RESUMO
The (6-maleimidocaproyl)hydrazone derivative of doxorubicin (DOXO-EMCH) is an albumin-binding prodrug of doxorubicin with acid-sensitive properties that demonstrates superior antitumor efficacy in murine tumor models, and has been evaluated in a phase I study. In order to establish the toxicity profile of this prodrug, acute and repeat-dose toxicity studies were performed with DOXO-EMCH in CD1-mice, Sprague-Dawley rats and Beagle dogs. Although the objective of the acute toxicity studies was not the determination of LD50 values, the LD50 of DOXO-EMCH was >60 mg/kg doxorubicin equivalents in both male and female mice (the LD50 of doxorubicin in CD-1 mice is -12 mg/kg). In Sprague-Dawley rats, the LD50 was 23.4 and 45.9 mg/kg doxorubicin equivalents for males and females, respectively. For comparison, the LD50 of doxorubicin in Sprague-Dawley rats is -10.5 mg/kg. The major clinical sign noted following intravenous administration of DOXO-EMCH in mice and rats was a dose-dependent peripheral neuropathy which, in general, developed as a delayed toxicity 1-3 weeks after application. The observed neurotoxicity has been well documented for Sprague-Dawley rats treated with doxorubicin at a dose of 5 and 10 mg/kg. In Beagle dogs, LD10 was not reached for DOXO-EMCH at 4.5 mg/kg doxorubicin equivalents. A four-cycle intravenous study with DOXO-EMCH at dose levels of 4 x 2.5, 5.0 or 7.5 mg/kg doxorubicin equivalents in rats revealed approximately three-fold less side effects on the hemolymphoreticular system when compared to 4 x 2.5 mg/kg doxorubicin dose, whereas effects on the testes/oligospermia seem to be comparable between both drugs at equitoxic dose. A No Observable Adverse Effect Level (NOAEL) for DOXO-EMCH of 4 x 2.5 mg/kg doxorubicin equivalents was established in this study. This dose is equivalent to the maximum tolerated dose (MTD) of doxorubicin in rats. In a two-cycle study over a period of 6 weeks in Beagle dogs (intravenous administration of DOXO-EMCH at dose levels of 1.5, 3.0 or 4.5 mg/kg doxorubicin equivalents), dose-related systemic histamine-like reactions within the first 3 hours after injection were noted in all treated groups. Only transient and temporary effects on hematology, urinary function, as well as on histopathology in mid- and/or high-dose animals, were observed. The low dose of 2 x 1.5 mg/kg was considered to be the NOAEL in this study, which is equivalent to twice the MTD o f doxorubicin i nBeagle dogs. In summary, the toxicity studies with DOXO-EMCH in mice, rats or dogs have not identified any other special toxicity when compared to the toxicity data for doxorubicin. Preclinical tolerance of DOXO-EMCH was higher in mice, rats and dogs compared to doxorubicin. A dose of 20 mg/m2 doxorubicin equivalents was recommended as the starting dose for a phase I study with DOXO-EMCH.
