RESUMO
Vitamin D3 (VD3) induces intestinal CYP3A that metabolizes orally administered anti-leukemic chemotherapeutic substrates dexamethasone (DEX) and dasatinib potentially causing a vitamin-drug interaction. To determine the impact of VD3 status on systemic exposure and efficacy of these chemotherapeutic agents, we used VD3 sufficient and deficient mice and performed pharmacokinetic and anti-leukemic efficacy studies. Female C57BL/6J and hCYP3A4 transgenic VD3 deficient mice had significantly lower duodenal (but not hepatic) mouse Cyp3a11 and hCYP3A4 expression compared to VD3 sufficient mice, while duodenal expression of Mdr1a, Bcrp and Mrp4 were significantly higher in deficient mice. When the effect of VD3 status on DEX systemic exposure was compared following a discontinuous oral DEX regimen, similar to that used to treat pediatric acute lymphoblastic leukemia patients, male VD3 deficient mice had significantly higher mean plasma DEX levels (31.7 nM) compared to sufficient mice (12.43 nM) at days 3.5 but not at any later timepoints. Following a single oral gavage of DEX, there was a statistically, but not practically, significant decrease in DEX systemic exposure in VD3 deficient vs. sufficient mice. While VD3 status had no effect on oral dasatinib's area under the plasma drug concentration-time curve, VD3 deficient male mice had significantly higher dasatinib plasma levels at t = 0.25 hr. Dexamethasone was unable to reverse the poorer survival of VD3 sufficient vs. deficient mice to BCR-ABL leukemia. In conclusion, although VD3 levels significantly altered intestinal mouse Cyp3a in female mice, DEX plasma exposure was only transiently different for orally administered DEX and dasatinib in male mice. Likewise, the small effect size of VD3 deficiency on single oral dose DEX clearance suggests that the clinical significance of VD3 levels on DEX systemic exposure are likely to be limited.
Assuntos
Dasatinibe , Vitamina D , Animais , Feminino , Masculino , CamundongosRESUMO
Abcg2/Bcrp and Abcb1a/Pgp are xenobiotic efflux transporters limiting substrate permeability in the gastrointestinal system and brain, and increasing renal and hepatic drug clearance. The systemic impact of Bcrp and Pgp ablation on metabolic homeostasis of endogenous substrates is incompletely understood. We performed untargeted metabolomics of cerebrospinal fluid (CSF) and plasma, transcriptomics of brain, liver and kidney from male Sprague Dawley rats (WT) and Bcrp/Pgp double knock-out (dKO) rats, and integrated metabolomic/transcriptomic analysis to identify putative substrates and perturbations in canonical metabolic pathways. A predictive Bayesian machine learning model was used to predict in silico those metabolites with greater substrate-like features for either transporters. The CSF and plasma levels of 169 metabolites, nutrients, signaling molecules, antioxidants and lipids were significantly altered in dKO rats, compared to WT rats. These metabolite changes suggested alterations in histidine, branched chain amino acid, purine and pyrimidine metabolism in the dKO rats. Levels of methylated and sulfated metabolites and some primary bile acids were increased in dKO CSF or plasma. Elevated uric acid levels appeared to be a primary driver of changes in purine and pyrimidine biosynthesis. Alterations in Bcrp/Pgp dKO CSF levels of antioxidants, precursors of neurotransmitters, and uric acid suggests the transporters may contribute to the regulation of a healthy central nervous system in rats. Microbiome-generated metabolites were found to be elevated in dKO rat plasma and CSF. The altered dKO metabolome appeared to cause compensatory transcriptional change in urate biosynthesis and response to lipopolysaccharide in brain, oxidation-reduction processes and response to oxidative stress and porphyrin biosynthesis in kidney, and circadian rhythm genes in liver. These findings present insight into endogenous functions of Bcrp and Pgp, the impact that transporter substrates, inhibitors or polymorphisms may have on metabolism, how transporter inhibition could rewire drug sensitivity indirectly through metabolic changes, and identify functional Bcrp biomarkers.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/deficiência , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/deficiência , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Animais , Encéfalo/metabolismo , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Histidina/metabolismo , Rim/metabolismo , Fígado/metabolismo , Masculino , Taxa de Depuração Metabólica , Metabolômica , Purinas/metabolismo , Pirimidinas/metabolismo , Ratos , Ratos TransgênicosRESUMO
The effects of vitamin A and/or vitamin D deficiency were studied in an Arf-/- BCR-ABL acute lymphoblastic leukemia murine model. Vitamin D sufficient mice died earlier (p = 0.003) compared to vitamin D deficient (VDD) mice. Vitamin A deficient (VAD) mice fared worst with more rapid disease progression and decreased survival. Mice deficient for vitamins A and D (VADD) had disease progression similar to VAD mice. Regulatory T cells, previously shown to associate with poor BCR-ABL leukemia control, were present at higher frequencies among CD4+ splenocytes of vitamin A deficient vs. sufficient mice. In vitro studies demonstrated 1,25-dihydroxyvitamin D (1,25(OH)2VD3) increased the number of BCR-ABL ALL cells only when co-cultured with bone marrow stroma. 1,25(OH)2VD3 induced CXCL12 expression in vivo and in vitro in stromal cells and CXCL12 increased stromal migration and the number of BCR-ABL blasts. Vitamin D plus leukemia reprogrammed the marrow increasing production of collagens, potentially trapping ALL blasts. Vitamin A (all trans retinoic acid, ATRA) treated leukemic cells had increased apoptosis, decreased cells in S-phase, and increased cells in G0/G1. ATRA signaled through the retinoid X receptor to decrease BCR-ABL leukemic cell viability. In conclusion, vitamin A and D deficiencies have opposing effects on mouse survival from BCR-ABL ALL.
