Potent interaction of flavopiridol with MRP1

The multidrug resistance protein 1 (MRP1) is an ATP-dependent transport protein for organic anions, as well as neutral or positively charged anticancer agents. In this study we show that flavopiridol, a synthetic flavonoid currently studied in phase 1 trials for its anti-proliferative characteristics, interacts with MRP1 in a potent way. Flavopiridol, as well as other (iso)flavonoids stimulate the ATPase activity of MRP1 in a dose-dependent way at low micromolar concentrations. A new specific monoclonal antibody against MRP1 (MIB6) inhibits the (iso)flavonoid-induced ATPase activity of plasma membrane vesicles prepared from the MRP1 overexpressing cell line GLC4/ADR. The accumulation of daunorubicin in GLC4/ADR cells is increased by flavopiridol and by other non-glycosylated (iso)flavonoids that interact with MRP1 ATPase activity. However, flavopiridol is the only tested compound that affects the daunorubicin accumulation when present at concentrations below 1 μM. Glycosylated (iso)flavonoids do not affect MRP1-mediated transport or ATPase activity. Finally, MRP1 overexpressing and transfected cells are resistant to flavopiridol, but not to other (iso)flavonoids tested. These findings may be of relevance for the development of anticancer therapies with flavopiridol. © 1999 Cancer Research Campaig

A Systems Approach for Tumor Pharmacokinetics

Recent advances in genome inspired target discovery, small molecule screens, development of biological and nanotechnology have led to the introduction of a myriad of new differently sized agents into the clinic. The differences in small and large molecule delivery are becoming increasingly important in combination therapies as well as the use of drugs that modify the physiology of tumors such as anti-angiogenic treatment. The complexity of targeting has led to the development of mathematical models to facilitate understanding, but unfortunately, these studies are often only applicable to a particular molecule, making pharmacokinetic comparisons difficult. Here we develop and describe a framework for categorizing primary pharmacokinetics of drugs in tumors. For modeling purposes, we define drugs not by their mechanism of action but rather their rate-limiting step of delivery. Our simulations account for variations in perfusion, vascularization, interstitial transport, and non-linear local binding and metabolism. Based on a comparison of the fundamental rates determining uptake, drugs were classified into four categories depending on whether uptake is limited by blood flow, extravasation, interstitial diffusion, or local binding and metabolism. Simulations comparing small molecule versus macromolecular drugs show a sharp difference in distribution, which has implications for multi-drug therapies. The tissue-level distribution differs widely in tumors for small molecules versus macromolecular biologic drugs, and this should be considered in the design of agents and treatments. An example using antibodies in mouse xenografts illustrates the different in vivo behavior. This type of transport analysis can be used to aid in model development, experimental data analysis, and imaging and therapeutic agent design.National Institutes of Health (U.S.) (grant T32 CA079443

A method for studying plasma membrane transport with intact cells using computerized fluorometry.

A new method is presented for measuring rapid efflux of fluorescent compounds from monolayer cells. Cells grown on a glass coverslip were loaded with a fluorescent substrate. Thereafter, the coverslip was installed outside the light path in a stirred and thermostated cuvette of a fluorometer. The efflux was recorded by measuring the changes of fluorescence in the extracellular medium. The method was used to study the kinetics of active and passive plasma membrane transport of the P-glycoprotein substrates rhodamine 123 and daunorubicin. The method has advantages over other methods: (1) no radioactively labeled substrate is needed, (2) fluorescence of the transported substrate is not compromised by the cells, (3) changes in the extracellular concentration of the substrate can be monitored continuously and therefore a substantial improvement of the kinetic resolution is obtained, and (4) the measurement setup is relatively simple and a standard fluorometer can be used. From the efflux data, cellular transport parameters could be calculated, such as passive permeation coefficients and active transport rates

Anthracyclines modulate multidrug resistance protein (MRP) mediated organic anion transport

