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1.
Methods Mol Biol ; 2342: 653-664, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34272710

RESUMO

In this chapter, we illustrate the criticality of proper fitting of enzyme kinetic data. Simple techniques are provided to arrive at meaningful kinetic parameters, illustrated using an example, nonmonotonic data set. In the initial analysis of this data set, derived Km and Vmax parameters incorporated into PBPK models resulted in outcomes that did not adequately describe clinical data. This prompted a re-review of the in vitro data set and curve-fitting procedures. During this review, it was found that the 3-parameter model was fitted on data that was improperly unweighted. Reanalysis of the data using a weighted model returned a better fit and resulted in kinetic parameters better aligning with clinical data. Tools and techniques used to identify and compare kinetic models of this data set are provided, including various replots, visual inspection, examination of residuals, and the Akaike information criterion.


Assuntos
Citocromo P-450 CYP3A/metabolismo , Preparações Farmacêuticas/análise , Algoritmos , Cromatografia , Análise de Dados , Humanos , Técnicas In Vitro , Cinética , Modelos Teóricos , Preparações Farmacêuticas/química
2.
Chem Biol Interact ; 345: 109559, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34153224

RESUMO

AIM: We aimed (i) to study the effects of genetic polymorphism of cytochrome P450 3A4 (CYP3A4) and drug interactions on acalabrutinib (ACA) metabolism and (ii) to investigate the mechanisms underlying the effects of CYP3A4 variants on the differential kinetic profiles of ACA and ibrutinib. METHOD: Recombinant human CYP3A4 and variants were expressed using a Bac-to-Bac baculovirus expression system. The cell microsome was prepared and subjected to kinetic study. The analyte concentrations were determined by UPLC-MS/MS. A molecular docking assay was employed to investigate the mechanisms leading to differences in kinetic profiles. RESULTS: The kinetic parameters of ACA, catalyzed by CYP3A4 and 28 of its variants, were determined, including Vmax, Km, and Ksi. CYP3A4.6-8, 12, 13, 17, 18, 20, and 30 lost their catalytic function. No significant differences were found for CYP3A4.4, 5, 10, 15, 31, and 34 compared with CYP3A4.1 with respect to intrinsic clearance (Vmax/Km, Clint). However, the Clint values of CYP3A4.9, 14, 16, 19, 23, 24, 28, 32 were obviously decreased, ranging from 0.02 to 0.05 µL/min/pmol. On the contrary, the catalytic activities of CYP3A4.2, 3, 11, 29, and 33 were increased dramatically. The Clint value of CYP3A4.11 was 5.95 times as high as that of CYP3A4.1. Subsequently, CYP3A4.1, 3, 11, 23, and 28 were chosen to study the kinetic changes in combination with ketoconazole. Interestingly, we found the inhibitory potency of ketoconazole varied in different variants. In addition, the kinetic parameters of ibrutinib and ACA were accordingly compared in different CYP3A4 variants. Significant differences in relative clearance were observed among variants, which would probably influence the distance between the redox site and the heme iron atom. CONCLUSION: Genetic polymorphism of CYP3A4 extensively changes its ACA-metabolizing enzymatic activity. In combination with a CYP inhibitor, its inhibitory potency also varied among different variants. Even the same variants exhibited different capabilities catalyzing ACA. Its enzymatic capabilities are probably determined by the distance between the substrate and the heme iron atom, which could be impacted by mutation.


Assuntos
Benzamidas/metabolismo , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Variação Genética , Pirazinas/metabolismo , Biocatálise , Citocromo P-450 CYP3A/química , Heme/metabolismo , Humanos , Simulação de Acoplamento Molecular , Oxirredução , Conformação Proteica
3.
Life Sci ; 280: 119666, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34087279

RESUMO

AIMS: The preclinical evaluation of innovative drugs plays an important role in the new drugs development. As a derivative of pirfenidone (PFD), mefunidone (MFD) has shown better anti-fibrosis and anti-inflammatory activity in both cell lines and animal models. To support the clinical investigations of MFD, the metabolic characterization of MFD was initially evaluated in preclinical models. MAIN METHODS: The potential metabolites of MFD were analyzed by LC-MS/MS methods. The induction effect of MFD on CYP1A2, CYP2B6, and CYP3A4 was performed in primary human hepatocytes, and the inhibition of CYP enzymes by MFD was also evaluated in human liver microsomes. Finally, the pharmacokinetic profiles of MFD were assessed in SD rats after the rats had received multiple doses (62.5 mg/kg) of MFD. KEY FINDINGS: MFD was metabolized in three pathways including oxidation, N-demethylation, and hydroxylation. Except for slight inhibition on the activity of CYP2D6, MFD exerted no effect on other CYP enzymes. Moreover, drug accumulation of MFD was not observed in rats after repeated dosing of MFD. SIGNIFICANCE: MFD was first discovered in preclinical investigations without inducing and inhibiting metabolic enzymes. This work provides some important information about the metabolic characterization of MFD for its further clinical investigations.


