|
1
|
Bilsborough J, Fiorino MF, Henkle BW. Select animal models of colitis and their value in predicting clinical efficacy of biological therapies in ulcerative colitis. Expert Opin Drug Discov 2020; 16:567-577. [PMID: 33245673 DOI: 10.1080/17460441.2021.1851185] [Citation(s) in RCA: 29] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
Introduction: Advancing new therapies from discovery to development usually requires proof-of-concept in animal models to justify the costs of continuing the program. While animal models are useful for understanding the mechanism of action (MOA) of a target, limitations of many published colitis models restrict their value to predict clinical efficacy.Areas covered: The authors focused their literature search on published studies of chronic animal models used to evaluate the pre-clinical efficacy of therapeutic molecules subsequently evaluated in clinical trials for UC. The UC therapies evaluated were anti-α4β7, anti-IL13, anti-IL12p40, and anti-IL23p19. The models of chronic colitis evaluating these molecules were: mdra1a-/-, chronic dextran sulfate sodium (DSS), chronic 2,4,6-trinitrobenzene sulfonic acid (TNBS), and the T cell transfer model.Expert opinion: While some models provide insight into target MOA in UC, none is consistently superior in predicting efficacy. Evaluation of multiple models, with varying mechanisms of colitis induction, is needed to understand potential drug efficacy. Additional models of greater complexity, reflecting the disease chronicity/heterogeneity seen in humans, are needed. Although helpful in prioritizing targets, animal models alone will likely not improve outcomes of UC clinical trials. Transformational changes to clinical efficacy will likely only occur when precision medicine approaches are employed.
Collapse
Affiliation(s)
- Janine Bilsborough
- IBD Drug Discovery and Development Unit, Cedars-Sinai Medical Center, Los Angeles, CA, USA
| | - Marie F Fiorino
- IBD Drug Discovery and Development Unit, F. Widjaja Foundation Inflammatory Bowel and Immunbiology Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA
| | - Bradley W Henkle
- IBD Drug Discovery and Development Unit, F. Widjaja Foundation Inflammatory Bowel and Immunbiology Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA
| |
Collapse
|
|
2
|
Damasceno S, Gómez-Nieto R, Garcia-Cairasco N, Herrero-Turrión MJ, Marín F, Lopéz DE. Top Common Differentially Expressed Genes in the Epileptogenic Nucleus of Two Strains of Rodents Susceptible to Audiogenic Seizures: WAR and GASH/Sal. Front Neurol 2020; 11:33. [PMID: 32117006 PMCID: PMC7031349 DOI: 10.3389/fneur.2020.00033] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2019] [Accepted: 01/10/2020] [Indexed: 11/16/2022] Open
Abstract
The Wistar Audiogenic Rat (WAR) and the Genetic Audiogenic Seizure Hamster from Salamanca (GASH/Sal) strains are audiogenic epilepsy models, in which seizures are triggered by acoustic stimulation. These strains were developed by selective reproduction and have a genetic background with minimal or no variation. In the current study, we evaluated the transcriptome of the inferior colliculus, the epileptogenic nucleus, of both audiogenic models, in order to get insights into common molecular aspects associated to their epileptic phenotype. Based on GASH/Sal RNA-Seq and WAR microarray data, we performed a comparative analysis that includes selection and functional annotation of differentially regulated genes in each model, transcriptional evaluation by quantitative reverse transcription PCR of common genes identified in both transcriptomes and immunohistochemistry. The microarray data revealed 71 genes with differential expression in WAR, and the RNA-Seq data revealed 64 genes in GASH/Sal, showing common genes in both models. Analysis of transcripts showed that Egr3 was overexpressed in WAR and GASH/Sal after audiogenic seizures. The Npy, Rgs2, Ttr, and Abcb1a genes presented the same transcriptional profile in the WAR, being overexpressed in the naïve and stimulated WAR in relation to their controls. Npy appeared overexpressed only in the naïve GASH/Sal compared to its control, while Rgs2 and Ttr genes appeared overexpressed in naïve GASH/Sal and overexpressed after audiogenic seizure. No statistical difference was observed in the expression of Abcb1a in the GASH/Sal model. Compared to control animals, the immunohistochemical analysis of the inferior colliculus showed an increased immunoreactivity for NPY, RGS2, and TTR in both audiogenic models. Our data suggest that WAR and GASH/Sal strains have a difference in the timing of gene expression after seizure, in which GASH/Sal seems to respond more quickly. The transcriptional profile of the Npy, Rgs2, and Ttr genes under free-seizure conditions in both audiogenic models indicates an intrinsic expression already established in the strains. Our findings suggest that these genes may be causing small changes in different biological processes involved in seizure occurrence and response, and indirectly contributing to the susceptibility of the WAR and GASH/Sal models to audiogenic seizures.
Collapse
Affiliation(s)
- Samara Damasceno
- Institute of Neurosciences of Castilla y León, University of Salamanca, Salamanca, Spain
| | - Ricardo Gómez-Nieto
- Institute of Neurosciences of Castilla y León, University of Salamanca, Salamanca, Spain.,Salamanca Institute for Biomedical Research, Salamanca, Spain
| | | | - Manuel Javier Herrero-Turrión
- Institute of Neurosciences of Castilla y León, University of Salamanca, Salamanca, Spain.,INCYL Neurological Tissue Bank (BTN-INCYL), Salamanca, Spain
| | - Faustino Marín
- Department of Human Anatomy and Psychobiology, School of Medicine, University of Murcia, Murcia, Spain
| | - Dolores E Lopéz
- Institute of Neurosciences of Castilla y León, University of Salamanca, Salamanca, Spain.,Salamanca Institute for Biomedical Research, Salamanca, Spain
| |
Collapse
|
|
3
|
Ankathil R. ABCB1 genetic variants in leukemias: current insights into treatment outcomes. Pharmgenomics Pers Med 2017; 10:169-181. [PMID: 28546766 PMCID: PMC5438075 DOI: 10.2147/pgpm.s105208] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Despite improvements in treatment of different types of leukemia, not all patients respond optimally for a particular treatment. Some treatments will work better for some, while being harmful or ineffective for others. This is due to genetic variation in the form of single-nucleotide polymorphisms (SNPs) that affect gene expression or function and cause inherited interindividual differences in the metabolism and disposition of drugs. Drug transporters are one of the determinants governing the pharmacokinetic profile of chemotherapeutic drugs. The ABCB1 transporter gene transports a wide range of drugs, including drugs used in leukemia treatment. Polymorphisms in the ABCB1 gene do affect intrinsic resistance and pharmacokinetics of several drugs used in leukemia treatment protocols and thereby affect the efficacy of treatment and event-free survival. This review focuses on the impact of three commonly occurring SNPs (1236C>T, 2677G>T/A, and 3435C>T) of ABCB1 on treatment response of various types of leukemia. From the literature available, some of the genotypes and haplotypes of these SNPs have been found to be potential determinants of interindividual variability in drug disposition and pharmacologic response in different types of leukemia. However, due to inconsistencies in the results observed across the studies, additional studies, considering novel genomic methodologies, comprehensive definition of clinical phenotypes, adequate sample size, and uniformity in all the confounding factors, are warranted.
Collapse
Affiliation(s)
- Ravindran Ankathil
- Human Genome Centre, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, Kelantan, Malaysia
| |
Collapse
|
|
4
|
Sakai-Kato K, Nanjo K, Kusuhara H, Nishiyama N, Kataoka K, Kawanishi T, Okuda H, Goda Y. Effect of Knockout of Mdr1a and Mdr1b ABCB1 Genes on the Systemic Exposure of a Doxorubicin-Conjugated Block Copolymer in Mice. Mol Pharm 2015; 12:3175-83. [PMID: 26194248 DOI: 10.1021/acs.molpharmaceut.5b00234] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
We previously elucidated that ATP-binding cassette subfamily B member 1 (ABCB1) mediates the efflux of doxorubicin-conjugated block copolymers from HeLa cells. Here, we investigated the role of ABCB1 in the in vivo behavior of a doxorubicin-conjugated polymer in Mdr1a/1b(-/-) mice. The area under the curve for intravenously administered polymer in Mdr1a/1b(-/-) mice was 2.2-fold greater than that in wild-type mice. The polymer was mostly distributed in the liver followed by spleen and less so in the brain, heart, kidney, and lung. The amount of polymer excreted in the urine was significantly decreased in Mdr1a/1b(-/-) mice. The amounts of polymers excreted in the feces were similar in both groups despite the higher systemic exposure in Mdr1a/1b(-/-) mice. Confocal microscopy images showed polymer localized in CD68(+) macrophages in the liver. These results show that knockout of ABCB1 prolonged systemic exposure of the doxorubicin-conjugated polymer in mice. Our results suggest that ABCB1 mediated the excretion of doxorubicin-conjugated polymer in urine and feces. Our results provide valuable information about the behavior of block copolymers in vivo, which is important for evaluating the pharmacokinetics of active substances conjugated to block copolymers or the accumulation of block copolymers in vivo.
Collapse
Affiliation(s)
- Kumiko Sakai-Kato
- Division of Drugs, National Institute of Health Sciences , 1-18-1 Kamiyoga, Setagaya, Tokyo 158-8501, Japan
| | - Kunie Nanjo
- Division of Drugs, National Institute of Health Sciences , 1-18-1 Kamiyoga, Setagaya, Tokyo 158-8501, Japan
| | | | - Nobuhiro Nishiyama
- Polymer Chemistry Division, Chemical Resources Laboratory, Tokyo Institute of Technology , R1-11, 4259 Nagatsuda, Midori, Yokohama 226-8503, Japan
| | - Kazunori Kataoka
- Department of Materials Engineering, Graduate School of Engineering, The University of Tokyo , 7-3-1 Hongo, Bunkyo, Tokyo 113-8656, Japan
| | - Toru Kawanishi
- National Institute of Health Sciences , 1-18-1 Kamiyoga, Setagaya, Tokyo 158-8501, Japan
| | - Haruhiro Okuda
- National Institute of Health Sciences , 1-18-1 Kamiyoga, Setagaya, Tokyo 158-8501, Japan
| | - Yukihiro Goda
- Division of Drugs, National Institute of Health Sciences , 1-18-1 Kamiyoga, Setagaya, Tokyo 158-8501, Japan
| |
Collapse
|
|
5
|
Germann UA, Chambers TC. Molecular analysis of the multidrug transporter, P-glycoprotein. Cytotechnology 2012; 27:31-60. [PMID: 19002782 DOI: 10.1023/a:1008023629269] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Inherent or acquired resistance of tumor cells to cytotoxic drugs represents a major limitation to the successful chemotherapeutic treatment of cancer. During the past three decades dramatic progress has been made in the understanding of the molecular basis of this phenomenon. Analyses of drug-selected tumor cells which exhibit simultaneous resistance to structurally unrelated anti-cancer drugs have led to the discovery of the human MDR1 gene product, P-glycoprotein, as one of the mechanisms responsible for multidrug resistance. Overexpression of this 170 kDa N-glycosylated plasma membrane protein in mammalian cells has been associated with ATP-dependent reduced drug accumulation, suggesting that P-glycoprotein may act as an energy-dependent drug efflux pump. P-glycoprotein consists of two highly homologous halves each of which contains a transmembrane domain and an ATP binding fold. This overall architecture is characteristic for members of the ATP-binding cassette or ABC superfamily of transporters. Cell biological, molecular genetic and biochemical approaches have been used for structure-function studies of P-glycoprotein and analysis of its mechanism of action. This review summarizes the current status of knowledge on the domain organization, topology and higher order structure of P-glycoprotein, the location of drug- and ATP binding sites within P-glycoprotein, its ATPase and drug transport activities, its possible functions as an ion channel, ATP channel and lipid transporter, its potential role in cholesterol biosynthesis, and the effects of phosphorylation on P-glycoprotein activity.
