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1.
Drug Metab Dispos ; 52(9): 949-956, 2024 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-38866474

RESUMO

The role of the kidney as an excretory organ for exogenous and endogenous compounds is well recognized, but there is a wealth of data demonstrating that the kidney has significant metabolizing capacity for a variety of exogenous and endogenous compounds that in some cases surpass the liver. The induction of drug-metabolizing enzymes by some chemicals can cause drug-drug interactions and intraindividual variability in drug clearance. In this study, we evaluated the expression and induction of cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT) isoforms in 3D-cultured primary human renal proximal tubule epithelial cells (RPTEC) to elucidate their utility as models of renal drug metabolism. CYP2B6, CYP2E1, CYP3A4, CYP3A5, and all detected UGTs (UGT1A1, UGT1A4, UGT1A6, UGT1A9, and UGT2B7) mRNA levels in 3D-RPTEC were significantly higher than those in 2D-RPTEC and HK-2 cells and were close to the levels in the human kidney cortex. CYP1B1 and CYP2J2 mRNA levels in 3D-RPTEC were comparable to those in 2D-RPTEC, HK-2 cells, and the human kidney cortex. Midazolam 1'-hydroxylation, trifluoperazine N-glucuronidation, serotonin O-glucuronidation, propofol O-glucuronidation, and morphine 3-glucuronidation in the 3D-RPTEC were significantly higher than the 2D-RPTEC and comparable to those in the HepaRG cells, although bupropion, ebastine, and calcitriol hydroxylations were not different between the 2D- and 3D-RPTEC. Treatment with ligands of the aryl hydrocarbon receptor and farnesoid X receptor induced CYP1A1 and UGT2B4 expression, respectively, in 3D-RPTEC compared with 2D-RPTEC. We provided information on the expression, activity, and induction abilities of P450s and UGTs in 3D-RPTEC as an in vitro human renal metabolism model. SIGNIFICANCE STATEMENT: This study demonstrated that the expression of cytochrome P450s (P450s) and UDP-glucuronosyltransferases (UGTs) in 3D-cultured primary human renal proximal tubule epithelial cells (3D-RPTEC) was higher than those in 2D-cultured primary human renal proximal tubule epithelial cells and HK-2 cells. The results were comparable to that in the human kidney cortex. 3D-RPTEC are useful for evaluating the induction of kidney P450s, UDP-glucuronosyltransferases, and human renal drug metabolism in cellulo.


Assuntos
Sistema Enzimático do Citocromo P-450 , Células Epiteliais , Glucuronosiltransferase , Túbulos Renais Proximais , Humanos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Glucuronosiltransferase/metabolismo , Glucuronosiltransferase/genética , Células Epiteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Células Cultivadas , Indução Enzimática/efeitos dos fármacos , Linhagem Celular , Técnicas de Cultura de Células/métodos , RNA Mensageiro/metabolismo , RNA Mensageiro/genética
2.
Chem Res Toxicol ; 37(5): 671-674, 2024 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-38626399

RESUMO

Autoinduction of cytochrome P450 (P450) 3A4-mediated metabolism of thalidomide was investigated in humanized-liver mice and human hepatocyte-derived HepaSH cells. The mean plasma ratios of 5-hydroxythalidomide and glutathione adducts to thalidomide were significantly induced (3.5- and 6.0-fold, respectively) by thalidomide treatment daily at 1000 mg/kg for 3 days and measured at 2 h after the fourth administration (on day 4). 5-Hydroxythalidomide was metabolically activated by P450 3A4 in HepaSH cells pretreated with 300 and 1000 µM thalidomide, and 5,6-dihydroxythalidomide was detected. Significant induction of P450 3A4 mRNA expression (4.1-fold) in the livers of thalidomide-treated mice occurred. Thalidomide exerts a variety of actions through multiple mechanisms following bioactivation by induced human P450 3A enzymes.


Assuntos
Citocromo P-450 CYP3A , Hepatócitos , Talidomida , Citocromo P-450 CYP3A/metabolismo , Citocromo P-450 CYP3A/genética , Humanos , Animais , Talidomida/farmacologia , Talidomida/análogos & derivados , Camundongos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Linhagem Celular , RNA Mensageiro/metabolismo , Indução Enzimática/efeitos dos fármacos , Masculino , Indutores do Citocromo P-450 CYP3A/farmacologia
3.
Arch Toxicol ; 98(8): 2541-2556, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38713375

RESUMO

Rifampicin is a strong inducer of cytochrome P450 (CYP3A4) and P-glycoprotein (P-gp/ABCB1), leading to profound drug-drug interactions. In contrast, the chemically related rifabutin does not show such pronounced induction properties in vivo. The aim of our study was to conduct a comprehensive analysis of the different induction potentials of rifampicin and rifabutin in primary human hepatocytes and to analyze the mechanism of potential differences. Therefore, we evaluated CYP3A4/ABCB1 mRNA expression (polymerase chain reaction), CYP3A4/P-gp protein expression (immunoaffinity-liquid chromatography-mass spectrometry, IA-LC-MS/MS), CYP3A4 activity (testosterone hydroxylation), and considered intracellular drug uptake after treatment with increasing rifamycin concentrations (0.01-10 µM). Furthermore, rifamycin effects on the protein levels of CYP2C8, CYP2C9, and CYP2C19 were analyzed (IA-LC-MS/MS). Mechanistic analysis included the evaluation of possible suicide CYP3A4 inhibition (IC50 shift assay) and drug impact on translational efficiency (cell-free luminescence assays). Rifabutin accumulated 6- to 15-fold higher in hepatocytes than rifampicin, but induced CYP3A4 mRNA comparably to rifampicin (e. g. rifampicin 61-fold vs. rifabutin 44-fold, 72 h). While rifampicin for example enhanced protein (10 µM: 21-fold) and activity levels considerably (53-fold), rifabutin only slightly increased CYP3A4 protein expression (10 µM: 3.3-fold) or activity (11-fold) compared to rifampicin after 72 h. Both rifamycins similarly influenced expression of other eliminating proteins. A potential CYP3A4 suicide inhibition by a specific rifabutin metabolite or disruption of ribosome function were excluded experimentally. In conclusion, the lack of protein enhancement, could explain rifabutin's weaker induction-related drug-drug interaction risk in vivo.


