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This study aimed to elucidate the impact of variations in liver enzyme activity, particularly CYP3A4, on the metabolism of furmonertinib. An in vitro enzyme incubation system was established for furmonertinib using liver microsomes and recombinant CYP3A4 baculosomes, with analytes detected by LC-MS/MS. The pharmacokinetic characteristics of furmonertinib were studied in vivo using Sprague-Dawley rats. It was found that telmisartan significantly inhibited the metabolism of furmonertinib, as demonstrated by a significant increase in the AUC of furmonertinib when co-administered with telmisartan, compared to the furmonertinib-alone group. Mechanistically, it was noncompetitive in rat liver microsomes, while it was mixed competitive and noncompetitive in human liver microsomes and CYP3A4. Considering the genetic polymorphism of CYP3A4, the study further investigated its effect on the kinetics of furmonertinib. The results showed that compared to CYP3A4.1, CYP3A4.29 had significantly increased activity in catalyzing furmonertinib, whereas CYP3A4.7, 9, 10, 12, 13, 14, 18, 23, 33, and 34 showed markedly decreased activity. The inhibitory activity of telmisartan varied in CYP3A4.1 and CYP3A4.18, with IC50 values of 8.56 ± 0.90⯵M and 27.48 ± 3.52⯵M, respectively. The key loci affecting the inhibitory effect were identified as ARG105, ILE301, ALA370, and LEU373. Collectively, these data would provide a reference for the quantitative application of furmonertinib.
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Citocromo P-450 CYP3A , Microsomas Hepáticos , Ratas Sprague-Dawley , Animales , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Humanos , Masculino , Ratas , Polimorfismo Genético , Telmisartán/farmacología , Inhibidores del Citocromo P-450 CYP3A/farmacología , Interacciones FarmacológicasRESUMEN
Clothianidin, classified as a second-generation neonicotinoid, has achieved extensive application due to its high efficacy against insect pests. This broad-spectrum usage has resulted in its frequent detection in environmental surveys. CYP2C19 and CYP3A4 are crucial for converting clothianidin to desmethyl-clothianidin (dm-clothianidin). The expression of these CYP450s can be significantly influenced by genetic polymorphisms. The objective of our research was to examine the catalytic effects of 27 CYP3A4 variants and 31 CYP2C19 variants on the metabolism of clothianidin within recombinant insect microsomes. These variants were assessed through a well-established incubation procedure. In addition, the concentration of its metabolite dm-clothianidin was quantified by employing an ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Lastly, the kinetic parameters of these CYP3A4 and CYP2C19 variants were calculated by applying Michaelis-Menten kinetic analysis to fit the data. The observed changes in enzyme activity were related to the metabolic transformation of clothianidin to dm-clothianidin. In the CYP2C19 metabolic pathway, one variant (CYP2C19.23) showed no notable change in intrinsic clearance (CLint), four variants (CYP2C19.29, .30, .31 and L16F) demonstrated a marked increase in CLint (110.86-183.46 %), and the remaining 25 variants exhibited a considerable decrease in CLint (26.38-89.79 %), with a maximum decrease of 73.62 % (CYP2C19.6). In the CYP3A4 metabolic pathway, 26 variants demonstrated significantly reduced CLint (10.54-52.52 %), with a maximum decrease of 89.46 % (CYP3A4.20). Our results suggested that most variants of CYP3A4 and CYP2C19 significantly altered the enzymatic activities associated with clothianidin metabolism to various degrees. This study provides new insights into assessing the metabolic behavior of pesticides and delivers crucial data that can guide clinical detoxification strategies.