Assuntos
Proteínas de Fusão bcr-abl/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Vitamina A/metabolismo , Vitamina D/metabolismo , Animais , Apoptose , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células Cultivadas , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Receptores X de Retinoides/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Vitamina A/genética , Vitamina A/farmacologia , Vitamina D/genética , Vitamina D/farmacologiaRESUMO
We investigated whether pheophorbide A (PhA) could serve as a selective breast cancer resistance protein (BCRP) substrate (victim) to screen in vivo using fluorescent live animal imaging for transporter-mediated interactions with orally administered inhibitors (perpetrators), and whether that could be coupled with serum sampling to measure the systemic concentration of PhA with a fast-throughput in vitro fluorescent assay. PhA is a breakdown product of chlorophyll and is highly fluorescent in the near-infrared (NIR) spectrum. Whole-body NIR fluorescence was greater in the Bcrp KO compared with wild-type (WT) mice fed a regular diet containing chlorophyll and PhA, with fluorescence in WT mice confined to the intestine. PhA intestinal enterocyte fluorescence, after removing lumen contents, was greater in Bcrp knockout (KO) mice versus WT mice due to PhA enterocyte absorption and lack of PhA efflux by Bcrp. This difference was eliminated by maintaining the mice on an alfalfa (chlorophyll/PhA)-free diet. The area under the fluorescence ratio-time curve up to 6 hours (AUCFL 0-6 h) of orally administrated PhA was 3.5 times greater in the Bcrp KO mice compared with WT mice, and the PhA serum concentration was 50-fold higher in KO mice. Pretreatment with known BCRP inhibitors lapatinib, curcumin, elacridar, pantoprazole, and sorafenib, at clinically relevant doses, significantly increased PhA AUCFL 0-6 h by 2.4-, 2.3-, 2.2-, 1.5-, and 1.4-fold, respectively, whereas the area under PhA serum concentration-time curve calculated up to 6 hours (AUCSerum 0-6 h) increased by 13.8-, 7.8-, 5.2-, 2.02-, and 1.45-fold, respectively, and corresponded to their hierarchy as in vitro BCRP inhibitors. Our results demonstrate that live animal imaging using PhA can be used to identify BCRP inhibitors and to assess the potential for BCRP-mediated clinical drug-drug interactions.
Assuntos
Clorofila/análogos & derivados , Interações Medicamentosas/fisiologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Transporte Biológico/fisiologia , Linhagem Celular , Clorofila/metabolismo , Cães , Fluorescência , Células Madin Darby de Rim Canino , Masculino , Camundongos , Camundongos KnockoutRESUMO
Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 engineering of the CYP3A5 *3 locus (rs776746) in human liver cell line HuH-7 (CYP3A5 *3/*3) has led to three CYP3A5 *1 cell lines by deletion of the exon 3B splice junction or point mutation. Cell lines CYP3A5 *1/*3 sd (single deletion), CYP3A5 *1/*1 dd (double deletion), or CYP3A5 *1/*3 pm (point mutation) expressed the CYP3A5 *1 mRNA and had elevated CYP3A5 mRNA (P < 0.0005 for all engineered cell lines) and protein expression compared with HuH-7. In metabolism assays, HuH-7 had less tacrolimus (all P < 0.05) or midazolam (MDZ) (all P < 0.005) disappearance than all engineered cell lines. HuH-7 had less 1-OH MDZ (all P < 0.0005) or 4-OH (all P < 0.005) production in metabolism assays than all bioengineered cell lines. We confirmed CYP3A5 metabolic activity with the CYP3A4 selective inhibitor CYP3CIDE. This is the first report of genomic CYP3A5 bioengineering in human cell lines with drug metabolism analysis.
