to 7.2 whereas intact dendrimers do not. Dendrimers with defective branching have been synthesized and also have improved transfection activity compared to that of the intact dendrimers. For a series of heat-treated dendrimers we observe a correlation between transfection activity and the degree of flexibility, computed with a random cleavage simulation of the degradation process. We suggest that the increased transfection after the heating process is principally due to the increase in flexibility that enables the fractured dendrimer to be compact when complexed with DNA and swell when released from DNA.

Synthesis of carboxyacyl derivatives of phosphatidylethanolamine and use as an efficient method for conjugation of protein to liposomes.

Carboxyacyl derivatives of phosphatidylethanolamine with different chain length were synthesized. These compounds were generally prepared by conversion of an appropriate dicarboxylic acid to its anhydride with dicyclohexylcarbodiimide, then reaction with phosphatidylethanolamine (PE) and triethylamine, followed by acidification. These derivatives, when incorporated into liposomes, were highly efficient in conjugating protein to liposomes. Liposomes with PE amide derivative incorporated were activated with water-soluble carbodiimide, and subsequently reacted with protein. The protein to lipid coupling efficiency was shown to be dependent on the chain length of the derivative, and the optimum coupling efficiency was achieved with PE amide of 1,12-dodecanedicarboxylic acid. Up to 60% covalent coupling efficiency of mouse IgG to liposomes was demonstrated with little non-covalent binding. This method will be of great importance in the liposome-targeting field.

Use of a reversed-phase evaporation vesicle formulation for a homogeneous liposome immunoassay.

Complement-mediated release of enzyme molecules from reversed-phase evaporation vesicles serves as the basis of the sensitive homogeneous immunoassay reported here. We found it necessary to co-entrap the substrate glucose 6-phosphate with the bacterial enzyme glucose-6-phosphate dehydrogenase (EC 1.1.1.49) to protect enzyme activity during liposome preparation. Enzyme can be released specifically from these liposomes by incubation with antibody and complement. the enzyme is not merely available to substrate but is actually physically free of the liposomes. Inhibition of this complement-mediated lysis by theophylline is the basis for the homogeneous liposome immunoassay described. The assay results vary linearly with theophylline concentrations in plasma in the clinically relevant range, and serum components do not interfere. The reagents in the assay kit are stable for at least seven months when stored at 5 degrees C. No nontheophylline compounds reacted significantly with the antiserum used. The assay can be run in a kinetic format, with either ultraviolet or colorimetric detection.