Transporter targeted gatifloxacin prodrugs: synthesis, permeability, and topical ocular delivery

Abstract

In this work, we aim to design and synthesize prodrugs of gatifloxacin targeting organic cation transporter (OCT), monocarboxylate transporter (MCT), and ATB (0, +) transporters and to identify a prodrug with enhanced delivery to the back of the eye. Dimethylamino-propyl, carboxy-propyl, and amino-propyl(2-methyl) derivatives of gatifloxacin (GFX), DMAP-GFX, CP-GFX, and APM-GFX, were designed and synthesized to target OCT, MCT, and ATB (0, +) transporters, respectively. An LC-MS method was developed to analyze drug and prodrug levels in various studies. Solubility and log D (pH 7.4) were measured for prodrugs and the parent drug. The permeability of the prodrugs was determined in the cornea, conjunctiva, and sclera-choroid-retinal pigment epitheluim (SCRPE) and compared with gatifloxacin using an Ussing chamber assembly. Permeability mechanisms were elucidated by determining the transport in the presence of transporter specific inhibitors. 1-Methyl-4-phenylpyridinium iodide (MPP+), nicotinic acid sodium salt, and α-methyl-DL-tryptophan were used to inhibit OCT, MCT, and ATB (0, +) transporters, respectively. A prodrug selected based on in vitro studies was administered as an eye drop to pigmented rabbits, and the delivery to various eye tissues including vitreous humor was compared with gatifloxacin dosing. DMAP-GFX exhibited 12.8-fold greater solubility than GFX. All prodrugs were more lipophilic, with the measured log D (pH 7.4) values ranging from 0.05 to 1.04, when compared to GFX (log D: -1.15). DMAP-GFX showed 1.4-, 1.8-, and 1.9-fold improvement in permeability across the cornea, conjunctiva, and SCRPE when compared to GFX. Moreover, it exhibited reduced permeability in the presence of MPP+ (competitive inhibitor of OCT), indicating OCT-mediated transport. CP-GFX showed 1.2-, 2.3-, and 2.5-fold improvement in permeability across the cornea, conjunctiva, and SCRPE, respectively. In the presence of nicotinic acid (competitive inhibitor of MCT), the permeability of CP-GFX was reduced across the conjunctiva. However, the cornea and SCRPE permeability of CP-GFX was not affected by nicotinic acid. APM-GFX did not show any improvement in permeability when compared to GFX across the cornea, conjunctiva, and SCRPE. Based on solubility and permeability, DMAP-GFX was selected for in vivo studies. DMAP-GFX showed 3.6- and 1.95-fold higher levels in vitreous humor and CRPE compared to that of GFX at 1 h after topical dosing. In vivo conversion of DMAP-GFX prodrug to GFX was quantified in tissues isolated at 1 h after dosing. The parent drug-to-prodrug ratio was 8, 70, 24, 21, 29, 13, 55, and 60% in the cornea, conjunctiva, iris-ciliary body, aqueous humor, sclera, CRPE, retina, and vitreous humor, respectively. In conclusion, DMAP-GFX prodrug enhanced solubility, log D, as well as OCT mediated delivery of gatifloxacin to the back of the eye.

Figure 1

Cumulative % transport of DMAP-GFX prodrug across a) cornea, b) conjunctiva, and c) SCRPE. Cornea and SCRPE were from NZW rabbit and conjunctiva was from bovine eyes. Cumulative % transport of DMAP-GFX was significantly increased across all tissues (p < 0.01), and DMAP-GFX transport was significantly inhibited by MPP+ across all tissues (p < 0.005). GFX levels formed in the prodrug group were below the detection limits. Data is expressed as mean ± SD for n = 4.

Figure 2

Cumulative % transport of CP-GFX was significantly higher (p <0.01) than GFX across all tissues a) cornea, b) conjunctiva, and c) SCRPE. Cornea and SCRPE were from NZW rabbit and conjunctiva was from bovine eyes. CP-GFX transport was significantly inhibited by nicotinic acid (competitive inhibitor of MCT) across conjunctiva, but was not inhibited across cornea and SCRPE. GFX levels formed in the prodrug group were below the detection limits. Data is expressed as mean ± SD for n = 4.

Figure 3

No improvement of cumulative % transport of APM-GFX prodrug in comparison to GFX across a) cornea, b) conjunctiva, and c) SCRPE. APM-GFX transport was not inhibited by α-methyl-DL-tryptophan (ATB inhibitor) across a) cornea and b) conjunctiva, and c) SCRPE. Cornea and SCRPE were from NZW rabbit and conjunctiva was from bovine eyes. GFX levels formed in the prodrug group were below the detection limits. Data is expressed as mean ± SD for n = 4.

Figure 4

Permeability coefficients of gatifloxacin prodrugs in comparison to gatifloxacin across cornea, conjunctiva, and SCRPE tissues. Cornea and SCRPE were from NZW rabbit and conjunctiva was isolated from bovine eyes. Data is expressed as mean ± SD for n = 4.

Figure 5

Ocular distribution of GFX and DMAP-GFX prodrug at 1 hour after their topical eye drop application in NZW pigmented rabbits. Levels of prodrug represent the sum of the GFX formed and unchanged prodrug in equivalents of prodrug. Vitreous levels were 3.6-fold higher with DMAP-GFX compared to GFX (* p < 0.05 calculated by Student's t-test). Data is expressed as mean ± SD for n = 4 animals.

Figure 6

Amount of GFX formed and DMAP-GFX prodrug remaining from in vivo ocular pharmacokinetics studies in pigmented rabbits at the end of 1 hour. Levels of the drug formed and prodrug in cornea and conjunctiva were plotted in the inset. Data is expressed as mean ± SD for n = 4.

Scheme 1

Synthesis of dimethylamino-propyl-gatifloxacin (DMAP-GFX) and carboxy-propyl-gatifloxacin (CP-GFX) prodrugs targeting OCT and MCT transporters, respectively.

Scheme 2

Synthesis of aminopropyl(2-methyl)-gatifloxacin (APM-GFX) prodrug intended for targeting ATB (0, +) transporter.