Assuntos
Antibióticos Antineoplásicos/toxicidade , Doxorrubicina/análogos & derivados , Hidrazonas/toxicidade , Pró-Fármacos/toxicidade , Albumina Sérica/metabolismo , Testes de Toxicidade Aguda , Testes de Toxicidade Crônica , Animais , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/metabolismo , Cães , Doxorrubicina/administração & dosagem , Doxorrubicina/metabolismo , Doxorrubicina/toxicidade , Feminino , Hidrazonas/administração & dosagem , Hidrazonas/metabolismo , Dose Letal Mediana , Masculino , Camundongos , Camundongos Endogâmicos , Pró-Fármacos/administração & dosagem , Pró-Fármacos/metabolismo , Ligação Proteica , Ratos , Ratos Sprague-DawleyRESUMO
The multidrug resistance protein 1 (MRP1) belonging to the ATP-binding cassette (ABC) superfamily of transport proteins can confer resistance to multiple natural product drugs and methotrexate in human tumor cells. In addition, MRP1 is expressed in normal tissues acting as an efflux pump for glutathione, glucuronate, and sulfate conjugates and may thus influence the pharmacokinetic properties of many drugs. Using polymerase chain reaction-single-strand conformation polymorphism analysis, we screened 36 Caucasian volunteers for mutations in the coding exons of the MRP1 gene, including the adjacent intron sequences. Among several mutations found, two are expected to cause amino acid substitutions. One of these mutations (G671V) was of special interest because it is located near the first nucleotide binding domain. To determine whether this mutation caused a change in the MRP1 phenotype, a mutant MRP1 expression vector was constructed and transfected into SV40-transformed human embryonic kidney cells (HEKSV293T) and the transport properties of the mutant protein were examined. Transport of the MRP1 substrates leukotriene C4, 17beta-estradiol 17beta-(D)-glucuronide, and estrone sulfate by membrane vesicles prepared from transiently transfected HEKSV293T cells was comparable to that of wild-type MRP1.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Substituição de Aminoácidos , Mutação , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Adulto , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Sequência Conservada , Éxons , Humanos , Íntrons , Rim , Cinética , Mutagênese Sítio-Dirigida , Ontário , Mutação Puntual , Polimorfismo Conformacional de Fita Simples , Alinhamento de Sequência , Transfecção , População Branca/genéticaRESUMO
The most important biliary efflux transporter known so far is the multidrug resistance protein 2 (MRP2). Previously, we isolated and characterized the 5'-flanking region of the rat mrp2 gene. In the present study, we performed site-directed mutagenesis experiments indicating that both a Y-Box and a GC-Box in the rat mrp2 promoter are essential for the full basal expression of the gene, but have no significant relevance for its inducibility by the chemical carcinogen 2-acetylaminofluorene. Gel mobility shift experiments demonstrated the binding of the transcription factor CBF/NF-Y, but not of EFIA/YB-1, to the Y-Box. Site-directed mutations in the Y-Box decreasing reporter gene activity of a promoter construct prevented the binding of NF-Y. Consequently, NF-Y contributes substantially to the basal expression of the gene. A site-directed mutation in the GC-Box also reduced basal expression and resulted in a reduced complex formation with the transcription factor Sp1. The corresponding region of the human MRP2 promoter comprises no Sp1 site, but a Y-Box-like element binding YB-1 but not NF-Y, which, however, does not contribute to basal expression. In conclusion, NF-Y and Sp1 binding sites play a decisive role in the basal expression of the rat mrp2 gene, while the human MRP2 gene is regulated differently.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/biossíntese , Transportadores de Cassetes de Ligação de ATP , Fator de Ligação a CCAAT/metabolismo , Proteínas de Transporte/biossíntese , Regulação Neoplásica da Expressão Gênica , Proteínas de Membrana Transportadoras , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Fator de Transcrição Sp1/metabolismo , 2-Acetilaminofluoreno/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Sítios de Ligação , Carcinógenos/farmacologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/metabolismo , Proteína 2 Associada à Farmacorresistência Múltipla , Mutagênese Sítio-Dirigida/fisiologia , Regiões Promotoras Genéticas , Ratos , Transfecção , Células Tumorais CultivadasRESUMO
