Novel biotinylated lipid prodrugs of acyclovir for the treatment of herpetic keratitis (HK): transporter recognition, tissue stability and antiviral activity

Abstract

Purpose: Biotinylated lipid prodrugs of acyclovir (ACV) were designed to target the sodium dependent multivitamin transporter (SMVT) on the cornea to facilitate enhanced cellular absorption of ACV.

Methods: All the prodrugs were screened for in vitro cellular uptake, interaction with SMVT, docking analysis, cytotoxicity, enzymatic stability and antiviral activity.

Results: Uptake of biotinylated lipid prodrugs of ACV (B-R-ACV and B-12HS-ACV) was significantly higher than biotinylated prodrug (B-ACV), lipid prodrugs (R-ACV and 12HS-ACV) and ACV in corneal cells. Transepithelial transport across rabbit corneas indicated the recognition of the prodrugs by SMVT. Average Vina scores obtained from docking studies further confirmed that biotinylated lipid prodrugs possess enhanced affinity towards SMVT. All the prodrugs studied did not cause any cytotoxicity and were found to be safe and non-toxic. B-R-ACV and B-12HS-ACV were found to be relatively more stable in ocular tissue homogenates and exhibited excellent antiviral activity.

Conclusions: Biotinylated lipid prodrugs demonstrated synergistic improvement in cellular uptake due to recognition of the prodrugs by SMVT on the cornea and lipid mediated transcellular diffusion. These biotinylated lipid prodrugs appear to be promising drug candidates for the treatment of herpetic keratitis (HK) and may lower ACV resistance in patients with poor clinical response.

Fig. 1

Cellular accumulation of B-R-ACV, B-12HS-ACV, B-ACV, R-ACV, 12HS-ACV and ACV on HCEC cells. Uptake was performed at 37°C with DPBS buffer for 30 min. Values represent mean ± standard deviation (n= 4). A P-value of less than 0.05 was considered to be statistically significant and denoted by asterisk (*).

Fig. 2

Cellular accumulation of B-R-ACV, B-12HS-ACV and B-ACV on HCEC cells in the presence and absence of sodium ions in DPBS buffer at 37°C. Data represents mean ± standard deviation (n= 4). A P-value of less than 0.05 was considered to be statistically significant and denoted by asterisk (*).

Fig. 3

Transepithelial transport of [³H] biotin across freshly excised rabbit corneas in the absence and presence of 50μM unlabeled biotin, B-ACV, B-R-ACV, and B-12HS-ACV. Cumulative amount of [³H] biotin transported was measured in DPBS buffer at 34°C for 180 min. Data represents mean ± standard deviation (n= 4-6).

Fig. 4

Comparison of permeabilities (cm/sec) of [³H] biotin alone, in the presence of 50μM unlabeled biotin, B-ACV, B-R-ACV, and B-12HS-ACV across freshly excised rabbit corneas. Data represents mean ± standard deviation (n= 4-6). A P-value of less than 0.05 was considered to be statistically significant and denoted by asterisk (*).

Fig. 5

Biotinylated prodrugs of ACV are shown in their predicted binding site on SMVT with all amino acid residues within 4 Å of the ligand. A) B-ACV; B) B-R-ACV and C) B-12HS-ACV.

Fig. 6

Cytotoxicity assay in the presence of B-R-ACV, B-12HS-ACV, B-ACV and ACV on HCEC and rPCEC cells for 48 h. DMSO (10% v/v) served as a positive control. Data represent mean percentage of viable cells ± standard deviation (n= 4). A P-value of less than 0.05 was considered to be statistically significant and denoted by asterisk (*).

Fig. 7

Comparison of hydrolytic rate constants for (A) B-R-ACV and (B) B-12HS-ACV in various ocular homogenates (cornea, ICB and lens). Data represents mean ± standard deviation (n= 4).