Assuntos
Citocromo P-450 CYP3A , Interações Medicamentosas , Hepatócitos , RNA Mensageiro , Rifabutina , Rifampina , Rifabutina/análogos & derivados , Rifabutina/toxicidade , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Citocromo P-450 CYP3A/metabolismo , Citocromo P-450 CYP3A/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Rifampina/farmacologia , Rifampina/toxicidade , Células Cultivadas , Indutores do Citocromo P-450 CYP3A/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Indução Enzimática/efeitos dos fármacos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Masculino , Espectrometria de Massas em Tandem
4.
J Appl Toxicol ; 44(11): 1773-1783, 2024 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-39039701

RESUMO

Hepatic enzyme induction, an inherent defense system against xenobiotics, is known to simultaneously affect endocrine system functions in mammals under specific conditions, particularly thyroid hormone (TH) regulation. While this phenomenon has been studied extensively, the pathway leading to this indirect thyroid effect in mammals has unclear applicability to amphibians, despite the importance of amphibian species in assessing thyroid-disruptive chemicals. Here, we investigated the effects of three well-known mammalian enzyme inducers-ß-naphthoflavone (BNF), pregnenolone carbonitrile (PCN), and sodium phenobarbital (NaPB)-on the gene expression of phase-I and phase-II metabolizing enzymes in Xenopus laevis tadpoles. Waterborne exposure to BNF and PCN significantly induced the expression of both phase-I (cytochrome P450, CYP) and phase-II enzymes (UDP-glucuronosyltransferase, UGT and sulfotransferase, SULT), but in different patterns, while NaPB exposure induced CYP2B expression without affecting phase-II enzymes in tadpoles, in contrast to mammals. Furthermore, an ex vivo hepatic enzyme activity assay confirmed that BNF treatment significantly increased phase-II metabolic activity (glucuronidation and sulfation) toward TH. These results suggest the potential for certain mammalian enzyme inducers to influence TH clearance in X. laevis tadpoles. Our findings provide insights into the profiles of xenosensing activity and enzyme induction in amphibians, which can facilitate a better understanding of the mechanisms of indirect effects on the thyroid system via hepatic enzyme induction in nonmammalian species.


Assuntos
Sistema Enzimático do Citocromo P-450 , Larva , Fígado , Metamorfose Biológica , Hormônios Tireóideos , Xenopus laevis , beta-Naftoflavona , Animais , Larva/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/metabolismo , Metamorfose Biológica/efeitos dos fármacos , Hormônios Tireóideos/metabolismo , beta-Naftoflavona/farmacologia , beta-Naftoflavona/toxicidade , Sistema Enzimático do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Glucuronosiltransferase/metabolismo , Glucuronosiltransferase/genética , Indução Enzimática/efeitos dos fármacos , Carbonitrila de Pregnenolona/farmacologia , Fenobarbital/farmacologia , Sulfotransferases/metabolismo , Sulfotransferases/genética
5.
Physiol Rev ; 96(1): 307-64, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26681794

RESUMO

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are generated from aerobic metabolism, as a result of accidental electron leakage as well as regulated enzymatic processes. Because ROS/RNS can induce oxidative injury and act in redox signaling, enzymes metabolizing them will inherently promote either health or disease, depending on the physiological context. It is thus misleading to consider conventionally called antioxidant enzymes to be largely, if not exclusively, health protective. Because such a notion is nonetheless common, we herein attempt to rationalize why this simplistic view should be avoided. First we give an updated summary of physiological phenotypes triggered in mouse models of overexpression or knockout of major antioxidant enzymes. Subsequently, we focus on a series of striking cases that demonstrate "paradoxical" outcomes, i.e., increased fitness upon deletion of antioxidant enzymes or disease triggered by their overexpression. We elaborate mechanisms by which these phenotypes are mediated via chemical, biological, and metabolic interactions of the antioxidant enzymes with their substrates, downstream events, and cellular context. Furthermore, we propose that novel treatments of antioxidant enzyme-related human diseases may be enabled by deliberate targeting of dual roles of the pertaining enzymes. We also discuss the potential of "antioxidant" nutrients and phytochemicals, via regulating the expression or function of antioxidant enzymes, in preventing, treating, or aggravating chronic diseases. We conclude that "paradoxical" roles of antioxidant enzymes in physiology, health, and disease derive from sophisticated molecular mechanisms of redox biology and metabolic homeostasis. Simply viewing antioxidant enzymes as always being beneficial is not only conceptually misleading but also clinically hazardous if such notions underpin medical treatment protocols based on modulation of redox pathways.