Assuntos
Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacocinética , Hepatócitos/metabolismo , Piperazinas/metabolismo , Piperazinas/farmacocinética , Piridonas/metabolismo , Piridonas/farmacocinética , Animais , Anti-Inflamatórios/farmacologia , Células Cultivadas , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP3A/metabolismo , Hepatócitos/efeitos dos fármacos , Humanos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Piperazinas/farmacologia , Piridonas/farmacologia , Ratos , Ratos Sprague-Dawley
4.
Molecules ; 26(10)2021 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-34069400

RESUMO

Acacetin, apigenin, chrysin, and pinocembrin are flavonoid aglycones found in foods such as parsley, honey, celery, and chamomile tea. Flavonoids can act as substrates and inhibitors of the CYP3A4 enzyme, a heme containing enzyme responsible for the metabolism of one third of drugs on the market. The aim of this study was to investigate the inhibitory effect of selected flavonoids on the CYP3A4 enzyme, the kinetics of inhibition, the possible covalent binding of the inhibitor to the enzyme, and whether flavonoids can act as pseudo-irreversible inhibitors. For the determination of inhibition kinetics, nifedipine oxidation was used as a marker reaction. A hemochromopyridine test was used to assess the possible covalent binding to the heme, and incubation with dialysis was used in order to assess the reversibility of the inhibition. All the tested flavonoids inhibited the CYP3A4 enzyme activity. Chrysin was the most potent inhibitor: IC50 = 2.5 ± 0.6 µM, Ki = 2.4 ± 1.0 µM, kinact = 0.07 ± 0.01 min-1, kinact/Ki = 0.03 min-1 µM-1. Chrysin caused the highest reduction of heme (94.5 ± 0.5% residual concentration). None of the tested flavonoids showed pseudo-irreversible inhibition. Although the inactivation of the CYP3A4 enzyme is caused by interaction with heme, inhibitor-heme adducts could not be trapped. These results indicate that flavonoids have the potential to inhibit the CYP3A4 enzyme and interact with other drugs and medications. However, possible food-drug interactions have to be assessed clinically.


Assuntos
Citocromo P-450 CYP3A/efeitos dos fármacos , Flavonoides/farmacologia , Citocromo P-450 CYP3A/metabolismo , Flavonoides/química , Humanos , Cinética , Estrutura Molecular , Nifedipino/metabolismo , Oxirredução
5.
J Med Chem ; 64(12): 8437-8446, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34097831

RESUMO

Paclitaxel (PTX) is a first-line treatment in breast cancer, though resistance develops quickly and frequently. Cytochrome P450 enzymes CYP3A4 and CYP2C8, which metabolically inactivate PTX in hepatic tissue, are overexpressed in malignant breast tissues. CYP3A4 expression correlates with PTX therapy failure and poor outcomes, though no direct evidence of CYP3A4 contributing to PTX sensitivity exists. Because CYP3A4/2C8 is susceptible to carbon monoxide (CO)-mediated inhibition and CO (a gaseous signaling molecule) has previously exhibited drug-sensitizing effects in cancer cells, we hypothesized that CO-mediated inhibition of CYP3A4/2C8 could lead to enhanced drug sensitivity. Using a photo-activated CO-releasing molecule, we have assessed the ability of CO to alter the pharmacokinetics of PTX in breast cancer cells via inhibition of CYP3A4/2C8 and determined that CO does enhance sensitivity of breast cancer cells to PTX. Inhibition of CYP3A4/2C8 by CO could therefore be a promising therapeutic strategy to enhance PTX response in breast cancer.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Monóxido de Carbono/farmacologia , Inibidores do Citocromo P-450 CYP2C8/farmacologia , Inibidores do Citocromo P-450 CYP3A/farmacologia , Paclitaxel/farmacologia , Antineoplásicos/farmacocinética , Monóxido de Carbono/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cloranfenicol/farmacologia , Complexos de Coordenação/farmacologia , Complexos de Coordenação/efeitos da radiação , Citocromo P-450 CYP2C8/metabolismo , Inibidores do Citocromo P-450 CYP2C8/efeitos da radiação , Citocromo P-450 CYP3A/metabolismo , Inibidores do Citocromo P-450 CYP3A/efeitos da radiação , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Luz , Manganês/química , Paclitaxel/farmacocinética
6.
Int J Mol Sci ; 22(11)2021 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-34072457