Expression of a variety of ABC efflux pumps including certain conjugate transporters of the multidrug resistance protein (MRP) subfamily is inducible in primate and rodent tissues, and in a variety of cell lines and primary cells in culture. In human cell lines (HepG2, MCF-7), we studied the inducibility of MRPs 1-5. Similar to the rat mrp2 gene, human mrp2 is inducible by the chemical carcinogen 2-AAF, the chemotherapeutic drug cisplatin and the barbiturate phenobarbital, as demonstrated in Northern and Western Blots. Furthermore, the antibiotic rifampicin was identified as MRP2 inducer in HepG2 cells. MRP1 and 4 mRNAs being expressed in human liver at a very low level could not be detected in HepG2 cells after treatment with various agents. However, MRP3 and 5 mRNAs were detected in addition to MRP2 and their expression was found to be increased by 2-AAF, cisplatin and rifampicin. MRP1 expression was studied in MCF-7 cells where the chemotherapeutic drug vinblastine and tert-butyl hydroquinone but not the MRP2 inducing agents described above acted as inducers.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Transporte/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Membrana Transportadoras , Toxinas Biológicas/toxicidade , Animais , Proteínas de Transporte de Ânions , Humanos , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Regulação para CimaRESUMO
The effects of xenobiotic drugs and toxic compounds depend largely on their kinetic properties, which can be influenced by transmembrane drug transporters like MDR1/P-glycoprotein and the drug-conjugate transporters multidrug resistance protein (MRP) 1 and 2. As the dog is a preferential species used in the pharmacological and toxicological evaluation of new drugs, we sequenced the canine MRP2 cDNA and investigated its expression in various canine tissues compared with the related transporters MRP1 and P-glycoprotein. The tissue distribution pattern of these ABC-transporters differs partially from the distribution described in humans. So we found relatively high renal and low hepatic canine MRP2 expression levels, relatively high hepatic canine MRP1 expression levels, and quite high levels of MRP1 and P-glycoprotein in the dog brain. The knowledge of the tissue distribution pattern of these transporters will aid to interpret pharmacokinetic and toxicokinetic data gained from dog studies and to extrapolate them to humans.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/genética , Resistência a Múltiplos Medicamentos/genética , Genes MDR/genética , Proteínas de Membrana Transportadoras , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , DNA Complementar/análise , Cães , Humanos , Rim/metabolismo , Fígado/metabolismo , Masculino , Dados de Sequência Molecular , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos , RNA/análise , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Especificidade da Espécie , Distribuição TecidualRESUMO
AIM: To develop a fast fluorometric screening assay based on vincristine resistant Caco-2 cells (Caco-2VCR) in order to elucidate potential P-glycoprotein (Pgp) interactions of compounds, and to characterise Caco-2VCR cells with regard to their expression of the efflux transporters Pgp, MRP1 and MRP2. METHODS: We applied the Caco-2VCR cells to a 96-well plate-based calcein AM extrusion assay. The Caco-2VCR cells were cultured as monolayers and incubated with calcein AM with/without addition of Pgp modulators. Fourteen known Pgp modulators were tested in the assay (chloropromazine, cyclosporin A, domperidone, digoxin, ivermectin, ketoconazole, loperamide, metoprolol, propranolol, progesterone, quinidine, quinine, verapamil and vincristine). For each compound an EC50 value was calculated. Protein and mRNA levels of the efflux transporters were analysed by Western blot and polymerase chain reaction techniques. RESULTS: All compounds with the exception of digoxin displayed increased calcein levels. Protein and mRNA analysis showed increased levels of Pgp after vincristine exposure, while expression of the efflux transporters MRP1 and MRP2 remained unchanged. CONCLUSIONS: The calcein AM extrusion assay applied to Caco-2VCR cells can be a valuable tool as a screening assay for new compounds and their potential interaction with P-glycoprotein.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Fluoresceínas/metabolismo , Corantes Fluorescentes/metabolismo , Vincristina/farmacologia , Algoritmos , Western Blotting , Células CACO-2 , Avaliação Pré-Clínica de Medicamentos , Resistencia a Medicamentos Antineoplásicos , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Humanos , Indicadores e Reagentes , Modelos Biológicos , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The importance of the ATP-dependent transporter P-glycoprotein, which is expressed in the brush border membrane of enterocytes and in other tissues with excretory function, for overall drug disposition is well recognized. For example, induction of intestinal P-glycoprotein by rifampin appears to be the underlying mechanism of decreased plasma concentrations of P-glycoprotein substrates such as digoxin with concomitant rifampin therapy. The contribution of transporter proteins other than P-glycoprotein to drug interactions in humans has not been elucidated. Therefore, we tested in this study the hypothesis whether the conjugate export