Collapse
Affiliation(s)
- U A Germann
- Vertex Pharmaceuticals Incorporated, 130 Waverly Street, Cambridge, MA, 02139-4242, U.S.A.,
| | | |
Collapse
|
|
6
|
Hackenberger BK, Velki M, Stepić S, Hackenberger DK. First evidence for the presence of efflux pump in the earthworm Eisenia andrei. ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY 2012; 75:40-45. [PMID: 22033226 DOI: 10.1016/j.ecoenv.2011.06.024] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/11/2011] [Revised: 06/22/2011] [Accepted: 06/27/2011] [Indexed: 05/31/2023]
Abstract
Efflux pumps are transport proteins involved in the extrusion of toxic substrates from cells to the external environment. Activities of efflux pumps have been found in many organisms, however such activity has not been evidenced in earthworms. Adult Eisenia andrei earthworms were exposed to efflux modulators - verapamil (a known inhibitor of efflux pump protein) and dexamethasone (a known inducer of efflux activity) - and the amount of absorbed fluorescent dye rhodamine B was measured. The results showed that verapamil inhibited efflux activity and decreased removal of rhodamine B, whereas dexamethasone induced efflux activity and increased removal of rhodamine B. This is the first evidence of the presence of efflux pump in earthworm Eisenia andrei. Since earthworms are often used as test organisms due to their sensitive reactions towards environmental influences, the discovery of efflux pump activity can contribute to the better understanding of toxicity of certain pollutants.
Collapse
|
|
7
|
Strober W, Fuss IJ. Experimental models of mucosal inflammation. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2009; 579:55-97. [PMID: 16620012 DOI: 10.1007/0-387-33778-4_5] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Affiliation(s)
- Warren Strober
- Mucosal Immunity Section, Laboratory of Host Defense NIAID, National Institutes of Health, Bethesda, MD, USA
| | | |
Collapse
|
|
8
|
Abstract
In vivo and in vitro studies have demonstrated that P-glycoprotein (P-gp) plays a very significant role in the ADME processes (absorption, distribution, metabolism, excretion) and drug-drug interaction (DDI) of drugs in humans. P-gp is the product of multidrug resistance gene (MDR1/ABCB1). Pharmacogenomics and pharmacogenetics studies have revealed that genetic polymorphisms of MDR1 are associated with alteration in P-gp expression and function in different ethnicities and subjects. By now, 50 single nucleotide polymorphisms (SNPs) and 3 insertion/deletion polymorphisms have been found in the MDR1 gene. Some of them, such as C3435T, have been identified to be a risk factor for numerous diseases. It is believed that further understanding of the physiology and biochemistry of P-gp with respect to its genetic variations may be important for individualized pharmacotherapy. Therefore, based on the latest public information and our studies, this review focuses on the following four aspects: 1) the impact of P-gp on pharmacokinetics; 2) MDR1 genetic polymorphisms and their impacts on pharmacogenetics; 3) relationship between altered P-gp expression and function and the MDR1(C3435T) SNP, and 4) relevance of MDR1 polymorphisms to certain human diseases.
Collapse
Affiliation(s)
- Yan-Hong Li
- Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
| | | | | | | |
Collapse
|
|
9
|
Bonhomme-Faivre L, Benyamina A, Reynaud M, Farinotti R, Abbara C. Disposition of Delta tetrahydrocannabinol in CF1 mice deficient in mdr1a P-glycoprotein. Addict Biol 2008; 13:295-300. [PMID: 18331373 DOI: 10.1111/j.1369-1600.2008.00096.x] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023]
Abstract
P-glycoprotein (P-gp) plays a major role in drug efflux. All the transported substrates are more or less hydrophobic and amphiphatic in nature. Being lipophilic, Delta(9) tetrahydrocannabinol (THC), the main cannabis component, could be a potential P-gp substrate. The aim of this project was to determine the contribution of the mdr1a gene product to THC disposition. Therefore, oral THC and digoxin (substrate test for P-gp) pharmacokinetics have been investigated in the intestinal epithelium and in the brain capillary endothelium of CF1 mdr1a-/- mice (mice naturally deficient in P-gp). These pharmacokinetics were compared to THC and digoxin oral pharmacokinetics in wild type mice mdr1a+/+ (not P-gp deficient). The application of Bailer's method showed that THC total exposure measured by the area under the plasma concentration time curve was 2.17-fold higher in CF1 mice naturally deficient in P-gp than in wild type mice after oral administration of 25 mg/kg of THC, and 2.4-fold higher after oral administration of 33 microg/kg of digoxin. As a consequence, the oral bioavailability of THC and digoxin was higher in naturally P-gp-deficient mice. We concluded that P-gp limits THC oral uptake and mediates direct drug excretion from the systemic circulation into the intestinal lumen.
Collapse
Affiliation(s)
- Laurence Bonhomme-Faivre
- Department of Pharmacology and Pharmacy, Hôpital Paul Brousse, 14 Avenue Paul Vaillant Couturier, 94800 Villejuif, France.
| | | | | | | | | |
Collapse
|
|
10
|
Barile E, Corea G, Lanzotti V. Diterpenes from Euphorbia as Potential Leads for Drug Design. Nat Prod Commun 2008. [DOI: 10.1177/1934578x0800300629] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022] Open
Abstract
In the course of our chemical survey of bioactive plant metabolites, a large number of diterpenes have been isolated from Euphorbia species that showed interesting pharmacological activities. In particular, over sixty jatrophane, modified jatrophane, segetane, pepluane, and paraliane diterpenoids, fifty of them reported for the first time, were extracted, purified and characterized from Euphorbia dendroides, E. characias, E. peplus, E. amygdaloides, and E. paralias. The compounds based on jatrophane and modified jatrophane skeletons were shown to be potent inhibitors of P-glycoprotein, a membrane protein that confers upon cells the ability to resist lethal doses of certain cytotoxic drugs by pumping them out of the cells, thus resulting in a reduced cytotoxic effect. Those belonging to the rare classes of pepluane and paraliane were shown to be promising anti-inflammatory agents in vivo. In addition, by using LPS-stimulated J774 murine macrophages, it was demonstrated that the effect is ascribable to the reduction in the production of nitric oxide, prostaglandin E2 and TNF-α by inhibiting the expression of inducible nitric oxide synthase, cyclooxygenase-2 and TNF-α mRNA, respectively, through the down-regulation of NF-κB binding activity. The isolation of structurally-related analogues allowed us to perform SAR studies, which gave information on the key pharmacophoric elements of these new classes of promising drugs.
Collapse
Affiliation(s)
- Elisa Barile
- Dipartimento STAAM, Università del Molise, Via F. De Sanctis, 86100 Campobasso, Italy
| | - Gabriella Corea
- Dipartimento STAAM, Università del Molise, Via F. De Sanctis, 86100 Campobasso, Italy
| | - Virginia Lanzotti
- Dipartimento STAAM, Università del Molise, Via F. De Sanctis, 86100 Campobasso, Italy
| |
Collapse
|
|
11
|
Wilk JN, Bilsborough J, Viney JL. The mdr1a-/- mouse model of spontaneous colitis: a relevant and appropriate animal model to study inflammatory bowel disease. Immunol Res 2008; 31:151-9. [PMID: 15778512 DOI: 10.1385/ir:31:2:151] [Citation(s) in RCA: 54] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
There are many types of colitis models in animals that researchers use to elucidate the mechanism of action of human inflammatory bowel disease (IBD). These models are also used to test novel therapeutics and therapeutic treatment regimens. Here, we will review the characteristics of the mdr1a -/- model of spontaneous colitis that we believe make this model an important part of the IBD researcher's toolbox. We will also share new data that will reinforce the fact that this model is relevant in the study of IBD. Mdr1a -/- mice lack the murine multiple drug resistance gene for P-glycoprotein 170 that is normally expressed in multiple tissues including intestinal epithelial cells. These mice spontaneously develop a form of colitis at around 12 wk of age. The fact that the complexity of this model mirrors the complexity of disease in humans, as well as recent literature that links MDR1 polymorphisms in humans to Crohn's Disease and Ulcerative Colitis, makes this an appropriate animal model to study.
Collapse
|
|
12
|
|
|
13
|
Václavíková R, Boumendjel A, Ehrlichová M, Kovár J, Gut I. Modulation of paclitaxel transport by flavonoid derivatives in human breast cancer cells. Is there a correlation between binding affinity to NBD of P-gp and modulation of transport? Bioorg Med Chem 2006; 14:4519-25. [PMID: 16516478 DOI: 10.1016/j.bmc.2006.02.025] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2005] [Revised: 02/07/2006] [Accepted: 02/14/2006] [Indexed: 11/15/2022]
Abstract
We have investigated the effect of 13 flavonoid derivatives on [(14)C]paclitaxel transport in two human breast cancer cell lines, the adriamycin-resistant NCI/ADR-RES and sensitive MDA-MB-435. For this study, we selected representatives of aurones, chalcones, flavones, flavonols, chromones, and isoflavones with known binding affinity toward nucleotide-binding domain (NBD2) of P-glycoprotein and for which no reported work is available regarding paclitaxel transport. Aurones CB-284, CB-285, CB-287, and ML-50 most effectively inhibited P-gp related transport in the resistant line in comparison with chalcones, flavones, flavonols, chromones, and isoflavone derivatives and accordingly increased the accumulation of [(14)C]paclitaxel and decreased its efflux. Those agents efficiently modulated paclitaxel transport in P-gp highly expressing resistant human breast cancer cells and they could increase the efficiency of chemotherapy in paclitaxel-resistant tumors. In contrast, the sensitive cell line responded reversely in that CB-284, CB-285, CB-287, and ML-50 significantly inhibited accumulation of [(14)C]paclitaxel and especially CB-287, which significantly stimulated its efflux. Some, but not all, of the data correlated with the binding of flavonoid derivatives to P-gp, and indicated that even in the P-gp highly expressing NCI/ADR-RES cells, the binding was not the only factor influencing the transport of [(14)C]paclitaxel. Opposite effects of flavonoid derivatives on the P-gp highly expressing and MDA-MB-435 non-expressing cell lines indicate that paclitaxel is not only transported by P-gp and let us assume that Mrp2 or ABCC5 seem to be good transport-candidates in these cells. The inhibition of paclitaxel accumulation and stimulation of its efflux are potentially unfavorable for drug therapy and since they could be due to modulation of drug transporters other than P-gp, their expression in tumors is of great significance for efficient chemotherapy.
Collapse
Affiliation(s)
- Radka Václavíková
- Biotransformation Group, National Institute of Public Health, Praha, Czech Republic.
| | | | | | | | | |
Collapse
|
|
14
|
Abstract
Frequency of the 4-bp deletion mutant in canine mdr1 gene was examined in 193 dogs of eight breeds in Japan. The mutant allele was found in Collies, Australian Shepherds, and Shetland Sheepdogs, where its respective frequencies were 58.3%, 33.3%, and 1.2%. The MDR1 protein was detected on peripheral blood mononuclear cells (PBMC) from a MDR1/MDR1 dog, but not on PBMC from a mdr1-1Delta/mdr1-1Delta Collie. Rhodamine 123 was extruded from MDR1/MDR1 lymphocytes. That excretion was inhibited by a MDR1 inhibitor, verapamil. On the other hand, Rh123 excretion was not observed from lymphocytes derived from a mdr1-1Delta/mdr1-1Delta Collie. These results indicated that the mutant mdr1 allele also existed in Collie-breed dogs in Japan at high rates and that mdr1-1Delta /mdr1-1Delta dogs have no functional MDR1.
