Wilfred D. Stein

From MDR to MXR: new understanding of multidrug resistance systems, their properties and clinical significance

Abstract: The ATP binding cassette (ABC) superfamily of membrane transporters is one of the largest protein classes known, and counts numerous proteins involved in the trafficking of biological molecules across cell membranes. The first known human ABC transporter was P-glycoprotein (P-gp), which confers multidrug resistance (MDR) to anticancer drugs. In recent years, we have obtained an increased understanding of the mechanism of action of P-gp as its ATPase activity, substrate specificity and pharmacokinetic interactions have been investigated. This review focuses on the functional characterization of P-gp, as well as other ABC transporters involved in MDR: the family of multidrug-resistance-associated proteins (MRP1-7), and the recently discovered ABC half-transporter MXR (also known as BCRP, ABCP and ABCG2). We describe recent progress in the analysis of protein structure-function relationships, and consider the conceptual problem of defining and identifying substrates and inhibitors of MDR. An in-depth discussion follows of how coupling of nucleotide hydrolysis to substrate transport takes place, and we propose a scheme for the mechanism of P-gp function. Finally, the clinical correlations, both for reversal of MDR in cancer and for drug delivery, are discussed.

Structure-activity relationships of P-glycoprotein interacting drugs: kinetic characterization of their effects on ATPase activity

We have determined the kinetic parameters for stimulation and inhibition by 34 drugs of the P-glycoprotein ATPase in membranes derived from CR1R12 Chinese hamster ovary cells. The drugs chosen were sets of calmodulin antagonists, steroids, hydrophobic cations, hydrophobic peptides, chemotherapeutic substrates of P-glycoprotein, and some other drugs with lower affinity for P-glycoprotein. We studied how these kinetic parameters correlated with the partition coefficient and the Van der Waals surface area of the drugs. The maximum velocity of ATPase stimulation decreased with surface area and showed a suggestion of a maximum with increasing partition coefficient. The affinity of these drugs for P-glycoprotein showed no significant correlation with partition coefficient but was highly correlated with the surface area suggesting that binding between modulators and P-glycoprotein takes place across a wide interaction surface on the protein.

Tumor regression and growth rates determined in five intramural NCI prostate cancer trials. The growth rate constant as an indicator of therapeutic efficacy.

PURPOSE In solid tumors such as prostate cancer, novel paradigms are needed to assess therapeutic efficacy. We utilized a method estimating tumor growth and regression rate constants from serial PSA measurements, and assessed its potential in patients with metastatic castration resistant prostate carcinoma (mCRPC). EXPERIMENTAL DESIGN Patients were enrolled in five phase II studies, including an experimental vaccine trial, representing the evolution of therapy in mCRPC. PSA measurements obtained before, and during, therapy were used. Data analysis using a two-phase mathematical equation yielded concomitant PSA growth and regression rate constants. RESULTS Growth rate constants (g) can be estimated while patients receive therapy and in such patients g is superior to PSA-DT in predicting OS. Incremental reductions in growth rate constants were recorded in successive trials with a 10-fold slower g in the most recent combination therapy trial (log g = 10(-3.17)) relative to single-agent thalidomide (log g = 10(-2.08)) more than a decade earlier. Growth rate constants correlated with survival, except in patients receiving vaccine-based therapy where the evidence demonstrates prolonged survival presumably due to immunity developing subsequent to vaccine administration. CONCLUSION Incremental reductions in tumor growth rate constants suggest increased efficacy in successive chemotherapy trials. The derived growth rate constant correlates with survival, and may be used to assess efficacy. The PSA-TRICOM vaccine appears to have provided marked benefit not apparent during vaccination, but consistent with subsequent development of a beneficial immune response. If validated as a surrogate for survival, growth rate constants would offer an important new efficacy endpoint for clinical trials.Purpose: In solid tumors such as prostate cancer, novel paradigms are needed to assess therapeutic efficacy. We utilized a method estimating tumor growth and regression rate constants from serial PSA measurements, and assessed its potential in patients with metastatic castration resistant prostate carcinoma (mCRPC). Experimental Design: Patients were enrolled in five phase II studies, including an experimental vaccine trial, representing the evolution of therapy in mCRPC. PSA measurements obtained before, and during, therapy were used. Data analysis using a two-phase mathematical equation yielded concomitant PSA growth and regression rate constants. Results: Growth rate constants (g) can be estimated while patients receive therapy and in such patients g is superior to PSA-DT in predicting OS. Incremental reductions in growth rate constants were recorded in successive trials with a 10-fold slower g in the most recent combination therapy trial (log g = 10−3.17) relative to single-agent thalidomide (log g = 10−2.08) more than a decade earlier. Growth rate constants correlated with survival, except in patients receiving vaccine-based therapy where the evidence demonstrates prolonged survival presumably due to immunity developing subsequent to vaccine administration. Conclusion: Incremental reductions in tumor growth rate constants suggest increased efficacy in successive chemotherapy trials. The derived growth rate constant correlates with survival, and may be used to assess efficacy. The PSA-TRICOM vaccine appears to have provided marked benefit not apparent during vaccination, but consistent with subsequent development of a beneficial immune response. If validated as a surrogate for survival, growth rate constants would offer an important new efficacy endpoint for clinical trials. Clin Cancer Res; 17(4); 907–17. ©2010 AACR.

