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Category: Corneal permeability
Animals used for research:   Rabbit  

Assessment of the permeability of molecules across human corneal epithelial cell layer (in vitro) or across animal cornea (ex vivo) is important in evaluating molecules' ability to enter the eye by transcorneal route.

The apparent permeability coefficient (Papp, cm/s) values for the standard molecules across HCE-T cells.

Experimentica in-house

Reference values
Papp × 106 (cm/s)

6-carboxyfluorescein

Papp × 106 (cm/s)

6-carboxyfluorescein*

Mean

2.9

0.81

SD

1.1

0.41

SEM

0.3

0.21

Rhodamine B

Papp × 106 (cm/s)

Rhodamine B*

Mean

40.2

16.31

SD

8.0

4.01

SEM

3.0

1.41

FITC-dextran 4000 Da

Papp × 106 (cm/s)

FITC-dextran 4000 Da

Mean

1.4

0.0567

SD

0.9

0.0167

SEM

0.2

FITC-dextran 70000 Da

Papp × 106 (cm/s)

FITC-dextran 70000 Da

Mean

0.07

Impermeable8

SD

0.02

SEM

0.01

*Permeation studies were done in cell culture plates.

The apparent permeability coefficient (Papp, cm/s) values for the standard molecules across excised rabbit and pig corneas.

Experimentica in-house

Reference values
Papp × 106 (cm/s)

[3H]mannitol

Papp × 106 (cm/s)

mannitol

Mean

3.2

0.386

SD

1.2

2.45

SEM

0.54

4.04

[14C]diazepam

Papp × 106 (cm/s)

diazepam

Mean

16.3

21.94

SD

0.2

SEM

0.15

6-carboxyfluorescein

Papp × 106 (cm/s)

6-carboxyfluorescein

validation ongoing

0.462

Rhodamine B

Papp × 106 (cm/s)

Rhodamine B

validation ongoing

18.12

SUMMARY

Cells or tissues
  1. Human corneal epithelial cells1 (HCE-T, RIKEN, Japan);

  2. Excised rabbit cornea or sclera.

 

The HCE-T cell cultures2-3 and excised rabbit corneas4-6 are the most used and best characterized corneal permeability models in vitro and ex vivo, respectively.

Standard molecules

Low permeability: 6-carboxyfluorescein, FITC-dextran 4 000 Da, FITC-dextran 70 000 Da

High permeability: Rhodamine B

Permeability study

The permeability experiments across the HCE-T cell cultures or excised rabbit corneas are conducted either in NaviCyte vertical chamber system (Harvard Apparatus, Holliston, MA, USA; Figure 1) to measure the transport of study molecules and standard molecules.

The HCE-T cell culture or excised rabbit cornea is inserted between two chambers and the cumulative amount of study molecules in the receiver chamber versus time is measured.

Read-outs

The rate (apparent permeability coefficient, Papp) of the study molecules is compared to the Papp values of the low and high permeability markers.

In addition, the Papp values of study compounds and standard molecules can be compared to the Papp values of known generic drug molecules across the excised rabbit cornea from the literature2,4-6.

Please contact us, if you would like to receive more information: info@experimentica.com

IMAGES

Corneal permeability studies using the NaviCyte vertical diffusion chambers

Figure 1. The HCE-T cell cultures mounted in the NaviCyte vertical diffusion chambers and permeability experiments across corneas are conducted in triplicates.

Figure 2. Expression of tight junction proteins occluding (left) and ZO-1 (right) in HCE-T cells cultured on Lab-Tek™ chamber slides (Nunc™, Thermo Fisher Scientific, Waltham, MA, USA) detected by immunofluorescence staining (green). Nuclei were stained with DAPI (blue). Scale bar 50 µm.

SELECTED PUBLICATIONS

  • Reichl S. Cell culture models of the human cornea – a comparative evaluation of their usefulness to determine ocular drug absorption in-vitro. J. Pharm. Pharmacol. 2008;60(3):299-307.

REFERENCES

  1. Araki-Sasaki K, Ohashi Y, Sasabe T, Hayashi K, Watanabe H, Tano Y, Handa H. An SV40-immortalized human corneal epithelial cell line and its characterization. IOVS 1995,36:614–621.
  2. Toropainen et al. Culture model of human corneal epithelium for prediction of ocular drug absorption. Invest Ophthalmol Vis Sci 2001,42(12):2942-2948.
  3. Reichl S. Cell culture models of the human cornea – a comparative evaluation of their usefulness to determine ocular drug absorption in-vitro. J. Pharm. Pharmacol. 2008;60(3):299-307.
  4. Majumdar et al. Evaluation of active and passive transport processes in corneas extracted from preserved rabbit eyes. J Pharm Sci 2010, 99(4):1921-1930.
  5. Schoenwald. Ocular drug delivery. Pharmacokinetic considerations. Clin Pharmacokinet 1990, 18(4):255-269.
  6. Suhonen et al. Different effects of pH on the permeation of pilocarpine and pilocarpine prodrugs across the isolated rabbit cornea. Eur J Pharm Sci 1998, 6(3):169-176.
  7. Sasaki et al. Ocular permeability of FITC-dextran with absorption promoter for ocular delivery of peptide drug. J Drug Target. 1995;3(2):129-135.
  8. Huang et al. Paracellular permeability of corneal and conjunctival epithelia. Invest Ophthalmol Vis Sci. 1989;30(4):684-689.

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