Assuntos
Antioxidantes/metabolismo , Enzimas/metabolismo , Nível de Saúde , Estresse Oxidativo , Animais , Modelos Animais de Doenças , Indução Enzimática , Repressão Enzimática , Enzimas/biossíntese , Enzimas/genética , Técnicas de Silenciamento de Genes , Predisposição Genética para Doença , Humanos , Camundongos Transgênicos , Estado Nutricional , Oxirredução , Fenótipo , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fatores de Risco
6.
Drug Metab Dispos ; 51(3): 276-284, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36460477

RESUMO

Reliable in vitro to in vivo translation of cytochrome P450 (CYP) 3A4 induction potential is essential to support risk mitigation for compounds during pharmaceutical discovery and development. In this study, a linear correlation of CYP3A4 mRNA induction potential in human hepatocytes with the respective pregnane-X receptor (PXR) activation in a reporter gene assay using DPX2 cells was successfully demonstrated for 13 clinically used drugs. Based on this correlation, using rifampicin as a positive control, the magnitude of CYP3A4 mRNA induction for 71 internal compounds at several concentrations up to 10 µM (n = 90) was predicted within 2-fold error for 64% of cases with only a few false positives (19%). Furthermore, the in vivo area under the curve reduction of probe CYP substrates was reasonably predicted for eight marketed drugs (carbamazepine, dexamethasone, enzalutamide, nevirapine, phenobarbital, phenytoin, rifampicin, and rufinamide) using the static net effect model using both the PXR activation and CYP3A4 mRNA induction data. The liver exit concentrations were used for the model in place of the inlet concentrations to avoid false positive predictions and the concentration achieving twofold induction (F2) was used to compensate for the lack of full induction kinetics due to cytotoxicity and solubility limitations in vitro. These findings can complement the currently available induction risk mitigation strategy and potentially influence the drug interaction modeling work conducted at clinical stages. SIGNIFICANCE STATEMENT: The established correlation of CYP3A4 mRNA in human hepatocytes to PXR activation provides a clear cut-off to identify a compound showing an in vitro induction risk, complementing current regulatory guidance. Also, the demonstrated in vitro-in vivo translation of induction data strongly supports a clinical development program although limitations remain for drug candidates showing complex disposition pathways, such as involvement of auto-inhibition/induction, active transport and high protein binding.


Assuntos
Citocromo P-450 CYP3A , Receptores de Esteroides , Humanos , Citocromo P-450 CYP3A/metabolismo , Receptor de Pregnano X/metabolismo , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Rifampina/farmacologia , Rifampina/metabolismo , Indução Enzimática , Hepatócitos/metabolismo , RNA Mensageiro/metabolismo
7.
Ther Drug Monit ; 45(5): 653-659, 2023 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-36645709

RESUMO

BACKGROUND: Perampanel (PER) is an oral antiepileptic drug and its concomitant use with carbamazepine (CBZ) leads to decreased PER concentrations. However, the magnitude of its influence may vary, depending on the dynamics of the enzyme induction properties of CBZ. This study aimed to develop a population pharmacokinetic (PPK) model considering the dynamics of enzyme induction and evaluate the effect of CBZ on PER pharmacokinetics. METHODS: We retrospectively collected data on patient background, laboratory tests, and prescribed drugs from electronic medical records. We developed 2 PPK models incorporating the effect of CBZ-mediated enzyme induction to describe time-concentration profiles of PER using the following different approaches: (1) treating the concomitant use of CBZ as a categorical covariate (empirical PPK model) and (2) incorporating the time-course of changes in the amount of enzyme by CBZ-mediated induction (semimechanistic PPK model). The bias and precision of the predictions were investigated by calculating the mean error, mean absolute error, and root mean squared error. RESULTS: A total of 133 PER concentrations from 64 patients were available for PPK modelling. PPK analyses showed that the co-administration of CBZ increased the clearance of PER. Goodness-of-fit plots indicated a favorable description of the observed data and low bias. The mean error, mean absolute error, and root mean square error values based on the semimechanistic model were smaller than those obtained using the empirical PPK model for predicting PER concentrations in patients with CBZ. CONCLUSIONS: We developed 2 PPK models to describe PER pharmacokinetics based on different approaches, using electronic medical record data. Our PPK models support the use of PER in clinical practice.


Assuntos
Carbamazepina , Epilepsia , Humanos , Estudos Retrospectivos , Indução Enzimática , Carbamazepina/uso terapêutico , Epilepsia/tratamento farmacológico , Benzodiazepinas/uso terapêutico , Interações Medicamentosas
8.
J Chem Inf Model ; 63(1): 173-186, 2023 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-36473234

RESUMO

Three structurally closely related dopamine D1 receptor positive allosteric modulators (D1 PAMs) based on a tetrahydroisoquinoline (THIQ) scaffold were profiled for their CYP3A4 induction potentials. It was found that the length of the linker at the C5 position greatly affected the potentials of these D1 PAMs as CYP3A4 inducers, and the level of induction correlated well with the activation of the pregnane X receptor (PXR). Based on the published PXR X-ray crystal structures, we built a binding model specifically for these THIQ-scaffold-based D1 PAMs in the PXR ligand-binding pocket via an ensemble docking approach and found the model could explain the observed CYP induction disparity. Combined with our previously reported D1 receptor homology model, which identified the C5 position as pointing toward the solvent-exposed space, our PXR-binding model coincidentally suggested that structural modifications at the C5 position could productively modulate the CYP induction potential while maintaining the D1 PAM potency of these THIQ-based PAMs.


