Discover Clarity in Corneal Permeability with Human Corneal Orbs absorption.com Fany Guerra , Jackie Tusano , Lindsay Hartsock , Erica Weiskircher , Jibin Li , Ismael J. Hidalgo 1 1 2 Absorption Systems, Exton, Pennsylvania, USA 2 2 1 1 Lifeline Cell Technology, Frederick, Maryland, USA A wholly owned subsidiary of International Stem Cell Corporation Purpose To characterize corneal orbs, cultured from human embryonic stem cells, as an in vitro model to predict the corneal absorption of drugs compared with human corneal tissue and rabbit corneal tissue. Methods Results Figure 2. Table 1. A) Corneal orb in culture medium. B) H&E stained corneal orb (40X) with the epithelial layer (a) anterior basement (Bowman’s) membrane (b) stroma (substantia propria); (c), and endothelium (d). Permeability of model compounds across ex vivo and in vitro corneal models. In Vitro Corneal Orbs Human pluripotent stem cells were cultured to form hollow, fluid-filled corneal orbs. Intact orbs were incubated with 10 µM test compounds at 37˚C for 30 min. At the end of incubation the internal contents were carefully collected using a needle and syringe (uptake assay depicted in Fig. 1), and drug concentrations were quantified by LC-MS/MS. Apparent permeability coefficient (Papp) was calculated as: Papp = Q/(C0 x A x T) where Q is the amount of drug accumulated in the orbs, C0 is the initial drug concentration, A is the surface area of the orbs, and T is the incubation time. A B 1 Papp (× 10-6 cm/s)±SD ex vivo A Dutch Belted Rabbit Cornea New Zealand Rabbit Cornea Mean SD Mean SD Mean SD Mean SD Acebutolol 4.68 0.39 1.71 0.55 8.43 3.96 1.33 0.63 Atenolol 1.84 0.46 1.11 0.30 11.00 2.24 4.29 2.59 Betaxolol 32.02 4.42 16.50 1.91 6.34 1.74 Brimonidine 28.82 1.22 20.39 1.82 17.70 2.74 15.80 1.23 Ciprofloxacin 0.42 0.35 0.35 0.16 5.53 1.91 6.49 5.41 Dexamethasone total 19.5 2.29 28.5 9.80 11.78 1.45 12.73 1.18 Latanoprost total* 96.80 37.70 14.30 34.40 11.93 Timolol 37.0 17.30 1.31 17.00 2.70 Compound C Figure 1. Depiction of uptake assay in corneal orbs. Corneal orbs were incubated in dosing solution containing test compound (A – start; B – during incubation). C) Intact orbs were transferred to a new well and the contents of the orb collected using a needle and syringe. B b a Ex Vivo Excised Corneal Tissue Excised human and rabbit (male, Dutchbelted pigmented and male, New Zealand White) corneas were mounted in vertical diffusion chambers. Tissues were maintained at 37˚C. Test compounds were dosed at 10 µM into the mucosal (donor) chamber and samples were taken from the serosal (receiver) chamber. The concentrations of test compounds were quantified by LC-MS/MS. Apparent permeability Papp = (dCr/dt) x Vr / (A x C0), where dCr/dt is the slope of the cumulative concentration in the receiver compartment vs. time, Vr is the volume of the receiver chamber, A is the diffusion area, and C0 is the measured dosing concentration. c d in vitro 6.41 19.44 Human Cornea 5.17 Human Orbs * measured after latanoprost dosing Figure 4. Results Papp correlation between human corneal orbs and ex vivo human (a) and rabbit (b and c) excised corneal tissue. BCRP MRP2 1B1 1B3 2B1 MRP1 PepT1 100 75 Figure 3. 50 76 bp 62 bp 67 bp 63 bp 95 bp 94 bp 73bp 83 bp 69 bp Molecular expression of drug transporters in human corneal orbs R 2 = 0.8457 25 20 15 a 10 5 0 Corneal orbs cultured from human pluripotent stem cells are a unique human in vitro model for assessment of corneal absorption. This novel in vitro model exhibits comparable topical drug absorption barrier properties and enzymatic activity to excised human corneal tissue. These results demonstrate that human corneal orbs may represent an ideal in vitro model for testing corneal drug permeability, which circumvents the limited availability of healthy human corneal tissue and potentially reduces the used of rabbits for preclinical testing of ocular drugs. 5 80 70 60 50 40 30 b 20 10 10 15 20 25 30 35 40 20 R 2 = 0.7197 15 10 c 5 0 0 5 10 15 20 25 Human Orbs Papp (x10-6 cm/s) Human Orbs Papp (x10-6 cm/s) 30 35 40 0 5 10 15 20 Human Orbs Papp (x10-6 cm/s) Figure 5. Permeability of dexamethasone acetate in human corneal orbs and human and rabbit excised corneal tissue. Presence of base form indicates esterase activity in each of the models. Esterase activity in both DB and NZW rabbit excised corneal tissue was much higher (~100% conversion to base form) than that seen in excised human corneal tissue and corneal orbs. 30 Dexamethasone Acetate Dexamethasone Base Dexamethasone Total 25 Papp (x10-6cm/s) Conclusion R 2 = 0.8746 0 0 The apparent permeabilities of model compounds were determined and compared between the ex vivo rabbit and human corneal tissue and in vitro corneal orb models (Table 1). The permeability results from human corneal orbs were then compared to excised human and rabbit corneal tissue (Fig. 4). Corneal orbs showed good correlations to both human and DB rabbit excised tissue (R2 of 0.85 and 0.88, respectively). Corneal orbs showed moderate correlation to NZ rabbit excised tissue (R2 of 0.72). Esterase activity was assessed indirectly based on the permeability and hydrolysis of dexamethasone acetate to dexamethasone. As seen in Fig. 5, the esterase activity of corneal orbs is more similar to the esterase activity seen in human cornea than that in rabbit cornea. 100 90 NZ Rabbit Cornea vs. Human Orbs 25 In vitro NZW Rabbit Corneal Papp (x10-6 cm/s) P-gp 35 30 DB Rabbit Cornea vs. Human Orbs 110 In Vitro DB Rabbit Corneal Papp (x10-6 cm/s) B-actin Human Cornea vs. Human Orbs 40 Human Corneal Papp (x10-6 cm/s) Corneal orbs, a novel model for corneal absorption, were characterized histologically and for transporter gene expression prior to testing their similarity to the other model systems. H&E staining confirmed differentiation of the pluripotent stem cell-derived corneal orbs into the different tissue layers typical of the cornea (Fig. 2). For comparison of drug transporter expression, mRNA expression in corneal orbs was assessed using RT-PCR (Fig. 3). 20 15 10 5 0 DB Rabbit Cornea NZ Rabbit Cornea Human Cornea Human Corneal Orbs absorption.com 25 30