Corneal Permeation Assay

Summary: The exploration of a drug candidate’s ability to enter the eye by transcorneal route.

Model Description

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

Cells or tissues1. 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 moleculesLow permeability: 6-carboxyfluorescein, FITC-dextran 4 000 Da, FITC-dextran 70 000 Da
High permeability: Rhodamine B
Permeability studyThe 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-outsThe 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.

Outcomes and Read-Outs 

Fig. 1. The NaviCyte vertical diffusion chambers and permeability experiments across corneas are conducted in triplicates.
Fig. 1. The NaviCyte vertical diffusion chambers and permeability experiments across corneas are conducted in triplicates. 
Fig. 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.
Fig. 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. 

Reference values for standard molecules 

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 0.8 1 
SD 1.1 0.4 1 
SEM 0.3 0.2 1 
   
Rhodamine B Papp × 106 (cm/s) # Rhodamine B
Mean 40.2 16.3 1 
SD 8.0 4.0 1 
SEM 3.0 1.4 1 
   
FITC-dextran 4000 Da Papp × 106 (cm/s) # FITC-dextran 4400 Da 
Mean 1.4 0.056 7 
SD 0.9 0.016 7 
SEM 0.2  
   
FITC-dextran 70 000 Da Papp × 106 (cm/s) # FITC-dextran 70 000 Da 
Mean 0.07 Impermeable 8 
SD 0.02  
SEM 0.01  

# Permeation studies have been done in NaviCyte chambers. *Permeation studies were done in cell culture inserts. 

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

 Experimentica in-house Papp × 106 (cm/s)  Reference values Papp × 106 (cm/s) 
 6-carboxyfluorescein 6-carboxyfluorescein 
Mean 1.4 0.46 2 
SD 0.9  
SEM 0.2  
 Rhodamine B Rhodamine B 
Mean 9.9 18.1 2 
SD 3.1  
SEM 0.9  
 FITC dextran 4000 Da FITC-dextran 4400 Da 
Mean 0.2 0.056 7 
SD 0.1 0.016 7 
SEM 0.1  
Fig. 3. Reference values for Rabbit corneal permeability.
Fig. 3. Reference values for Rabbit corneal permeability. 

References

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