RESUMO

Cytochrome P450 3A7 (CYP3A7) is a fetal/neonatal liver enzyme that participates in estriol synthesis, clearance of all-trans retinoic acid, and xenobiotic metabolism. Compared to the closely related major drug-metabolizing enzyme in adult liver, CYP3A4, the ligand binding and catalytic capacity of CYP3A7 are substantially reduced. To better understand the structural basis for these functional differences, the 2.15 Å crystal structure of CYP3A7 has been solved. Comparative analysis of CYP3A enzymes shows that decreased structural plasticity rather than the active site microenvironment defines the ligand binding ability of CYP3A7. In particular, a rotameric switch in the gatekeeping amino acid F304 triggers local and long-range rearrangements that transmit to the F-G fragment and alter its interactions with the I-E-D-helical core, resulting in a more rigid structure. Elongation of the ß3-ß4 strands, H-bond linkage in the substrate channel, and steric constraints in the C-terminal loop further increase the active site rigidity and limit conformational ensemble. Collectively, these structural distinctions lower protein plasticity and change the heme environment, which, in turn, could impede the spin-state transition essential for optimal reactivity and oxidation of substrates.


Assuntos
Citocromo P-450 CYP3A/química , Ligantes , Sequência de Aminoácidos , Aminoácidos , Sítios de Ligação , Catálise , Domínio Catalítico , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Humanos , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Mutação , Polimorfismo Genético , Ligação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato
7.
Lab Invest ; 101(9): 1197-1209, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34031539

RESUMO

Uremic toxin accumulation is one possible reason for alterations in hepatic drug metabolism in patients with chronic kidney disease (CKD). However, the types of uremic toxins and underlying mechanisms are poorly understood. In this study, we report the role of advanced oxidation protein products (AOPPs), a modified protein uremic toxin, in the downregulation of cytochromes P450 1A2 (CYP1A2) and P450 3A4 (CYP3A4) expression levels and activities. We found that AOPP accumulation in plasma in a rat CKD model was associated with decreased protein levels of CYP1A2 and CYP3A4. CYP1A2 and CYP3A4 metabolites (acetaminophen and 6ß-hydroxytestosterone, respectively,) in liver microsomes were also significantly decreased. In human hepatocytes, AOPPs significantly decreased CYP1A2 and CYP3A4 protein levels in a dose- and time-dependent manner and downregulated their activities; however, bovine serum albumin (BSA), a synthetic precursor of AOPPs, had no effect on these parameters. The effect of AOPPs was associated with upregulation of p-IKKα/ß, p-IκBα, p-NF-κB, and inflammatory cytokines protein levels and increases in p-IKKα/ß/IKKα, p-IκBα/IκBα, and p-NF-κB/NF-κB phosphorylation ratios. Further, NF-kB pathway inhibitors BAY-117082 and PDTC abolished the downregulatory effects of AOPPs. These findings suggest that AOPPs downregulate CYP1A2 and CYP3A4 expression and activities by increasing inflammatory cytokine production and stimulating NF-κB-mediated signaling. Protein uremic toxins, such as AOPPs, may modify the nonrenal clearance of drugs in patients with CKD by influencing metabolic enzymes.


Assuntos
Produtos da Oxidação Avançada de Proteínas/farmacologia , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP3A/metabolismo , Regulação para Baixo/efeitos dos fármacos , NF-kappa B/metabolismo , Animais , Linhagem Celular , Modelos Animais de Doenças , Células Hep G2 , Humanos , Masculino , Microssomos Hepáticos/metabolismo , Ratos , Ratos Sprague-Dawley , Insuficiência Renal Crônica/metabolismo , Transdução de Sinais/efeitos dos fármacos
8.
Chem Biol Interact ; 345: 109527, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34058179

RESUMO

Several therapeutic options are available for type 1 Gaucher disease (GD1), including enzymatic replacement therapy (ERT) and substrate reduction therapy (SRT). Eliglustat is a selective inhibitor of glucosylceramide synthase that is extensively metabolized by CYP2D6 and, to a lesser extent by CYP3A4; it is also an inhibitor of the P-gp transporter. The aim of this study is to evaluate the metabolizer profile of these cytochrome isoforms in 61 GD1 patients, and to analyze interferences with concomitant therapies. Patients were selected from the Spanish Gaucher Disease Registry considering clinical data, GBA genotype, severity score index, comorbidities, concomitant drugs, type and response to therapy and adverse effects. The polymorphisms of CYP2D6, CYP3A4 and three ABCB1 transporter variants were analyzed by Polymerase Chain Reaction (PCR). The most frequent metabolizer profile was extensive or intermediate for CYP2D6, extensive for CYP3A4*1B and CYP3A4*22 and normal activity for ABCB1. Correlations between metabolizer profile and other variables were analyzed by multiple regression study. Twenty-eight patients received ERT, 17 eliglustat and seven miglustat. Forty-two patients (68.8%) had associated diseases and 54.5% were taking daily concomitant medication. Nine patients under eliglustat therapy received concomitant drugs that interact with the CYPs and/or ABCB1, five of these did not reach therapeutic goals and three presented mild or moderate adverse effects (headache and gastrointestinal disorders). Detailed analysis in four patients with TTT haplotype, corresponding to lack of activity of the transporter, was performed. In order to apply personalized medicine and avoid interferences and adverse effects, the individual CYP metabolizer profile and transporter must be considered when choosing the concomitant medication and/or making dose adjustments.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP3A/metabolismo , Doença de Gaucher/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP3A/genética , Feminino , Doença de Gaucher/genética , Doença de Gaucher/terapia , Haplótipos , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo Genético , Espanha , Adulto Jovem
9.
Biomed Pharmacother ; 139: 111631, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33940510