pump MRP2 (cMOAT), which is another member of the ABC transporter family, is inducible by rifampin in humans. Duodenal biopsies were obtained from 16 healthy subjects before and after nine days of oral treatment with 600 mg rifampin/day. MRP2 mRNA and protein were determined by reverse transcription-polymerase chain reaction and immunohistochemistry. Rifampin induced duodenal MRP2 mRNA in 14 out of 16 individuals. Moreover, MRP2 protein, which was expressed in the apical membrane of enterocytes, was significantly induced by rifampin in 10 out of 16 subjects. In summary, rifampin induces MRP2 mRNA and protein in human duodenum. Increased elimination of MRP2 substrates (eg, drug conjugates) into the lumen of the gastrointestinal tract during treatment with rifampin could be a new mechanism of drug interactions.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Mucosa Intestinal/metabolismo , Proteínas de Membrana Transportadoras , Rifampina/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Humanos , Masculino , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: P-Glycoprotein is an efflux pump in many epithelial cells with excretory function. It has been demonstrated that rifampin (INN, rifampicin) induces P-glycoprotein, particularly in the gut wall. We therefore hypothesized that rifampin affects pharmacokinetics of the P-glycoprotein substrate talinolol, a beta1-blocker without appreciable metabolic disposition but intense intestinal secretion in human beings. METHODS: Pharmacokinetics of talinolol (a single dose of 30 mg administered intravenously or 100 mg administered orally for 7 days) and duodenal expression of the MDR1 gene product P-glycoprotein as assessed by reverse transcriptase-polymerase chain reaction of the MDR1-messenger ribonucleic acid, by immunohistochemistry and Western blot analysis were analyzed before and after coadministration of rifampin (600 mg per day for 9 days) in 8 male healthy volunteers (age 22 to 26 years). RESULTS: During rifampin treatment, the areas under the curve of intravenous and oral talinolol were significantly lower (21% and 35%; P < .05). Treatment with rifampin resulted in a significantly increased expression of duodenal P-glycoprotein content 4.2-fold (2.9, 6.51) (Western blot) and messenger RNA was increased in six of the eight volunteers. P-Glycoprotein expression in biopsy specimens of gut mucosa correlated significantly with the systemic clearance of intravenous talinolol (rs = 0.74; P < .001). CONCLUSIONS: Rifampin induces P-glycoprotein-mediated excretion of talinolol predominantly in the gut wall. Moreover, clearance of talinolol from the blood into the lumen of the gastrointestinal tract may be predicted by the individual intestinal P-glycoprotein expression. Thus we describe a new type of steady-state drug interaction affecting compounds that are subject to transport rather than metabolism.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/efeitos dos fármacos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antagonistas Adrenérgicos beta/farmacocinética , Antituberculosos/farmacologia , Duodeno/metabolismo , Propanolaminas/farmacocinética , Rifampina/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Administração Oral , Antagonistas Adrenérgicos beta/administração & dosagem , Antagonistas Adrenérgicos beta/sangue , Adulto , Antiarrítmicos/farmacocinética , Anti-Hipertensivos/farmacocinética , Área Sob a Curva , Western Blotting , Endoscopia do Sistema Digestório , Indução Enzimática/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Infusões Intravenosas , Masculino , Propanolaminas/administração & dosagem , Propanolaminas/sangue , RNA Mensageiro/análise , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para CimaRESUMO
Gene expression of the canalicular conjugate transporter mrp2 is inducible by treatment with the DNA-damaging agents 2-acetylaminofluorene (50 and 100 microM), and cisplatin (20 microM) in primary rat hepatocytes as well as in the rat hepatoma cell line H4IIE. Furthermore, phenobarbital (1 and 2 mM) induces mrp2 gene expression, probably explaining the increase in bile-salt-independent bile flow caused by phenobarbital, while the cholestatic drug ethinyl estradiol (10(-6) M) leads to an increase in mrp2 mRNA but decreases Mrp2 protein level probably via a posttranscriptional mechanism. The 5'-flanking region of the rat mrp2 gene was sequenced and cloned into a luciferase reporter vector. Transient transfection assays with reporter vectors containing unidirectionally deleted 5'-flanking regions using H4IIE cells indicate that two different sequences of 17 and 37 bases comprising a Y-Box and a GC-Box are required for mrp2 gene basal expression. Sequences mediating 2-AAF induction are located within a region 250 bases upstream of the translation start site while the inducing effect of phenobarbital seems to be mediated by another domain located further upstream.