Assuntos
Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Citocromo P-450 CYP3A/genética , Hepatócitos/metabolismo , Midazolam/metabolismo , Tacrolimo/metabolismo , Linhagem Celular , Humanos , Fígado/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação Puntual/genética , RNA Mensageiro/genética , Deleção de Sequência/genéticaRESUMO
P-glycoprotein (Pgp) [the product of the MDR1 (ABCB1) gene] at the blood-brain barrier (BBB) limits central nervous system (CNS) entry of many prescribed drugs, contributing to the poor success rate of CNS drug candidates. Modulating Pgp expression could improve drug delivery into the brain; however, assays to predict regulation of human BBB Pgp are lacking. We developed a transgenic mouse model to monitor human MDR1 transcription in the brain and spinal cord in vivo. A reporter construct consisting of â¼10 kb of the human MDR1 promoter controlling the firefly luciferase gene was used to generate a transgenic mouse line (MDR1-luc). Fluorescence in situ hybridization localized the MDR1-luciferase transgene on chromosome 3. Reporter gene expression was monitored with an in vivo imaging system following D-luciferin injection. Basal expression was detectable in the brain, and treatment with activators of the constitutive androstane, pregnane X, and glucocorticoid receptors induced brain and spinal MDR1-luc transcription. Since D-luciferin is a substrate of ABCG2, the feasibility of improving D-luciferin brain accumulation (and luciferase signal) was tested by coadministering the dual ABCB1/ABCG2 inhibitor elacridar. The brain and spine MDR1-luc signal intensity was increased by elacridar treatment, suggesting enhanced D-luciferin brain bioavailability. There was regional heterogeneity in MDR1 transcription (cortex > cerebellum) that coincided with higher mouse Pgp protein expression. We confirmed luciferase expression in brain vessel endothelial cells by ex vivo analysis of tissue luciferase protein expression. We conclude that the MDR1-luc mouse provides a unique in vivo system to visualize MDR1 CNS expression and regulation.
Assuntos
Encéfalo/metabolismo , Genes Reporter/fisiologia , Luciferases de Vaga-Lume/biossíntese , Coluna Vertebral/metabolismo , Transcrição Gênica/fisiologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/biossíntese , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Diagnóstico por Imagem , Feminino , Humanos , Luciferases de Vaga-Lume/genética , Camundongos , Camundongos TransgênicosRESUMO
Human liver gene regulatory (Bayesian) network analysis was previously used to identify a cytochrome P450 (P450) gene subnetwork with Aldo-keto reductase 1D1 (AKR1D1) as a key regulatory driver of this subnetwork. This study assessed the biologic importance of AKR1D1 [a key enzyme in the synthesis of bile acids, ligand activators of farnesoid X receptor (FXR), pregnane X receptor (PXR), and constitutive androstane receptor (CAR), known transcriptional regulators of P450s] to hepatic P450 expression. Overexpression of AKR1D1 in primary human hepatocytes led to increased expression of CYP3A4, CYP2C8, CYP2C9, CYP2C19, and CYP2B6. Conversely, AKR1D1 knockdown decreased expression of these P450s. We resequenced AKR1D1 from 98 donor livers and identified a 3'-untranslated region (UTR) (rs1872930) single nucleotide polymorphism (SNP) significantly associated with higher AKR1D1 mRNA expression. AKR1D1 3'-UTR-luciferase reporter studies showed that the variant allele resulted in higher luciferase activity, suggesting that the SNP increases AKR1D1 mRNA stability and/or translation efficiency. Consistent with AKR1D1's putative role as a driver of the P450 subnetwork, the AKR1D1 3'-UTR SNP was significantly associated with increased hepatic mRNA expression of multiple P450s (CYP3A4, CYP2C8, CYP2C9, CYP2C19, and CYP2B6) and CYP3A4, CYP2C8, CYP2C19, and CYP2B6 activities. After adjusting for multiple testing, the association remained significant for AKR1D1, CYP2C9, and CYP2C8 mRNA expression and CYP2C8 activity. These results provide new insights into the variation in expression and activity of P450s that can account for interindividual differences in drug metabolism/efficacy and adverse drug events. In conclusion, we provide the first experimental evidence supporting a role for AKR1D1 as a key genetic regulator of the P450 network.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Regulação Enzimológica da Expressão Gênica , Oxirredutases/genética , Processamento Alternativo , Variação Genética , Células Hep G2 , Hepatócitos/enzimologia , Humanos , Fígado/enzimologia , Oxirredutases/fisiologia , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/análise , RNA Mensageiro/químicaRESUMO