RESUMO

The exposure to linezolid is characterized by a large inter-individual variability; age, renal dysfunction and body weight explain this variability only to a limited extent and a considerable portion of it remains unexplained; therefore, we decided to investigate the role of individual genetic background focusing in particular on the risk of linezolid underexposure. 191 patients in therapy with linezolid at the standard dose of 600 mg twice daily were considered. Linezolid plasma concentration was determined at the steady state and classified as "below", "within" or "above" reference range. Genetic polymorphisms for ATP Binding Cassette Subfamily B Member 1 (ABCB1), Cytochrome P450 (CYP) enzymes CYP3A4 and CYP3A5, and Cytochrome P450 Oxidoreductase (POR) were investigated. Age significantly correlated with drug exposure, and patients CYP3A5 expressers (GA and AA) were found at high risk to be underexposed to the drug when treated at standard dose. This association was confirmed even after correction with age. No association was found with ABCB1 polymorphism. Our data suggest that CYP3A5 polymorphisms might significantly affect linezolid disposition, putting patients at higher risk to be underexposed, while P-glycoprotein polymorphism seem not to play any role.


Assuntos
Anti-Infecciosos/farmacocinética , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Linezolida/farmacocinética , Farmacogenética , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/metabolismo , Anti-Infecciosos/administração & dosagem , Feminino , Regulação Enzimológica da Expressão Gênica , Genótipo , Humanos , Linezolida/administração & dosagem , Masculino , Pessoa de Meia-Idade , Polimorfismo Genético/genética , Estudos Retrospectivos , Adulto Jovem
10.
Eur J Med Chem ; 220: 113496, 2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-33933755

RESUMO

The synthesis of two isomeric testosterone dimers and an androstenedione dimer is reported. The design takes advantage of an efficient transformation of testosterone leading to the synthesis of the key diene, 7α-(buta-1,3-dienyl)-4-androsten-17ß-ol-3-one, through an elimination reaction. It was found that in some instances the same reaction led to partial epimerization of the 17ß-hydroxyl group into the 17α-hydroxyl group. The specific orientation of the hydroxyl function was confirmed by NMR spectroscopy. Capitalizing on this unforeseen side reaction, several dimers were assembled using an olefin metathesis reaction with Hoveyda-Grubbs catalyst. This led to the formation of two isomeric testosterone dimers with 17α-OH or 17ß-OH (14α and 14ß) as well as an androstenedione dimer (14). The new dimers and their respective precursors were tested on androgen-dependent (LNCaP) and androgen independent (PC3 and DU145) prostate cancer cells. It was discovered that the most active dimer was made of the natural hormone testosterone (14ß) with an average IC50 of 13.3 µM. In LNCaP cells, 14ß was ∼5 times more active than the antiandrogen drug cyproterone acetate (IC50 of 12.0 µM vs. 59.6 µM, respectively). At low concentrations (0.25-0.5 µM), 14α and 14ß were able to completely inhibit LNCaP cell growth induced by testosterone or dihydrotestosterone. Furthermore, cross-reactivity of androgen-based dimers with sterol-metabolizing cytochrome P450 3A4 was explored and the results are disclosed herein.


Assuntos
Androstenodiona/farmacologia , Antineoplásicos/farmacologia , Citocromo P-450 CYP3A/metabolismo , Desenho de Fármacos , Neoplasias da Próstata/tratamento farmacológico , Testosterona/farmacologia , Androstenodiona/síntese química , Androstenodiona/química , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dimerização , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Masculino , Estrutura Molecular , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Recombinantes , Relação Estrutura-Atividade , Testosterona/síntese química , Testosterona/química , Células Tumorais Cultivadas
11.
J Med Chem ; 64(10): 6413-6522, 2021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-34003642

RESUMO

This perspective discusses the role of pregnane xenobiotic receptor (PXR) in drug discovery and the impact of its activation on CYP3A4 induction. The use of structural biology to reduce PXR activity on drug discovery projects has become more common in recent years. Analysis of this work highlights several important molecular interactions, and the resultant structural modifications to reduce PXR activity are summarized. The computational approaches undertaken to support the design of new drugs devoid of PXR activation potential are also discussed. Finally, the SAR of empirical design strategies to reduce PXR activity is reviewed, and the key SAR transformations are discussed and summarized. In conclusion, this perspective demonstrates that PXR activity can be greatly diminished or negated on active drug discovery projects with the knowledge now available. This perspective should be useful to anyone who seeks to reduce PXR activity on a drug discovery project.