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Citocromo P-450 CYP2C19 , Citocromo P-450 CYP3A , Guanidinas , Neonicotinoides , Polimorfismo Genético , Tiazoles , Citocromo P-450 CYP2C19/genética , Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Tiazoles/metabolismo , Guanidinas/metabolismo , Neonicotinoides/metabolismo , Humanos , Animales , Cinética , Espectrometría de Masas en Tándem , Insecticidas/metabolismo , Microsomas/metabolismoRESUMEN
Tofacitinib can effectively improve the clinical symptoms of rheumatoid arthritis (RA) patients. In this current study, a recombinant human CYP2C19 and CYP3A4 system was operated to study the effects of recombinant variants on tofacitinib metabolism. Moreover, the interaction between tofacitinib and myricetin was analyzed in vitro. The levels of M9 (the main metabolite of tofacitinib) was detected by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The findings revealed that 11 variants showed significant changes in the levels of M9 compared to CYP3A4.1, while the other variants didn't reveal any remarkable significances. Compared with CYP2C19.1, 11 variants showed increases in the levels of M9, and 10 variants showed decreases. Additionally, it was demonstrated in vitro that the inhibition of tofacitinib by myricetin was a non-competitive type in rat liver microsomes (RLM) and human liver microsomes (HLM). However, the inhibitory mechanism was a competitive type in CYP3A4.18, and mixed type in CYP3A4.1 and .28, respectively. The data demonstrated that gene polymorphisms and myricetin had significant effects on the metabolism of tofacitinib, contributing to important clinical data for the precise use.
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Citocromo P-450 CYP2C19 , Citocromo P-450 CYP3A , Interacciones Farmacológicas , Flavonoides , Microsomas Hepáticos , Piperidinas , Pirimidinas , Humanos , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Flavonoides/farmacología , Flavonoides/metabolismo , Pirimidinas/farmacología , Pirimidinas/metabolismo , Animales , Microsomas Hepáticos/metabolismo , Microsomas Hepáticos/efectos de los fármacos , Citocromo P-450 CYP2C19/genética , Citocromo P-450 CYP2C19/metabolismo , Ratas , Piperidinas/farmacología , Piperidinas/farmacocinética , Piperidinas/metabolismo , Polimorfismo Genético , Pirroles/farmacología , Pirroles/metabolismoRESUMEN
This study aimed to investigate the interactions between icotinib/apatinib and oxycodone in rats and to unveil the underlying mechanism. An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to determine oxycodone and its demethylated metabolite simultaneously. In vivo, Sprague-Dawley (SD) male rats were administered oxycodone with or without icotinib or apatinib. Blood samples were collected and subjected to UPLC-MS/MS analysis. An enzyme incubation assay was performed to investigate the mechanism of drug-drug interaction using both rat and human liver microsomes (RLM and HLM). The results showed that icotinib markedly increased the AUC(0-t) and AUC(0-∞) of oxycodone but decreased the CLz/F. The Cmax of oxycodone increased significantly upon co-administration of apatinib. In vitro, the Km value of oxycodone metabolism was 101.7 ± 5.40 µM and 529.6 ± 19.60 µM in RLMs and HLMs, respectively. Icotinib and apatinib inhibited the disposition of oxycodone, with a mixed mechanism in RLM (IC50 = 3.29 ± 0.090 µM and 0.95 ± 0.88 µM, respectively) and a competitive and mixed mechanism in HLM (IC50 = 22.34 ± 0.81 µM and 0.48 ± 0.05 µM, respectively). In conclusion, both icotinib and apatinib inhibit the metabolism of oxycodone in vitro and in vivo. Therefore, the dose of oxycodone should be reconsidered when co-administered with icotinib or apatinib.