Collapse
Affiliation(s)
- Akiko Kawabata
- Department of Veterinary Internal Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Japan
| | | | | | | |
Collapse
|
|
15
|
Parker RB, Yates CR, Laizure SC, Weber KT. P-glycoprotein modulates aldosterone plasma disposition and tissue uptake. J Cardiovasc Pharmacol 2006; 47:55-9. [PMID: 16424786 DOI: 10.1097/01.fjc.0000194251.61554.6b] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
Aldosterone plays an important role in the pathophysiology of numerous cardiovascular disorders including heart failure and hypertension. Because aldosterone's actions are primarily mediated by its interaction with an intracellular mineralocorticoid receptor, factors affecting the cellular uptake and distribution of aldosterone may be important determinants of the hormone's activity. P-glycoprotein (P-gp) is an ATP-binding cassette efflux transporter encoded by the ABCB1 (also known as MDR1) gene in humans. P-gp is expressed on the luminal membrane of the capillary endothelial cells of tissues that are targets for aldosterone, including the brain and heart, where it attenuates cellular uptake of substrates. Recent in vitro evidence indicates P-gp transports aldosterone. Therefore, in this study we tested the hypothesis that P-gp modulates the uptake of aldosterone into the brain and heart by comparing the plasma and tissue distribution of [3H]-aldosterone in wild-type and P-gp-deficient [mdr1a/1b (-/-)] mice. Compared with wild-type mice, [3H]-aldosterone activity in the plasma, brain, and heart was significantly (P < 0.05) higher in the mdr1a/1b (-/-) animals. The area under the plasma or tissue concentration-time curves in the mdr1a/1b (-/-) mice was 2.0, 1.6, and 1.6-fold higher in the brain, heart, and plasma, respectively, than in wild-type controls. Our results demonstrate that P-gp plays an important role in aldosterone plasma disposition and modestly limits its uptake into the brain. The increased exposure of the brain and heart to aldosterone in the absence of P-gp suggests P-gp may play a key role in modulating aldosterone's effects in these organs.
Collapse
Affiliation(s)
- Robert B Parker
- University of Tennessee Health Science Center, Department of Pharmacy, Memphis, TN 38163, USA.
| | | | | | | |
Collapse
|
|
16
|
Lasagna N, Fantappiè O, Solazzo M, Morbidelli L, Marchetti S, Cipriani G, Ziche M, Mazzanti R. Hepatocyte growth factor and inducible nitric oxide synthase are involved in multidrug resistance-induced angiogenesis in hepatocellular carcinoma cell lines. Cancer Res 2006; 66:2673-82. [PMID: 16510587 DOI: 10.1158/0008-5472.can-05-2290] [Citation(s) in RCA: 51] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Based on literature, it is possible to hypothesize that multidrug resistance (MDR) and angiogenic phenotypes are linked to each other in human liver cancer cells. Our goal is to assess whether MDR cells trigger angiogenesis and to study the possible molecular mechanisms involved. Conditioned medium from parental drug-sensitive P5 cells (P5-CM) and MDR-positive P1(0.5) cells [P1(0.5)-CM] stimulated human umbilical vein endothelial cells (HUVEC) survival, proliferation, migration, and microtubular structure formation, but P1(0.5)-CM had a significantly greater effect than P5-CM. Cell implants were done in the rabbit avascular cornea to measure angiogenesis in vivo: P1(0.5) cells induced an important neovascular response in rabbit cornea after 1 week, whereas P5 cells had no effect. P1(0.5) and P5 cells produced vascular endothelial growth factor, but only P1(0.5) secreted hepatocyte growth factor (HGF) into the medium, and small interfering RNA specific for MDR1 clearly reduced HGF production in P1(0.5) cells. The transcription factor Ets-1 and the HGF receptor c-Met were up-regulated in P1(0.5) cells and in HUVEC cultured in P1(0.5)-CM. Inducible nitric oxide synthase (iNOS) seemed to play a major role in the proangiogenic effect of P1(0.5), and its inhibition by 1400W blunted the capacity of P1(0.5) cells to stimulate HUVEC proliferation, migration, and Ets-1 expression. In conclusion, these data show that development of MDR and angiogenic phenotypes are linked to each other in MDR cells. HGF production, Ets-1 and c-Met up-regulation, and iNOS expression can be part of the molecular mechanisms that enhance the angiogenic activity of the MDR-positive hepatocellular carcinoma cell line.
Collapse
Affiliation(s)
- Nadia Lasagna
- Department of Internal Medicine, Postgraduate School in Oncology, DENOthe, University of Florence, Azienda Ospedaliero-Universitaria Careggi, Florence, Italy
| | | | | | | | | | | | | | | |
Collapse
|
|
17
|
Akimoto M, Yoshikawa M, Ebara M, Sato T, Fukuda H, Kondo F, Saisho H. Relationship between therapeutic efficacy of arterial infusion chemotherapy and expression of P-glycoprotein and p53 protein in advanced hepatocellular carcinoma. World J Gastroenterol 2006; 12:868-73. [PMID: 16521213 PMCID: PMC4066150 DOI: 10.3748/wjg.v12.i6.868] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
AIM: To investigate the relationship between the chemotherapeutic drug efficacy and the expression of P-glycoprotein (PGP) and p53 protein in advanced hepatocellular carcinoma (HCC).
METHODS: The study was conducted on 41 patients with advanced HCC who were treated by repeated arterial infusion chemotherapy. Biopsy specimens from the tumor were collected before the start of treatment in all the patients, and the specimens were stored frozen until immunohistochemical staining, which was performed after the start of treatment, to detect PGP and p53 protein expressions. Twenty of the forty-one patients were treated with an anthracycline drug (epirubicin hydrochloride; anthracycline group), and the remaining 21 were treated with a non-anthracycline drug (mitoxantrone hydrochloride in 11 patients and carboplatin in 10 patients; non-anthracycline group). The relationship between the chemotherapeutic efficacy and the results of immunostaining were compared between the two groups.
RESULTS: Before the start of the treatment, PGP-positive rate was 90.2% (strongly-positive, 36.6%) and p53 protein-positive rate was 34.1% (strongly-positive, 19.5%). In the anthracycline group, the response rate was 40.0%. The number of patients showing poor response to the treatment was significantly larger in the patients with strongly positive PGP expression (P = 0.005), and their prognoses were poor (P = 0.001). In the non-anthracycline group, the response rate was 42.9%, and there was no significant relationship between the chemotherapeutic drug efficacy and the PGP or p53 protein expression. When only the data from the 11 patients treated with anthraquinone drug, mitoxantrone, were analyzed, however, the number of patients who showed poor response to treatment was significantly higher among the p53-positive patients (P = 0.012), irrespective of the survival outcome.
CONCLUSION: The chemotherapeutic efficacy with an anthracycline drug for advanced HCC can be predicted by immunohistochemical analysis of PGP expression. Similarly, immunostaining to evaluate p53 protein may be useful to predict the response in patients treated with an anthraquinone drug.
Collapse
Affiliation(s)
- Masahide Akimoto
- Department of Medicine and Clinical Oncology, School of Medicine, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba City, Chiba 260-0856, Japan.
| | | | | | | | | | | | | |
Collapse
|
|
18
|
Dalmas O, Do Cao MA, Lugo MR, Sharom FJ, Di Pietro A, Jault JM. Time-Resolved Fluorescence Resonance Energy Transfer Shows that the Bacterial Multidrug ABC Half-Transporter BmrA Functions as a Homodimer†. Biochemistry 2005; 44:4312-21. [PMID: 15766260 DOI: 10.1021/bi0482809] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Members of the ATP-binding cassette (ABC) transporters share the same basic architecture, with a four-core domain made of two transmembrane plus two nucleotide-binding domains. However, a supramolecular organization has been detected in some ABC transporters, which might be relevant to physiological regulation of substrate transport. Here, the oligomerization status of a bacterial half-ABC multidrug transporter, BmrA, was investigated. Each BmrA monomer containing a single cysteine residue introduced close to either the Walker A or the ABC signature motifs was labeled using two probes, 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid (fluorescence donor) or 4-dimethylaminophenylazophenyl-4'-maleimide (fluorescence acceptor). Reconstitution into proteoliposomes of BmrA monomers labeled separately with either the fluorescence donor or the fluorescence acceptor allowed measurement of time-resolved fluorescence resonance energy transfer between the two probes, showing that efficient reassociation of the singly labeled BmrA monomers occurred upon reconstitution. The efficiency of energy transfer studied as a function of increasing concentration of BmrA-labeled with the fluorescence acceptor argues for a dimeric association of BmrA instead of a tetrameric one. Furthermore, the efficiency of energy transfer allowed estimation of the distances between the two bound probes. Results suggest that, in the resting state, BmrA in a lipid bilayer environment preferentially adopts a closed conformation similar to that found in the BtuCD crystal structure and that the presence of different effectors does not substantially modify its global conformation.
Collapse
Affiliation(s)
- Olivier Dalmas
- Institut de Biologie et Chimie des Protéines, UMR 5086 CNRS-UCBL1 and IFR 128, 7 passage du Vercors, 69367 Lyon Cedex 07, France
| | | | | | | | | | | |
Collapse
|
|
19
|
Cummings AM, Kavlock RJ. Gene-environment interactions: a review of effects on reproduction and development. Crit Rev Toxicol 2005; 34:461-85. [PMID: 15609483 DOI: 10.1080/10408440490519786] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/26/2022]
Abstract
Polymorphisms in genes can lead to differences in the level of susceptibility of individuals to potentially adverse effects of environmental influences, such as chemical exposure, on prenatal development or male or female reproductive function. We have reviewed the literature in this area, with the caveat that papers involving straight gene knock-outs in experimental animals, without a clear human relevance, were largely excluded. This review represents current knowledge in this rapidly moving field, presenting both human epidemiological and animal data, where available. Among the polymorphic genes and environmental interactions discussed with respect to prenatal development are those for P-glycoprotein (multidrug resistance protein) and the avermectins; methylenetetrahydrofolate reductase (MTHFR), an enzyme in folate metabolism, and dietary folic acid; transforming growth factor alpha (TGFalpha) and cigarette smoke; and alcohol dehydrogenase (ADH) and cytochrome P-450 (CYP) 2E1 in association with alcohol consumption. Effects on male reproduction attributable to gene-environment interaction involve infertility seen as a result of either organophosphorous (OP) pesticide interaction with the polymorphic paraoxonase (PON1) gene or antiandrogenic agent interaction with the androgen receptor (AR). MTHFR, folate metabolism, and dietary folic acid are also considered in conjunction with preeclampsia and early pregnancy loss, and the effect of the interaction of glutathione S-transferase (GST) with exposure to benzene or cigarette smoke on pregnancy maintenance is explored. As a conclusion, we offer a discussion of lessons learned and suggested research needs.
Collapse
Affiliation(s)
- Audrey M Cummings
- Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711, USA.
| | | |
Collapse
|
|
20
|
Agrawal S, Panchagnula R. Implication of biopharmaceutics and pharmacokinetics of rifampicin in variable bioavailability from solid oral dosage forms. Biopharm Drug Dispos 2005; 26:321-34. [PMID: 16059874 DOI: 10.1002/bdd.464] [Citation(s) in RCA: 38] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Rifampicin is one of the oldest and most effective chemotherapeutic agents available for the treatment of tuberculosis but exhibits variable bioavailability from separate and fixed dose combination formulations, which has been identified as a major bottleneck in the effective treatment of tuberculosis. In this investigation, physico-chemical characterization, single dose pharmacokinetic studies and the permeability of rifampicin under physiological conditions in the rat were studied to trace the possible reasons for its variable absorption. Rifampicin exhibits very high solubility in acidic and basic pH, corresponding to the pH of the stomach and distal intestine, respectively, whereas it is moderately soluble at the jejunal pH. From single-dose pharmacokinetic studies and permeability characterization, rifampicin is a highly permeable molecule and thus according to BCS, it is a borderline class II drug. This investigation has ruled out the possibility of intrinsic solubility, effective permeability, drug decomposition, presystemic metabolism and interaction with other antituberculosis drugs as direct factors responsible for the variable bioavailability of rifampicin. However, it was found that the rate of dissolution in association with pH and the concentration-dependent absorption of rifampicin affects the in vivo performance of the dosage forms. In addition, this is the first report of methodology for correcting inlet concentration for permeability calculations of a chemically unstable molecule.