The subarachnoid space, where cerebrospinal fluid (CSF) flows over the brain and spinal cord, is lined on one side by arachnoid barrier (AB) cells that form part of the blood-CSF barrier. However, despite the fact that drugs are administered into the CSF and CSF drug concentrations are used as a surrogate for brain drug concentration following systemic drug administration, the tight-junctioned AB cells have never been examined for whether they express drug transporters that would influence CSF and central nervous system drug disposition. Hence, we characterized drug transporter expression and function in AB cells. Immunohistochemical analysis showed P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) in mouse AB cells but not other meningeal tissue. The Gene Expression Nervous System Atlas (GENSAT) database and the Allen Mouse Brain Atlas confirmed these observations. Microarray analysis of mouse and human arachnoidal tissue revealed expression of many drug transporters and some drug-metabolizing enzymes. Immortalized mouse AB cells express functional P-gp on the apical (dura-facing) membrane and BCRP on both apical and basal (CSF-facing) membranes. Thus, like blood-brain barrier cells and choroid plexus cells, AB cells highly express drug transport proteins and likely contribute to the blood-CSF drug permeation barrier.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Aracnoide-Máter/citologia , Barreira Hematoencefálica/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/biossíntese , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Transporte Biológico/genética , Encéfalo/metabolismo , Linhagem Celular , Expressão Gênica , Haplorrinos , Humanos , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/metabolismo , Medula Espinal/metabolismoRESUMO
BACKGROUND: Several microtubule targeting agents are capable of inducing CYP3A4 via activation of the pregnane X receptor (PXR; NR1I2). OBJECTIVE: To evaluate the CYP3A4 induction potential of vinblastine both clinically and in vitro and determine the involvement of the nuclear receptors NR1I2 and the constitutive androstane receptor (NR1I3). METHODS: Midazolam pharmacokinetics were evaluated in 6 patients who were enrolled in a Phase 1/2 study of infusional vinblastine given in combination with the ABCB1 (P-glycoprotein) antagonist valspodar (PSC 833) and received the CYP3A4 phenotyping probe midazolam on more than 1 occasion. Genotyping was conducted in CYP3A4, CYP3A5, and ABCB1 to rule out potential pharmacogenetic influences. Clinical data were followed-up by Western blotting and reporter assays in HepG2 and NIH3T3 cells treated with vinblastine over a dose range of 150-4800 ng/mL for 48 hours. RESULTS: In 6 patients with cancer, vinblastine increased the median (95% CI) clearance of the CYP3A4 phenotyping probe midazolam from 21.7 L/h (12.6 to 28.1) to 32.3 L/h (17.3 to 53.9) (p = 0.0156, Wilcoxon signed-rank test). No obvious effect of polymorphisms in CYP3A4, CYP3A5, and ABCB1 on midazolam clearance was observed. In vitro, vinblastine induced CYP3A4 protein. Furthermore, cell-based reporter gene assays using transiently transfected HepG2 and NIH3T3 cells indicated that vinblastine (150-4800 ng/mL) weakly activated human and mouse full-length NR1I2, but had no influence on NR1I3. CONCLUSIONS: Collectively, these findings suggest that vinblastine is able to induce CYP3A4, at least in part, via an NR1I2-dependent mechanism, and thus has the potential to facilitate its own elimination and cause interactions with other CYP3A4 substrates.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Citocromo P-450 CYP3A/biossíntese , Receptores de Esteroides/metabolismo , Vimblastina/farmacologia , Adulto , Idoso , Animais , Antineoplásicos Fitogênicos/administração & dosagem , Antineoplásicos Fitogênicos/uso terapêutico , Western Blotting , Carcinoma de Células Renais/tratamento farmacológico , Receptor Constitutivo de Androstano , Ciclosporinas/farmacologia , Relação Dose-Resposta a Droga , Indução Enzimática/efeitos dos fármacos , Feminino , Genes Reporter , Células Hep G2 , Humanos , Neoplasias Renais/tratamento farmacológico , Masculino , Camundongos , Midazolam/metabolismo , Pessoa de Meia-Idade , Células NIH 3T3 , Farmacogenética , Polimorfismo Genético , Receptor de Pregnano X , Receptores Citoplasmáticos e Nucleares/metabolismo , Vimblastina/administração & dosagem , Vimblastina/uso terapêuticoRESUMO
The pregnane X receptor (PXR; PXR.1) can be activated by structurally diverse lipophilic ligands. PXR.2, an alternatively spliced form of PXR, lacks 111 nucleotides encoding 37 amino acids in the ligand binding domain. PXR.2 bound a classic CYP3A4 PXR response element (PXRE) in electrophoretic mobility shift assays, but transfected PXR.2 failed to transactivate a CYP3A4-promoter-luciferase reporter plasmid in HepG2 cells treated with various PXR ligands. Cotransfection experiments showed that PXR.2 behaved as a dominant negative, interfering with PXR.1/rifampin activation of CYP3A4-PXRE-LUC. In HepG2 and LS180 cells stably transduced with PXR.1, PXR target genes (CYP3A4, MDR1, CYP2B6, and UGT1A1) were higher than mock-transduced cells in the absence of ligand and were further induced in the presence of rifampin. In contrast, PXR.2 stably introduced into the same host cells failed to induce target genes over levels in mock-transfected cells after drug treatment. Our homology modeling suggests that ligands bind PXR.1 more favorably, probably because of the presence of a key disordered loop region, which is missing in PXR.2. Yeast two-hybrid assays revealed that, even in the presence of ligand, the corepressors remain tightly bound to PXR.2, and coactivators are unable to bind at helix 12. In summary, PXR.2 can bind to PXREs but fails to transactivate target genes because ligands do not bind the ligand binding domain of PXR.2 productively, corepressors remain tightly bound, and coactivators are not recruited to PXR.2.