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Oxicodona , Espectrometría de Masas en Tándem , Ratas , Masculino , Humanos , Animales , Ratas Sprague-Dawley , Cromatografía Liquida , Oxicodona/farmacología , Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masas en Tándem/métodosRESUMEN
INTRODUCTION: Quercetin and apigenin are two common dietary flavonoids widely found in foods and fruits. Quercetin and apigenin can act as the inhibitors of CYP450 enzymes, which may affect the pharmacokinetics of clinical drugs. Vortioxetine (VOR), approved for marketing by the Food and Drug Administration (FDA) in 2013, is a novel clinical drug for treating major depressive disorder (MDD). OBJECTIVE: This study aimed to evaluate the effects of quercetin and apigenin on the metabolism of VOR in in vivo and in vitro experiments. METHOD: Firstly, 18 Sprague-Dawley rats were randomly divided into three groups: control group (VOR), group A (VOR + 30 mg/kg quercetin) and group B (VOR + 20 mg/kg apigenin). We collected the blood samples at different time points before and after the final oral administration of 2 mg/kg VOR. Subsequently, we further used rat liver microsomes (RLMs) to investigate the half-maximal inhibitory concentration (IC50) of the metabolism of vortioxetine. Finally, we evaluated the inhibitory mechanism of two dietary flavonoids on VOR metabolism in RLMs. RESULTS: In animal experiments, we found AUC (0-∞) (area under the curve from 0 to infinity) and CLz/F (clearance) to be obviously changed. Compared to controls, AUC (0-∞) of VOR in group A and group B was 2.22 and 3.54 times higher, respectively, while CLz/F of VOR in group A and group B was significantly decreased down to nearly two-fifth and one-third. In in vitro studies, the IC50 value of quercetin and apigenin in the metabolic rate of vortioxetine was 5.322 µM and 3.319 µM, respectively. Ki value of quercetin and apigenin was found to be 0.279 and 2.741, respectively, and the αKi value of quercetin and apigenin was 0.066 and 3.051 µM, respectively. CONCLUSION: Quercetin and apigenin exhibited inhibitory effects on the metabolism of vortioxetine in vivo and in vitro. Moreover, quercetin and apigenin non-competitively inhibited the metabolism of VOR in RLMs. Thus, we should pay more attention to the combination between these dietary flavonoids and VOR in the future clinical use.
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The purpose of this study was to (i) investigate the effect of CYP3A4 variants on tofacitinib metabolism, and (ii) investigate the interaction of tofacitinib with resveratrol and its underlying mechanisms. The concentration of M9, the main metabolite of tofacitinib, was determined by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The results showed that the clearance rate of CYP3A4.18 variant was significantly decreased compared with CYP3A4.1, and the CYP3A4.28 variant was changed, but not statistically significant. In addition, the potential interaction of resveratrol with tofacitinib was determined based on rat liver microsomes (RLM), human liver microsomes (HLM), and CYP3A4 response systems. Resveratrol has an IC50 of 15.67 µM in RLM with a non-competitive mechanism. In HLM with a non-competitive mechanism, the IC50 value was 8.88 µM. The IC50 values were 6.41 µM, 10.60 µM and 27.08 µM in CYP3A4.1, .18 and .28, respectively, all with a competitive mechanism. In the in vivo study, Sprague-Dawley (SD) rats were randomized into two groups (n = 6) to receive tofacitinib with or without resveratrol. We found that the AUC(0-∞) of tofacitinib in the experimental group increased to around 207.5% compared with the control group. And Cmax increased to 260.0%. In summary, our data showed that resveratrol significantly affect the metabolism of tofacitinib, thus providing basic data for the precise clinical application of tofacitinib.
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Citocromo P-450 CYP3A , Espectrometría de Masas en Tándem , Ratas , Humanos , Animales , Resveratrol/farmacología , Cromatografía Liquida , Ratas Sprague-Dawley , Citocromo P-450 CYP3A/metabolismo , Espectrometría de Masas en Tándem/métodos , Microsomas Hepáticos/metabolismo , Cromatografía Líquida de Alta Presión/métodosRESUMEN
This study aimed 1) to investigate the influence of CYP2D6 variants on the catalyzing of fluvoxamine, and 2) to study the interaction between fluvoxamine and apatinib. An enzymatic reaction system was setup and the kinetic profile of CYP2D6 in metabolizing fluvoxamine was determined. In vivo, drug-drug interaction was investigated using Sprague-Dawley (SD) rats. Fluvoxamine was given gavage with or without apatinib. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to determine the concentrations of fluvoxamine and desmethyl-fluvoxamine. The results demonstrated that the relative clearance rates of CYP2D6.A5V, V104A, D337G, F164L, V342M, R440C and R497C increased significantly compared with CYP2D6.1, ranging from 153.626% ± 6.718% to 394.310% ± 33.268%. The activities of other variants reduced to different extent, or even lost function, but there was no statistical difference. The IC50 of apatinib against fluvoxamine disposition was determined, which is 0.190 µM in RLM and 6.419 µM in HLM, respectively. In vivo, apatinib can enhance the plasma exposure of fluvoxamine remarkably characterized by increased AUC, Tmax and Cmax. Meanwhile, the produce of desmethyl fluvoxamine was dramatically inhibited, both AUC and Cmax decreased significantly. Mechanistically, apatinib inhibit the generation of fluvoxamine metabolite with a mixed manner both in RLM and HLM. Furthermore, there were differences in the potency of apatinib in suppressing fluvoxamine metabolism among CYP2D6.1, 2 and 10. In conclusion, CYP2D6 gene polymorphisms and drug-drug interaction can remarkably affect the plasma exposure of fluvoxamine. The present study provides basis data for guiding individual application of fluvoxamine.
