RESUMO
Isotretinoin [13-cis-retinoic acid (13cisRA)] is widely used for the treatment of neuroblastoma and acne. It acts via regulating gene transcription through binding to retinoic acid receptors. Yet, the potential for isotretinoin to cause transcriptionally mediated drug-drug interactions (DDIs) has not been fully explored. We hypothesized that isotretinoin and its active metabolites all-trans-retinoic acid (atRA) and 4-oxo-13cisRA would alter the transcription of enzymes and transporters in the human liver via binding to nuclear receptors. The goal of this study was to define the DDI potential of isotretinoin and its metabolites resulting from transcriptional regulation of cytochrome P450 and transporter mRNAs. In human hepatocytes (n = 3), 13cisRA, atRA, and 4-oxo-13cisRA decreased OATP1B1, CYP1A2, CYP2C9, and CYP2D6 mRNA and increased CYP2B6 and CYP3A4 mRNA in a concentration-dependent manner. The EC50 values for OATP1B1 mRNA downregulation ranged from 2 to 110 nM, with maximum effect (Emax ) ranging from 0.17- to 0.54-fold. Based on the EC50 and Emax values and the known circulating concentrations of 13cisRA and its metabolites after isotretinoin dosing, a 55% decrease in OATP1B1 activity was predicted in vivo. In vivo DDI potential was evaluated clinically in participants dosed with isotretinoin for up to 32 weeks using coproporphyrin-I (CP-I) as an OATP1B1 biomarker. CP-I steady-state serum concentrations were unaltered following 2, 8, or 16 weeks of isotretinoin treatment. These data show that isotretinoin and its metabolites alter transcription of multiple enzymes and transporters in vitro, but translation of these changes to in vivo drug-drug interactions requires clinical evaluation for each enzyme. SIGNIFICANCE STATEMENT: Isotretinoin and its metabolites alter the mRNA expression of multiple cytochrome P450s (CYPs) and transporters in human hepatocytes, suggesting that isotretinoin may cause clinically significant drug-drug interactions (DDIs). Despite the observed changes in organic anion transporting polypeptide 1B1 (OATP1B1) mRNA in human hepatocytes, no clinical DDI was observed when measuring a biomarker, coproporphyrin-I. Further work is needed to determine whether these findings can be extrapolated to a lack of a DDI with CYP1A2, CYP2B6, and CYP2C9 substrates.
Assuntos
Isotretinoína , Transportadores de Ânions Orgânicos , Biomarcadores , Coproporfirinas/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP2C9/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação para Baixo , Interações Medicamentosas , Humanos , Isotretinoína/metabolismo , Isotretinoína/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo , RNA Mensageiro/genéticaRESUMO
Pyrrolo[2,1-c][1,4]benzodiazepine dimer (PBD) has shown broad antitumor properties and potential as a therapeutic agent for cancers. During a routine drug-drug interaction assessment, it was found that PBD is a reversible inhibitor of CYP2C8 (IC50 = 1.1 µM) but not CYP1A2, 2B6, 2C9, 2C19, 2D6, or 3A4/5. Additionally, PBD is a classic time-dependent inhibition (TDI) of CYP3A4/5, with >30-fold shift in IC50 after a preincubation with NADPH. All other CYPs tested did not show evidence for TDI, but potent inhibition of CYP2B6 (IC50 = 1.5 µM) was observed after a preincubation with or without (w/wo) NADPH, which was an unexpected observation given the fact that no inhibition was observed in the direct inhibition assay. No other CYP isoforms were susceptible to this apparent non-NADPH-dependent inhibition, suggesting that PBD may selectively inactivate CYP2B6 without metabolic activation. The washing of the human liver microsome pellet after incubation with PBD did not fully recover CYP2B6 activity, indicating that PBD is covalently bound to CYP2B6, leading to inactivation of the enzyme. To further investigate the mechanism of NADPH-independent inhibition, the IC50 shift was determined for several PBD analogs, and it was found that the compounds without both reactive imines did not show NADPH-independent inhibition of CYP2B6, implying that NADPH-independent inactivation was likely caused by direct covalent binding of PBD to the enzyme in a highly structure-specific manner. These data clearly highlight the need to assess direct and time-dependent inhibition w/wo NADPH to adequately characterize the in vitro CYP inhibitory properties of drug candidates with reactive moieties. SIGNIFICANCE STATEMENT: We described a very unique in vitro CYP inhibition profile of pyrrolo[2,1-c][1,4]benzodiazepine dimer as a potent reversible CYP2C8 inhibitor, an NADPH-dependent CYP3A4/5 time-dependent inhibition (TDI) inhibitor, and an NADPH-independent CYP2B6 TDI inhibitor, and inhibition of CYPs occurs through three distinct mechanisms: reversible drug-enzyme binding, enzyme inactivation via bioactivation, and enzyme inactivation by covalent binding via chemical reactions. Our results suggest that, for compounds with reactive functional moieties, false positives can be reported when the conventional TDI assay is utilized.