Assuntos
Descoberta de Drogas , Receptor de Pregnano X/metabolismo , Sítios de Ligação , Citocromo P-450 CYP3A/metabolismo , Desenho de Fármacos , Humanos , Ligantes , Simulação de Dinâmica Molecular , Receptor de Pregnano X/antagonistas & inibidores , Rifampina/química , Rifampina/metabolismo , Relação Estrutura-Atividade
12.
Eur J Pharm Sci ; 162: 105826, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-33813039

RESUMO

Resiquimod (R-848) is an immune response modifier activating toll-like receptor 7 and 8. Its potential to cause pharmacokinetic interactions with concurrently administered drugs is unknown. To study the time course of the effect of resiquimod in LS180 cells as a model for intestinal tissue, luciferase-based reporter gene assays and reverse transcription polymerase chain reaction were used to investigate whether resiquimod affects the activities of nuclear factor kappa B (NF-ĸB), pregnane x receptor (PXR) or the transcription of selected central genes for drug disposition (cytochrome P-450 isozyme 3A4 (CYP3A4), CYP1A1, UDP-glucuronosyltransferase 1A1 (UGT1A1), ATP-binding cassette transporters ABCC2, ABCB1). Its impact on the activities of organic anion transporting polypeptides 1 or 3 (OATP1B1/3), breast cancer resistance protein (BCRP), P-glycoprotein (P-gp) or CYP3A4 was evaluated using fluorescence- or luminescence-based activity assays. Resiquimod irrelevantly increased NF-ĸB activity after 2 h (1 µM: 1.07-fold, P = 0.0188; 10 µM: 1.09-fold, P = 0.0142), and diminished it after 24 h (1 µM: 0.64-fold, P < 0.0001; 10 µM: 0.68-fold, P < 0.0001) and 30 h (10 µM: 0.68-fold, P = 0.0003). Concurrently, PXR activity after 24 h was marginally increased by 10 µM (1.05-fold, P = 0.0019). Resiquimod did not alter mRNA expression levels, activities of uptake or efflux transporters, or CYP3A4 activity. Given the marginal effects on NF-ĸB, PXR, expression levels of selected PXR target genes, and activities of important drug transporters and CYP3A4 in vitro, resiquimod is not expected to cause major pharmacokinetic drug-drug interactions in vivo.


Assuntos
Imidazóis/farmacologia , Receptores de Esteroides , Receptor 7 Toll-Like/agonistas , Receptor 8 Toll-Like/agonistas , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Proteínas de Neoplasias , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo
13.
J Biol Chem ; 296: 100668, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33865853

RESUMO

CYP24A1-deficient (Cyp24a1 KO) rats were generated using the CRISPER/Cas9 system to investigate CYP24A1-dependent or -independent metabolism of 25(OH)D3, the prohormone of calcitriol. Plasma 25(OH)D3 concentrations in Cyp24a1 KO rats were approximately twofold higher than in wild-type rats. Wild-type rats showed five metabolites of 25(OH)D3 in plasma following oral administration of 25(OH)D3, and these metabolites were not detected in Cyp24a1 KO rats. Among these metabolites, 25(OH)D3-26,23-lactone was identified as the second major metabolite with a significantly higher Tmax value than others. When 23S,25(OH)2D3 was administered to Cyp24a1 KO rats, neither 23,25,26(OH)3D3 nor 25(OH)D3-26,23-lactone was observed. However, when 23S,25R,26(OH)3D3 was administered to Cyp24a1 KO rats, plasma 25(OH)D3-26,23-lactone was detected. These results suggested that CYP24A1 is responsible for the conversion of 25(OH)D3 to 23,25,26(OH)3D3 via 23,25(OH)2D3, but enzyme(s) other than CYP24A1 may be involved in the conversion of 23,25,26(OH)3D3 to 25(OH)D3-26,23-lactone. Enzymatic studies using recombinant human CYP species and the inhibitory effects of ketoconazole suggested that CYP3A plays an essential role in the conversion of 23,25,26(OH)3D3 into 25(OH)D3-26,23-lactone in both rats and humans. Taken together, our data indicate that Cyp24a1 KO rats are valuable for metabolic studies of vitamin D and its analogs. In addition, long-term administration of 25(OH)D3 to Cyp24a1 KO rats at 110 µg/kg body weight/day resulted in significant weight loss and ectopic calcification. Thus, Cyp24a1 KO rats could represent an important model for studying renal diseases originating from CYP24A1 dysfunction.


