(M1330-03-18) Permeability of Small Molecular Weight Compounds and Macromolecules through Ex Vivo Rabbit Ocular Tissues

Purpose: Currently, intravitreal injections (IVT) are the method of choice for retinal diseases. However, these invasive injections require frequent visits to the hospital which is very expensive and often leads to poor patient compliance. One of the alternative approaches to address this issue is topical drug delivery. After the administration of an eye drop, the drug enters the back of the eye through either corneal or non-corneal route. This study explores the potential of topical drug delivery to the retina through the non-corneal route. The aim is to determine the apparent permeability (Papp) of small molecular weight compounds as well as a peptide and an antibody through ex vivo rabbit sclera, conjunctiva, and sclera-choroid-RPE (SCR).

Methods: 13 compounds were selected for the cassette to represent a variety of physicochemical properties (table 1). The peptide used in the experiments was anti-VEGF peptide VII4* (VEPNc[CDIHVnLWEWEC]FERL; Guryanov et al. 2021) and the antibody bevacizumab (Avastin 25 mg/ml). The permeability experiment was performed using Ussing diffusion chamber system. Silicone adapters with a circular opening of 0.64 cm² were placed on the chambers. Next, freshly dissected mixed breed pigmented rabbit ocular tissues were placed on the adapters, between the chambers. Balanced salt solution (BSS plus) was used as an experiment buffer for conjunctiva and sclera, and Neurobasal medium supplemented with 1 % N2 and 0.4 % Glutamax for SCR. The chambers were placed in a heater block base connected to a circulating water bath to maintain temperature at 37 °C. They were mixed by bubbling with a gas mix of 5 % CO2, 10 % O2, 85 % N2 for sclera and SCR, or 5 % CO2, 10 % O2 for conjunctiva. Donor concentrations of the small molecular weight compounds were 2 or 20 µg/ml, peptide 20 µg/ml or 200 µg/ml, and bevacizumab 3.5 mg/ml. Samples of 500 µl were drawn at timepoints 15, 30, 45, 60, 75, 90, 120, 150, 180, 210, 240, 270, 300, 330 and 360 minutes for conjunctiva and sclera, or at 1, 2, 6, 9, 19, 20, 21, 22, 23 and 24 hours for SCR. To ensure tissue integrity, transepithelial potential difference (TEP) measurements were conducted with a Voltage-current clamp (VCC MC6) and used for the calculations of the transepithelial electrical resistance with equation TER = ΔV/ΔI * A, where ΔV is the difference in voltage, ΔI the applied current, and A area of the exposed tissue. Permeation of atenolol was also used as an integrity marker. The same experiment was also conducted with a Franz cells with sclera. The sample analysis of the cassette compounds were performed using liquid chromatogram mass spectrometer triple quadrupole (LC-MS QQQ) and the resulting data were analyzed with MassHunter QQQ quantitative analysis program. The peptide samples were analyzed ultra high performance LC (UHPLC) coupled with QQQ MS. The samples containing bevacizumab were analyzed using an in-house ELISA. The absorbance was read at 450 nm and 570 nm with a plate reader (Cytation 3 imaging reader, Biotek; AH diagnostics). The apparent permeability was calculated using equation Papp(cm/s) = J / (C0 * A), where J (ng/s) is the drug flux across the tissue after lag time (linear period), C0 (ng/cm3) is the initial donor concentration, and A (0.64 cm2) is the area of the exposed tissue. The Papp of RPE has been calculated with the equation 1/Papp(RPE) = 1/Papp(SCR) – 1/Papp(sclera) (Flynn et al. 1974).

Results: The Papp values of the cassette compounds through sclera in the Ussing system ranged from 1.86 ± 2.42 *10-6 cm/s (n=11) to 7.79 ± 2.19 *10-6 cm/s (n=12) propranolol being the least and atenolol the most permeable. In the Franz system, Papp values were 2-4 times higher (p < 0.01). Papp through conjunctiva were on a range from 6.38 ± 5.54 *10-6 cm/s (propranolol) to 13.39 ± 8.24 *10-6 cm/s (acetazolamide) (n=7). Corresponding values through RPE were 0.04 ± 0.02 (n=5) and 1.45 ± 1.05 *10-6 cm/s (n=17) for propranolol and fluconazole respectively, indicating that the sclera and conjunctiva are significantly more permeable to small MW drugs than the RPE (p < 0.05). The Papp of the anti-VEGF peptide was at the same range with the small molecular weight compound, SCR being the most restricting membrane as expected. For bevacizumab the corresponding Papp values were approximately 10 times lower than the Papp of the cassette compounds (P < 0.01). Tissues were confirmed to be intact.

Conclusion: In summary, comparing the Ussing chamber with the Franz cell is challenging due to experimental setup differences. Also, the Papp values showed minor variation within the small molecular weight compounds considering the diverse compound properties. The results give valuable information about the rate-limiting barrier RPE in topical drug delivery and for the future development of less invasive administration routes to treat retinal diseases.

References: 1. Flynn G., Yalkowsky S. and Roseman T. Mass transport phenomena and models: theoretical concepts. J Pharm Sci. 1974 Apr; 63(4):479-510
2. Guryanov I, Korzhikov-Vlakh V, Bhattacharya M, Biondi B, Masiero G, Formaggio F, Tennikova T, Urtti A. Conformationally Constrained Peptides with High Affinity to the Vascular Endothelial Growth Factor. J Med Chem. 2021 Aug 12;64(15):10900-10907. DOI: 10.1021/acs.jmedchem.1c00219
3. Pelkonen L, et al. Melanin binding study of clinical drugs with cassette dosing and rapid equilibrium dialysis inserts. Eur J Pharm Sci. 2017 Nov 15;109:162-168

Acknowledgements: This study was financially supported by Johnson & Johnson Innovative Medicine and Government of Flander by VLAIO (Vlaams Agentschap Innoveren & Ondernemen, HBC.2021.1130)


Table 1. Properties of the cassette compounds (Pelkonen et al. 2017). L = low binder, I = intermediate binder, H = high binder