Assuntos
Processamento Alternativo , Citocromo P-450 CYP3A/química , Isoformas de Proteínas/farmacologia , Receptores de Esteroides/química , Ativação Transcricional/efeitos dos fármacos , Domínio Catalítico , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Humanos , Microssomos Hepáticos , Receptor de Pregnano X , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ligação Proteica , Conformação Proteica , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Rifampina/farmacologia , Transfecção , Células Tumorais CultivadasRESUMO
We have investigated several in silico and in vitro methods to improve our ability to predict potential drug interactions of antibiotics. Our focus was to identify those antibiotics that activate pregnane X receptor (PXR) and induce CYP3A4 in human hepatocytes and intestinal cells. Human PXR activation was screened using reporter assays in HepG2 cells, kinetic measurements of PXR activation were made in DPX-2 cells, and induction of CYP3A4 expression and activity was verified by quantitative polymerase chain reaction, immunoblotting, and testosterone 6beta-hydroxylation in primary human hepatocytes and LS180 cells. We found that in HepG2 cells CYP3A4 transcription was activated strongly (> 10-fold) by rifampin and troleandomycin; moderately (> or = 7-fold) by dicloxacillin, tetracycline, clindamycin, griseofulvin, and (> or = 4-fold) erythromycin; and weakly (> 2.4-fold) by nafcillin, cefaclor, sulfisoxazole, and (> 2-fold) cefadroxil and penicillin V. Similar although not identical results were obtained in DPX-2 cells. CYP3A4 mRNA and protein expression were induced by these antibiotics to differing extents in both liver and intestinal cells. CYP3A4 activity was significantly increased by rifampin (9.7-fold), nafcillin and dicloxacillin (5.9-fold), and weakly induced (2-fold) by tetracycline, sufisoxazole, troleandomycin, and clindamycin. Multiple pharmacophore models and docking indicated a good fit for dicloxacillin and nafcillin in PXR. These results suggest that in vitro and in silico methods can help to prioritize and identify antibiotics that are most likely to reduce exposures of medications (such as oral contraceptive agents) which interact with enzymes and transporters regulated by PXR. In summary, nafcillin, dicloxacillin, cephradine, tetracycline, sulfixoxazole, erythromycin, clindamycin, and griseofulvin exhibit a clear propensity to induce CYP3A4 and warrant further clinical investigation.
Assuntos
Antibacterianos/farmacologia , Citocromo P-450 CYP3A/biossíntese , Intestinos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Receptores de Esteroides/agonistas , Sequência de Bases , Linhagem Celular , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Primers do DNA , Indução Enzimática , Genes Reporter , Humanos , Técnicas In Vitro , Intestinos/enzimologia , Fígado/enzimologia , Receptor de Pregnano X , RNA Mensageiro/genéticaRESUMO
The patient was a 62-year-old man with small-cell lung cancer (limited disease). He was treated with cisplatin (CDDP) plus etoposide(ETP) and concurrent radiotherapy as first-line treatment. Although a complete response was achieved, his pro-GRP elevated 10 months later. He was treated with several anticancer agents including, CDDP plus irinotecan (CPT-11), CPT-11, paclitaxel and amrubicin (AMR). Although, as a monotherapy, the administration of AMR (30 mg/m(2)) for 3 consecutive days (3-day schedule) seemed to be most effective, severe myelosuppression was observed, and this treatment was discontinued. We changed the treatment schedule to biweekly administration of AMR (30 mg/m(2)). The level of pro-GRP decreased, and it was maintained at a similar level for 7 months. No severe toxicity was observed during this period. This case suggests that the bi-weekly administration of AMR may be a useful option for the treatment of small-cell lung cancer when a 3-day AMR schedule is highly myelosuppressive.