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AIM: This study investigated into the effect of CYP3A4 genetic polymorphism on istradefylline metabolism. Moreover, the potential drug-drug interaction with istradefylline was determined as well as underlied mechanism. METHOD: In vitro, enzymatic reaction was performed to determine the kinetic parameters of CYP3A4 and its variants on catalyzing istradefylline. Meanwhile, the rat liver microsomes incubation assay was applied to screen interacting drugs. In vivo, SD rats were used to investigate the selected drug interaction. UPLC-MS/MS was used to detect the metabolite M1. RESULT: The results demonstrated that the relative clearance rate of CYP3A4.29 decrease significantly compared with CYP3A4.1. But there is no statistically diverse in activities among CYP3A4.1, 2 and 3. The relative clearance rates of the remaining variants are significantly decreased compared with CYP3A4.1. In addition, 148 drugs were screened to determine the potential interaction with istradefylline, among which calcium channel blockers were identified. It's indicated that nimodipine has a significant inhibitory effect on metabolizing istradefylline with IC50 of 6.927 ± 0.372 µM, which via competitive and non-competitive mixed mechanism. In vivo, when istradefylline and nimodipine was co-administered to SD rats, we found the main pharmacokinetic parameters of M1 reduced remarkably, including AUC, MRT, Cmax and CLz/F. CONCLUSION: CYP3A4 genetic polymorphism and nimodipine affect the metabolism of istradefylline. Thus, the present study provided reference data for clinical individualized medicine of istradefylline.
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Citocromo P-450 CYP3A , Nimodipina , Animales , Bloqueadores de los Canales de Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Cromatografía Liquida , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Interacciones Farmacológicas , Microsomas Hepáticos/metabolismo , Nimodipina/metabolismo , Nimodipina/farmacología , Polimorfismo Genético , Purinas , Ratas , Ratas Sprague-Dawley , Espectrometría de Masas en TándemRESUMEN
Objectives: This study aims to evaluate the catalytic activities of 31 CYP2C19 alleles and their effects on the metabolism of tapentadol in vitro. Materials and Methods: Insect microsomes expressing the CYP2C19 alleles were incubated with 50-1250 µM tapentadol for 40 min at 37 °C and terminated by cooling to -80 °C, immediately. Tapentadol and N-desmethyl tapentadol were analyzed by a UPLC-MS/MS system. The kinetic parameters Km, Vmax, and intrinsic clearance (Vmax/Km) of N-desmethyl tapentadol were determined. Results: As a result, the intrinsic clearance (Vmax/Km) values of most variants were significantly altered, while CYP2C19.3 and 35FS had no detectable enzyme activity. Only one variant, N277K, showed no significant difference from CYP2C19.1B. Two variants CYP2C19.29 and L16F displayed markedly increased intrinsic clearance values of 302.22% and 199.97%, respectively; whereas 24 variants exhibited significantly decreased relative clearance ranging from 0.32% to 79.15% of CYP2C19.1B. Especially, CYP2C19.2G, 2H, R124Q, and R261W exhibited a drastic decrease in clearance (>80%) compared with wild-type CYP2C19.1B. Conclusion: As the first study of all aforementioned alleles for tapentadol metabolism, the comprehensive data in vitro may provide novel insights into the allele-specific and substrate-specific activity of CYP2C19.