Assuntos
Citocromo P-450 CYP3A , Receptores de Esteroides , Receptor de Pregnano X/metabolismo , Citocromo P-450 CYP3A/metabolismo , Receptores de Esteroides/química , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Indução Enzimática
9.
J Appl Toxicol ; 43(6): 828-844, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36549901

RESUMO

The fungicide boscalid induces thyroid histopathological and hormone effects in the rat, secondary to liver enzyme induction. To assess the human relevance of liver enzyme induction presumably leading to thyroid hormone disruption, a species comparative in vitro study on T4-glucuronidation was conducted. Currently, no guidelines how to evaluate Phase II induction are in place. Therefore, we investigated the optimal conditions to evaluate Phase I and Phase II induction potential of boscalid in primary rat (PRH) and human (PHH) hepatocytes. Endpoints included mRNA gene expression and enzyme activities (cytochrome P450 isozymes [CYPs] and uridine diphosphate-glucuronosyltransferases [UGTs]), measured after 3 (D3) and 7 (D7) days of exposure to reference compounds and to 5, 10, and 20 µM boscalid, focusing on T4-glucuronidation. Basal CYP activities and T4 glucuronidation were similar or higher on D7 than D3. The highest induction responses of CYPs were on D3, whereas UGT induction and T4-glucuronidation increases were highest on D7. Boscalid induced CYP1A, CYP2B, and CYP3A mRNA and/or increased related activities in PRH and PHH. Species differences in the induction pattern of UGT genes by reference inducers (ß-naphthoflavone [BNF], 5-pregnen-3ß-ol-20-one-16α-carbonitirile [PCN], rifampicin [RIF], and phenobarbital [PB]) and boscalid were seen: UGT1A1, UGT1A3, and UGT1A9 were predominantly induced in PHH, while UGT2B1 was predominantly induced in PRH. Basal activity levels for T4-glucuronidation were very low in humans and an order of magnitude higher in rat, for this reason increases in activities were assessed as delta activity to the control. Significant increases in T4-glucuronidation occurred with boscalid in rat but not in human hepatocytes.


Assuntos
Microssomos Hepáticos , Tiroxina , Ratos , Humanos , Animais , Tiroxina/metabolismo , Microssomos Hepáticos/metabolismo , Hepatócitos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , RNA Mensageiro/genética , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Indução Enzimática
10.
Genes Dev ; 29(23): 2490-503, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26637528

RESUMO

Glucose-rich diets shorten the life spans of various organisms. However, the metabolic processes involved in this phenomenon remain unknown. Here, we show that sterol regulatory element-binding protein (SREBP) and mediator-15 (MDT-15) prevent the life-shortening effects of a glucose-rich diet by regulating fat-converting processes in Caenorhabditis elegans. Up-regulation of the SREBP/MDT-15 transcription factor complex was necessary and sufficient for alleviating the life-shortening effect of a glucose-rich diet. Glucose feeding induced key enzymes that convert saturated fatty acids (SFAs) to unsaturated fatty acids (UFAs), which are regulated by SREBP and MDT-15. Furthermore, SREBP/MDT-15 reduced the levels of SFAs and moderated glucose toxicity on life span. Our study may help to develop strategies against elevated blood glucose and free fatty acids, which cause glucolipotoxicity in diabetic patients.


Assuntos
Envelhecimento/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Ácidos Graxos/metabolismo , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo , Fatores de Transcrição/metabolismo , Envelhecimento/efeitos dos fármacos , Envelhecimento/metabolismo , Animais , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/enzimologia , Proteínas de Caenorhabditis elegans/genética , Dieta , Sacarose Alimentar/farmacologia , Indução Enzimática/efeitos dos fármacos , Ácidos Graxos Dessaturases/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Estudo de Associação Genômica Ampla , Glucose/metabolismo , Glucose/farmacologia , Glucose/toxicidade , Interferência de RNA , Proteínas de Ligação a Elemento Regulador de Esterol/genética , Fatores de Transcrição/genética
11.
J Neurosci ; 41(35): 7340-7349, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34290083

RESUMO

Alzheimer's disease (AD) is a progressive neurodegenerative disease marked by the accumulation of amyloid-ß (Aß) plaques and neurofibrillary tangles. Aß oligomers cause synaptic dysfunction early in AD by enhancing long-term depression (LTD; a paradigm for forgetfulness) via metabotropic glutamate receptor (mGluR)-dependent regulation of striatal-enriched tyrosine phosphatase (STEP61). Reelin is a neuromodulator that signals through ApoE (apolipoprotein E) receptors to protect the synapse against Aß toxicity (Durakoglugil et al., 2009) Reelin signaling is impaired by ApoE4, the most important genetic risk factor for AD, and Aß-oligomers activate metabotropic glutamate receptors (Renner et al., 2010). We therefore asked whether Reelin might also affect mGluR-LTD. To this end, we induced chemical mGluR-LTD using DHPG (Dihydroxyphenylglycine), a selective mGluR5 agonist. We found that exogenous Reelin reduces the DHPG-induced increase in STEP61, prevents the dephosphorylation of GluA2, and concomitantly blocks mGluR-mediated LTD. By contrast, Reelin deficiency increased expression of Ca2+-permeable GluA2-lacking AMPA receptors along with higher STEP61 levels, resulting in occlusion of DHPG-induced LTD in hippocampal CA1 neurons. We propose a model in which Reelin modulates local protein synthesis as well as AMPA receptor subunit composition through modulation of mGluR-mediated signaling with implications for memory consolidation or neurodegeneration.SIGNIFICANCE STATEMENT Reelin is an important neuromodulator, which in the adult brain controls synaptic plasticity and protects against neurodegeneration. Amyloid-ß has been shown to use mGluRs to induce synaptic depression through endocytosis of NMDA and AMPA receptors, a mechanism referred to as LTD, a paradigm of forgetfulness. Our results show that Reelin regulates the phosphatase STEP, which plays an important role in neurodegeneration, as well as the expression of calcium-permeable AMPA receptors, which play a role in memory formation. These data suggest that Reelin uses mGluR LTD pathways to regulate memory formation as well as neurodegeneration.