Assuntos
Antraciclinas/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma de Células Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Medula Óssea/efeitos dos fármacos , Camptotecina/administração & dosagem , Camptotecina/análogos & derivados , Cisplatino/administração & dosagem , Esquema de Medicação , Etoposídeo/administração & dosagem , Humanos , Irinotecano , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: The nuclear hormone receptor (NR) superfamily complement in humans is composed of 48 genes with diverse roles in metabolic homeostasis, development, and detoxification. In general, NRs are strongly conserved between vertebrate species, and few examples of molecular adaptation (positive selection) within this superfamily have been demonstrated. Previous studies utilizing two-species comparisons reveal strong purifying (negative) selection of most NR genes, with two possible exceptions being the ligand-binding domains (LBDs) of the pregnane X receptor (PXR, NR1I2) and the constitutive androstane receptor (CAR, NR1I3), two proteins involved in the regulation of toxic compound metabolism and elimination. The aim of this study was to apply detailed phylogenetic analysis using maximum likelihood methods to the entire complement of genes in the vertebrate NR superfamily. Analyses were carried out both across all vertebrates and limited to mammals and also separately for the two major domains of NRs, the DNA-binding domain (DBD) and LBD, in addition to the full-length sequences. Additional functional data is also reported for activation of PXR and the vitamin D receptor (VDR; NR1I1) to gain further insight into the evolution of the NR1I subfamily. RESULTS: The NR genes appear to be subject to strong purifying selection, particularly in the DBDs. Estimates of the ratio of the non-synonymous to synonymous nucleotide substitution rates (the omega ratio) revealed that only the PXR LBD had a sub-population of codons with an estimated omega ratio greater than 1. CAR was also unusual in showing high relative omega ratios in both the DBD and LBD, a finding that may relate to the recent appearance of the CAR gene (presumably by duplication of a pre-mammalian PXR gene) just prior to the evolution of mammals. Functional analyses of the NR1I subfamily show that human and zebrafish PXRs show similar activation by steroid hormones and early bile salts, properties not shared by sea lamprey, mouse, or human VDRs, or by Xenopus laevis PXRs. CONCLUSION: NR genes generally show strong sequence conservation and little evidence for positive selection. The main exceptions are PXR and CAR, genes that may have adapted to cross-species differences in toxic compound exposure.
RESUMO
The pregnane X receptor (PXR) is a transcriptional regulator of xenobiotic metabolizing enzymes, including cytochrome P450 3A (CYP3A), and transporters. Pretreatment of mice and rats with inducers of CYP3A increases acetaminophen (APAP) hepatotoxicity. In untreated mice, the amount of hepatic CYP3A11 mRNA is 4-fold greater in PXR(-/-) mice compared to wild-type mice (Guo et al., 2003), a finding anticipated to increase APAP hepatotoxicity in PXR(-/-) mice. We investigated APAP hepatotoxicity in wild-type and PXR(-/-) mice in a C57BL/6 background, with APAP administered by gavage. Despite a 2.5-fold higher level of total hepatic CYP3A protein and a 3.6-fold higher level of CYP3A activity compared to wild-type mice, PXR(-/-) mice were less sensitive to APAP hepatotoxicity. Hepatic levels of CYP2E1 were identical in the two mouse lines, but hepatic CYP1A2 levels were 3-fold greater in wild-type mice compared to PXR(-/-) mice. Caffeine, an inhibitor of CYP1A2 activity and an enhancer of CYP3A activity, decreased APAP hepatotoxicity in wild-type mice. APAP uptake was 1.5-fold greater in wild-type mice compared to PXR(-/-) mice. No significant differences in the formation of APAP glucuronide and sulfate-conjugated metabolites were observed between wild-type and PXR(-/-) mice. Glutathione levels were similar in the two mouse lines and were transiently decreased to similar amounts after APAP administration. Our finding that APAP hepatotoxicity was decreased in PXR(-/-) mice indicates that PXR is an important modulator of APAP hepatotoxicity, through positive modulation of constitutive CYP1A2 expression and possibly through increased APAP absorption.