We studied the ATP-dependent uptake of dinitrophenyl-glutathione (GS-DNP) into plasma membrane vesicles derived from parental GLC4 cells and from multidrug resistant GLC4/ADR cells. The latter have a high expression of the multidrug resistance protein (MRP). Uptake of GS-DNP into membrane vesicles from GLC4/ADR cells was highly stimulated by the addition of ATP, compared to the uptake into membrane vesicles from GLC4 cells. This ATP-dependent uptake into membrane vesicles from GLC4/ADR cells was saturable with a K(m) of 1.2 ± 0.2 μM and a V(max) of 560 ± 80 pmol/mg prot./min. ATP stimulated GS-DNP uptake with a K(m) of 187 ± 4 μM. This uptake was specifically inhibited by a polyclonal serum raised against a fusion protein containing a segment of MRP. The ATP-dependent uptake of GS-DNP was not only inhibited by organic anions, such as oxidized glutathione (GSSG), methotrexate (MTX) and some bile acids, but also by non-anionic natural product drugs, such as anthracyclines, vinca alkaloids and etoposide (VP-16). Uptake of GSSG and MTX into membrane vesicles from GLC4/ADR cells could be stimulated by ATP. The ATP-dependent uptake of GSSG had a K(m) of 43 ± 3 μM and a V(max) of 900 ± 200 nmol/mg protein/min. The ATP-dependent uptake of GS-DNP seemed to be non-competitively inhibited by the anthracycline daunorubicin (DNR), whereas the ATP-dependent GSSG uptake seemed to be competitively inhibited by DNR. A substrate binding site on MRP is proposed that comprises a pocket in which both DNR and GS-DNP or GSSG bind in random order to different, only partly overlapping sites. In this pocket binding of a second compound is influenced by the compound which was bound first

REDISTRIBUTION OF CANALICULAR ORGANIC ANION TRANSPORT ACTIVITY IN ISOLATED AND CULTURED RAT HEPATOCYTES

The hepatocanalicular transport of a large number of organic anions, such as bilirubin glucuronides and glutathione conjugates in the rat, is mediated by an adenosine triphosphate (ATP)-dependent transport system, which is termed canalicular multispecific organic anion transporter (cMOAT). This system is mainly de fined by its deficiency in mutant TR(-) rats. We have previously reported that in cultured hepatocytes the fluorescent organic anion glutathione-bimane (GS-B) accumulates in intracellular vesicles and that this transport is mediated by cMOAT. We now show that this intracellular accumulation of fluorescent organic anion is largely absent in freshly isolated hepatocytes but appears when cells are incubated in suspension at 37 degrees C or cultured for periods of 1 to 24 hours. The appearance of intracellular cMOAT activity coincides with the disappearance of 70% of cMOAT activity from the plasma membrane as measured by the transport activity of the cells for the organic anion dinitrophenyl-glutathione (GS-DNP). Both the appearance of intracellular cMOAT and the disappearance of transport activity from the plasma membrane were completely inhibited at temperatures below 20 degrees C. Residual cMOAT activity in 24-hour cultured hepatocytes could be further diminished by incubation of the cells with 1 mu mol/L monensin or 10 mmol/L methylamine. We conclude that after disruption of the cell polarity by collagenase isolation of the hepatocytes, remnants of apical membrane containing cMOAT are rapidly endocytosed when the cells are kept at 37 degrees C. Evidence suggests that at least part of the transporters may recycle back to the plasma membrane after endocytosis. These observations may be relevant for the understanding of regulation of canalicular transport

Canalicular multispecific organic anion transporter/multidrug resistance protein 2 mediates low-affinity transport of reduced glutathione.