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PURPOSE: Cancer patients experience pain during medical treatment. Therefore, anticancer drugs and painkillers are often prescribed together. This study aims to determine the interaction between anlotinib and oxycodone and reveal the underlying mechanism. METHODS: UPLC-MS/MS, an efficient and sensitive method, was used for the simultaneous determination of oxycodone and oxycodone metabolites. Sprague-Dawley rats were given oxycodone with or without anlotinib. Then, UPLC-MS/MS was used to determine the blood concentration of oxycodone. To study the interaction mechanism, rat and human liver microsomes (HLMs) were used for determining enzyme kinetics. RESULTS: Long-term administration of oxycodone combined with anlotinib resulted in significantly increased pharmacokinetic parameters AUC(0-t), AUC(0-∞), and Cmax for oxycodone, indicating that anlotinib inhibited oxycodone. In vitro kinetic measurements indicated that anlotinib inhibited the metabolism of oxycodone through a mixed mechanism. Further studies indicated that in HLMs, anlotinib strongly inhibited the metabolism of oxycodone. CONCLUSION: This study showed that anlotinib inhibited the metabolism of oxycodone both in vitro and in vivo. It is recommended that the dose of oxycodone should be reconsidered when oxycodone is combined with anlotinib in clinical practice.
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Oxicodona , Espectrometría de Masas en Tándem , Animales , Cromatografía Liquida , Humanos , Indoles , Oxicodona/efectos adversos , Oxicodona/farmacocinética , Quinolinas , Ratas , Ratas Sprague-Dawley , Espectrometría de Masas en Tándem/métodosRESUMEN
Upadacitinib, as a selective and reversible Janus kinase (JAK) inhibitor, has been widely used in the treatment of atopic dermatitis, ulcerative colitis and other inflammatory bowel diseases and other immune-mediated diseases. The combination of methotrexate and upadacitinib is a common clinical treatment strategy for rheumatoid arthritis (RA) in recent years. In this study, we established an ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay for quantitative measurement of upadacitinib and methotrexate, by which we successfully determined pharmacokinetic parameters of them in rat plasma. In order to pretreat the samples, we used acetonitrile as the precipitant, and for the internal standard (IS), we chose tofacitinib. The Acquity BEHC18 (2.1 mm × 50 mm, 1.7 µm) column, with acetonitrile and 0.1% formic acid aqueous solution composed mobile phases, was used to separate upadacitinib, methotrexate and tofacitinib. A Xevo TQ-S triple quadrupole tandem mass spectrometer was used as the detecting instrument in the positive ion mode. For upadacitinib, excellent linearity was shown of this assay in the calibration range with 0.1-200 ng/mL, and as for methotrexate, the range was 0.05-100 ng/mL. As the results indicated, the lower limit of quantification (LLOQ) was respectively 0.1 and 0.05 ng/mL for upadacitinib and methotrexate, the intra- and inter-day precision were ≤ 13.3%, and the accuracy of all the analytes ranged from -4.1% to 12.7%. The recovery of each analyte was > 80.2% in this experiment, and matrix effects we observed were unobvious. The establishment of this method and its successful application in rat plasma can provide a theoretical and technical support for the deeper study of pharmacodynamics and the clinical medication strategies.
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Cromatografía Líquida de Alta Presión/métodos , Compuestos Heterocíclicos con 3 Anillos/sangre , Metotrexato/sangre , Espectrometría de Masas en Tándem/métodos , Animales , Compuestos Heterocíclicos con 3 Anillos/química , Compuestos Heterocíclicos con 3 Anillos/farmacocinética , Límite de Detección , Modelos Lineales , Masculino , Metotrexato/química , Metotrexato/farmacocinética , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los ResultadosRESUMEN
We aim to study the effects of CYP2D6 variants and drug-drug interaction on the metabolism of dacomitinib. CYP2D6 variants were incubated with 25-1000 µM dacomitinib for 40 min at 37 °C, and the reaction was terminated by cooling to -80 °C immediately. For an in vivo experiment, 18 male Sprague-Dawley rats were randomly divided into three groups (n = 6): a single dose of 5 mg/kg dacomitinib (group A), a single dose of 6 mg/kg trazodone (group B), and a combined group (group C). Processed samples were analyzed by ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS.) The relative clearance of dacomitinib was reduced for most of the variants. Moreover, the inhibitory potency of classic CYP inhibitors on dacomitinib metabolism was significantly different among the main subtypes of CYP2D6. Interestingly, compared with gefitinib, even the same CYP2D6 variants showed significant differences in metabolic activity, suggesting that the activity of CYP2D6 has strong variability. In addition, the interaction between trazodone and dacomitinib was determined both in vitro and in vivo. When dacomitinib was given in combination with trazodone, the blood exposure to these two drugs increased remarkably. The mechanistic study revealed that the interaction followed the noncompetitive inhibition. We demonstrated that the activity of CYP2D6 variants to metabolize dacomitinib was significantly reduced. In combination with the CYP2D6 inhibitor, the degree of activity inhibition of different variants obviously differed. When trazodone and dacomitinib were used in combination, the body exposure to the two drugs increased significantly. This study provides data for the precise use of dacomitinib in clinical settings.