Collapse
Affiliation(s)
- Shrutidevi Agrawal
- Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, Phase-X, Mohali-160062, Punjab, India
| | | |
Collapse
|
|
21
|
Lee BD, Li Z, French KJ, Zhuang Y, Xia Z, Smith CD. Synthesis and Evaluation of Dihydropyrroloquinolines That Selectively Antagonize P-Glycoprotein. J Med Chem 2004; 47:1413-22. [PMID: 14998330 DOI: 10.1021/jm0303204] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
In a search for improved multiple drug resistance (MDR) modulators, we identified a novel series of substituted pyrroloquinolines that selectively inhibits the function of P-glycoprotein (Pgp) without modulating multidrug resistance-related protein 1 (MRP1). These compounds were evaluated for their toxicity toward drug-sensitive tumor cells (i.e. MCF-7, T24) and for their ability to antagonize Pgp-mediated drug-resistant cells (i.e. NCI/ADR) and MRP1-mediated resistant cells (i.e. MCF-7/VP). Cytotoxicity and drug accumulation assays demonstrated that the dihydropyrroloquinolines inhibit Pgp to varying degrees, without any significant inhibition of MRP1. The compound termed PGP-4008 was the most effective at inhibiting Pgp in vitro and was further evaluated in vivo. PGP-4008 inhibited tumor growth in a murine syngeneic Pgp-mediated MDR solid tumor model when given in combination with doxorubicin. PGP-4008 was rapidly absorbed after intraperitoneal administration, with its plasma concentrations exceeding the in vitro effective dose for more than 2 h. PGP-4008 did not alter the plasma distribution of concomitantly administered anticancer drugs and did not cause systemic toxicity as was observed for cyclosporin A. Because of their enhanced selectivity toward Pgp, these substituted dihydropyrroloquinolines may be effective MDR modulators in a clinical setting.
Collapse
Affiliation(s)
- Brian D Lee
- Department of Pharmacology, The Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033, USA
| | | | | | | | | | | |
Collapse
|
|
22
|
Corea G, Fattorusso E, Lanzotti V, Motti R, Simon PN, Dumontet C, Di Pietro A. Jatrophane Diterpenes as Modulators of Multidrug Resistance. Advances of Structure−Activity Relationships and Discovery of the Potent Lead Pepluanin A. J Med Chem 2004; 47:988-92. [PMID: 14761200 DOI: 10.1021/jm030951y] [Citation(s) in RCA: 67] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
From the whole plant of Euphorbia peplus L., five new diterpenes based on a jatrophane skeleton (pepluanins A-E, 1-5) were isolated, together with two known analogues (6 and 7), which served to divulge in detail the structure-activity relationships within this class of P-glycoprotein inhibitors. The results revealed the importance of substitutions on the medium-sized ring (carbons 8, 9, 14, and 15). In particular, the activity is collapsed by the presence of a free hydroxyl at C-8, while it increases with a carbonyl at C-14, an acetoxyl at C-9, and a free hydroxyl at C-15. The most potent compound of the series, pepluanin A, showed a very high activity for a jatrophane diterpene, outperforming cyclosporin A by a factor of at least 2 in the inhibition of Pgp-mediated daunomycin transport.
Collapse
Affiliation(s)
- Gabriella Corea
- Dipartimento di Chimica delle Sostanze Naturali, Università di Napoli "Federico II", Via D. Montesano 49, I-80131 Napoli, Italy
| | | | | | | | | | | | | |
Collapse
|
|
23
|
Abstract
Despite the introduction of newer antiepileptic drugs (AEDs), nonresponsiveness to AEDs remains a common problem in epilepsy clinics. There may be important genetic determinants for responsiveness, and this discussion focused on some potential areas: drug transporters; drug-metabolizing enzymes, and ion channels. We review the literature and speculate the contribution of each of these factors in management of patients with epilepsy in the future.
Collapse
|
|
24
|
Parker RB, Yates CR, Soberman JE, Laizure SC. Effects of grapefruit juice on intestinal P-glycoprotein: evaluation using digoxin in humans. Pharmacotherapy 2003; 23:979-87. [PMID: 12921244 DOI: 10.1592/phco.23.8.979.32881] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
STUDY OBJECTIVES To determine the effects of grapefruit juice on the pharmacokinetics of oral digoxin, a P-glycoprotein substrate not metabolized by cytochrome P450 3A4, in healthy volunteers, and to assess whether polymorphic multidrug-resistance-1 (MDR1) expression contributes to interindividual variability in digoxin disposition. DESIGN Prospective, open-label, unblinded, crossover study. SETTING University research center. SUBJECTS Seven healthy adult volunteers (four men, three women). INTERVENTION Each subject received a single oral dose of digoxin 1.0 mg with water or grapefruit juice with at least a 2-week washout between treatments. During the grapefruit juice phase, juice was administered 3 times/day for 5 days before digoxin administration to maximize any effect on P-glycoprotein. MEASUREMENTS AND MAIN RESULTS Digoxin pharmacokinetics in the presence and absence of grapefruit juice were compared. The MDR1 exon 26 C3435T genotype was determined by real-time polymerase chain reaction. Compared with water, grapefruit juice significantly reduced the digoxin absorption rate constant (3.0 +/- 2.4 to 1.2 +/- 1.0 hr(-1), p<0.05) and increased absorption lag time (0.32 +/- 0.12 to 0.53 +/- 0.34 hr, p<0.05). Grapefruit juice did not affect digoxin maximum concentration (Cmax), area under the curve (AUC), elimination half-life, or renal clearance. The effect of grapefruit juice on digoxin Cmax (-45% to +41%) and AUC(0-4) (-29% to +25%) varied substantially among subjects and was inversely correlated with the values during the water phase. Trends toward higher digoxin Cmax AUC, and absorption rate constant during the water phase were found in CC homozygotes compared with subjects carrying a T allele. CONCLUSION Inhibition of intestinal P-glycoprotein does not appear to play an important role in drug interactions involving grapefruit juice. Interindividual variability in response to grapefruit juice may be related to the balance of intestinal drug uptake and efflux transport.
Collapse
Affiliation(s)
- Robert B Parker
- Department of Pharmacy, University of Tennessee Health Science Center, Memphis 38163, USA
| | | | | | | |
Collapse
|
|
25
|
Corea G, Fattorusso E, Lanzotti V, Taglialatela-Scafati O, Appendino G, Ballero M, Simon PN, Dumontet C, Di Pietro A. Jatrophane diterpenes as P-glycoprotein inhibitors. First insights of structure-activity relationships and discovery of a new, powerful lead. J Med Chem 2003; 46:3395-402. [PMID: 12852769 DOI: 10.1021/jm030787e] [Citation(s) in RCA: 68] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
The Mediterranean spurge Euphorbia dendroides L. afforded a series of 10 closely related jatrophane polyesters, nine of which are new, which served as a base for the establishment of structure-activity relationships within this class of P-glycoprotein inhibitors. The results, while pointing to the general role of lipophilicity for activity, also highlighted the relevance of the substitution pattern at the positions 2, 3, and 5, suggesting the involvement of this fragment in binding. The most powerful compound of the series, euphodendroidin D (4), outperformed cyclosporin by a factor of 2 to inhibit Pgp-mediated daunomycin transport.
Collapse
Affiliation(s)
- Gabriella Corea
- Dipartimento di Chimica delle Sostanze Naturali, Università di Napoli Federico II, Via D. Montesano 49, I-80131 Naples, Italy
| | | | | | | | | | | | | | | | | |
Collapse
|
|
26
|
Abstract
Successful drug delivery will achieve an appropriate drug concentration at the target to elicit a desired level of response. The concentration of circulating free drug is dependent upon the pharmacokinetic processes of absorption, distribution, metabolism and elimination. The response usually results from interaction of a drug with a target protein. The genetic basis that underlies pharmacokinetic and pharmacodynamic interindividual variability is an important consideration in the design of drug delivery systems. Genetic polymorphism has been identified in drug-metabolizing enzymes, transporters and targets. Potentially, these pharmacogenomic factors can affect the concentration of free drug available for delivery and the subsequent elicited response.
Collapse
Affiliation(s)
- Mayssa Attar
- Department of Pharmaceutical Sciences, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA 90089-9121, USA
| | | |
Collapse
|
|
27
|
Roulet A, Puel O, Gesta S, Lepage JF, Drag M, Soll M, Alvinerie M, Pineau T. MDR1-deficient genotype in Collie dogs hypersensitive to the P-glycoprotein substrate ivermectin. Eur J Pharmacol 2003; 460:85-91. [PMID: 12559367 DOI: 10.1016/s0014-2999(02)02955-2] [Citation(s) in RCA: 98] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
Multidrug resistance (MDR) phenotypes in cancer cells are associated with overexpression of the drug carrier P-glycoprotein. The antiparasitic drug ivermectin, one of its substrates, abnormally accumulates in the brain of transgenic mice lacking the P-glycoprotein, resulting in neurotoxicity. Similarly, an enhanced sensitivity to ivermectin has been reported in certain dogs of the Collie breed. To explore the basis of this phenotype, we analyzed the canine P-glycoprotein-encoding MDR1 gene, and we report the first characterization of the cDNA for wild-type (Beagle) P-glycoprotein. The corresponding transcripts from ivermectin-sensitive Collies revealed a homozygous 4-bp exonic deletion. We established, by genetic testings, that the MDR1 frame shift is predictable. Accordingly, no P-glycoprotein was detected in the homozygote-deficient dogs. In conclusion, we characterized a unique case of naturally occurring gene invalidation. This provides a putative novel model that remains to be exploited in the field of human therapeutics and that might significantly affect tissue distribution and drug bioavailability studies.