Assuntos
Carcinoma Hepatocelular/química , Regulação Neoplásica da Expressão Gênica/genética , Genes MDR/genética , 2-Acetilaminofluoreno/farmacologia , Animais , Sítios de Ligação/genética , Cisplatino/farmacologia , Clofibrato/farmacologia , Clonagem Molecular , Etinilestradiol/farmacologia , Genes Reporter/genética , Fenobarbital/farmacologia , RNA Mensageiro/metabolismo , Ratos , Análise de Sequência de DNA , Transfecção/genética , Células Tumorais CultivadasRESUMO
The multidrug resistance protein 2 (Mrp2) also called canalicular multidrug resistance protein (cMrp) or canalicular multispecific organic anion transporter (cMoat) is a transmembrane export pump located at the canalicular domain of hepatocytes. Mrp2 transports a broad spectrum of organic anions including glucuronides, glutathione conjugates, and organic sulphates into bile. Based on previous observations in rat hepatocytes, the inducibility of mrp2 gene expression in primate liver was investigated in rhesus monkeys treated with tamoxifen or rifampicin. It was found that treatment with tamoxifen (25 mg/kg per day; over 7 days) or rifampicin (15 mg/kg per day; over 7 days) leading to an induction of cytochrome P450 3A4, resulted in a strong increase in mrp2 mRNA in the liver of male and female rhesus monkeys. On the protein level, tamoxifen also was a highly effective inducer, while rifampicin showed some inducing effect in a female and was inactive in a male monkey. In sections of paraffin-embedded liver tissue, immunofluorescence staining confirmed the results of Western blot analysis. Induced Mrp2 was localized to the canalicular domain of the hepatocytes. In conclusion, our data show inducibility of mrp2 gene expression in the liver of primates which may represent an adaptive response aimed at the enhanced biliary elimination of the inducing drugs and/or their metabolites.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Genes MDR , Fígado/efeitos dos fármacos , Rifampina/farmacologia , Tamoxifeno/farmacologia , Animais , Northern Blotting , Western Blotting , Feminino , Fígado/metabolismo , Macaca mulatta , Masculino , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The human multidrug-resistance-associated protein (MRP), a member of the adenosine triphosphate (ATP)-binding cassette transporter superfamily, is frequently overexpressed in tumor cells resistant to antineoplastic drugs. In the rat, two Mrp isoforms have been identified, Mrp and cMrp. cMrp, also called Mrp2 or cMoat (canalicular multispecific organic anion transporter), is expressed in the canalicular membrane of rat hepatocytes and mediates the excretion of glucuronate, sulfate, and glutathione conjugates into bile. We investigated the expression of cMrp and Mrp in rat hepatocytes in primary culture. Treatment with the chemical carcinogen 2-acetylaminofluorene (2-AAF), the antineoplastic drug cisplatin, and the protein-synthesis inhibitor cycloheximide led to a dose-dependent and time-dependent increase in cmrp gene expression. A 347-base pair cmrp complementary DNA (cDNA) probe served to demonstrate the induction of cmrp messenger RNA (mRNA) with 40 micromol/L 2-AAF, 5 micromol/L cisplatin, or 5 micromol/L cycloheximide. An analogous response was obtained for the increase in cMrp protein. Mrp mRNA was below the detection limit in Northern blots of RNA from liver and hepatocyte cultures, in contrast to rat testis mRNA which served as a positive control. Immunofluorescence microscopy of cultured hepatocytes was used to visualize cMrp in the plasma membrane. Treatment with 2-AAF led to a marked increase in the immunofluorescence signal confirming the cMrp-inducing potency of 2-AAF. In conclusion, the inducing effect of the compounds studied may reflect a general inducibility of hepatic cMrp by a variety of cytotoxic, carcinogenic, and chemotherapeutic agents which is likely to be of relevance for the acquisition of multidrug resistance during chemotherapy and in the process of chemical carcinogenesis in the liver.