Assuntos
Acetaminofen/toxicidade , Fígado/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/fisiologia , Receptores de Esteroides/fisiologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/fisiologia , Acetaminofen/metabolismo , Animais , Benzoquinonas/metabolismo , Transporte Biológico , Cafeína/farmacologia , Citocromo P-450 CYP1A2/análise , Citocromo P-450 CYP2E1/análise , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/análise , Glutationa/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Iminas/metabolismo , Absorção Intestinal , Fígado/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Receptor de Pregnano X , Transcrição Gênica/efeitos dos fármacosRESUMO
The pregnane X receptor (PXR) regulates the metabolism and elimination of bile salts, steroids, and xenobiotics. The sequence of the PXR ligand-binding domain diverges extensively between different animals, suggesting interspecies differences in ligands. Of the endogenous ligands known to activate PXR, biliary bile salts vary the most across vertebrate species, ranging from 27-carbon (C27) bile alcohol sulfates (early fish, amphibians) to C24 bile acids (birds, mammals). Using a luciferase-based reporter assay, human PXR was activated by a wide variety of bile salts. In contrast, zebrafish PXR was activated efficiently only by cyprinol sulfate, the major zebrafish bile salt, but not by recent bile acids. Chicken, mouse, rat, and rabbit PXRs were all activated by species-specific bile acids and by early fish bile alcohol sulfates. In addition, phylogenetic analysis using maximum likelihood demonstrated evidence for nonneutral evolution of the PXR ligand-binding domain. PXR activation by bile salts has expanded from narrow specificity for C27 bile alcohol sulfates (early fish) to a broader specificity for recent bile acids (birds, mammals). PXR specificity for bile salts has thus paralleled the increasing complexity of the bile salt synthetic pathway during vertebrate evolution, an unusual example of ligand-receptor coevolution in the nuclear hormone receptor superfamily.
Assuntos
Ácidos e Sais Biliares/farmacologia , Evolução Molecular , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/classificação , Receptores de Esteroides/agonistas , Receptores de Esteroides/classificação , Animais , Ácidos e Sais Biliares/química , Ácidos e Sais Biliares/fisiologia , Colestanóis/farmacologia , Humanos , Camundongos , Estrutura Molecular , Filogenia , Receptor de Pregnano X , Coelhos , Ratos , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Esteroides/genética , Especificidade da Espécie , Proteínas de Xenopus/efeitos dos fármacos , Xenopus laevis , Peixe-ZebraRESUMO
To gain insight on the expression of pregnane X receptor (PXR), we analyzed PXR.1 and PXR alternatively spliced transcripts in a panel of 36 human tissues. PXR.1 was expressed in many more tissues than previously determined, including human bone marrow and select regions of the human brain. In each of these tissues, we observed alternative splicing of various exons of PXR that generated multiple distinct PXR isoforms. The most abundant PXR alternative mRNA transcripts lacked 111 nucleotides, deleting 37 amino acids from the PXR LBD (PXR.2), or lacked 123 nt, deleting 41 amino acids from the PXR LBD (PXR.3). CYP3A4, a gene transcriptionally regulated by PXR, showed incomplete overlap with PXR in its tissue distribution. Quantitation of PXR mRNAs in human liver demonstrated that PXR.2 and PXR.3 represented 6.7% and 0.32% of total PXR mRNA transcripts. Brain expression of PXR prompted analysis of whether some brain acting chemicals were PXR ligands. The neurosteroids allopregnanolone and pregnanolone activated PXR and induced transcription of a CYP3A4-luciferase reporter. Nicotine, the psychoactive and addictive chemical in cigarettes, and a known inducer of brain CYP2B6, was an efficacious activator of PXR and inducer of CYP3A4 transcription. Because nicotine activation of PXR will enhance metabolism of nicotine to the non-psychoactive cotinine, these results provide one molecular mechanism for the development of tolerance to nicotine. Moreover, the identification of PXR in many human tissues, such as brain, and activation by tissue specific ligands (such as neurosteroids) suggests additional biological roles for this receptor in these tissues.
