Abstract
Purpose: :
To study the aqueous stability, transporter interaction, metabolism and cytotoxicity of stereoisomeric dipeptide monoester prodrugs of Gancyclovir for the treatment of Ocular Herpes Simplex virus infections.
Methods: :
Four stereoisomeric dipeptide prodrugs L-Valine-D-Valine-Gancyclovir (LDGCV), D-Valine-L-Valine-Gancyclovir (DLGCV), L-Valine-L-Valine-Gancyclovir (LLGCV), D-Valine-D-Valine- Gancyclovir (DDGCV) and two amino acid prodrugs D-Val-Gancyclovir (DGCV), and L-Val-Gancyclovir (LGCV) were synthesized and characterized using mass and NMR spectrometry. MDCK and SIRC cell lines were used as model cell lines to study the interaction of these prodrugs with Peptide Transporter (PEPT). The aqueous stability studies of these prodrugs were performed in Dulbecco’s phosphate buffered Saline (DPBS) at different pH (6.5, 7.4 and 8.5) at 340C for 10 days. Metabolism of these prodrugs were carried out in ocular tissue homogenates. Cytotoxicity of the prodrugs was analyzed using CtoTox-ONETM Homogenous Membrane Integrity Assay kit from Promega. Corneal transport of these prodrugs was carried out using a side-by-side diffusion apparatus. All the samples were analyzed using HPLC and LC-MS/MS.
Results: :
No detectable degradation was observed for LDGCV, DGCV at pH 6.5. However, the degradation rate was found to be higher at alkaline pH. The stability in increasing order for the prodrugs was found to be DDGCV>LDGCV>DLGCV>DGCV>LLGCV>LGCV. The interaction of these prodrugs with PEPT was confirmed by their significant inhibition of [3H] Glysar uptake in vitro. All prodrugs other than DDGCV and DGCV were found to interact with PEPT1. Cytotoxicity of the prodrugs was significantly less than the marketed drug Trifluorothymidine. Corneal transport sample concentrations were not detectable by HPLC detection limit. Extraction and analytical method were optimized in LC-MS/MS to analyze these samples.
Conclusions: :
Our results indicate that stereoisomeric dipeptide monoester prodrugs of GCV can not only enhance the aqueous stability but also retain their affinity towards Peptide transporter as well as improving the enzymatic stability. Hence these can be a better substitute to conventional peptide prodrugs of GCV in treatment of Herpes Simplex virus infections.
Keywords: herpes simplex virus • keratitis • antiviral drugs