COMPETITIVE, NON-COMPETITIVE AND COOPERATIVE INTERACTIONS BETWEEN SUBSTRATES OF P-GLYCOPROTEIN AS MEASURED BY ITS ATPASE ACTIVITY

We have studied the interaction between verapamil and other modulators of the P-glycoprotein ATPase from membranes of CR1R12 Chinese hamster ovary cells. Four major categories of interaction were identified. (i) Non-competitive inhibition of verapamils stimulation of enzyme activity was found with vanadate. (ii) Competitive inhibition of the ATPase was found for the pair verapamil and cyclosporin A. (iii) Allosteric inhibition with an increase in the Hill number for verapamil was found in the cases of daunorubicin, epirubicin, gramicidin S and D, vinblastine, amiodarone, and colchicine. (iv) Cooperative stimulation of verapamil-induced ATPase activity was found with progesterone, diltiazem, amitriptyline, and propranolol. At high levels, progesterone and verapamil mutually enhanced each others inhibitory action on the ATPase. Our data show that the substrate binding behavior of P-glycoprotein is complex with more than one binding site being present. This information could form the basis for the development of improved modulators of P-glycoprotein.

Testing and characterizing the simple carrier

Abstract 1. 1. We treat the simple carrier at the level of steady-state analysis, making no assumptions as to equilibrium or symmetry and taking into account specifically the effect of electric fields and unstirred layers. 2. 2. We develop tests and rejection criteria applicable under these conditions. 3. 3. For those transport systems which pass the tests for the simple carrier, we show how to obtain from the experimental data (i) the permeabilities of any unstirred layers which may be present at the membrane surfaces and (ii) a complete characterization of the transport system itself in terms of only four independent parameters. 4. 4. The interpretation of these parameters in terms of molecular rate constants is discussed.

Co-operative, competitive and non-competitive interactions between modulators of P-glycoprotein.

We measured the effects of individual modulators and of pairs of modulators of the multidrug resistance pump, P-glycoprotein, on the accumulation of labelled daunomycin into multidrug-resistant P388 leukemia cells at 37 degrees C and developed a kinetic analysis which enables such data to be modelled in terms of co-operative, competitive or non-competitive interaction between pairs of modulators. The modulators verapamil, cyclosporin and trifluoperazine interacted with P-glycoprotein as single molecules, while vinblastine, mefloquine, dipyridamole, tamoxifen and quinidine displayed Hill numbers close to 2, suggesting that pairs of modulator molecules need to act together in order to bring about effective reversal of P-glycoprotein. When the modulators were presented to P-glycoprotein in pairs, we found examples of both competitive and non-competitive behaviour. We interpret these results on a model in which two modulatory sites exit on the MDR pump. To one of these, mefloquine, vinblastine and tamoxifen bind preferentially; to the other, verapamil, dipyridamole, trifluoperazine and quinidine bind (but mefloquine and tamoxifen only weakly if at all). Cyclosporin A can interact with both sites.