Assuntos
Depressão Sináptica de Longo Prazo/fisiologia , Neurônios/fisiologia , Proteínas Tirosina Fosfatases não Receptoras/fisiologia , Receptores de Glutamato Metabotrópico/fisiologia , Proteína Reelina/fisiologia , 2-Amino-5-fosfonovalerato/farmacologia , Animais , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/efeitos dos fármacos , Cálcio/fisiologia , Células Cultivadas , Córtex Cerebral/citologia , Indução Enzimática/efeitos dos fármacos , Depressão Sináptica de Longo Prazo/efeitos dos fármacos , Memória/fisiologia , Metoxi-Hidroxifenilglicol/análogos & derivados , Metoxi-Hidroxifenilglicol/farmacologia , Camundongos , Degeneração Neural/fisiopatologia , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Fosforilação/efeitos dos fármacos , Picrotoxina/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/metabolismo , Receptores de Glutamato Metabotrópico/agonistas , Proteínas Recombinantes/metabolismo , Proteína Reelina/deficiência , Proteína Reelina/genética
12.
Antimicrob Agents Chemother ; 66(10): e0227721, 2022 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-36069614

RESUMO

Tuberculosis is the most common cause of death in HIV-infected individuals. Rifampin and isoniazid are the backbones of the current first-line antitubercular therapy. The aim of the present study was to describe the time-dependent pharmacokinetics and pharmacogenetics of rifampin and isoniazid and to quantitatively evaluate the drug-drug interaction between rifampin and isoniazid in patients coinfected with HIV. Plasma concentrations of isoniazid, acetyl-isoniazid, isonicotinic acid, rifampin, and 25-desacetylrifampin from 40 HIV therapy-naive patients were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after the first dose and at steady state of antitubercular therapy. Patients were genotyped for determination of acetylator status and CYP2C19 phenotype. Nonlinear mixed-effects models were developed to describe the pharmacokinetic data. The model estimated an autoinduction of both rifampin bioavailability (0.5-fold) and clearance (2.3-fold). 25-Desacetylrifampin clearance was 2.1-fold higher at steady state than after the first dose. Additionally, ultrarapid CYP2C19 metabolizers had a 2-fold-higher rifampin clearance at steady state than intermediate or extensive metabolizers. An induction of isonicotinic acid formation from isoniazid dependent on total rifampin dose was estimated. Simulations indicated a 30% lower isoniazid exposure at steady state during administration of standard rifampin doses than isoniazid exposure in noninduced individuals. Rifampin exposure was correlated with CYP2C19 polymorphism, and rifampin administration may increase exposure to toxic metabolites by isoniazid in patients. Both findings may influence the risk of treatment failure, resistance development, and toxicity and require further investigation, especially with regard to ongoing high-dose rifampin trials.


Assuntos
Antituberculosos , Infecções por HIV , Isoniazida , Rifampina , Tuberculose , Humanos , Antituberculosos/farmacocinética , Cromatografia Líquida , Citocromo P-450 CYP2C19/genética , Indução Enzimática , Infecções por HIV/tratamento farmacológico , Infecções por HIV/microbiologia , Isoniazida/farmacocinética , Rifampina/farmacocinética , Espectrometria de Massas em Tandem , Tuberculose/tratamento farmacológico , Tuberculose/virologia
13.
Am J Pathol ; 191(12): 2072-2079, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34560064

RESUMO

Bone homeostasis depends on the balance between bone resorption by osteoclasts (OCs) and bone formation by osteoblasts. Bone resorption can become excessive under various pathologic conditions, including rheumatoid arthritis. Previous studies have shown that OC formation is promoted under hypoxia. However, the precise mechanisms behind OC formation under hypoxia have not been elucidated. The present study investigated the role of inducible nitric oxide synthase (iNOS) in OC differentiation under hypoxia. Primary bone marrow cells obtained from mice were stimulated with receptor activator of NF-κB ligand and macrophage colony-stimulating factor to induce OC differentiation. The number of OCs increased in culture under hypoxia (oxygen concentration, 5%) compared with that under normoxia (oxygen concentration, 20%). iNOS gene and protein expression increased in culture under hypoxia. Addition of an iNOS inhibitor under hypoxic conditions suppressed osteoclastogenesis. Addition of a nitric oxide donor to the normoxic culture promoted osteoclastogenesis. Furthermore, insulin-like growth factor 2 expression was significantly altered in both iNOS inhibition experiments and nitric oxide donor experiments. These data might provide clues to therapies for excessive osteoclastogenesis under several hypoxic pathologic conditions, including rheumatoid arthritis.