Collapse
MESH Headings
- ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics
- ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism
- Alleles
- Amino Acid Sequence
- Animals
- Base Sequence
- Blotting, Western
- DNA/chemistry
- DNA/genetics
- DNA, Complementary/chemistry
- DNA, Complementary/genetics
- Dogs
- Drug Resistance, Multiple/genetics
- Female
- Genotype
- Humans
- Ivermectin/metabolism
- Ivermectin/pharmacology
- Male
- Molecular Sequence Data
- Polymerase Chain Reaction
- Sequence Alignment
- Sequence Analysis, DNA
- Sequence Homology, Amino Acid
- Sequence Homology, Nucleic Acid
- Tumor Cells, Cultured
Collapse
Affiliation(s)
- Alain Roulet
- Laboratoire de Pharmacologie et Toxicologie, INRA-180 Chemin de Tournefeuille, BP 3, 31931 Toulouse Cedex 9, France
| | | | | | | | | | | | | | | |
Collapse
|
|
28
|
Song P, Li S, Meibohm B, Gaber AO, Honaker MR, Kotb M, Yates CR. Detection of MDR1 single nucleotide polymorphisms C3435T and G2677T using real-time polymerase chain reaction: MDR1 single nucleotide polymorphism genotyping assay. AAPS PHARMSCI 2002; 4:E29. [PMID: 12646001 PMCID: PMC2751318 DOI: 10.1208/ps040429] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/16/2023]
Abstract
The objective of this study was to develop a real-time polymerase chain reaction (PCR) method to detect MDR1 (human multidrug resistance gene) single nucleotide polymorphisms (SNPs) C3435T and G2677T. C3435T and G2677T are linked to MDR1*2, which is associated with enhanced efflux activity in vitro. Using the Smart Cycler, an allele-specific real-time PCR-based genotyping method was developed to detect C3435T and G2677T. The MDR1 genotype of human genomic DNA templates was determined by direct DNA sequencing. PCR reactions for genotyping C3435T and G2677T by using allele-specific primers were conducted in separate tubes. An additional nucleotide mismatch at the third position from the 3' end of each allele-specific primer was used to abrogate nonspecific PCR amplification. The fluorescence emitted by SYBR Green I was monitored to detect formation of specific PCR products. PCR growth curves exceeding the threshold cycle were considered positive. Fluorescence melt-curve analysis was used to corroborate results from PCR growth curves. Using PCR growth curves, our assay accurately determined hetero- and homozygosity for C3435T and G2677T. Genotype assignments based on PCR growth curve, melt-curve analysis, agarose gel electrophoresis, and direct DNA sequencing results of PCR products were in perfect agreement. We have developed a rapid MDR1 genotyping method that can be used to assess the contribution of MDR1*2 to pharmacokinetic and pharmacodynamic variability of P-glycoprotein substrates.
Collapse
Affiliation(s)
- Pengfei Song
- Department of Pharmaceutical Sciences, University of Tennessee, 38163 Memphis, TN
| | - Shen Li
- Department of Pharmaceutical Sciences, University of Tennessee, 38163 Memphis, TN
| | - Bernd Meibohm
- Department of Pharmaceutical Sciences, University of Tennessee, 38163 Memphis, TN
| | - A. Osama Gaber
- Transplant Division, University of Tennessee, 38163 Memphis, TN
| | | | - Malak Kotb
- Transplant Division, University of Tennessee, 38163 Memphis, TN
| | - Charles R. Yates
- Department of Pharmaceutical Sciences, University of Tennessee, 38163 Memphis, TN
| |
Collapse
|
|
29
|
Garrigues A, Escargueil AE, Orlowski S. The multidrug transporter, P-glycoprotein, actively mediates cholesterol redistribution in the cell membrane. Proc Natl Acad Sci U S A 2002; 99:10347-52. [PMID: 12145328 PMCID: PMC124917 DOI: 10.1073/pnas.162366399] [Citation(s) in RCA: 153] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
P-glycoprotein (P-gp) is a plasma membrane ATP-binding cassette transporter, responsible for multidrug resistance in tumor cells. P-gp catalyzes the ATP hydrolysis-dependent efflux of numerous amphiphilic compounds of unrelated chemical structures. In the absence of any identified substrate, P-gp exhibits an apparently futile, basal ATPase activity. By using native membrane vesicles containing high amounts of P-gp, we show here that (i) this basal ATPase activity is tightly dependent on the presence of cholesterol in the membrane; (ii) the stimulation of P-gp ATPase activity by drugs transported by P-gp is higher in the absence than in the presence of cholesterol and, conversely, the stimulation of P-gp ATPase activity by cholesterol is higher in the absence than in the presence of known P-gp substrates; (iii) P-gp mediates the ATP-dependent relocation of cholesterol from the cytosolic leaflet to the exoplasmic leaflet of the plasma membrane; and (iv) the decrease of the cholesterol dependence of P-gp ATPase activity induced by known P-gp substrates is correlated with the inhibition of the ATP-dependent cholesterol redistribution within the membrane. These data are highly evocative of a coupling between the basal ATPase activity of P-gp and its intramembrane cholesterol-redistribution function, and they are fully consistent with the possibility that P-gp may actively translocate cholesterol in the membrane. Finally, this P-gp-mediated cholesterol redistribution in the cell membrane makes it likely that P-gp contributes in stabilizing the cholesterol-rich microdomains, rafts and caveolae, and that it is involved in the regulation of cholesterol trafficking in cells.
Collapse
Affiliation(s)
- Alexia Garrigues
- Département de Biologie Joliot-Curie/Direction des Sciences du Vivant, Commissariat à l'Energie Atomique, Unité de Recherche Associée 2096 Centre National de la Recherche Scientifique and LRA17V Université Paris-Sud, 91191 Gif-sur-Yvette, France
| | | | | |
Collapse
|
|
30
|
Seegers U, Potschka H, Löscher W. Expression of the multidrug transporter P-glycoprotein in brain capillary endothelial cells and brain parenchyma of amygdala-kindled rats. Epilepsia 2002; 43:675-84. [PMID: 12102668 DOI: 10.1046/j.1528-1157.2002.33101.x] [Citation(s) in RCA: 51] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
PURPOSE Based on data from brain biopsy samples of patients with pharmacoresistant partial epilepsy, overexpression of the multidrug transporter P-glycoprotein (PGP) in brain capillary endothelium has recently been proposed as a potential mechanism of resistance to antiepileptic drugs (AEDs). We examined whether PGP is overexpressed in brain regions of amygdala-kindled rats, a widely used model of temporal lobe epilepsy (TLE), which is often resistant to AEDs. METHODS Rats were kindled by stimulation of the basolateral amygdala (BLA); electrode-implanted but nonkindled rats and naive (not implanted) rats served as controls. PGP was determined by immunohistochemistry either 1 or 2 weeks after the last kindled seizure, by using a monoclonal anti-PGP antibody. Six brain regions were examined ipsi- and contralateral to the BLA electrode: the BLA, the hippocampal formation, the piriform cortex, the substantia nigra, the frontal and parietal cortex, and the cerebellum. RESULTS In both kindled rats and controls, PGP staining was observed mainly in microvessel endothelial cells and, to a much lesser extent, in parenchymal cells. The distribution of PGP expression across brain regions was not homogeneous, but significant differences were found in both the endothelial and parenchymal expression of this protein. In kindled rats, ipsilateral PGP expression tended to be higher than contralateral expression in several brain regions, which was statistically significant in the piriform cortex and parietal cortex. However, compared with controls, no significant overexpression of PGP in capillary endothelial cells or brain parenchyma of kindled rats was seen in any ipsilateral brain region, including the BLA. For comparison with kindled rats, kainate-treated rats were used as positive controls. As reported previously, kainate-induced seizures significantly increased PGP expression in the hippocampus and other limbic brain regions. CONCLUSIONS Amygdala-kindling does not induce any lasting overexpression of PGP in several brain regions previously involved in the kindling process. In view of the many pathophysiologic and pharmacologic similarities between the kindling model and TLE, these data may indicate that PGP overexpression in pharmacoresistant patients with TLE is a result of uncontrolled seizures but not of the processes underlying epilepsy. It remains to be determined whether transient PGP overexpression is present in kindled rats shortly after a seizure, and whether pharmacoresistant subgroups of kindled rats exhibit an increased expression of PGP. Furthermore, other multidrug transporters, such as multidrug resistance-associated protein, might be involved in the resistance of kindled rats to AEDs.
Collapse
MESH Headings
- ATP Binding Cassette Transporter, Subfamily B, Member 1/analysis
- ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism
- Amygdala/metabolism
- Amygdala/physiology
- Animals
- Anticonvulsants/therapeutic use
- Brain/metabolism
- Brain/physiology
- Brain Chemistry
- Dentate Gyrus/chemistry
- Dentate Gyrus/metabolism
- Dentate Gyrus/physiology
- Drug Resistance, Multiple/physiology
- Endothelium, Vascular/chemistry
- Endothelium, Vascular/cytology
- Epilepsies, Partial/drug therapy
- Epilepsies, Partial/metabolism
- Epilepsies, Partial/physiopathology
- Female
- Kindling, Neurologic/metabolism
- Kindling, Neurologic/physiology
- Rats
- Rats, Wistar
Collapse
Affiliation(s)
- Ulrike Seegers
- Department of Pharmacology, Toxicology and Pharmacy, School of Veterinary Medicine, Hanover, Germany
| | | | | |
Collapse
|
|
31
|
Brady JM, Cherrington NJ, Hartley DP, Buist SC, Li N, Klaassen CD. Tissue distribution and chemical induction of multiple drug resistance genes in rats. Drug Metab Dispos 2002; 30:838-44. [PMID: 12065443 DOI: 10.1124/dmd.30.7.838] [Citation(s) in RCA: 123] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
Multiple drug resistance (mdr) genes encode P-glycoprotein, which is responsible for resistance to some cancer chemotherapeutic drugs and efflux of xenobiotics of cells. Thus, mdr can protect organs from xenobiotics. In rats, there are two mdr1 genes capable of xenobiotic transport, mdr1a and mdr1b. The purpose of this study was to determine the tissue distribution of rat mdr1a and mdr1b mRNA and whether microsomal enzyme inducers that increase phase I and II drug-metabolizing enzymes coordinately regulate mdr1a and/or mdr1b. The mRNA levels of mdr1a and mdr1b were determined using branched-DNA signal amplification technology. The highest level of expression of mdr1a mRNA was observed in the gastrointestinal tract, with levels increasing, respectively, from duodenum, jejunum, and ileum to large intestine. Expression levels of mdr1a mRNA in the cerebral cortex, cerebellum, kidney, lung, and liver were less than one-tenth of that in the ileum. The tissue distribution of mdr1b mRNA was similar to mdr1a with highest expression in the gastrointestinal tract but only about 3-fold higher than in most other tissues. The induction of mdr1a and mdr1b mRNA transcripts in liver, kidney, and ileum by treatment of rats with 18 chemicals representing aryl hydrocarbon receptor ligands, constitutive androstane receptor ligands, pregnane X receptor ligands, peroxisome proliferator-activated receptor ligands, electrophile-response-element activators, and CYP4502E1 inducers was assessed. Hepatic, renal, and intestinal expression of mdr1a and mdr1b mRNA were not significantly altered by treatment of rats with any of these classes of ligands. In conclusion, the primary expression of rat mdr1 genes is in the gastrointestinal tract where they are thought to function to decrease the absorption of some xenobiotics. Rat mdr1 gene expression is not readily increased by microsomal enzyme inducers in rats through coordinate mechanisms with phase I and II drug-metabolizing enzymes.
Collapse
Affiliation(s)
- James M Brady
- Deptartment of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas 66160, USA
| | | | | | | | | | | |
Collapse
|
|
32
|
Abstract
In recent years the status of the inflammatory bowel diseases (IBDs) as canonical autoimmune diseases has risen steadily with the recognition that these diseases are, at their crux, abnormalities in mucosal responses to normally harmless antigens in the mucosal microflora and therefore responses to antigens that by their proximity and persistence are equivalent to self-antigens. This new paradigm is in no small measure traceable to the advent of multiple models of mucosal inflammation whose very existence is indicative of the fact that many types of immune imbalance can lead to loss of tolerance for mucosal antigens and thus inflammation centered in the gastrointestinal tract. We analyze the immunology of the IBDs through the lens of the murine models, first by drawing attention to their common features and then by considering individual models at a level of detail necessary to reveal their individual capacities to provide insight into IBD pathogenesis. What emerges is that murine models of mucosal inflammation have given us a road map that allows us to begin to define the immunology of the IBDs in all its complexity and to find unexpected ways to treat these diseases.
