Membrane Assays to Characterize Interaction of Drugs with ABCB1.

ATP-binding cassette sub-family B member 1 (ABCB1) [P-glycoprotein (P-gp), multidrug resistance protein 1 (MDR1)] can affect the pharmacokinetics, safety, and efficacy of drugs making it important to identify compounds that interact with ABCB1. The ATPase assay and vesicular transport (VT) assay are membrane based assays that can be used to measure the interaction of compounds with ABCB1 at a lower cost and higher throughput compared to cellular-based assays and therefore can be used earlier in the drug development process. To that end, we tested compounds previously identified as ABCB1 substrates and inhibitors for interaction with ABCB1 using the ATPase and VT assays. All compounds tested interacted with ABCB1 in both the ATPase and VT assays. All compounds previously identified as ABCB1 substrates activated ABCB1-mediated ATPase activity in the ATPase assay. All compounds previously identified as ABCB1 inhibitors inhibited the ABCB1-mediated transport in the VT assay. Interestingly, six of the ten compounds previously identified as ABCB1 inhibitors activated the basal ATPase activity in activation assays suggesting that the compounds are substrates of ABCB1 but can inhibit ABCB1 in inhibition assays. Importantly, for ATPase activators the EC50 of activation correlated with the IC50 values from the VT assay showing that interactions of compounds with ABCB1 can be measured with similar levels of potency in either assay. For ATPase nonactivators the IC50 values from the ATPase inhibition and VT inhibition assay showed correlation. These results demonstrate the utility of membrane assays as tools to detect and rank order drug-transporter interactions.

Structure and function of BCRP, a broad specificity transporter of xenobiotics and endobiotics.

The discovery and characterization of breast cancer resistance protein (BCRP) as an efflux transporter conferring multidrug resistance has set off a remarkable trajectory in the understanding of its role in physiology and disease. While the relevance in drug resistance and general pharmacokinetic properties quickly became apparent, the lack of a characteristic phenotype in genetically impaired animals and humans cast doubt on the physiological importance of this ATP-binding cassette family member, similarly to fellow multidrug transporters, despite well-known endogenous substrates. Later, high-performance genetic analyses and fine resolution tissue expression data forayed into unexpected territories concerning BCRP relevance, and ultimately, the rise of quantitative proteomics allows putting observed interactions into absolute frameworks for modeling and insight into interindividual and species differences. This overview summarizes existing knowledge on the BCRP transporter on molecular, tissue and system level, both in physiology and disease, and describes a selection of experimental procedures that are the most widely applied for the identification and characterization of substrate and inhibitor-type interactions.

A P-gp vesicular transport inhibition assay - optimization and validation for drug-drug interaction testing.

Accurate determination of potential drug-drug interaction mediated by efflux transporters (tDDI) is crucial to assess the risk of pharmacokinetic interaction and toxicity of drugs. Passive permeability and uptake transporter mediated transport are important covariates of cell-based inhibition assays that need to be taken into consideration when performing kinetic analysis of data. Vesicular uptake inhibition has been considered by regulatory agencies as a viable alternative for testing tDDI potential of low passive permeability drugs in particular. Membranes prepared from a P-gp overexpressing human cell line has superior transport properties over membranes prepared from Sf9 cells and cholesterol enriched Sf9 membranes. P-gp expressed in this membrane effluxes N-methyl-quinidine (NMQ) with high affinity (K(m) is 3.65 µM) and a high rate (V(max) is 656 pmol/mg protein/min). Digoxin, vinblastine and paclitaxel, established P-gp substrates inhibited transport of NMQ with estimated K(i) values of 250, 0.1 and 0.6 µM, respectively. A panel of 11 drugs that have been listed by regulatory agencies as reference inhibitors were used to validate the assay to predict clinical inhibition potential. All the drugs that have been implicated in P-gp mediated DDI were found to be inhibitors in a relevant concentration range.

Prediction of Therapy Response and Prognosis in Leukemias by Flow Cytometric MDR Assays.

Multidrug resistance (MDR) is an unwanted phenomenon, that may cause therapy failure in several neoplasms including hematological malignancies. The purpose of any type of laboratory MDR assay is to reliably identify such patients and to provide useful data to clinicians with a relatively short turnaround time. MDR can be multicausal and several previous data identified a group of transmembrane proteins - the ATP-binding casette (ABC) proteins - that may be involved in MDR in various hematological malignancies. The prototype of these proteins is the P-glycoprotein (Pgp, MDR1, ABCB1) that is a seven-membrane spanning transmembrane protein capable of extruding several cytotoxic drugs that are of key importance in the treatment of hematological disorders. Similarly other ABC proteins - Multidrug resistance associated protein 1 (ABCC1) and breast cancer resistance protein (ABCG2) are both capable of pumping out cytotoxic drugs. Here, we present flow cytometric methods to identify MDR proteins by antigen and activity assays. The advantage of flow technology is the short turnaround time and its relative easiness compared to nucleic acid based technologies. However, for the activity assays, it should be noted, that these functional tests require live cells, thus adequate results can only be provided if the specimen transport can be completed within 6 hours of sample collection. Identification of MDR proteins provides prognostic information and may modulate therapy, thus signifies a clinically useful information in the evaluation of patients with leukemias.