Assuntos
Hipóxia Celular/fisiologia , Óxido Nítrico Sintase Tipo II/fisiologia , Osteoclastos/fisiologia , Animais , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Hipóxia Celular/efeitos dos fármacos , Células Cultivadas , Indução Enzimática/efeitos dos fármacos , Indução Enzimática/genética , Hipóxia/genética , Hipóxia/metabolismo , Hipóxia/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Oxigênio/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , ômega-N-Metilarginina/farmacologia
14.
Blood ; 136(1): 93-105, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32202637

RESUMO

Inhibition of the B-cell receptor pathway, and specifically of Bruton tyrosine kinase (BTK), is a leading therapeutic strategy in B-cell malignancies, including chronic lymphocytic leukemia (CLL). Target occupancy is a measure of covalent binding to BTK and has been applied as a pharmacodynamic parameter in clinical studies of BTK inhibitors. However, the kinetics of de novo BTK synthesis, which determines occupancy, and the relationship between occupancy, pathway inhibition and clinical outcomes remain undefined. This randomized phase 2 study investigated the safety, efficacy, and pharmacodynamics of a selective BTK inhibitor acalabrutinib at 100 mg twice daily (BID) or 200 mg once daily (QD) in 48 patients with relapsed/refractory or high-risk treatment-naïve CLL. Acalabrutinib was well tolerated and yielded an overall response rate (ORR) of partial response or better of 95.8% (95% confidence interval [CI], 78.9-99.9) and an estimated progression-free survival (PFS) rate at 24 months of 91.5% (95% CI, 70.0-97.8) with BID dosing and an ORR of 79.2% (95% CI, 57.9-92.9) and an estimated PFS rate at 24 months of 87.2% (95% CI, 57.2-96.7) with QD dosing. BTK resynthesis was faster in patients with CLL than in healthy volunteers. BID dosing maintained higher BTK occupancy and achieved more potent pathway inhibition compared with QD dosing. Small increments in occupancy attained by BID dosing relative to QD dosing compounded over time to augment downstream biological effects. The impact of BTK occupancy on long-term clinical outcomes remains to be determined. This trial was registered at www.clinicaltrials.gov as #NCT02337829.


Assuntos
Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Antineoplásicos/uso terapêutico , Benzamidas/uso terapêutico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Proteínas de Neoplasias/antagonistas & inibidores , Inibidores de Proteínas Quinases/uso terapêutico , Pirazinas/uso terapêutico , Tirosina Quinase da Agamaglobulinemia/biossíntese , Tirosina Quinase da Agamaglobulinemia/genética , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Benzamidas/administração & dosagem , Benzamidas/efeitos adversos , Esquema de Medicação , Indução Enzimática , Feminino , Cefaleia/induzido quimicamente , Doenças Hematológicas/induzido quimicamente , Humanos , Leucemia Linfocítica Crônica de Células B/enzimologia , Leucemia Linfocítica Crônica de Células B/genética , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Dor/induzido quimicamente , Intervalo Livre de Progressão , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/efeitos adversos , Pirazinas/administração & dosagem , Pirazinas/efeitos adversos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , RNA-Seq , Transcriptoma , Resultado do Tratamento
15.
Drug Metab Dispos ; 50(4): 374-385, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35094979

RESUMO

The proteomes of ordered and disordered lipid microdomains in rat liver microsomes from control and phenobarbital (PB)-treated rats were determined after solubilization with Brij 98 and analyzed by tandem mass tag (TMT)-liquid chromatography-mass spectrometry (LC-MS). This allowed characterization of the liver microsomal proteome and the effects of phenobarbital-mediated induction, focusing on quantification of the relative levels of the drug-metabolizing enzymes._The microsomal proteome from control rats was represented by 333 (23%) proteins from ordered lipid microdomains, 517 (36%) proteins from disordered lipid domains, and 587 (41%) proteins that uniformly distributed between lipid microdomains. Most enzymes related to drug metabolism were mainly localized in disordered lipid microdomains. However, cytochrome P450 (CYP) 1A2, multiple forms of CYP2D, and several forms of UDP glucuronosyltransferases (UGT) 1A1 and 1A6) localized to ordered lipid microdomains. Other drug-metabolizing enzymes, including several forms of cytochromes P450, were uniformly distributed between the ordered and disordered regions. The redox partners, NADPH-cytochrome P450 reductase and cytochrome b5, localized to disordered microdomains. PB induction resulted in only modest changes in protein localization. Less than five proteins were variably associated with the ordered and disordered membrane microdomains in PB and control microsomes. PB induction was associated with fewer proteins localizing in the disordered membranes and more being uniformly distributed or localized to ordered domains. Ingenuity Pathway Analysis (IPA) was used to ascertain the effect of PB on cellular pathways, resulting in attenuation of pathways related to energy storage/utilization and overall cellular signaling and an increase in those related to degradative pathways. SIGNIFICANCE STATEMENT: This work identifies the lipid microdomain localization of the proteome from control and phenobarbital-induced rat liver microsomes. Thus, it provides an initial framework to understand how lipid/protein segregation influences protein-protein interactions in a tissue extract commonly used for studies in drug metabolism and uses bioinformatics to elucidate the effects of phenobarbital induction on cellular pathways.