Assuntos
Processamento Alternativo/genética , Química Encefálica/genética , Neurotransmissores/farmacologia , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Esteroides/agonistas , Receptores de Esteroides/genética , Esteroides/farmacologia , Adulto , Idoso , Sequência de Aminoácidos , Animais , Evolução Biológica , Sequência Conservada , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/metabolismo , Primers do DNA , Cães , Feminino , Peixes , Humanos , Isomerismo , Fígado/metabolismo , Macaca mulatta , Masculino , Camundongos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Plasmídeos/genética , Receptor de Pregnano X , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Coelhos , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fumar/metabolismo , Suínos , Distribuição Tecidual , TransfecçãoRESUMO
The constitutive androstane receptor (CAR) NR1I3 is a transcription factor that upon activation by xenobiotics induces transcription of drug-metabolizing and drug transporter genes. Our goal was to identify whether alternative splicing of CAR makes an important contribution to the generation of novel CAR proteins. The wild-type CAR mRNA (CAR.1) and splice variants (SVs) were amplified from human liver cDNAs and in a panel of cDNAs from 36 human tissues, using exon 1- and 3'-untranslated region primers, cloned and sequenced. Twenty-two unique hCAR splice variants (CAR-SVs) containing different combinations of splicing (deletion of exons 2, 4, 5, 7, partial deletion of exon 9, or insertion of 12 or 15 base pairs from introns 6 or 7) were identified. CAR mRNAs were expressed in small intestine, kidney, testis, adrenal, and brain caudate nucleus. Intestine expressed only CAR.1 mRNA, whereas spleen, heart, and prostate expressed only CAR-SVs. In vitro transcription and translation of CAR-SVs lacking exon 2 (missing ATG start site) generated CAR proteins that differed in M(r) from CAR.1. These CAR-SVs used a translation initiation site in exon 1, generating CAR with a unique amino-terminal sequence. Transcripts lacking part of exon 9 altered the CAR reading frame generating CAR proteins with a unique carboxy-terminal end. CAR-SVs demonstrated compromised binding to CYP2B6 NR elements and transcriptional activation of a CYP2B6 luciferase reporter. The expression of CAR in additional human cell types increases the range of tissue specific transcriptional responses regulated by this receptor, suggesting additional biological roles for CAR and CAR-SV proteins in these tissues.
Assuntos
Processamento Alternativo , Receptores Citoplasmáticos e Nucleares/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Sequência de Bases , Biologia Computacional , Receptor Constitutivo de Androstano , DNA/análise , DNA/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Isoformas de Proteínas , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Ativação TranscricionalRESUMO
The ABC transporter, Mrp4, transports the sulfated steroid DHEA-s, and sulfated bile acids interact with Mrp4 with high affinity. Hepatic Mrp4 levels are low, but increase under cholestatic conditions. We therefore inferred that up-regulation of Mrp4 during cholestasis is a compensatory mechanism to protect the liver from accumulation of hydrophobic bile acids. We determined that the nuclear receptor CAR is required to coordinately up-regulate hepatic expression of Mrp4 and an enzyme known to sulfate hydroxy-bile acids and steroids, Sult2a1. CAR activators increased Mrp4 and Sult2a1 expression in primary human hepatocytes and HepG2, a human liver cell line. Sult2a1 was down-regulated in Mrp4-null mice, further indicating an inter-relation between Mrp4 and Sult2a1 gene expression. Based on the hydrophilic nature of sulfated bile acids and the Mrp4 capability to transport sulfated steroids, our findings suggest that Mrp4 and Sult2a1 participate in an integrated pathway mediating elimination of sulfated steroid and bile acid metabolites from the liver.
Assuntos
Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Sulfotransferases/metabolismo , Fatores de Transcrição/metabolismo , Animais , Ácidos e Sais Biliares/metabolismo , Western Blotting , Linhagem Celular , Colestase , Receptor Constitutivo de Androstano , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Genes Reporter , Genótipo , Proteínas de Fluorescência Verde , Hepatócitos/metabolismo , Humanos , Hidroxiácidos/metabolismo , Imuno-Histoquímica , Ligantes , Fígado/citologia , Fígado/metabolismo , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Modelos Genéticos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Células NIH 3T3 , Análise de Sequência com Séries de Oligonucleotídeos , Oligonucleotídeos/química , Ligação Proteica , RNA/metabolismo , Retroviridae/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esteroides/metabolismo , Transcrição Gênica , Transfecção , Regulação para CimaRESUMO
Elevated serum total homocysteine (tHcy) levels are associated with increased risk for cardiovascular disease and dementia. The prevalence rates of homozygous mutants among Japanese women (n = 300) were 17.3%, 1.3%, 18.6%, and 5.3% for methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C, reduced folate carrier (RFC-1) A80G, and methionine synthase (MS) A2756G, respectively. The tHcy value was significantly lower (p < 0.001) in young women with CC or CT of MTHFR than with TT (10.9+/-4.7 micromol/L) (n =250). Diversities of serum folate and tHcy in women with 23 combinations of different alleles at low folate intake converged to the highest (34.0+/-8.6 nmol/L) and lowest (7.6+/-1.5 micromol/L) levels, respectively, after folic acid (400 microg/day) supplementation. In the regression equation ( y= ax + b) of serum folate ( y nmol/L) plotted against mean folate intake ( x microg/day), the values of "a" were 0.032, 0.037, and 0.045 for individuals with CC, CT, and TT alleles, respectively, of MTHFR.