Required from Customer

• A study design—the customer must specify the value of the assay variables listed in the Options section
• Either a minimum of 300 μL of test compound at 10 mM in DMSO, or 10 mg of powder
• Exact molecular mass of test compound and its salt form
• Solubility of test compound in Glutathione-bicarbonated Ringer’s (GBR) buffer
• Stability of test compound in GBR buffer at 37°C
• MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

• Apparent permeability (P_app) and recovery of test compound
• Post-experiment tissue integrity results (P_app of control compound)

Substrate

• Test compound at one concentration (typically 5 to 10 µM) in GBR with maximum DMSO concentration <0.3%

Assay System

• Freshly excised corneal and/or conjunctival tissue from rabbit eyes, mounted in vertical Ussing chambers at 37°C

Assay Conditions

• Run the assay with tissue from N=3 eyes (from a total of 2 donors), and report the data from at least N=2
• Pre-incubate the tissue with GBR buffer for 30 minutes prior to initiation of the permeability assay
• Dose the test compound on the anterior side
• Sample receiver side at 60 and 120 minutes
• Sample donor side at 0 and 120 minutes
• For post-experiment tissue integrity testing, dose with atenolol on the anterior side and sample the receiver side at 30 minutes
• Freeze and retain tissue samples for up to 2 weeks
• Determine the concentration of test compound using a generic LC-MS/MS method with a minimum 6 point calibration curve

Assay QC

• The post-experiment P_app of atenolol must be less than a pre-determined value to verify the integrity of the tissue

Notes

1. The results from this assay are provided to the customer in the standard report format.
2. Apparent permeability is calculated from the following equation: P_app = (dC_r/dt) x V_r / (A x C₀), where dC_r/dt is the slope of the cumulative concentration of test compound in the receiver chamber over time, Vr is the volume of the receiver chamber, A is the diffusion area, and C₀ is the initial (dosing) concentration of test compound in the donor chamber.
3. Percent recovery = ((V_r x C_r-final) + (V_d x C_d-final))/(V_d x C₀) x 100, where V_r and V_d are the volumes of the receiver and donor chambers, respectively; C_r-final is the cumulative concentration of test compound in the receiver chamber at the end of the incubation; C_d-final is the concentration of test compound in the donor chamber at the end of the incubation; and C₀ is the initial (dosing) concentration.

Options

1. The customer must specify:
   • the strain of rabbit: Dutch-belted or New Zealand white
   • corneal and/or conjunctival tissue