The Impact of Endogenous Breast Cancer Resistance Protein on Human P-Glycoprotein-Mediated Transport Assays Using LLC-PK1 Cells Transfected With Human P-Glycoprotein.

Lilly Laboratories cell porcine kidney 1 (LLC-PK1) cells transfected with human P-glycoprotein (LLC-PK1-P-gp) are widely used in transport assays to identify drug candidates that function as substrates of this efflux transporter. Endogenous transporters expressed in LLC-PK1 cells may complicate the interpretation of findings from P-gp-mediated transport assays. We investigated the impact of porcine breast cancer resistance protein (Bcrp) in P-gp-mediated transport assays in LLC-PK1 cells. Porcine Bcrp mRNA was detected in both LLC-PK1 wildtype (WT) and LLC-PK1-P-gp cells by quantitative RT-PCR. To investigate the activity and impact of porcine Bcrp, we conducted transport assays using 6 typical BCRP substrates in LLC-PK1 cells. Efflux ratios (ER) of the 6 BCRP substrates in LLC-PK1 WT cells were >2, and were reduced in the presence of the BCRP inhibitor Ko143. The efflux activities of the 6 BCRP substrates were confirmed using MDCKII cells transfected with human BCRP. Net ERs of prazosin and fluvastatin, dual substrates of P-gp and BCRP, determined by dividing ERs in LLC-PK1-P-gp cells by those in LLC-PK1 WT cells, were <2, but increased to >2 in the presence of Ko143. These results indicated that endogenous Bcrp in LLC-PK1 cells was involved in the transport of BCRP substrates and may interfere with the identification of P-gp substrates.

Development and validation of semiautomated 96-well transport assay using LLC-PK1 cells transfected with human P-glycoprotein for high-throughput screening.

Transport assays using P-gp-expressing cell lines are commonly used to identify P-gp substrates and inhibitors in drug discovery. The P-gp cell-based assay is performed manually in 12- or 24-well plates and requires improvement for high-throughput screening. In this study, we established an efficient semiautomated 96-well transport assay using LLC-PK1 cells transfected with human P-gp. The protocol was optimized with a microplate washer for exchanging media and buffer to enhance throughput. P-gp substrates and inhibitors, and the paracellular marker Dextran Texas Red® were used to validate the 96-well transport assay. Cell monolayer integrity after washing by a microplate washer was confirmed by measuring paracellular permeability of Dextran Texas Red. Permeability and net flux ratio of the P-gp substrates and the inhibitory potency of the P-gp inhibitors were comparable in 24- and 96-well plates. The regression value of net flux ratio of P-gp substrates was high between the two formats (r²=0.99). The optimized 96-well transport assay using the microplate washer was found to be an efficient high-throughput screening tool that provided the same quality data as the 24-well plate for the identification of P-gp substrates and inhibitors in drug discovery.

Enhanced target-specific accumulation of radiolabeled antibodies by conjugating arginine-rich peptides as anchoring molecules.

We have devised and estimated a new strategy to prolong the residence time of radiolabeled antibodies in tumor in which an octaarginine peptide (R8) was used as an anchoring molecule to fix antibodies against CD20 (NuB2; IgG2a) on tumor cells. Conjugation of R8 with antibodies was performed by maleimide-thiol chemistry using thiol groups generated by reducing the disulfide bonds of the antibody. The R8-conjugated NuB2 was then reacted with succinimidyl meta-[¹²5I]iodobenzoate to prepare [¹²5I]SIB-NuB2(I) (0.92 R8/NuB2) and [¹²5I]SIB-NuB2(III) (3.38 R8/NuB2). Both SIB-NuB2(I) and SIB-NuB2(III) exhibited size-exclusion HPLC elution profiles and immunoreactivity to CD20-positive cells similar to those of NuB2. NuB2(I) also possessed isoelectric focusing (IEF) profile similar to NuB2. However, NuB2(III) registered a broad IEF band toward higher pI. When incubated with CD20-positive cells, [¹²5I]SIB-NuB2(I) and [¹²5I]SIB-NuB2(III) exhibited 1.4 and 4.0 times higher cell-associated radioactivity than [¹²5I]SIB-NuB2. After the cells were washed and reincubated in a fresh medium for 3 h, [¹²5I]SIB-NuB2(I) and [¹²5I]SIB-NuB2(III) exhibited significantly higher cell-associated radioactivity than [¹²5I]SIB-NuB2. In biodistribution studies in normal mice, while both [¹²5I]SIB-NuB2(I) and [¹²5I]SIB-NuB2 exhibited similar biodistribution profiles, [¹²5I]SIB-NuB2(III) showed faster clearance from the blood and higher hepatic radioactivity levels than [¹²5I]SIB-NuB2. In SCID mice bearing CD20-positive xenografts, [¹³¹I]SIB-NuB2(I) exhibited significantly higher radioactivity in xenografts than those of [¹²5I]SIB-NuB2 with no significant increase being observed in other tissues. The findings indicate that appropriate R8 modification of antibodies satisfies both specific targeting ability of antibody and strong cell-association property of R8, which was reflected in the increased radioactivity levels in tumor. These findings supported the applicability of this approach to enhance target-specific accumulation of radiolabeled antibodies.