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Inhibidores del Citocromo P-450 CYP2D6/farmacología , Citocromo P-450 CYP2D6/metabolismo , Polimorfismo Genético/efectos de los fármacos , Quinazolinonas/farmacología , Animales , Citocromo P-450 CYP2D6/genética , Inhibidores del Citocromo P-450 CYP2D6/química , Relación Dosis-Respuesta a Droga , Masculino , Estructura Molecular , Polimorfismo Genético/genética , Quinazolinonas/química , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
AIM: To investigate the enzymatic properties of cytochrome P450 3A4 (CYP3A4) variants and their ability to metabolize vandetanib (VNT) in vitro, and to study potential drug interactions in combination with VNT. METHOD: Recombinant CYP3A4 cell microsomes were prepared using a Bac-to-Bac baculovirus expression system. Enzymatic reactions were carried out, and the metabolites were determined by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). RESULTS: The activities of 27 CYP3A4 variants were determined to assess the degree of VNT metabolism that occurred. Analysis indicated that there was enhanced intrinsic clearance (Vmax/Km, CLint) for eight variants (CYP3A4.2, 3, 9, 15, 16, 29, 32, and 33), while there was a significant decrease in CYP3A4.5, 7, 8, 10-14, 17-20, 23, 24, 28, 31, and 34. Compared with CYP3A4.1, no significant differences were found for CYP3A4.6 and 30. Furthermore, the relative clearances were compared between VNT and cabozantinib, which were all metabolized by CYP3A4 with the same indications. When combined with ketoconazole, which is a CYP inhibitor, obvious differences were observed in the potency of VNT between different variants, including CYP3A4.2, 15, and 18. CONCLUSION: This comprehensive assessment of CYP3A4 variants provides significant insights into the allele-specific metabolism of VNT and drug interactions in vitro. We hope that these comprehensive data will provide references and predictions for the clinical application of VNT.
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Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Piperidinas/metabolismo , Inhibidores de Proteínas Quinasas/metabolismo , Quinazolinas/metabolismo , Alelos , Biotransformación , Inhibidores del Citocromo P-450 CYP3A/administración & dosificación , Interacciones Farmacológicas , Estudios de Asociación Genética , Variación Genética , Humanos , Técnicas In Vitro , Cetoconazol/administración & dosificación , Cinética , Tasa de Depuración Metabólica , Piperidinas/administración & dosificación , Piperidinas/farmacocinética , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacocinética , Quinazolinas/administración & dosificación , Quinazolinas/farmacocinética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
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.
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Benzamidas/metabolismo , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Variación Genética , Pirazinas/metabolismo , Biocatálisis , Citocromo P-450 CYP3A/química , Hemo/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Oxidación-Reducción , Conformación ProteicaRESUMEN
The Gamma interferon inducible lysosomal thiol reductase (GILT) plays a key biological role in the immune responses and involves in the processing of class II MHC-restricted antigen by stimulating disulfide bond reduction in mammals. To determine the biological function of GILT in the innate immune system of crustaceans, we sequenced and cloned GILT gene from red swamp crayfish, Procambarus clarkii (Pc-GILT). The deduced amino acid sequence of Pc-GILT contained the putative conserved structures of the GILT family proteins: the GILT signature (CQHGX2ECX2NX4C) sequence and the active site (CXXS) motif. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis suggested that a recombinant Pc-GILT protein was successfully expressed in Escherichia coli (E. coli). Quantitative real-time PCR analysis showed that Pc-GILT transcript level was highest in the hepatopancreas followed by the gut, heart and muscles. Additionally, we analyzed the transcription level of Pc-GILT gene in hepatopancreas of red swamp crayfish under biotic stress conditions. The expression of Pc-GILT gene upregulated after viral (poly I:C) and bacterial (peptidoglycan, lipopolysaccharide) infection. The suppression of Pc-GILT by double stranded RNA influenced the transcript levels of various immune-related genes. These observations indicate that the Pc-GILT probably plays a key biological role in the innate immune responses of red swamp crayfish, since it modulates the expression of genes associated with immune pathways.