Transporters as mediators of drug resistance in Plasmodium falciparum.

Drug resistance represents a major obstacle in the radical control of malaria. Drug resistance can arise in many different ways, but recent developments highlight the importance of mutations in transporter molecules as being major contributors to drug resistance in the human malaria parasite Plasmodium falciparum. While approximately 2.5% of the P. falciparum genome encodes membrane transporters, this review concentrates on three transporters, namely the chloroquine resistance transporter PfCRT, the multi-drug resistance transporter 1 PfMDR1, and the multi-drug resistance-associated protein PfMRP, which have been strongly associated with resistance to the major antimalarial drugs. The studies that identified these entities as contributors to resistance, and the possible molecular mechanisms that can bring about this phenotype, are discussed. A deep understanding of the underpinning mechanisms, and of the structural specificities of the players themselves, is a necessary basis for the development of the new drugs that will be needed for the future armamentarium against malaria.

Kinetic modelling of chloroquine uptake by malaria-infected erythrocytes: Assessment of the factors that may determine drug resistance

The antimalarial chloroquine, by virtue of its weak base properties, concentrates in the acidic compartment(s) of the intraerythrocytic parasite. Drug accumulation is essential for it to exert its pharmacological activity. Drug resistance has been thought to result from insufficient acidification of drug-accumulating organelle(s), (due to weakened proton pump activity and/or proton leak) or to result from the action of the recently suggested active efflux drug pump. In this work we have devised a kinetic model which takes into account the various processes that have been postulated to account for acidification and drug fluxes. Using this model to analyse the time-course of chloroquine uptake and the steady-state levels of drug accumulation, in strains of Plasmodium falciparum which display variable drug resistance, we demonstrate that drug resistance is compatible with the existence of a weakened proton pump in the resistant parasite strains. Consistent with recent molecular studies that show no correlation between the presence of the multidrug efflux pump gene and the phenotypic expression of chloroquine resistance, our analysis fails to detect any such pump activity. We also show that analysis of drug efflux kinetics cannot distinguish between the possible modes of drug resistance.

Polymorphisms within PfMDR1 alter the substrate specificity for anti-malarial drugs in Plasmodium falciparum.

Resistance to several anti‐malarial drugs has been associated with polymorphisms within the P‐glycoprotein homologue (Pgh‐1, PfMDR1) of the human malaria parasite Plasmodium falciparum. Pgh‐1, coded for by the gene pfmdr1, is predominately located at the membrane of the parasites digestive vacuole. How polymorphisms within this transporter mediate alter anti‐malarial drug responsiveness has remained obscure. Here we have functionally expressed pfmdr1 in Xenopus laevis oocytes. Our data demonstrate that Pgh‐1 transports vinblastine, an established substrate of mammalian MDR1, and the anti‐malarial drugs halofantrine, quinine and chloroquine. Importantly, polymorphisms within Pgh‐1 alter the substrate specificity for the anti‐malarial drugs. Wild‐type Pgh‐1 transports quinine and chloroquine, but not halofantrine, whereas polymorphic Pgh‐1 variants, associated with altered drug responsivenesses, transport halofantrine but not quinine and chloroquine. Our data further suggest that quinine acts as an inhibitor of Pgh‐1. Our data are discussed in terms of the model that Pgh‐1‐mediates, in a variant‐specific manner, import of certain drugs into the P. falciparum digestive vacuole, and that this contributes to accumulation of, and susceptibility to, the drug in question.

Non-Stokesian nature of transverse diffusion within human red cell membranes.

Although much research has recently been carried out on lateral diffusion in biological membranes (see ref. 35 for a recent review), far less attention has been given to the problem of transverse diffusion-diffusion perpendicular to the plane of the membrane. Yet transverse diffusion is of paramount importance not only for understanding the dynamic properties of lipids in membranes but also for predicting the permeabilities of both single cells and epithelia (e.g., kidney, gut, skin and the blood-brain barrier) to physiologically and pharmacologically active agents.