The effect of icotinib or apatinib on the pharmacokinetic profile of oxycodone in rats and the underlying mechanism.

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.

Combination of Hairy Root and Whole-Plant Transformation Protocols to Achieve Efficient CRISPR/Cas9 Genome Editing in Soybean.

The new gene-editing technology CRISPR/Cas system has been widely used for genome engineering in various organisms. Since the CRISPR/Cas gene-editing system has a certain possibility of low efficiency and the whole plant transformation of soybean is time-consuming and laborious, it is important to evaluate the editing efficiency of designed CRISPR constructs before the stable whole plant transformation process starts. Here, we provide a modified protocol for generating transgenic hairy soybean roots to assess the efficiency of guide RNA (gRNA) sequences of the CRISPR/Cas constructs within 14 days. The cost- and space-effective protocol was first tested in transgenic soybean harboring the GUS reporter gene for the efficiency of different gRNA sequences. Targeted DNA mutations were detected in 71.43-97.62% of the transgenic hairy roots analyzed as evident by GUS staining and DNA sequencing of the target region. Among the four designed gene-editing sites, the highest editing efficiency occurred at the 3' terminal of the GUS gene. In addition to the reporter gene, the protocol was tested for the gene-editing of 26 soybean genes. Among the gRNAs selected for stable transformation, the editing efficiency of hairy root transformation and stable transformation ranged from 5% to 88.8% and 2.7% to 80%, respectively. The editing efficiencies of stable transformation were positively correlated with those of hairy root transformation with a Pearson correlation coefficient (r) of 0.83. Our results demonstrated that soybean hairy root transformation could rapidly assess the efficiency of designed gRNA sequences on genome editing. This method can not only be directly applied to the functional study of root-specific genes, but more importantly, it can be applied to the pre-screening of gRNA in CRISPR/Cas gene editing.

CYP2D6 gene polymorphism and apatinib affect the metabolic profile of fluvoxamine.

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.

The effect of anlotinib on the pharmacokinetic profile of oxycodone and the underlying mechanism.

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.

The Influence of CYP3A4 Genetic Polymorphism and Proton Pump Inhibitors on Osimertinib Metabolism.

The aim of this study was to 1) investigate the effects of 27 CYP3A4 variants on the metabolism of osimertinib and 2) study the interactions between osimertinib and others as well as the underlying mechanism. A recombinant human CYP3A4 enzymatic incubation system was developed and employed to determine the kinetic profile of CYP3A4 variants. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was applied to detect the concentration of the main metabolite, AZ5104. The results demonstrated that the relative clearance rates of CYP3A4.19, 10, 18, 5, 16, 14, 11, 2, 13, 12, 7, 8, and 17 in catalyzing osimertinib were significantly reduced to a minimum of 25.68% compared to CYP3A4.1, while those of CYP3A4.29, 32, 33, 28, 15, 34, and 3 were obviously enhanced, ranging from 114.14% to 284.52%. The activities of the remaining variants were almost equal to those of CYP3A4.1. In addition, 114 drugs were screened to determine the potential interaction with osimertinib based on the rat liver microsome (RLM) reaction system. Sixteen of them inhibited the production of AZ5104 to 20% or less, especially proton pump inhibitors, among which the IC50 of rabeprazole was 6.49 ± 1.17 µM in RLM and 20.39 ± 2.32 µM in human liver microsome (HLM), with both following competitive and non-competitive mixed mechanism. In an in vivo study, Sprague-Dawley (SD) rats were randomly divided into groups, with six animals per group, receiving osimertinib with or without rabeprazole, omeprazole, and lansoprazole. We found that the AUC(0-t), AUC(0-∞), and Cmax of osimertinib decreased significantly after co-administration with rabeprazole orally, but they increased remarkably when osimertinib was administered through intraperitoneal injection. Taken together, our data demonstrate that the genetic polymorphism and proton pump inhibitors remarkably influence the disposition of osimertinib, thereby providing basic data for the precise application of osimertinib.

Effects of CYP2D6 Genetic Polymorphism and Drug Interaction on the Metabolism of Dacomitinib.

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.

Functional evaluation of cyclosporine metabolism by CYP3A4 variants and potential drug interactions.