Assuntos
Sistemas CRISPR-Cas , Calcifediol/metabolismo , Citocromo P-450 CYP3A/metabolismo , Metaboloma/efeitos dos fármacos , Vitamina D3 24-Hidroxilase/antagonistas & inibidores , Vitaminas/metabolismo , Animais , Animais Geneticamente Modificados , Calcifediol/administração & dosagem , Ratos , Vitamina D3 24-Hidroxilase/genética , Vitamina D3 24-Hidroxilase/metabolismo , Vitaminas/administração & dosagem
14.
J Chem Inf Model ; 61(5): 2418-2426, 2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-33884878

RESUMO

Human cytochrome P450 3A4 (CYP3A4) is responsible for the metabolism of ∼50% clinically used drugs. Midazolam (MDZ) is a commonly used sedative drug and serves as a marker substrate for the CYP3A4 activity assessment. MDZ is metabolized by CYP3A4 to two hydroxylation products, 1'-OH-MDZ and 4-OH-MDZ. It has been reported that the ratio of 1'-OH-MDZ and 4-OH-MDZ is dependent on the MDZ concentration, which reflects the homotropic cooperative behavior in MDZ metabolism by CYP3A4. Here, we used quantum chemistry (QC), molecular docking, conventional molecular dynamics (cMD), and Gaussian accelerated molecular dynamics (GaMD) approaches to investigate the mechanism of the interactions between CYP3A4 and MDZ. QC calculations suggest that C1' is less reactive for hydroxylation than C4, which is a pro-chirality carbon. However, the 4-OH-MDZ product is likely to be racemic due to the chirality inversion in the rebound step. The MD simulation results indicate that MDZ at the peripheral allosteric site is not stable and the binding modes of the MDZ molecules at the productive site are in line with the experimental observations.


Assuntos
Citocromo P-450 CYP3A , Midazolam , Sítio Alostérico , Citocromo P-450 CYP3A/metabolismo , Humanos , Simulação de Acoplamento Molecular , Oxirredução
15.
Int J Mol Sci ; 22(6)2021 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799598

RESUMO

We sought to develop a cell-based cytotoxicity assay using human hepatocytes, which reflect the effects of drug-metabolizing enzymes on cytotoxicity. In this study, we generated luminescent human hepatoblastoma HepG2 cells using the mouse artificial chromosome vector, in which click beetle luciferase alone or luciferase and major drug-metabolizing enzymes (CYP2C9, CYP2C19, CYP2D6, and CYP3A4) are expressed, and monitored the time-dependent changes of CYP-mediated cytotoxicity expression by bioluminescence measurement. Real-time bioluminescence measurement revealed that compared with CYP-non-expressing cells, the luminescence intensity of CYP-expressing cells rapidly decreased when the cells were treated with low concentrations of aflatoxin B1 or primaquine, which exhibits cytotoxicity in the presence of CYP3A4 or CYP2D6, respectively. Using kinetics data obtained by the real-time bioluminescence measurement, we estimated the time-dependent changes of 50% inhibitory concentration (IC50) values in the aflatoxin B1- and primaquine-treated cell lines. The first IC50 value was detected much earlier and at a lower concentration in primaquine-treated CYP-expressing HepG2 cells than in primaquine-treated CYP-non-expressing cells, and the decrease of IC50 values was much faster in the former than the latter. Thus, we successfully monitored time- and concentration-dependent dynamic changes of CYP-mediated cytotoxicity expression in CYP-expressing luminescent HepG2 cells by means of real-time bioluminescence measurement.


Assuntos
Aflatoxina B1/toxicidade , Efeito Fundador , Medições Luminescentes/métodos , Primaquina/toxicidade , Imagem com Lapso de Tempo/métodos , Xenobióticos/toxicidade , Animais , Linhagem Celular Tumoral , Citocromo P-450 CYP2C19/genética , Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP2C9/genética , Citocromo P-450 CYP2C9/metabolismo , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células Hep G2 , Humanos , Concentração Inibidora 50 , Luciferases/genética , Luciferases/metabolismo , Luminescência , Camundongos
16.
Xenobiotica ; 51(7): 752-763, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33896369

RESUMO

The induction of cytochrome P450s can result in reduced drug efficacy and lead to potential drug-drug interactions. The xenoreceptors-aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), and pregnane X receptor (PXR)-play key roles in CYP induction by xenobiotics. In order to be able to rapidly screen for the induction of three enzymes (CYP1A1, CYP2B6, and CYP3A4), we generated a stable AhR-responsive HepG2 cell line, a stable CAR-responsive HepG2 cell line, and a stable PXR-responsive HepG2 cell line.To validate these stable xenoreceptor-responsive HepG2 cell lines, we evaluated the induction of the different Gaussia reporter activities, as well as the mRNA and protein expression levels of endogenous CYPs in response to different inducers.The induction of luciferase activity in the stable xenoreceptor-responsive HepG2 cell lines by specific inducers occurred in a concentration dependent manner. There was a positive correlation between the induction of luciferase activities and the induction endogenous CYP mRNA expression levels. These xenoreceptor-responsive HepG2 cell lines were further validated with known CYP1A1, CYP2B6, and CYP3A4 inducers.These stable xenoreceptor-responsive HepG2 cell lines may be used in preclinical research for the rapid and sensitive detection of AhR, CAR, and PXR ligands that induce CYP450 isoforms.


