1. IMPACT OF EXPERIMENTAL CONDITIONS ON IN VITRO PERMEABILITY: (1) CORRELATIONS IN INTER-LABORATORY Caco-2 Y.H. Lee 1, K.J. Lee 1, N. Johnson 1, J. Castelo 1, R. Mower 1, K. Click 1, R. Christopher 1, P. Gordon 1, T. Hollenbeck 1, D. Moylan 1, V. Ereso 1, K. Holme 1, G. Grass 1, P.J. Sinko 2 1 LION bioscience, San Diego, CA; 2 College of Pharmacy, Rutgers University, Piscataway, NJ.APS Annual Meeting, Oct. 21, 2001, Denver, CO IMPACT OF EXPERIMENTAL CONDITIONS ON IN VITRO PERMEABILITY: (1) CORRELATIONS IN INTER-LABORATORY Caco-2 Y.H. Lee 1, K.J. Lee 1, N. Johnson 1, J. Castelo 1, R. Mower 1, K. Click 1, R. Christopher 1, P. Gordon 1, T. Hollenbeck 1, D. Moylan 1, V. Ereso 1, K. Holme 1, G. Grass 1, P.J. Sinko 2 1 LION bioscience, San Diego, CA; 2 College of Pharmacy, Rutgers University, Piscataway, NJ.APS Annual Meeting, Oct. 21, 2001, Denver, CO Purpose. To investigate inter-laboratory correlation of Caco-2 permeability using marker compounds. Methods. In vitro permeability of 21 marker compounds representing various transport processes was studied in a standardized Caco-2 monolayer (24-well transwell format/ 20-24 day culture/ passage 30-40). A B permeability was studied at 37 C, 50 opm, 95% humidity, and 5% CO 2 using a 100 M donor concentration with 1% DMSO. In vitro permeability values of marker compounds were also collected from 10 references with various culture conditions (passage #, serum, filter type/size, seeding density, monolayer age) and various transport conditions (agitation, pH, buffer), and they were correlated to the data studied at Lion. Results. The study results at Lion showed reproducible permeability for 4 transport markers at different Caco-2 monolayer batches (Mannitol: 0.45 – 0.47 x 10 -6 cm/s (n=3); atenolol: 0.69 – 1.80 x 10 -6 cm/s (n=12); propranolol: 18.5 – 36.8 x 10 -6 cm/s (n=12); and verapamil: 18.7 – 23.6 x 10 -6 cm/s (n=3)). However, the in vitro permeability values varied 11 fold (0.38 – 4.0 x 10 -6 cm/s) for mannitol, 45 fold (0.1 – 4.5 x 10 -6 cm/s) for atenolol, 10 fold (11.5 – 110 x 10 -6 cm/s) for propranolol, and 8 fold (9.2 – 69.4 x 10 -6 cm/s) for verapamil in inter-laboratory Caco- 2. It is also known that in vitro mannitol Papp values vary 345 fold (0.019 – 6.55 x 10 -6 cm/s) from 58 published references 1. By contrast, the correlation of Caco-2 permeability between Lion and each of 10 references was good (r 2 > 0.8, 8 of 10). Conclusions. The in vitro permeability of marker compounds is reproducible under a standardized experimental condition, but can vary significantly between laboratories due to the differences in culture and experimental conditions. This work suggests that correlating inter-laboratory Caco-2 permeability for certain transport markers may be a valuable standardization method to normalize inter-laboratory differences of Caco-2 permeability. ABSTRACT INTRODUCTION METHODS RESULTS CONCLUSION The assessment of a compounds intestinal permeability is one of the key factors for the successful prediction of oral absorption in the drug discovery and development processes. In vitro determination of permeability using a Caco-2 monolayer is a currently recognized method to predict the extent of in vivo oral absorption. To evaluate chemical candidates for their absorption potential faster and cheaper, in silico absorption models using in vitro Caco-2 permeability have been developed and used in drug discovery and development processes. However, it is well known that in vitro Caco-2 permeability can vary lab-to-lab due to the differences in culture and transport conditions. As cited in 58 references, in vitro mannitol permeability values vary 345 fold (0.019 – 6.55 x 10 -6 cm/s) 1. Therefore, an external validation is needed to compare data between labs. The key issue is the quantitative comparison of Caco-2 permeability between labs, but there is no standardized method to evaluate inter-lab variations. In this report, in vitro permeability of 21 marker compounds representing various transport processes was studied in a standardized Caco-2 protocol at Lion to investigate inter-lab variations of Caco-2 permeability. In vitro permeability values of marker compounds were also collected from 10 references with various culture conditions (passage #, serum, filter type/size, seeding density, monolayer age) and various transport conditions (agitation, pH, buffer), and they were compared and correlated to the data studied at Lion. Finally, the adjusted Caco-2 permeability of 4 marker compounds was back-calculated using a log linear correlation curve. Atenolol, mannitol, propranolol, and verapamil were chosen as 4 marker compounds as they were most frequently used in 10 references. Correlating inter-lab Caco-2 permeability using certain transport markers may be a valuable standardization method to normalize inter-lab differences of Caco-2 permeability. Reference: 1. D.A. Volpe and A.S. Hussain. Impact of experimental conditions on the in vitro permeability of mannitol: An artificial neural network analysis. 2000 AAPS *The number in parenthesis: number of markers employed in correlation