Collapse
Affiliation(s)
- Warren Strober
- Mucosal Immunity Section, Laboratory of Clinical Investigation, NIAID, NIH, Bethesda, Maryland 20892-1890, USA.
| | | | | |
Collapse
|
|
33
|
Löscher W, Potschka H. Role of multidrug transporters in pharmacoresistance to antiepileptic drugs. J Pharmacol Exp Ther 2002; 301:7-14. [PMID: 11907151 DOI: 10.1124/jpet.301.1.7] [Citation(s) in RCA: 288] [Impact Index Per Article: 12.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Epilepsy, one of the most common neurologic disorders, is a major public health issue. Despite more than 20 approved antiepileptic drugs (AEDs), about 30% of patients are refractory to treatment. An important characteristic of pharmacoresistant epilepsy is that most patients with refractory epilepsy are resistant to several, if not all, AEDs, even though these drugs act by different mechanisms. This argues against epilepsy-induced alterations in specific drug targets as a major cause of pharmacoresistant epilepsy, but rather points to nonspecific and possibly adaptive mechanisms, such as decreased drug uptake into the brain by intrinsic or acquired over-expression of multidrug transporters in the blood-brain barrier (BBB). There is accumulating evidence demonstrating that multidrug transporters such as P-glycoprotein (PGP) and members of the multidrug resistance-associated protein (MRP) family are over-expressed in capillary endothelial cells and astrocytes in epileptogenic brain tissue surgically resected from patients with medically intractable epilepsy. PGP and MRPs in the BBB are thought to act as an active defense mechanism, restricting the penetration of lipophilic substances into the brain. A large variety of compounds, including many lipophilic drugs, are substrates for either PGP or MRPs or both. It is thus not astonishing that several AEDs, which have been made lipophilic to penetrate into the brain, seem to be substrates for multidrug transporters in the BBB. Over-expression of such transporters in epileptogenic tissue is thus likely to reduce the amount of drug that reaches the epileptic neurons, which would be a likely explanation for pharmacoresistance. PGP and MRPs can be blocked by specific inhibitors, which raises the option to use such inhibitors as adjunctive treatment for medically refractory epilepsy. However, although over-expression of multidrug transporters is a novel and reasonable hypothesis to explain multidrug resistance in epilepsy, further studies are needed to establish this concept. Furthermore, there are certainly other mechanisms of pharmacoresistance that need to be identified.
Collapse
Affiliation(s)
- Wolfgang Löscher
- Department of Pharmacology, Toxicology, and Pharmacy, School of Veterinary Medicine, Hannover, Germany.
| | | |
Collapse
|
|
34
|
Schipper NG, Osterberg T, Wrange U, Westberg C, Sokolowski A, Rai R, Young W, Sjöström B. In vitro intestinal permeability of factor Xa inhibitors: influence of chemical structure on passive transport and susceptibility to efflux. Pharm Res 2001; 18:1735-41. [PMID: 11785694 DOI: 10.1023/a:1013378731183] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
PURPOSE To study the in vitro intestinal permeability of a number of newly synthesised factor Xa inhibitors to better understand the poor oral absorption of these compounds. METHODS The bidirectional transport of the fXa inhibitors was studied in the Caco-2 cell model and isolated rat ileal tissue. An attempt was made to characterize efflux mechanisms with the help of commonly used substrates and inhibitors of various transport proteins. In addition, the transport of the fXa inhibitors was studied in MDCK cells transfected with the human MDR1 gene and expressing large amounts of P-glycoprotein (Pgp). RESULTS The in vitro absorptive permeability was low for all but one of the fXa inhibitors. For compounds with non-substituted amidine, a charge (due to ionisation at neutral pH) may have resulted in poor membrane partitioning. Neutral compounds with substituted amidines were effluxed from the epithelial cells. The significance of the secretion process was illustrated by the results obtained for a neutral analogue showing high absorptive Caco-2 cell permeability that was not obviated by efflux. Transport inhibition studies in Caco-2 and permeability studies in the MDR1-transfected MDCK cells consistently showed that Pgp is not involved in the secretion of fXa inhibitors. Besides efflux, metabolic liability limited the permeation of the neutral lipophilic analogues with a carbamate ester. CONCLUSIONS Poor intestinal permeability may be an important factor in the incomplete oral absorption of the bisbenzimidazole-type fXa inhibitors. Poor permeability may be related to poor membrane partitioning for hydrophilic analogues, whereas susceptibility to efflux transports and gastro-intestinal enzymatic degradation may limit the permeability of some of the neutral less hydrophilic derivatives.
Collapse
Affiliation(s)
- N G Schipper
- Pharmacia Corporation, Division Biovitrum, Stockholm, Sweden
| | | | | | | | | | | | | | | |
Collapse
|
|
35
|
Wagner D, Spahn-Langguth H, Hanafy A, Koggel A, Langguth P. Intestinal drug efflux: formulation and food effects. Adv Drug Deliv Rev 2001; 50 Suppl 1:S13-31. [PMID: 11576693 DOI: 10.1016/s0169-409x(01)00183-1] [Citation(s) in RCA: 73] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]
Abstract
The intestine, primarily regarded as an absorptive organ, is also prepared for the elimination of certain organic acids, bases and neutral compounds depending on their affinity to intestinal carrier systems. Several of the transport systems known to mediate efflux in the major clearing organs--liver and kidney--are also expressed in the intestine. Examples of secretory transporters in the intestine are P-glycoprotein, members of the multidrug resistance associated protein family, breast cancer resistance protein, organic cation transporters and members of the organic anion polypeptide family. In this communication, the P-glycoprotein mediated intestinal secretion of talinolol, a model compound showing metabolic stability, has been investigated in the jejunum, ileum and colon of rat intestine by single-pass perfusion. A model has been developed which demonstrates an increase in carrier-mediated secretion in the order jejunum<ileum<colon. Furthermore, the potency of common excipients in peroral drug products towards inhibition of P-gp mediated secretion has been investigated using a radioligand-binding assay and transport studies in Caco-2 cell monolayers. Finally, evidence is provided which demonstrates that constituents of grapefruit juice not only may influence intestinal drug metabolism, but can also interfere with secretory transport systems, leading to a new and yet undescribed mechanism in drug-food interactions.
Collapse
Affiliation(s)
- D Wagner
- Department of Biopharmaceutics and Pharmaceutical Technology, School of Pharmacy, Johannes Gutenberg-University, Staudingerweg 5, 55099 Mainz, Germany
| | | | | | | | | |
Collapse
|
|
36
|
Abstract
Mobilization of dendritic cells into lymphatic vessels requires cytokine stimulation and induction of the chemokine receptor CCR7. The respective roles of the CCR7 ligands CCL19 and CCL21 in mediating migration are not fully defined, but chemotaxis to CCL19 mediates Langerhans cell exit from the epidermis. Optimal chemotaxis to CCL19 occurs when DCs are triggered with exogenous leukotriene C(4), an eicosanoid transported out of the cell via the ATP binding cassette (ABC) transporter multidrug resistance related protein 1 (MRP1, ABCC1). Indeed, MRP1 and the related multidrug resistance protein 1 (MDR1, p-glycoprotein, ABCB1) may control the intracellular and extracellular accumulation of key signaling lipids that regulate dendritic cell migration.
Collapse
Affiliation(s)
- G J Randolph
- Institute for Gene Therapy and Molecular Medicine, Mt. Sinai School of Medicine, 1425 Madison Avenue, Box 1496, New York, NY 10029, USA.
| |
Collapse
|
|
37
|
Potschka H, Löscher W. In vivo evidence for P-glycoprotein-mediated transport of phenytoin at the blood-brain barrier of rats. Epilepsia 2001; 42:1231-40. [PMID: 11737157 DOI: 10.1046/j.1528-1157.2001.01901.x] [Citation(s) in RCA: 149] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
PURPOSE The multidrug transporter P-glycoprotein (P-gp) is expressed at high levels in a variety of tissues such as the endothelial cells of the blood-brain barrier (BBB) capillaries, where it is thought to be involved in the exclusion of various drugs from the capillary endothelial cells, blocking their entry into brain. It was previously shown that pharmacoresistant partial epilepsy is associated with an increased expression of P-gp in brain capillary endothelium and astrocytes, leading to the hypothesis that increased P-gp expression may be involved in medically intractable epilepsy. However, it is not known whether the distribution of antiepileptic drugs (AEDs) into the brain is limited by P-gp. We used in vivo microdialysis in freely moving rats to study whether the concentration of the major AED phenytoin (PHT) in the extra-cellular fluid (ECF) of the cerebral cortex can be enhanced by inhibition of P-gp. METHODS Three different P-gp inhibitors, sodium cyanide, verapamil, and PSC 833, were used. These drugs were given via the microdialysis probe in the right frontal cortex, while a probe in the left cortex served as vehicle control side. Perfusion with the inhibitor started 15-60 min before systemic (i.p.) administration of PHT, 50 mg/kg. RESULTS PHT rapidly entered the brain ECF compartment, but ECF plasma ratios at time of maximal ECF levels were only approximately 0.04. All P-gp inhibitors significantly increased the ECF concentrations of PHT after local administration, indicating that P-gp in the BBB normally limits the distribution of PHT into the brain parenchyma. Cremorphor EL, the vehicle used to administer PSC, also was able to increase ECF PHT, which is explained by the previously reported inhibitory effect of cremophor on P-gp. CONCLUSIONS Provided that multidrug transporters such as P-gp also are involved in the BBB outward transport of other AEDs, increased expression of multidrug transporters, leading to inadequate accumulation of AEDs in the brain, would be a likely explanation for pharmacoresistant epilepsy.
Collapse
Affiliation(s)
- H Potschka
- Department of Pharmacology, Toxicology, and Pharmacy, School of Veterinary Medicine, Hannover, Germany
| | | |
Collapse
|
|
38
|
Carlsson C, Pärt P. 7-Ethoxyresorufin O-deethylase induction in rainbow trout gill epithelium cultured on permeable supports: asymmetrical distribution of substrate metabolites. AQUATIC TOXICOLOGY (AMSTERDAM, NETHERLANDS) 2001; 54:29-38. [PMID: 11451423 DOI: 10.1016/s0166-445x(00)00184-3] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/23/2023]
Abstract
The induction of 7-ethoxyresorufin O-deethylase (EROD) has been measured in cultured epithelia from rainbow trout gills. Epithelia incubated with water on the apical side and culture media at the basolateral side were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), beta-naphthoflavone (betaNF), benzo[k]fluoranthene (B(k)F), and 3,3',4,4',5-pentachlorobiphenyl (PCB#126) from the water. EROD activity was measured as the formation of resorufin from 7-ethoxyresorufin over time in intact epithelia. The EC(50) values obtained after 24 h of exposure (mean+/-S.D.) were for TCDD (n=9) 4.1+/-3.2x10(-11) M, for betaNF (n=6) 1.6+/-3.8x10(-9) M, for B(k)F (n=4) 5.4+/-3.0x10(-9) M and for PCB#126 (n=4) 6.15+/-10.1x10(-9) M. When assaying for EROD activity, it was found that the resorufin concentrations differed between the apical and the basolateral compartments, indicating an asymmetrical distribution of the enzymatically formed resorufin molecules. Generally, the resorufin concentration was highest in the basolateral compartment, but there were differences between epithelia obtained from different fish individuals. Of a total of 13 preparations 10 had the highest resorufin concentration in the basolateral compartment, while in three preparations, the resorufin was uniformly distributed or slightly higher in the apical compartment. The reasons for this asymmetrical distribution of substrate metabolites are not known, and the addition of multidrug resistance inhibitors (verapamil and cyclosporin A) did not alter the asymmetrical pattern. The transepithelial electrical resistance (TER) was also measured to diagnose the tightness of the epithelia. The change from culture media to experimental water (containing TCDD, betaNF, or DMSO as control) in the apical compartment resulted in a large increase in TER, followed by a decline, measured after 24 h. The cytochrome P450 1A (CYP1A) inducers had no effect on the TER and were judged, therefore, not to affect the tightness of the epithelia.