Asunto(s)
Proteínas de Artrópodos/inmunología , Astacoidea/inmunología , Inmunidad Innata/genética , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/inmunología , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos/antagonistas & inhibidores , Proteínas de Artrópodos/química , Proteínas de Artrópodos/genética , Astacoidea/clasificación , Astacoidea/genética , Secuencia de Bases , Expresión Génica , Regulación de la Expresión Génica/inmunología , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/antagonistas & inhibidores , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/química , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Filogenia , Interferencia de ARN , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Alineación de Secuencia , Estrés Fisiológico/genética , Estrés Fisiológico/inmunología , Distribución TisularRESUMEN
Peroxiredoxin 6 (Prx6) is an important member of the peroxiredoxin family that plays critical roles in protecting host against the toxicity of oxidative stress and participates in cell signaling. Herein, we report Prx6 gene from red swamp crayfish, Procambarus clarkii. The cDNA fragment of PcPrx6 was 660 bp, encoding a 219 amino acid residues protein. The quantitative real time PCR analysis showed ubiquitous expression of PcPrx6 mRNA in the tested tissues. The challenge with peptidoglycan and Poly I:C remarkably suppressed the mRNA level of PcPrx6 in hepatopancreas at 3, 12, 48â¯h compared with the PBS control. However, the expression level significantly increased after 36â¯h of their treatment. The knockdown of PcPrx6 by small interference RNA significantly enhanced the transcript levels of Toll pathway-responsive genes at 24â¯h. Recombinant PcPrx6 protein was purified using affinity chromatography and analyzed for its biological role. The results revealed that the recombinant PcPrx6 protein manifested the ability to protect supercoiled DNA damage from oxidative stress elicited by mixed function oxidative assay. Altogether, PcPrx6 may have multiple functional roles in the physiology of P. clarkii, since it negatively regulates the Toll signaling transduction and protects supercoiled DNA damage from oxidative stress.
Asunto(s)
Astacoidea/genética , Astacoidea/inmunología , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , Peroxiredoxina VI/genética , Peroxiredoxina VI/inmunología , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos/química , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/inmunología , Secuencia de Bases , Cromatografía de Afinidad , Daño del ADN , ADN Superhelicoidal/fisiología , Perfilación de la Expresión Génica , Estrés Oxidativo , Peptidoglicano/farmacología , Peroxiredoxina VI/química , Filogenia , Poli I-C/farmacología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Alineación de SecuenciaRESUMEN
Anti-lipopolysaccharide factors are a group of small proteins with broad spectrum antiviral property and antibacterial activity. Herein, we obtained the genomic sequence of the Procambarus clarkii anti-lipopolysaccharide factor (PcALF) gene by using polymerase chain reaction to investigate its expression pattern in various tissues and in the immune tissues (Hepatopancreas) following exposure to pathogens. The deduced protein of PcALF was conserved; it displayed the signal peptides and putative lipo-polysaccharide binding domain, particularly the two conserved cysteine amino acid residues at both ends of the domain. The recombinant protein of PcALF was successfully expressed in Escherichia coli and rabbit anti-PcALF polyclonal antibodies were prepared. The qRT-PCR analysis showed unequal distribution of PcALF transcript in the examined tissues, however the transcript level was greatest in hepatopancreas. The challenge with peptidoglycan (PGN), lipo-polysaccharide (LPS) and Poly I:C significantly enhanced expression level of PcALF in hepatopancreas when compared with the PBS control. RNA interference of PcALF affected the mRNA expression levels of immune-related genes. Taken together, our data suggested that PcALF is an inducible protein and could play a key biological role in the innate immune defense of P. clarkii.