Assuntos
Antineoplásicos/farmacocinética , Benzodiazepinas/farmacocinética , Inibidores do Citocromo P-450 CYP2B6/farmacocinética , Inibidores do Citocromo P-450 CYP3A/farmacocinética , NADP/metabolismo , Pirróis/farmacocinética , Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP3A/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Interações Medicamentosas , Feminino , Humanos , Masculino , Microssomos Hepáticos , Proteínas Recombinantes/metabolismo , Fatores de TempoRESUMO
All-trans-retinoic acid (atRA) downregulates cytochrome P450 (CYP)2D6 in several model systems. The aim of this study was to determine whether all active retinoids downregulate CYP2D6 and whether in vitro downregulation translates to in vivo drug-drug interactions (DDIs). The retinoids atRA, 13cisRA, and 4-oxo-13cisRA all decreased CYP2D6 mRNA in human hepatocytes in a concentration-dependent manner. The in vitro data predicted ~ 50% decrease in CYP2D6 activity in humans after dosing with 13cisRA. However, the geometric mean area under plasma concentration-time curve (AUC) ratio for dextromethorphan between treatment and control was 0.822, indicating a weak induction of dextromethorphan clearance following 13cisRA treatment. Similarly, in mice treatment with 4-oxo-13cisRA-induced mRNA expression of multiple mouse Cyp2d genes. In comparison, a weak induction of CYP3A4 in human hepatocytes translated to a weak in vivo induction of CYP3A4. These data suggest that in vitro CYP downregulation may not translate to in vivo DDIs, and better understanding of the mechanisms of CYP downregulation is needed.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Regulação para Baixo , Interações Medicamentosas , Isotretinoína/farmacologia , Adulto , Idoso de 80 Anos ou mais , Animais , Biomarcadores/sangue , Simulação por Computador , Sistema Enzimático do Citocromo P-450/metabolismo , Dextrometorfano/farmacocinética , Dextrorfano/farmacocinética , Regulação para Baixo/efeitos dos fármacos , Feminino , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de TempoRESUMO
BACKGROUND: A recent advancement in isolation and cryopreservation has resulted in commercially available primary human enterocytes that express various drug metabolizing enzymes (DMEs) and transporters. The main objective of this study was to further evaluate the utility of pooled cryopreserved enterocytes, specifically MetMax™ cryopreserved human enterocytes (In vitro ADMET Laboratories), as an in vitro model for assessing intestinal clearance in comparison to hepatocytes. METHODS: It was found that, for CYP3A4/5 substrates such as midazolam, amprenavir and loperamide, in vitro metabolic clearance is generally lower in enterocytes compared to that of hepatocytes, which is consistent with the relative abundance of the enzyme between the intestine and liver. In contrast, raloxifene, a surrogate UGT activity substrate, showed 3-fold greater turnover in enterocytes than hepatocytes, which is likely attributed to the differential expression of individual UGTs in human liver and intestine. For procaine, a known CES2 substrate, the measured apparent clearance was higher in hepatocytes, but formation of 4-aminobenzoic acid, a CE2-specific metabolite, was more pronounced in enterocytes, suggesting that CE2 is more active in enterocytes. Salbutamol, a SULT1A3 substrate, showed little turnover in both enterocytes and hepatocytes, and more abundant sulfate conjugate was detected in enterocytes, indicating higher SULT activity in enterocytes than hepatocytes. As expected, ketoconazole inhibited CYP3A4/5-mediated metabolite formation in enterocytes for midazolam, amprenavir and loperamide, suggesting that cryopreserved enterocytes may be useful in determining intestinal CYP3A inhibition parameters. Interestingly, elacridar, a P-gp inhibitor, suppressed metabolite formation in enterocytes for loperamide, a substrate of CYP3A4 and P-gp, suggesting that enterocytes in suspension do not have active P-gp efflux functions, and the suppression of metabolism in enterocytes is probably caused by inhibition of CYP3A4/5 by elacridar. RESULTS: Our results suggest that pooled cryopreserved human enterocytes, specifically the MetMax™ cryopreserved human enterocytes, represent a valuable in vitro model for assessing first-pass clearance and potential drug interactions in human intestine.