The aim of this study is to investigate the effects of CYP3A4 genetic polymorphisms on the metabolism of cyclosporine (CsA) in vitro and identify drugs that interact with CsA. An enzymatic incubation system was developed to evaluate the kinetic parameters of CYP3A4 on CsA catalysis. A total of 132 drugs were screened to identify potential drug-drug interactions. Sprague-Dawley rats were used to determine the interaction between CsA and nimodipine and nisoldipine. The metabolite AM1 was measured by ultra-performance liquid chromatography-tandem mass spectrometry. The results demonstrate that 16 CYP3A4 variants (CYP3A4.7, 8, 9, 12, 13, 14, 16, 18, 19, 23, 24, 28, 31, 32, 33, and 34) have a lower metabolic capacity for CsA, ranging from 7.19% to 72.10%, than CYP3A4.1. In contrast, the relative clearance rate of CYP3A4.5 is significantly higher than that of CYP3A4.1. Moreover, CYP3A4.20 loses its catalytic ability, and five other variants have no significant difference. A total of 12 drugs, especially calcium channel blockers, were found to remarkably inhibit the metabolism of CsA with an inhibitory rate of over 80%. Nimodipine inhibits the activity of CsA in rat liver microsomes with an IC50 of 20.54 ± 0.93 µM, while nisoldipine has an IC50 of 16.16 ± 0.78 µM. In in vivo, three groups of Sprague-Dawley rats were administered CsA with or without nimodipine or nisoldipine; the AUC(0-t) and AUC(0-∞) of CsA were significantly increased in the nimodipine group but not obviously in the nisoldipine group. Mechanistically, the inhibition mode of nimodipine on cyclosporine metabolism is a mixed inhibition. Our data show that gene polymorphisms of CYP3A4 and nimodipine remarkably affect the metabolism of CsA, thus providing a reference for the precise administration of CsA.

A Vacuolar Membrane Ferric-Chelate Reductase, OsFRO1, Alleviates Fe Toxicity in Rice (Oryza sativa L.).

Ferric reductase oxidase (FRO), the enzyme that reduced ferric iron [Fe (III)] into ferrous iron [Fe (II)], is known to play important roles in Fe absorption and homeostasis in plants that utilize a strategy I mechanism to obtain iron. Rice can use both strategies I and II for Fe uptake depending on the growth conditions. FRO is encoded by two genes in rice genome. Amino acid sequence alignment shows that OsFRO1 contains all necessary predicted motifs for a functional FRO enzyme, whereas OsFRO2 lacks a complete transmembrane domain at the N-terminal. Transient expression of OsFRO1: GFP protein fusion revealed that OsFRO1 is localized to the vacuolar membrane in rice protoplast. OsFRO1 is primarily expressed in leaves and transcript abundance was decreased under excess Fe conditions. Transgenic plants overexpressing OsFRO1 were more sensitive to Fe toxicity, in contrast RNA interference lines showed more tolerance to Fe excess stress. Furthermore, RNAi lines showed decreased Fe concentrations compared to wild type plants under Fe excess condition. Together these data show that OsFRO1 is involved in reducing ferric Fe into ferrous Fe in the vacuole, and makes the vacuolar stored Fe available to the cytoplasm through Fe (II) or chelated Fe (II) transporters. Under Fe excess condition, the downregulation of OsFRO1 in the RNAi plants reduced the amount of Fe (II) available for cytoplasm, to alleviate Fe excess toxicity. This indicates that OsFRO1 plays an important role to maintain Fe homeostasis between the cytoplasm and vacuole in rice.

Two soybean bHLH factors regulate response to iron deficiency.

Iron is an indispensable micronutrient for plant growth and development. Limited bioavailability of Fe in the soil leads to iron deficiency chlorosis in plants and yield loss. In this study, two soybean basic helix-loop-helix transcription factors, GmbHLH57 and GmbHLH300, were identified in response to Fe-deficiency. Both transcription factors are expressed in roots and nodules, and are induced by Fe deficiency; these patterns were confirmed in transgenic hairy roots expressing constructs of the endogenous promoters fused to a GUS reporter gene. Bimolecular fluorescence complementation, yeast two-hybrid and coimmunoprecipitation (co-IP) assays indicated a physical interaction between GmbHLH57 and GmbHLH300. Studies on transgenic soybeans overexpressing GmbHLH57 and GmbHLH300 revealed that overexpression of each transcription factor, alone, results in no change of the responses to Fe deficiency, whereas overexpression of both transcription factors upregulated the downstream Fe uptake genes and increased the Fe content in these transgenic plants. Compared to wild type, these double overexpression transgenic plants were more tolerant to Fe deficiency. Taken together, our findings establish that GmbHLH57 and GmbHLH300 are important transcription factors involved in Fe homeostasis in soybean.