Studies on human insulin adsorption kinetics at an organic-aqueous interface determined using a label-free electroanalytical approach.

Protein adsorption represents a considerable challenge in the development and production of macromolecular drugs. From an analytical point of view the adsorption process is difficult to study in an efficient way using currently available techniques. In this work potential and time dependent adsorption and adsorption kinetics of human insulin at an 1,2-dichloroethane-aqueous interface were studied using a novel electroanalytical approach based on measurements of interfacial capacitance. Two different types of measurements were performed; potential scans and time scans. In the potential scans, the capacitance was measured over a range of applied potential differences across the interface. The interfacial potential difference is linked to the charge at the interface. Adsorption of human insulin was detectable at a bulk phase insulin concentration as low as 0.1 microM as a negative shift in the potential of zero charge (pzc). Adsorption kinetics were further studied using time scans in which the interfacial capacitance was measured at a fixed applied interfacial potential difference. Using this approach it was possible to study how the adsorption kinetics and the shape of the time scan curves were related to the bulk concentration of insulin and the interfacial potential difference. The changes in capacitance could be described phenomenologically by pseudo-first-order kinetics at low concentrations of insulin except at positive interfacial potential differences and high insulin concentrations (> or =0.25 microM) where a more complex shape of the time scans curves was observed.

Acyclovir prodrug for the intestinal di/tri-peptide transporter PEPT1: comparison of in vivo bioavailability in rats and transport in Caco-2 cells.

It has previously been shown that the prodrug Glu(acyclovir)-Sar has a high affinity for PEPT1 in Caco-2 cells. However, affinity does not necessarily lead to translocation by the transporter which is necessary for achieving an increased oral bioavailability. Therefore i.v. and p.o. doses of Glu(acyclovir)-Sar, acyclovir and valacyclovir were given to rats and the collected blood samples were analysed via LC-MS-MS. Furthermore, Caco-2 cell monolayers were exposed apically to Glu(acyclovir)-Sar, acyclovir, and valacyclovir and the concentration of drug and prodrugs in the cell extracts were determined and taken as a measure for intracellular accumulation. In addition, bi-directional transport studies of Glu(acyclovir)-Sar across Caco-2 cell monolayers and in vitro metabolism studies of Glu(acyclovir)-Sar in various media of rat origin were performed. For these purposes HPLC-UV analysis was applied. Oral administration of Glu(acyclovir)-Sar to rats resulted in low bioavailabilities of acyclovir (<2%) and intact prodrug (<5%). Studies performed on Caco-2 cell monolayers showed that in contrast to valacyclovir Glu(acyclovir)-Sar did not result in a detectable amount of acyclovir or Glu(acyclovir)-Sar in the cell extracts. Bi-directional flux across Caco-2 cell monolayers apical to basolateral (FluxA-->B) and basolateral to apical (FluxB-->A) was measured and the FluxB-->A/FluxA-->B ratios of approximately 0.8 indicate that apical efflux mechanisms may not explain this lack of intracellular accumulation. These data indicate that Glu(acyclovir)-Sar may not be translocated by PEPT1.

Prodrugs of purine and pyrimidine analogues for the intestinal di/tri-peptide transporter PepT1: affinity for hPepT1 in Caco-2 cells, drug release in aqueous media and in vitro metabolism.

A general drug delivery approach for increasing oral bioavailability of purine and pyrimidine analogues such as acyclovir may be to link these compounds reversibly to stabilized dipeptide pro-moieties with affinity for the human intestinal di/tri-peptide transporter, hPepT1. In the present study, novel L-Glu-Sar and D-Glu-Ala ester prodrugs of acyclovir and 1-(2-hydroxyethyl)-linked thymine were synthesized and their affinities for hPepT1 in Caco-2 cells were determined. Furthermore, the degradation of the prodrugs was investigated in various aqueous and biological media and compared to the corresponding hydrolysis of the prodrug valaciclovir. Affinity studies showed that the L-Glu-Sar prodrugs had high affinity for hPepT1 (K(i) approximately 0.2-0.3 mM), whereas the D-Glu-Ala prodrugs had poor affinity (K(i) approximately 50 mM). The pH-rate profiles of the prodrugs D-Glu[1-(2-hydroxyethyl)thymine]-Ala and L-Glu[acyclovir]-Sar showed specific base catalyzed degradation at pH above 4.5 and 5.5, respectively. This implicates that the degradation rates at pH approximately 7.4 (t(1/2) approximately 3.5 and 5.5 h) are approximately 25 times faster than at upper small intestinal pH approximately 6.0. In 10% porcine intestinal homogenate and 80% human plasma the half-lives of the L-Glu-Sar prodrugs were approximately between 45 and 90 min indicating a limited enzyme catalyzed degradation. In contrast, valaciclovir underwent extensive enzyme catalyzed hydrolysis in 10% porcine intestinal homogenate (t(1/2) approximately 1 min). In conclusion, L-Glu-Sar may potentially function as pro-moiety for purine and pyrimidine analogues, where release of parent compound primarily is controlled by a specific base catalyzed hydrolysis. Acyclovir is quantitatively released at the relevant pH 7.4, whereas the 1-(2-hydroxyethyl)-linked thymine is released instead of the parent compound thymine.