Hydrophilic prodrug approach for reduced pigment binding and enhanced transscleral retinal delivery of celecoxib

Abstract

Transscleral retinal delivery of celecoxib, an anti-inflammatory and anti-VEGF agent, is restricted by its poor solubility and binding to the melanin pigment in choroid-RPE. The purpose of this study was to develop soluble prodrugs of celecoxib with reduced pigment binding and enhanced retinal delivery. Three hydrophilic amide prodrugs of celecoxib, celecoxib succinamidic acid (CSA), celecoxib maleamidic acid (CMA), and celecoxib acetamide (CAA) were synthesized and characterized for solubility and lipophilicity. In vitro melanin binding to natural melanin (Sepia officinalis) was estimated for all three prodrugs. In vitro transport studies across isolated bovine sclera and sclera-choroid-RPE (SCRPE) were performed. Prodrug with the highest permeability across SCRPE was characterized for metabolism and cytotoxicity and its in vivo transscleral delivery in pigmented rats. Aqueous solubilities of CSA, CMA, and CAA were 300-, 182-, and 76-fold higher, respectively, than celecoxib. Melanin binding affinity and capacity were significantly lower than for celecoxib for all three prodrugs. Rank order for the % in vitro transport across bovine sclera and SCRPE was CSA > CMA ~ CAA ~ celecoxib, with the transport being 8-fold higher for CSA than celecoxib. CSA was further assessed for its metabolic stability and in vivo delivery. CSA showed optimum metabolic stability in all eye tissues with only 10-20% conversion to parent celecoxib in 30 min. Metabolic enzymes responsible for bioconversion included amidases, esterase, and cytochrome P-450. In vivo delivery in pigmented BN rats showed that CSA had 4.7-, 1.4-, 3.3-, 6.0-, and 4.5-fold higher delivery to sclera, choroid-RPE, retina, vitreous, and lens than celecoxib. CSA has no cytotoxicity in ARPE-19 cells in the concentration range of 0.1 to 1000 μM. Celecoxib succinamidic acid, a soluble prodrug of celecoxib with reduced melanin binding, enhances transscleral retinal delivery of celecoxib.

Fig. 1

Schematic illustration of synthesis of celecoxib-acetamide, celecoxib-maleamidic acid and celecoxib succinamidic acid.

Fig. 2

Mass spectra of A) celecoxib, B) celecoxib acetamide (CAA), C) celecoxib-maleamidic acid (CMA), and D) celecoxib succinamidic acid (CSA).

Fig. 3

Cumulative % transport is significantly higher for celecoxib succinamidic acid (CSA) than celecoxib across bovine sclera and SCRPE. Cumulative % transport celecoxib and its amide prodrugs across bovine A) sclera and B) sclera-choroid-RPE. Data are expressed as mean ± SD for n = 6.

Fig. 4

In vitro metabolic stability of celecoxib succinamidic acid (CSA) in rat ocular tissues and plasma samples, A) % CSA remaining in reaction mixture and B) % celecoxib formed from CSA at the end of 30 min of incubation. Data are expressed as mean ± SD for n = 6.

Fig. 5

Effect of metabolic enzyme inhibitors on conversion of celecoxib succinamidic acid (CSA) to celecoxib in rat ocular tissue and plasma samples. Comparison of formation of celecoxib normalized to tissue weight in the presence of A) bis (4-nitro-phenyl) phosphate, inhibitor of amidases, B) paraxon, inhibitor of esterases, C) 1-aminobenzotriazole, inhibitor of cytochrome P-450, and D) cocktail of all three inhibitors. White unshaded bars represent the formation of celecoxib in the absence of any inhibitor and gray shaded bars show formation of celecoxib in the presence of metabolic enzyme inhibitors. Data are expressed as mean ± SD for n = 6. *, p < 0.05 compared with control.

Fig. 6

Ocular tissue levels of celecoxib succinamidic acid (CSA) (shaded) and celecoxib (unshaded) levels in posterior ocular tissues at 15 min following posterior subconjunctival administration of 25 μg of CSA or celecoxib. Data are expressed as mean ± SD for n =4. *, p < 0.05 compared with the celecoxib group.

Fig. 7

Ratio of ocular tissue concentration between celecoxib succinamidic acid (CSA) and celecoxib after subconjunctival injection in pigmented BN rats. Data are expressed as mean for n =4. Black thick line at one is the reference line indicating equal delivery between CSA and celecoxib groups.

Fig. 8

In vitro cytotoxicity of celecoxib succinamidic acid (CSA) on ARPE-19 at concentration range of 0.1 μM to 1 mM. Data are expressed as mean ± s.d for n =8.