This assay is used to determine the unidirectional permeability (anterior-to-posterior) of a test compound through rabbit corneal or conjunctival tissue
Catalog number EA227
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 and stability in GBR buffer at 37°C
- MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.
- Apparent permeability (Papp) and recovery of test compound
- Post-experiment tissue integrity results (Papp of control compound)
- Non-specific binding of test compound
- Test compound at one concentration (typically 5 to 10 µM) in GBR with maximum DMSO concentration <0.3%
- Freshly excised corneal and/or conjunctival tissue from rabbit eyes, mounted in vertical Ussing chambers at 37°C
- Non-specific binding assessment:
- Use three chambers (N=3)
- Dose a tissue-free device with 1% of the dosing concentration used for permeability assessment
- Sample at 0 and 120 minutes
- Recover <80% may trigger special handling procedures for the permeability assessment
- Permeability Assessment
- Run the assay with tissue from N=4 eyes (from a total of 2 donors), and report the data from at least N=3
- 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 and 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
- The post-experiment Papp of atenolol must be less than a pre-determined value to verify the integrity of the tissue
- The results from this assay are provided to the customer in the standard report format.
- Apparent permeability is calculated from the following equation: Papp = (dCr/dt) x Vr / (A x C0), where dCr/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.
- Percent recovery = ((Vr x Cr-final) + (Vd x Cd-final))/(Vd x C0) x 100, where Vr and Vd are the volumes of the receiver and donor chambers, respectively; Cr-final is the cumulative concentration of test compound in the receiver chamber at the end of the incubation; Cd-final is the concentration of test compound in the donor chamber at the end of the incubation; and C0 is the initial (dosing) concentration.
- Optional: Upon request, a reference compound can be run in parallel for an additional cost.
- The customer must specify:
• the strain of rabbit: Dutch-belted or New Zealand white
• corneal and/or conjunctival tissue