The canalicular multispecific organic anion transporter (cMOAT), a member of the ATP-binding cassette transporter family, mediates the transport of a broad range of non-bile salt organic anions from liver into bile. cMOAT-deficient Wistar rats (TR-) are mutated in the gene encoding cMOAT, leading to defective hepatobiliary transport of a whole range of substrates, including bilirubin glucuronide. These mutants also have impaired hepatobiliary excretion of GSH and, as a result, the bile flow in these animals is reduced. In the present work we demonstrate a role for cMOAT in the excretion of GSH both in vivo and in vitro. Biliary GSH excretion in rats heterozygous for the cMOAT mutation (TR/tr) was decreased to 63% of controls (TR/TR) (114+/-24 versus 181+/-20 nmol/min per kg body weight). Madin-Darby canine kidney (MDCK) II cells stably expressing the human cMOAT protein displayed >10-fold increase in apical GSH excretion compared with wild-type MDCKII cells (141+/-6.1 pmol/min per mg of protein versus 13.2+/-1.3 pmol/min per mg of protein in wild-type MDCKII cells). Similarly, MDCKII cells expressing the human multidrug resistance protein 1 showed a 4-fold increase in GSH excretion across the basolateral membrane. In several independent cMOAT-transfectants, the level of GSH excretion correlated with the expression level of the protein. Furthermore, we have shown, in cMOAT-transfected cells, that GSH is a low-affinity substrate for the transporter and that its excretion is reduced upon ATP depletion. In membrane vesicles isolated from cMOAT-expressing MDCKII cells, ATP-dependent S-(2,4-dinitrophenyl)glutathione uptake is competitively inhibited by high concentrations of GSH (Ki approximately 20 mM). We concluded that cMOAT mediates low-affinity transport of GSH. However, since hepatocellular GSH concentrations are high (5-10 mM), cMOAT might serve an important physiological function in maintenance of bile flow in addition to hepatic GSH turnover

The protective effect of cardiac gene transfer of CuZn-sod in comparison with the cardioprotector monohydroxyethylrutoside against doxorubicin-induced cardiotoxicity in cultured cells

The protective effect of cardiac gene transfer of CuZn-sod in comparison with the cardioprotector monohydroxyethylrutoside against doxorubicin-induced cardiotoxicity in cultured cells. Abou El Hassan MA, Heijn M, Rabelink MJ, van der Vijgh WJ, Bast A, Hoeben RC. Department of Medical Oncology, Free University Medical Center, PO Box 7057, 1007 MB Amsterdam, The Netherlands. [email protected] Doxorubicin-induced cardiotoxicity is related to its production of free radicals that specifically affect heart tissue because of its low antioxidant status. Monohydroxyethylrutoside (monoHER), a potent antioxidant flavonoid, is under development as a protector against doxorubicin-induced cardiotoxicity. The overexpression of high levels of superoxide dismutase (sod) protects against free radical damage in transgenic mice. Seeking alternatives besides the few cardioprotectors that are presently under investigation, the aim of the present study was to investigate the protective effect of cardiac gene transfer of CuZn-sod compared with that of the presently most promising cardioprotector monoHER against doxorubicin-induced cardiotoxic effects on neonatal rat cardiac myocytes (NeRCaMs) in vitro. NeRCaMs were infected with different multiplicity of infections (MOIs) of adenovirus encoding CuZn-sod (AdCuZn-sod). A control infection with an adenovirus vector encoding a nonrelated protein was included. The overexpression of CuZn-sod was characterized within 3 days postinfection. For doxorubicin treatment, NeRCaMs were divided into three groups. The first group was infected with AdCuZn-sod before treatment with doxorubicin (0-50 microM). The second and third groups were treated with doxorubicin (0-50 microM) alone and with 1 mM monoHER, respectively. The LDH release and survival of treated cells were measured 24 and 48 hours after doxorubicin treatment. The beating rate was followed during the 3 days after doxorubicin (0-100 microM) treatment. At the third day after infection with an MOI of 25 plaque-forming unit (PFU) of AdCuZn-sod/cell, the activity of CuZn-sod significantly increased (five-fold, P=.029). Higher MOI produced cytopathic effects (CPEs). Doxorubicin alone produced significant concentration- and time-dependent reduction in NeRCaMs beating rate and survival (P or =50 microM)-treated cells ceased to beat after 24 hours. This cytotoxicity was associated with an increase in the LDH release from the treated cells (P 72 hours in the presence of monoHER.The present study showed the lack of adenoviral CuZn-sod gene-transfer to protect myocardiocytes against doxorubicin-induced toxicity and confirms the efficacy of monoHER cardioprotection. Thus, a gene-therapy strategy involving overexpression of CuZn-sod to protect against doxorubicin-induced cardiotoxicity is not feasible with the currently available adenovirus vectors