Assuntos
Lipídeos de Membrana , Microssomos Hepáticos , Animais , Biologia Computacional , Sistema Enzimático do Citocromo P-450/metabolismo , Indução Enzimática , Lipídeos de Membrana/metabolismo , Microssomos Hepáticos/metabolismo , Fenobarbital/metabolismo , Fenobarbital/farmacologia , Óleos de Plantas , Polietilenoglicóis , Proteômica , Ratos
16.
Drug Metab Dispos ; 50(2): 105-113, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34857529

RESUMO

Screening for cytochrome P450 (CYP) induction potential is routine in drug development. Induction results in a net increase in CYP protein and is assessed typically by measuring indirect endpoints, i.e., enzyme activity and mRNA in vitro. Recent methodological advancements have made CYP protein quantification by liquid chromatography-mass spectrometry in vitro induction studies more accessible and amenable to routine testing. In this study, we evaluated CYP3A4 concentration dependence of induction response for 11 compounds (rifampin, rifabutin, carbamazepine, efavirenz, nitrendipine, flumazenil, pioglitazone, rosiglitazone, troglitazone, pazopanib, and ticagrelor) in plated hepatocytes from two or three donors incorporating in the assessment all three endpoints. In addition, the time-dependence of the induction was examined over 1, 2, or 3 days of treatment. For most compounds, mRNA, enzyme activity, and protein endpoints exhibited similarity in induction responses. Pazopanib and ticagrelor were notable exceptions as neither protein nor enzyme activity were induced despite mRNA induction of a magnitude similar to efavirenz, pioglitazone, or rosiglitazone, which clearly induced in all three endpoints. Static modeling of clinical induction responses supported a role for protein as a predictive endpoint. These data highlight the value of including CYP protein quantification as an induction assay endpoint to provide a more comprehensive assessment of induction liability. SIGNIFICANCE STATEMENT: Direct, liquid chromatography-mass spectrometry (LC-MS)-based quantification of cytochrome P450 (CYP) protein is a desirable induction assay endpoint; however such application has been limited due to inefficient workflows. Here, we incorporate recent advancements in protein quantitation methods to efficiently quantify CYP3A4 protein in in vitro induction assays with 11 compounds in up to 3 donors. The data indicate induction responses from mRNA do not always align with those of protein suggesting assessment of induction liability is more complex than thought previously.


Assuntos
Citocromo P-450 CYP3A , Hepatócitos , Células Cultivadas , Cromatografia Líquida/métodos , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Indução Enzimática , Hepatócitos/metabolismo , Humanos , Espectrometria de Massas , RNA Mensageiro/metabolismo
17.
Drug Metab Dispos ; 50(7): 1000-1001, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35504657

RESUMO

The concept of hepatic induction of drug-metabolizing cytochrome P450s (P450s) by xenobiotics, including therapeutic drugs, was proposed in the early 1960s. A polycyclic aromatic hydrocarbon and phenobarbital have been the two major inducers used to investigate this induction mechanism. Currently, the mechanisms mediated by aryl hydrocarbon receptor and constitutive androstane receptor are well-established. In addition to mammals, insects and fungi also express P450s and induce them following exposure to insecticides. These inductions may have environmental consequences. Finding the molecular mechanism regulating these inductions will be of major interest in the future. SIGNIFICANCE STATEMENT: This paper summarizes present and future of investigations into induction of drug-metabolizing enzymes.


Assuntos
Sistema Enzimático do Citocromo P-450 , Microssomos Hepáticos , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Indução Enzimática , Fígado/metabolismo , Mamíferos/metabolismo , Microssomos Hepáticos/metabolismo , Xenobióticos/farmacologia
18.
J Am Soc Nephrol ; 32(11): 2834-2850, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34716244

RESUMO

BACKGROUND: CKD, characterized by retained uremic solutes, is a strong and independent risk factor for thrombosis after vascular procedures . Urem ic solutes such as indoxyl sulfate (IS) and kynurenine (Kyn) mediate prothrombotic effect through tissue factor (TF). IS and Kyn biogenesis depends on multiple enzymes, with therapeutic implications unexplored. We examined the role of indoleamine 2,3-dioxygenase-1 (IDO-1), a rate-limiting enzyme of kynurenine biogenesis, in CKD-associated thrombosis after vascular injury. METHODS: IDO-1 expression in mice and human vessels was examined. IDO-1-/- mice, IDO-1 inhibitors, an adenine-induced CKD, and carotid artery injury models were used. RESULTS: Both global IDO-1-/- CKD mice and IDO-1 inhibitor in wild-type CKD mice showed reduced blood Kyn levels, TF expression in their arteries, and thrombogenicity compared with respective controls. Several advanced IDO-1 inhibitors downregulated TF expression in primary human aortic vascular smooth muscle cells specifically in response to uremic serum. Further mechanistic probing of arteries from an IS-specific mouse model, and CKD mice, showed upregulation of IDO-1 protein, which was due to inhibition of its polyubiquitination and degradation by IS in vascular smooth muscle cells. In two cohorts of patients with advanced CKD, blood IDO-1 activity was significantly higher in sera of study participants who subsequently developed thrombosis after endovascular interventions or vascular surgery. CONCLUSION: Leveraging genetic and pharmacologic manipulation in experimental models and data from human studies implicate IS as an inducer of IDO-1 and a perpetuator of the thrombotic milieu and supports IDO-1 as an antithrombotic target in CKD.