Assuntos
Citocromo P-450 CYP3A , Receptores de Esteroides , Citocromo P-450 CYP1A1 , Citocromo P-450 CYP2B6/genética , Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP3A/metabolismo , Indução Enzimática , Genes Reporter , Hepatócitos/metabolismo , Luciferases/genética , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo
17.
J Med Chem ; 64(9): 5323-5344, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33872507

RESUMO

Herein we describe the discovery, mode of action, and preclinical characterization of the soluble guanylate cyclase (sGC) activator runcaciguat. The sGC enzyme, via the formation of cyclic guanosine monophoshphate, is a key regulator of body and tissue homeostasis. sGC activators with their unique mode of action are activating the oxidized and heme-free and therefore NO-unresponsive form of sGC, which is formed under oxidative stress. The first generation of sGC activators like cinaciguat or ataciguat exhibited limitations and were discontinued. We overcame limitations of first-generation sGC activators and identified a new chemical class via high-throughput screening. The investigation of the structure-activity relationship allowed to improve potency and multiple solubility, permeability, metabolism, and drug-drug interactions parameters. This program resulted in the discovery of the oral sGC activator runcaciguat (compound 45, BAY 1101042). Runcaciguat is currently investigated in clinical phase 2 studies for the treatment of patients with chronic kidney disease and nonproliferative diabetic retinopathy.


Assuntos
Desenho de Fármacos , Ativadores de Enzimas/química , Guanilil Ciclase Solúvel/química , Animais , Sítios de Ligação , Cristalografia por Raios X , Citocromo P-450 CYP3A/química , Citocromo P-450 CYP3A/metabolismo , Cães , Ativadores de Enzimas/metabolismo , Ativadores de Enzimas/farmacologia , Ativadores de Enzimas/uso terapêutico , Meia-Vida , Frequência Cardíaca/efeitos dos fármacos , Hemodinâmica/efeitos dos fármacos , Hipertensão/tratamento farmacológico , Hipertensão/patologia , Simulação de Dinâmica Molecular , Ratos , Ratos Endogâmicos SHR , Solubilidade , Guanilil Ciclase Solúvel/metabolismo , Relação Estrutura-Atividade
18.
Biochem Biophys Res Commun ; 555: 1-6, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-33812052

RESUMO

Cigarette smoke (CS) contains many toxins that collectively harm nearly every organ in the body, and smoking is a key risk factor for many chronic diseases. Aside from its toxic actions, CS may alter expression of the drug- and steroid-binding pregnane X receptor (PXR), which when activated upregulates expression of cytochrome P450 (CYP) enzymes, glutathione transferases (GSTs), and multidrug resistance protein 1 (MDR1), an adaptive metabolic array that mediates clearance of CS component toxins. We sought to identify new PXR agonists that may be useful for restoring PXR activity in conditions wherein it is suppressed, and their mechanisms of PXR binding and activation. PXR has a uniquely larger, hydrophobic, and highly flexible ligand-binding domain (LBD) vs. other nuclear receptors, enabling it to interact with structurally diverse molecules. We tested certain calcium channel blockers (CCBs) as a pharmacological subset of potential PXR ligands, analyzing by molecular docking methods, and identified a putative active site in the PXR LBD, along with the relevant bonds and bonding energies. We analyzed felodipine binding and agonist activity in detail, as it showed the lowest binding energy among CCBs tested. We found felodipine was a potent PXR agonist as measured by luciferase reporter assay, whereas CCBs with higher binding energies were less potent (amlodipine) or nearly inactive (manidipine), and it induced CYP3A4 expression in HepG2 cells, a known target of PXR agonism. Felodipine also both induced PXR mRNA in HepG2 hepatocytes and reduced CS extract-induced diminution of PXR levels, indicating it modulates PXR expression. The results illuminate mechanisms of ligand-induced PXR activation and identify felodipine as a novel PXR agonist.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Fumar Cigarros/efeitos adversos , Felodipino/farmacologia , Receptor de Pregnano X/agonistas , Receptor de Pregnano X/metabolismo , Sítios de Ligação , Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/metabolismo , Simulação por Computador , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Indutores do Citocromo P-450 CYP3A/farmacologia , Regulação para Baixo/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Felodipino/química , Felodipino/metabolismo , Células Hep G2 , Humanos , Ligantes , Simulação de Acoplamento Molecular , Receptor de Pregnano X/química
19.
Chemotherapy ; 66(1-2): 47-52, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33677444