Collapse
Affiliation(s)
- C Carlsson
- Department of Environmental Toxicology, Uppsala University, Norbyvägen 18 A, S-752 36 Uppsala, Sweden.
| | | |
Collapse
|
|
39
|
Song IS, Chung SJ, Shim CK. Contribution of ion pair complexation with bile salts to biliary excretion of organic cations in rats. Am J Physiol Gastrointest Liver Physiol 2001; 281:G515-25. [PMID: 11447032 DOI: 10.1152/ajpgi.2001.281.2.g515] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
The objective of this study was to examine whether ion pair complexation with endogenous bile salts in hepatocytes contributes to the preferential biliary excretion of organic cations (OCs). Tributylmethylammonium (TBuMA; mol wt 200) and triethylmethylammonium (TEMA; mol wt 116) were selected as model OCs that exhibit significant and negligible biliary excretion, respectively, in rats. The apparent lipophilicity of TBuMA, but not that of TEMA, was increased by the presence of either rat bile or specific bile salts, suggesting the formation of lipophilic ion pair complexes for TBuMA with bile salts in the liver. The uptake of TBuMA into canalicular liver plasma membrane (cLPM) vesicles, but not that of TEMA, was increased in the presence of bile salts, with a significant increase for both ATP-dependent transport and passive diffusion. The uptake of TBuMA in the presence of the bile salts was inhibited by representative P-glycoprotein (P-gp) substrates and vice versa, suggesting the involvement of P-gp in the canalicular excretion of TBuMA-bile salt complexes in vivo. Increased affinity toward P-gp is suggested as the mechanism responsible for the increased ATP-dependent transport for the ion pair complexes. We propose that ion pair formation with bile slats in hepatocytes may be responsible for the preferential biliary excretion of high-molecular-weight OCs including TBuMA.
Collapse
Affiliation(s)
- I S Song
- Department of Pharmaceutics, College of Pharmacy, Seoul National University, Seoul 151-742, Korea
| | | | | |
Collapse
|
|
40
|
Comte G, Daskiewicz JB, Bayet C, Conseil G, Viornery-Vanier A, Dumontet C, Di Pietro A, Barron D. C-Isoprenylation of flavonoids enhances binding affinity toward P-glycoprotein and modulation of cancer cell chemoresistance. J Med Chem 2001; 44:763-8. [PMID: 11262086 DOI: 10.1021/jm991128y] [Citation(s) in RCA: 89] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Previous studies have shown that flavones bind to P-glycoprotein (Pgp) with higher affinity than isoflavones, flavanones, and glycosylated derivatives. In the present work, a series of C- or O-substituted hydrophobic derivatives of chrysin were synthesized to further investigate structural requirements of the A ring toward Pgp modulation. Increasing hydrophobicity at either position 6, 8, or 7 increased the affinity of in vitro binding to a purified cytosolic domain of Pgp, but only benzyl and 3,3-dimethylallyl C-substitution produced a high maximal quenching of the protein intrinsic fluorescence. Inhibition of membrane Pgp within leukemic cells, characterized by intracellular drug accumulation, was specifically produced by isoprenylated derivatives, with 8-(3,3-dimethylallyl)chrysin being even more efficient than the commonly used cyclosporin A.
Collapse
Affiliation(s)
- G Comte
- Laboratoire des Produits Naturels, CNRS-UMR 5013, UFR de Chimie-Biochimie, Université Claude Bernard Lyon 1, 43 Boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, France
| | | | | | | | | | | | | | | |
Collapse
|
|
41
|
Lawrence DS, Copper JE, Smith CD. Structure-activity studies of substituted quinoxalinones as multiple-drug-resistance antagonists. J Med Chem 2001; 44:594-601. [PMID: 11170649 DOI: 10.1021/jm000282d] [Citation(s) in RCA: 116] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
A significant problem in the clinical treatment of cancer relates to the development of tumor resistance to many chemotherapeutic agents. Acquired drug resistance is often mediated through overexpression of membrane transport proteins that effectively efflux anticancer agents. Two of the best-studied transporters, P-glycoprotein (Pgp) and MRP1, have pharmacological properties that only partially overlap. In our search for improved drug-resistance antagonists, we have identified a family of substituted quinoxalines that selectively antagonizes Pgp over MRP1. Consequently, a focused library of congeners was designed and synthesized starting with a parent bromomethylquinoxalinone. This parent quinoxalinone was then condensed with a series of phenols to yield a family of substituted phenoxymethylquinoxalinones. These compounds were evaluated for their toxicity toward drug-sensitive MCF-7 breast carcinoma cells and for their abilities to antagonize Pgp and MRP1 in drug-resistant cell lines (NCI/ADR and MCF-7/VP, respectively). The results of this structure-activity study indicate that compounds with carbonyl substitutions of the phenoxy group (ester, amide, or ketone moieties) demonstrate excellent antagonism of Pgp while having relatively low toxicity toward drug-sensitive cells. Importantly, none of these compounds antagonized MRP1. Because of their transporter selectivity, we predict that substituted quinoxalinones may be more effective MDR modulators in vivo than are nonselective transporter antagonists.
Collapse
Affiliation(s)
- D S Lawrence
- Department of Pharmacology, Pennsylvania State University, Hershey, Pennsylvania 17033, USA
| | | | | |
Collapse
|
|
42
|
Robbiani DF, Finch RA, Jäger D, Muller WA, Sartorelli AC, Randolph GJ. The leukotriene C(4) transporter MRP1 regulates CCL19 (MIP-3beta, ELC)-dependent mobilization of dendritic cells to lymph nodes. Cell 2000; 103:757-68. [PMID: 11114332 DOI: 10.1016/s0092-8674(00)00179-3] [Citation(s) in RCA: 367] [Impact Index Per Article: 14.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/26/2022]
Abstract
Adaptive immune responses begin after antigen-bearing dendritic cells (DCs) traffic from peripheral tissues to lymph nodes. Here, we show that DC migration from skin to lymph nodes utilizes the leukotriene C(4) (LTC(4)) transporter multidrug resistance-associated protein 1 (MRP1). DC mobilization from the epidermis and trafficking into lymphatic vessels was greatly reduced in MRP1(-/-) mice, but migration was restored by exogenous cysteinyl leukotrienes LTC(4) or LTD(4). In vitro, these cysteinyl leukotrienes promoted optimal chemotaxis to the chemokine CCL19, but not to other related chemokines. Antagonism of CCL19 in vivo prevented DC migration out of the epidermis. Thus, MRP-1 regulates DC migration to lymph nodes, apparently by transporting LTC(4), which in turn promotes chemotaxis to CCL19 and mobilization of DCs from the epidermis.
Collapse
Affiliation(s)
- D F Robbiani
- Department of Pathology and Immunology Program Weill Medical College and Graduate School of Medical Sciences of Cornell University, New York, NY 10029, USA
| | | | | | | | | | | |
Collapse
|
|
43
|
Abstract
PURPOSE Previous studies have suggested that P-glycoprotein (P-gp) modulates opioid antinociception for selected mu-and delta-agonists. This study was undertaken to assess morphine antinociception in mice lacking the mdr1a gene for expression of P-gp in the CNS. METHODS Morphine (n = 4-5/group) was administered as a single s.c. dose to mdr1a(-/-) mice (3-5 mg/kg) or wild-type FVB controls (8-10 mg/kg). Tail-flick response to radiant heat, expressed as percent of maximum response (%MPR), was used to determine the antinociceptive effect of morphine. Concentrations in serum, brain tissue, and spinal cord samples obtained immediately after the tail-flick test were determined by HPLC with fluorescence detection. Parallel experiments with R(+)-verapamil, a chemical inhibitor of P-gp, also were performed to further investigate the effect of P-gp on morphine-associated antinociception. RESULTS Morphine-associated antinociception was increased significantly in the mdr1a(-/-) mice. The ED50 for morphine was > 2-fold lower in mdr1a(-/-) (3.8+/-0.2 mg/kg) compared to FVB (8.8+/-0.2 mg/kg) mice. However, the EC50 derived from the brain tissue was similar between the two mouse strains (295 ng/g vs. 371 ng/g). Pretreatment with R(+)-verapamil produced changes similar to those observed in gene-deficient mice. P-gp does not appear to affect morphine distribution between spinal cord and blood, as the spinal cord:serum morphine concentration ratio was similar between gene-deficient and wild-type mice (0.47+/-0.03 vs. 0.56+/-0.04, p>0.05). CONCLUSIONS The results of this study are consistent with the hypothesis that P-gp attenuates the antinociceptive action of morphine by limiting the brain:blood partitioning of the opioid.
Collapse
Affiliation(s)
- J Zong
- Division of Drug Delivery and Disposition, School of Pharmacy, University of North Carolina at Chapel Hill 27599-7360, USA
| | | |
Collapse
|
|
44
|
Litman T, Brangi M, Hudson E, Fetsch P, Abati A, Ross DD, Miyake K, Resau JH, Bates SE. The multidrug-resistant phenotype associated with overexpression of the new ABC half-transporter, MXR (ABCG2). J Cell Sci 2000; 113 ( Pt 11):2011-21. [PMID: 10806112 DOI: 10.1242/jcs.113.11.2011] [Citation(s) in RCA: 348] [Impact Index Per Article: 13.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Mechanisms of drug resistance other than P-glycoprotein are of increasing interest as the list of newly identified members of the ABC transport family has grown. We sought to characterize the phenotype of the newly discovered ABC transporter encoded by the mitoxantrone resistance gene, MXR, also known as ABCP1 or BCRP. The pharmacodynamics of mitoxantrone and 12 other fluorescent drugs were evaluated by confocal microscopy in four multidrug-resistant human colon (S1) and breast (MCF-7) cancer cell lines. We utilized two sublines, MCF-7 AdVp3000 and S1-M1-80, and detected overexpression of MXR by PCR, immunoblot assay and immunohistochemistry. These MXR overexpressing sublines were compared to cell lines with P-glycoprotein- and MRP-mediated resistance. High levels of cross-resistance were observed for mitoxantrone, the anthracyclines, bisantrene and topotecan. Reduced levels of mitoxantrone, daunorubicin, bisantrene, topotecan, rhodamine 123 and prazosin were observed in the two sublines with high MXR expression. Neither the P-glycoprotein substrates vinblastine, paclitaxel, verapamil and calcein-AM, nor the MRP substrate calcein, were extruded from MCF-7 AdVp3000 and S1-M1-80 cells. Thus, the multidrug-resistant phenotype due to MXR expression is overlapping with, but distinct from, that due to P-glycoprotein. Further, cells that overexpress the MXR protein seem to be more resistant to mitoxantrone and topotecan than cells with P-glycoprotein-mediated multidrug resistance. Our studies suggest that the ABC half-transporter, MXR, is a potent, new mechanism for conferring multiple drug resistance. Definition of its mechanism of transport and its role in clinical oncology is required.
