In-vitro dynamic dissolution/bioconversion/permeation of fosamprenavir using a novel tool with an artificial biomimetic permeation barrier and microdialysis-sampling

Fosamprenavir is a phosphate ester prodrug that, upon dissolution, is cleaved to the poorly soluble yet readily absorbable parent drug amprenavir. In this study, a novel cell-free in vitro setup with quasi-continuous monitoring of the dynamic dissolution/bio-conversion/permeation of fosamprenavir was designed and tested. It consists of side-by-side diffusion cells, where the donor and acceptor compartments are separated by the biomimetic barrier PermeaPad®, and sampling from the donor compartment is accomplished via a microdialysis probe. Externally added bovine alkaline phosphatase induced bioconversion in the donor compartment. Microdialysis sampling allowed to follow the enzymatic conversion of fosamprenavir to amprenavir by the bovine alkaline phosphatase in an (almost) real-time manner eliminating the need to remove or inactivate the enzyme. Biomimetic conversion rates in the setup were established by adding appropriate amounts of the alkaline phosphatase. A substantial (6.5-fold) and persistent supersaturation of amprenavir was observed due to bioconversion at lower (500 µM) concentrations, resulting in a substantially increased flux across the biomimetic barrier, nicely reflecting the situation in vivo. At conditions with an almost 10-fold higher dose than the usual human dose, some replicates showed premature precipitation and collapse of supersaturation, while others did not. In conclusion, the proposed novel tool appears very promising in gaining an in-depth mechanistic understanding of the bioconversion/permeation interplay, including transient supersaturation of phosphate-ester prodrugs like fosamprenavir.

 

Comments：

This study describes the development and testing of a novel cell-free in vitro setup for evaluating the dynamic dissolution, bio-conversion, and permeation of fosamprenavir. The setup consists of side-by-side diffusion cells with a biomimetic barrier and a microdialysis probe for real-time monitoring of the enzymatic conversion of fosamprenavir to the active drug amprenavir. The conversion rate was established by adding bovine alkaline phosphatase, which induced the bioconversion of fosamprenavir to amprenavir. The results showed that bioconversion led to a substantial and persistent supersaturation of amprenavir, resulting in a substantially increased flux across the biomimetic barrier. The proposed tool appears promising for gaining an in-depth understanding of the bioconversion and permeation of phosphate-ester prodrugs like fosamprenavir, including the impact of transient supersaturation on their permeation behavior.

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