RESUMO

INTRODUCTION: Patients treated with midostaurin and chemotherapy are at risk of invasive fungal disease. Prophylactic posaconazole is recommended for these patients, but posaconazole strongly inhibits the CYP3A4 isozyme that metabolizes midostaurin. Posaconazole therefore introduces a risk of patient's overexposure to midostaurin. METHODS: Blood samples were obtained from 4 patients treated with midostaurin for newly diagnosed FLT3-mutAML. Patients had received a concomitant treatment with posaconazole, isavuconazole, or micafungin, respectively. All blood samples were drawn before daily dose administration of midostaurin. RESULTS: Posaconazole caused a ≥8-fold increase of midostaurin plasma levels at through, which was accompanied by a decreased plasma exposure to O-demethylated or hydroxylated midostaurin metabolites. We also show that hematologists react to risk perception by replacing posaco-nazole with antifungals like micafungin or isavuconazole, which lack a strong inhibition of CYP3A4 and fail to modify midostaurin pharmacokinetics but are not formally recommended in these settings. DISCUSSION: In real-life scenarios, concerns about CYP3A4 inhibition may outweigh compliance with recommendations. Large studies are needed to survey the risk:benefit of hematologist's decision to replace posaconazole with other antifungals.


Assuntos
Antifúngicos/uso terapêutico , Citocromo P-450 CYP3A/metabolismo , Micoses/tratamento farmacológico , Estaurosporina/análogos & derivados , Adulto , Idoso , Antineoplásicos/efeitos adversos , Antineoplásicos/sangue , Antineoplásicos/metabolismo , Antineoplásicos/uso terapêutico , Citocromo P-450 CYP3A/química , Diarreia/etiologia , Feminino , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Estaurosporina/efeitos adversos , Estaurosporina/sangue , Estaurosporina/metabolismo , Estaurosporina/uso terapêutico , Triazóis/uso terapêutico , Tirosina Quinase 3 Semelhante a fms/genética
20.
Cancer Chemother Pharmacol ; 88(1): 81-88, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33772633

RESUMO

PURPOSE: Pamiparib is an investigational, selective, oral poly(ADP-ribose) polymerase 1/2 (PARP1/2) inhibitor that has demonstrated PARP-DNA complex trapping and CNS penetration in preclinical models, as well as preliminary anti-tumor activity in early-phase clinical studies. We investigated whether the single-dose pharmacokinetic (PK) profile of pamiparib is altered by coadministration of a strong CYP3A inducer (rifampin) or a strong CYP3A inhibitor (itraconazole) in patients with solid tumors. METHODS: In this open-label, phase 1 study, adults with advanced solid tumors received either oral pamiparib 60 mg (days 1 and 10) and once-daily oral rifampin 600 mg (days 3-11) or oral pamiparib 20 mg (days 1 and 7) and once-daily oral itraconazole 200 mg (days 3-8). Primary endpoints included pamiparib maximum observed concentration (Cmax), and area under the plasma concentration-time curve from zero to last quantifiable concentration (AUC0-tlast) and infinity (AUC0-inf). Secondary endpoints included safety and tolerability. RESULTS: Rifampin coadministration did not affect pamiparib Cmax (geometric least-squares [GLS] mean ratio 0.94; 90% confidence interval 0.83-1.06), but reduced its AUC0-tlast (0.62 [0.54-0.70]) and AUC0-inf (0.57 [0.48-0.69]). Itraconazole coadministration did not affect pamiparib Cmax (1.05 [0.95-1.15]), AUC0-tlast (0.99 [0.91-1.09]), or AUC0-inf (0.99 [0.90-1.09]). There were no serious treatment-related adverse events. CONCLUSIONS: Pamiparib plasma exposure was reduced 38-43% with rifampin coadministration but was unaffected by itraconazole coadministration. Pamiparib dose modifications are not considered necessary when coadministered with CYP3A inhibitors. Clinical safety and efficacy data will be used with these results to recommend dose modifications when pamiparib is coadministered with CYP3A inducers.


Assuntos
Indutores do Citocromo P-450 CYP3A/uso terapêutico , Inibidores do Citocromo P-450 CYP3A/uso terapêutico , Fluorenos/farmacocinética , Fluorenos/uso terapêutico , Itraconazol/uso terapêutico , Neoplasias/tratamento farmacológico , Rifampina/uso terapêutico , Adulto , Idoso , Área Sob a Curva , Citocromo P-450 CYP3A/metabolismo , Interações Medicamentosas/fisiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacocinética , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico
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