Assuntos
Indicã/fisiologia , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Indolamina-Pirrol 2,3,-Dioxigenase/sangue , Cinurenina/fisiologia , Terapia de Alvo Molecular , Complicações Pós-Operatórias/enzimologia , Insuficiência Renal Crônica/enzimologia , Trombose/enzimologia , Procedimentos Cirúrgicos Vasculares/efeitos adversos , Animais , Aorta , Lesões das Artérias Carótidas/complicações , Trombose das Artérias Carótidas/etiologia , Trombose das Artérias Carótidas/prevenção & controle , Meios de Cultura/farmacologia , Indução Enzimática/efeitos dos fármacos , Retroalimentação Fisiológica , Feminino , Células HEK293 , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/deficiência , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Cinurenina/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos de Músculo Liso/efeitos dos fármacos , Complicações Pós-Operatórias/sangue , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/prevenção & controle , Insuficiência Renal Crônica/tratamento farmacológico , Tromboplastina/metabolismo , Trombose/sangue , Trombose/etiologia , Trombose/prevenção & controle , Triptofano/metabolismo , Uremia/sangue
19.
Int J Mol Sci ; 23(7)2022 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-35408925

RESUMO

In cattle, phenobarbital (PB) upregulates target drug-metabolizing enzyme (DME) mRNA levels. However, few data about PB's post-transcriptional effects are actually available. This work provides the first, and an almost complete, characterization of PB-dependent changes in DME catalytic activities in bovine liver using common probe substrates and confirmatory immunoblotting investigations. As expected, PB increased the total cytochrome P450 (CYP) content and the extent of metyrapone binding; moreover, an augmentation of protein amounts and related enzyme activities was observed for known PB targets such as CYP2B, 2C, and 3A, but also CYP2E1. However, contradictory results were obtained for CYP1A, while a decreased catalytic activity was observed for flavin-containing monooxygenases 1 and 3. The barbiturate had no effect on the chosen hydrolytic and conjugative DMEs. For the first time, we also measured the 26S proteasome activity, and the increase observed in PB-treated cattle would suggest this post-translational event might contribute to cattle DME regulation. Overall, this study increased the knowledge of cattle hepatic drug metabolism, and further confirmed the presence of species differences in DME expression and activity between cattle, humans, and rodents. This reinforced the need for an extensive characterization and understanding of comparative molecular mechanisms involved in expression, regulation, and function of DMEs.


Assuntos
Fenobarbital , Xenobióticos , Animais , Bovinos , Sistema Enzimático do Citocromo P-450/metabolismo , Indução Enzimática , Fígado/metabolismo , Microssomos Hepáticos/metabolismo , Fenobarbital/farmacologia , Xenobióticos/metabolismo
20.
Int J Mol Sci ; 23(5)2022 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-35269594

RESUMO

The abnormal accumulation of methylglyoxal (MG) leading to increased glycation of protein and DNA has emerged as an important metabolic stress, dicarbonyl stress, linked to aging, and disease. Increased MG glycation produces inactivation and misfolding of proteins, cell dysfunction, activation of the unfolded protein response, and related low-grade inflammation. Glycation of DNA and the spliceosome contribute to an antiproliferative and apoptotic response of high, cytotoxic levels of MG. Glyoxalase 1 (Glo1) of the glyoxalase system has a major role in the metabolism of MG. Small molecule inducers of Glo1, Glo1 inducers, have been developed to alleviate dicarbonyl stress as a prospective treatment for the prevention and early-stage reversal of type 2 diabetes and prevention of vascular complications of diabetes. The first clinical trial with the Glo1 inducer, trans-resveratrol and hesperetin combination (tRES-HESP)-a randomized, double-blind, placebo-controlled crossover phase 2A study for correction of insulin resistance in overweight and obese subjects, was completed successfully. tRES-HESP corrected insulin resistance, improved dysglycemia, and low-grade inflammation. Cell permeable Glo1 inhibitor prodrugs have been developed to induce severe dicarbonyl stress as a prospective treatment for cancer-particularly for high Glo1 expressing-related multidrug-resistant tumors. The prototype Glo1 inhibitor is prodrug S-p-bromobenzylglutathione cyclopentyl diester (BBGD). It has antitumor activity in vitro and in tumor-bearing mice in vivo. In the National Cancer Institute human tumor cell line screen, BBGD was most active against the glioblastoma SNB-19 cell line. Recently, potent antitumor activity was found in glioblastoma multiforme tumor-bearing mice. High Glo1 expression is a negative survival factor in chemotherapy of breast cancer where adjunct therapy with a Glo1 inhibitor may improve treatment outcomes. BBGD has not yet been evaluated clinically. Glycation by MG now appears to be a pathogenic process that may be pharmacologically manipulated for therapeutic outcomes of potentially important clinical impact.


Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Glutationa/análogos & derivados , Hesperidina/uso terapêutico , Lactoilglutationa Liase/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Resveratrol/uso terapêutico , Animais , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Quimioterapia Combinada , Indução Enzimática/efeitos dos fármacos , Glutationa/química , Glutationa/uso terapêutico , Glicosilação/efeitos dos fármacos , Hesperidina/química , Humanos , Resistência à Insulina/fisiologia , Lactoilglutationa Liase/antagonistas & inibidores , Camundongos , Estrutura Molecular , Neoplasias Experimentais/metabolismo , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Obesidade/fisiopatologia , Aldeído Pirúvico/química , Aldeído Pirúvico/metabolismo , Resveratrol/química
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