Assuntos
Citocromo P-450 CYP3A/metabolismo , Interações Medicamentosas , Enterócitos/metabolismo , Mucosa Intestinal/metabolismo , Farmacocinética , Células Cultivadas , Criopreservação , Feminino , Hepatócitos/metabolismo , Humanos , Mucosa Intestinal/citologia , Masculino , Taxa de Depuração MetabólicaRESUMO
Vector-based systems comprised of exogenous nucleic acid sequences remain the standard for ectopic expression of a particular gene. Such systems offer robust overexpression, but have inherent drawbacks such as the tedious process of construction, excluding sequences (e.g. introns and untranslated regions) important for gene function and potential insertional mutagenesis of host genome associated with the use of viral vectors. We and others have recently reported that short double-stranded RNAs (dsRNAs) can induce endogenous gene expression by targeting promoter sequences in a phenomenon referred to as RNA activation (RNAa) and such dsRNAs are termed small activating RNAs (saRNAs). To date, RNAa has been successfully utilized to induce the expression of different genes such as tumor suppressor genes. Here, we describe a detailed protocol for target selection and dsRNA design with associated experiments to facilitate RNAa in cultured cells. This technique may be applied to selectively activate endogenous gene expression for studying gene function, interrogating molecular pathways and reprogramming cell fate.
RESUMO
Rev-Erb-α and Rev-Erb-ß are nuclear receptors that regulate the expression of genes involved in the control of circadian rhythm, metabolism and inflammatory responses. Rev-Erbs function as transcriptional repressors by recruiting nuclear receptor co-repressor (NCoR)-HDAC3 complexes to Rev-Erb response elements in enhancers and promoters of target genes, but the molecular basis for cell-specific programs of repression is not known. Here we present evidence that in mouse macrophages Rev-Erbs regulate target gene expression by inhibiting the functions of distal enhancers that are selected by macrophage-lineage-determining factors, thereby establishing a macrophage-specific program of repression. Remarkably, the repressive functions of Rev-Erbs are associated with their ability to inhibit the transcription of enhancer-derived RNAs (eRNAs). Furthermore, targeted degradation of eRNAs at two enhancers subject to negative regulation by Rev-Erbs resulted in reduced expression of nearby messenger RNAs, suggesting a direct role of these eRNAs in enhancer function. By precisely defining eRNA start sites using a modified form of global run-on sequencing that quantifies nascent 5' ends, we show that transfer of full enhancer activity to a target promoter requires both the sequences mediating transcription-factor binding and the specific sequences encoding the eRNA transcript. These studies provide evidence for a direct role of eRNAs in contributing to enhancer functions and suggest that Rev-Erbs act to suppress gene expression at a distance by repressing eRNA transcription.
Assuntos
Regulação para Baixo/genética , Elementos Facilitadores Genéticos/genética , Macrófagos/metabolismo , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , Transcrição Gênica/genética , Alelos , Animais , Sequência de Bases , Sítios de Ligação , Técnicas de Silenciamento de Genes , Camundongos , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/deficiência , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , Especificidade de Órgãos , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Elementos de Resposta/genéticaRESUMO
RNA activation (RNAa) is a mechanism of gene activation triggered by promoter-targeted small double-stranded RNA (dsRNA), also known as small activating RNA (saRNA). p21(WAF1/CIP1) (p21) is a putative tumor suppressor gene due to its role as a key negative regulator of the cell cycle and cell proliferation. It is frequently downregulated in cancer including hepatocellular carcinoma (HCC), but is rarely mutated or deleted, making it an ideal target for RNAa-based overexpression to restore its tumor suppressor function. In the present study, we investigated the antigrowth effects of p21 RNAa in HCC cells. Transfection of a p21 saRNA (dsP21-322) into HepG2 and Hep3B cells significantly induced the expression of p21 at both the mRNA and protein levels, and inhibited cell proliferation and survival. Further analysis of dsP21-322 transfected cells revealed that dsP21-322 arrested the cell cycle at the G(0)/G(1) phase in HepG2 cells but at G(2)/M phase in Hep3B cells which lack functional p53 and Rb genes, and induced both early and late stage apoptosis by activating caspase 3 in both cell lines. These results demonstrated that RNAa of p21 has in vitro antigrowth effects on HCC cells via impeding cell cycle progression and inducing apoptotic cell death. This study suggests that targeted activation of p21 by RNAa may be explored as a novel therapy for the treatment of HCC.