Collapse
Affiliation(s)
- T Litman
- National Cancer Institute, Medicine Branch, National Institutes of Health, Bethesda, MD 20892, USA
| | | | | | | | | | | | | | | | | |
Collapse
|
|
45
|
Ferté J. Analysis of the tangled relationships between P-glycoprotein-mediated multidrug resistance and the lipid phase of the cell membrane. EUROPEAN JOURNAL OF BIOCHEMISTRY 2000; 267:277-94. [PMID: 10632698 DOI: 10.1046/j.1432-1327.2000.01046.x] [Citation(s) in RCA: 151] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
P-glycoprotein (Pgp), the so-called multidrug transporter, is a plasma membrane glycoprotein often involved in the resistance of cancer cells towards multiple anticancer agents in the multidrug-resistant (MDR) phenotype. It has long been recognized that the lipid phase of the plasma membrane plays an important role with respect to multidrug resistance and Pgp because: the compounds involved in the MDR phenotype are hydrophobic and diffuse passively through the membrane; Pgp domains involved in drug binding are located within the putative transmembrane segments; Pgp activity is highly sensitive to its lipid environment; and Pgp may be involved in lipid trafficking and metabolism. Unraveling the different roles played by the membrane lipid phase in MDR is relevant, not only to the evaluation of the precise role of Pgp, but also to the understanding of the mechanism of action and function of Pgp. With this aim, I review the data from different fields (cancer research, medicinal chemistry, membrane biophysics, pharmaceutical research) concerning drug-membrane, as well as Pgp-membrane, interactions. It is emphasized that the lipid phase of the membrane cannot be overlooked while investigating the MDR phenotype. Taking into account these aspects should be useful in the search of ways to obviate MDR and could also be relevant to the study of other multidrug transporters.
Collapse
Affiliation(s)
- J Ferté
- Service de Biophysique des Protéines et des Membranes, DSV-DBCM-SBPM, CEA, Centre de Saclay, Gif-sur-Yvette, France.
| |
Collapse
|
|
46
|
Sharom FJ, Yu X, Lu P, Liu R, Chu JW, Szabó K, Müller M, Hose CD, Monks A, Váradi A, Seprôdi J, Sarkadi B. Interaction of the P-glycoprotein multidrug transporter (MDR1) with high affinity peptide chemosensitizers in isolated membranes, reconstituted systems, and intact cells. Biochem Pharmacol 1999; 58:571-86. [PMID: 10413294 DOI: 10.1016/s0006-2952(99)00139-2] [Citation(s) in RCA: 89] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
P-glycoprotein-mediated multidrug resistance can be reversed by the action of a group of compounds known as chemosensitizers. The interactions with P-glycoprotein of two novel hydrophobic peptide chemosensitizers (reversins 121 and 205) have been studied in model systems in vitro, and in a variety of MDR1-expressing intact tumor cells. The reversins bound to purified P-glycoprotein with high affinity (77-154 nM), as assessed by a quenching assay using fluorescently labeled purified protein. The peptides modulated P-glycoprotein ATPase activity in Sf9 insect cell membranes expressing human MDR1, plasma membrane vesicles from multidrug-resistant cells, and reconstituted proteoliposomes. Both peptides induced a large stimulation of ATPase activity; however, higher concentrations, especially of reversin 205, led to inhibition. This pattern was different from that of simple linear peptides, and resembled that of chemosensitizers such as verapamil. In both membrane vesicles and reconstituted proteoliposomes, 1-2 microM reversins were more effective than cyclosporin A at blocking colchicine transport. Reversin 121 and reversin 205 restored the uptake of [3H]daunorubicin and rhodamine 123 in MDR1-expressing cells to the level observed in the drug-sensitive parent cell lines, and also effectively inhibited the extrusion of calcein acetoxymethyl ester from intact cells. In cytotoxicity assays, reversin 121 and reversin 205 eliminated the resistance of MDR1-expressing tumor cells against MDR1-substrate anticancer drugs, and they had no toxic effects in MDR1-negative control cells. We suggest that peptides of the reversin type interact with the MDR1 protein with high affinity and specificity, and thus they may be good candidates for the development of MDR1-modulating agents to sensitize drug resistance in cancer.
Collapse
Affiliation(s)
- F J Sharom
- Guelph-Waterloo Centre for Graduate Work in Chemistry and Biochemistry, Department of Chemistry and Biochemistry, University of Guelph, Ontario, Canada.
| | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
47
|
Di Pietro A, Dayan G, Conseil G, Steinfels E, Krell T, Trompier D, Baubichon-Cortay H, Jault J. P-glycoprotein-mediated resistance to chemotherapy in cancer cells: using recombinant cytosolic domains to establish structure-function relationships. Braz J Med Biol Res 1999; 32:925-39. [PMID: 10454753 DOI: 10.1590/s0100-879x1999000800001] [Citation(s) in RCA: 41] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Resistance to chemotherapy in cancer cells is mainly mediated by overexpression of P-glycoprotein (Pgp), a plasma membrane ATP-binding cassette (ABC) transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis. Pgp consists of two homologous halves each containing a transmembrane domain and a cytosolic nucleotide-binding domain (NBD) which contains two consensus Walker motifs, A and B, involved in ATP binding and hydrolysis. The protein also contains an S signature characteristic of ABC transporters. The molecular mechanism of Pgp-mediated drug transport is not known. Since the transporter has an extraordinarily broad substrate specificity, its cellular function has been described as a "hydrophobic vacuum cleaner". The limited knowledge about the mechanism of Pgp, partly due to the lack of a high-resolution structure, is well reflected in the failure to efficiently inhibit its activity in cancer cells and thus to reverse multidrug resistance (MDR). In contrast to the difficulties encountered when studying the full-length Pgp, the recombinant NBDs can be obtained in large amounts as soluble proteins. The biochemical and biophysical characterization of recombinant NBDs is shown here to provide a suitable alternative route to establish structure-function relationships. NBDs were shown to bind ATP and analogues as well as potent modulators of MDR, such as hydrophobic steroids, at a region close to the ATP site. Interestingly, flavonoids also bind to NBDs with high affinity. Their binding site partly overlaps both the ATP-binding site and the steroid-interacting region. Therefore flavonoids constitute a new promising class of bifunctional modulators of Pgp.
Collapse
Affiliation(s)
- A Di Pietro
- Laboratoire de Biochimie Structurale et Fonctionnelle, Institut de Biologie et Chimie des Protéines, Lyon, France.
| | | | | | | | | | | | | | | |
Collapse
|
|
48
|
Morris AP. The regulation of epithelial cell cAMP- and calcium-dependent chloride channels. ADVANCES IN PHARMACOLOGY 1999; 46:209-51. [PMID: 10332504 DOI: 10.1016/s1054-3589(08)60472-x] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/13/2023]
Abstract
This chapter has focused on two types of chloride conductance found in epithelial cells. The leap from the Ussing chamber to patch-clamp studies has identified yet other conductances present which have also been electrophysiologically characterized. In the case of the swelling activated wholecell chloride current, a physiological function is apparent and a single-channel basis found, but its genetic identity remains unknown (see reviews by Frizzell and Morris, 1994; and Strange et al., 1996). The outwardly rectified chloride channel has been the subject of considerable electrophysiological interest over the past 10 years and is well characterized at the single-channel level, but its physiological function remains controversial (reviewed by Frizzell and Morris, 1994; Devidas and Guggino, 1997). Yet other conductances related to the CLC gene family also appear to be present in epithelial cells of the kidney (reviewed by Jentsch, 1996; Jentsch and Gunter, 1997) where physiological functions for some isoforms are emerging. Clearly, there remain many unknowns. Chief among these is the molecular basis of GCa2+Cl and many of other the conductances. As sequences become available it is expected that the wealth of information gained by investigation into CFTR function will provide a conceptual blueprint for similar studies in these later channel clones.
Collapse
Affiliation(s)
- A P Morris
- Department of Integrative Biology, University of Texas-Houston Health Science Center 77030, USA
| |
Collapse
|
|
49
|
Romsicki Y, Sharom FJ. The membrane lipid environment modulates drug interactions with the P-glycoprotein multidrug transporter. Biochemistry 1999; 38:6887-96. [PMID: 10346910 DOI: 10.1021/bi990064q] [Citation(s) in RCA: 175] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
The P-glycoprotein multidrug transporter functions as an ATP-driven efflux pump for a large number of structurally unrelated hydrophobic compounds. Substrates are believed to gain access to the transporter after partitioning into the membrane, rather than from the extracellular aqueous phase. The binding of drug substrates to P-glycoprotein may thus be modulated by the properties of the lipid bilayer. The interactions with P-glycoprotein of two drugs (vinblastine and daunorubicin) and a chemosensitizer (verapamil) were characterized by quenching of purified fluorescently labeled protein in the presence of various phospholipids. Biphasic quench curves were observed for vinblastine and verapamil, suggesting that more than one molecule of these compounds may bind to the transporter simultaneously. All three drugs bound to P-glycoprotein with substantially higher affinity in egg phosphatidylcholine (PC), compared to brain phosphatidylserine (PS) and egg phosphatidylethanolamine (PE). The nature of the lipid acyl chains also modulated binding, with affinity decreasing in the order egg PC > dimyristoyl-PC (DMPC) > dipalmitoyl-PC (DPPC). Following reconstitution of the transporter into DMPC, all three compounds bound to P-glycoprotein with 2-4-fold higher affinity in gel phase lipid relative to liquid-crystalline phase lipid. The P-glycoprotein ATPase stimulation/inhibition profiles for the drugs were also altered in different lipids, in a manner consistent with the observed changes in binding affinity. The ability of the drugs to partition into bilayers of phosphatidylcholines was determined. All of the drugs partitioned much better into egg PC relative to DMPC and DPPC. The binding affinity increased (i.e., the value of Kd decreased) as the drug-lipid partition coefficient increased, supporting the proposal that the effective concentration of the drug substrate in the membrane is important for interaction with the transporter. These results provide support for the vacuum cleaner model of P-glycoprotein action.
Collapse
Affiliation(s)
- Y Romsicki
- Guelph-Waterloo Centre for Graduate Work in Chemistry and Biochemistry, Department of Chemistry, University of Guelph, Ontario, Canada
| | | |
Collapse
|
|
50
|
Hayes JH, Soroka CJ, Rios-Velez L, Boyer JL. Hepatic sequestration and modulation of the canalicular transport of the organic cation, daunorubicin, in the Rat. Hepatology 1999; 29:483-93. [PMID: 9918926 DOI: 10.1002/hep.510290216] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/07/2022]
Abstract
In contrast to organic anions, substrates for the canalicular mdr1a and b are usually organic cations and are often sequestered in high concentrations in intracellular acidic compartments. Because many of these compounds are therapeutic agents, we investigated if their sequestration could be regulated. We used isolated perfused rat liver (IPRL), isolated rat hepatocyte couplets (IRHC), and WIF-B cells to study the cellular localization and biliary excretion of the fluorescent cation, daunorubicin (DNR). Despite rapid (within 15 minutes) and efficient (>90%) cellular uptake in the IPRL, only approximately 10% of the dose administered (0.2-20 micromol) was excreted in bile after 85 minutes. Confocal microscopy revealed fluorescence predominantly in vesicles in the pericanalicular region in IPRL, IRHC, and WIF-B cells. Treatment of these cells with chloroquine and bafilomycin A, agents that disrupt the pH gradient across the vesicular membrane, resulted in a loss of vesicular fluorescence, reversible in the case of bafilomycin A. Taurocholate (TC) and dibutyryl cAMP (DBcAMP), stimulators of transcytotic vesicular transport, increased the biliary recovery of DNR significantly above controls, by 70% and 35%, respectively. The microtubule destabilizer, nocodazole, decreased biliary excretion of DNR. No effect on secretion was noted in TR- mutant rats deficient in mrp2. Coadministration of verapamil, an inhibitor of mdr1, also decreased DNR excretion. While TC and DBcAMP did not affect the fluorescent intensity or pattern of distribution in IRHC, nocodazole resulted in redistribution of DNR to peripheral punctuate structures. These findings suggest that the organic cation, DNR, is largely sequestered in cells such as hepatocytes, yet its excretion can still be modulated.
Collapse
Affiliation(s)
- J H Hayes
- Department of Medicine and Liver Center, Yale University School of Medicine, New Haven, CT, USA
| | | | | | | |
Collapse
|