Rutin attenuates ensartinib-induced hepatotoxicity by non-transcriptional regulation of TXNIP.

Ensartinib, an approved ALK inhibitor, is used as a first-line therapy for advanced ALK-positive non-small cell lung cancer in China. However, the hepatotoxicity of ensartinib seriously limits its clinical application and the regulatory mechanism is still elusive. Here, through transcriptome analysis we found that transcriptional activation of TXNIP was the main cause of ensartinib-induced liver dysfunction. A high TXNIP level and abnormal TXNIP translocation severely impaired hepatic function via mitochondrial dysfunction and hepatocyte apoptosis, and TXNIP deficiency attenuated hepatocyte apoptosis under ensartinib treatment. The increase in TXNIP induced by ensartinib is related to AKT inhibition and is mediated by MondoA. Through screening potential TXNIP inhibitors, we found that the natural polyphenolic flavonoid rutin, unlike most reported TXNIP inhibitors can inhibit TXNIP by binding to TXNIP and partially promoting its proteasomal degradation. Further studies showed rutin can attenuate the hepatotoxicity of ensartinib without antagonizing its antitumor effects. Accordingly, we suggest that TXNIP is the key cause of ensartinib-induced hepatotoxicity and rutin is a potential clinically safe and feasible therapeutic strategy for TXNIP intervention.

Effects of dacomitinib on the pharmacokinetics of poziotinib in vivo and in vitro.

CONTEXT: Dacomitinib and poziotinib, irreversible ErbB family blockers, are often used for treatment of non-small cell lung cancer (NSCLC) in the clinic. OBJECTIVE: This study investigates the effect of dacomitinib on the pharmacokinetics of poziotinib in rats. MATERIALS AND METHODS: Twelve Sprague-Dawley rats were randomly divided into two groups: the test group (20 mg/kg dacomitinib for 14 consecutive days) and the control group (equal amounts of vehicle). Each group was given an oral dose of 10 mg/kg poziotinib 30 min after administration of dacomitinib or vehicle at the end of the 14 day administration. The concentration of poziotinib in plasma was quantified by UPLC-MS/MS. Both in vitro effects of dacomitinib on poziotinib and the mechanism of the observed inhibition were studied in rat liver microsomes and human liver microsomes. RESULTS: When orally administered, dacomitinib increased the AUC, Tmax and decreased CL of poziotinib (p < 0.05). The IC50 values of M1 in RLM, HLM and CYP3A4 were 11.36, 30.49 and 19.57 µM, respectively. The IC50 values of M2 in RLM, HLM and CYP2D6 were 43.69, 0.34 and 0.11 µM, respectively, and dacomitinib inhibited poziotinib by a mixed way in CYP3A4 and CYP2D6. The results of the in vivo experiments were consistent with those of the in vitro experiments. CONCLUSIONS: This research demonstrates that a drug-drug interaction between poziotinib and dacomitinib possibly exists when readministered with poziotinib; thus, clinicians should pay attention to the resulting changes in pharmacokinetic parameters and accordingly, adjust the dose of poziotinib in clinical settings.

Further delineation of primary B cell immunodeficiency caused by novel variants of the BLNK gene in two Chinese patients.

Biallelic variants in BLNK cause primary B-cell immunodeficiency that usually results in absence of B cells and immunoglobulin. Here, we identified disease-causing variant(s) in two unrelated Chinese patients with agammaglobulinemia. Patient 1 showed a moderate reduction in total B-cell count but demonstrated both extremely low levels of memory B-cells and lower levels of memory T cells relative to those in healthy controls. Whole-exome sequencing (WES) revealed a novel heterozygous splice variant (c.676+1G>A), and suggested exon 9 deletion from BLNK, which was subsequently validated by quantitative polymerase chain reaction. For Patient 2, WES revealed novel compound heterozygous of a frameshift variant (p.T152Pfs*6) and a synonymous variant (c.525G>A) that resulted in exon 6 skipping, according to cDNA sequencing. These findings represent the first report of a BLNK-deficient patient presenting with impaired memory B-cell and memory T-cell development. Furthermore, this study is the first reporting a pathogenic synonymous splice variant in BLNK.

Effects of ticagrelor on the pharmacokinetics of rivaroxaban in rats.

CONTEXT: Rivaroxaban and ticagrelor are two common drugs for the treatment of atrial fibrillation and acute coronary syndrome. However, the drug-drug interaction between them is still unknown. OBJECTIVE: To investigate the effects of ticagrelor on the pharmacokinetics of rivaroxaban in rats both in vivo and in vitro. MATERIALS AND METHODS: A sensitive and reliable UPLC-MS/MS method was developed for the determination of rivaroxaban in rat plasma. Ten Sprague-Dawley rats were randomly divided into ticagrelor pre-treated group (10 mg/kg/day for 14 days) and control group. The pharmacokinetics of orally administered rivaroxaban (10 mg/kg, single dose) with or without ticagrelor pre-treatment was investigated with developed UPLC-MS/MS method. Additionally, Sprague-Dawley rat liver microsomes were also used to investigate the drug-drug interaction between these two drugs in vitro. RESULTS: The C max (221.34 ± 53.33 vs. 691.18 ± 238.31 ng/mL) and the AUC(0-t) (1060.97 ± 291.21 vs. 3483.03 ± 753.83 µg·h/L) of rivaroxaban increased significantly (p < 0.05) with ticagrelor pre-treatment. The MRT(0-∞) of rivaroxaban increased from 4.41 ± 0.79 to 5.97 ± 1.11 h, while the intrinsic clearance decreased from 9.93 ± 2.55 to 2.89 ± 0.63 L/h/kg (both p < 0.05) after pre-treated with ticagrelor. Enzyme kinetic study indicated that ticagrelor decreased rivaroxaban metabolic clearance with the IC50 value of 14.04 µmol/L. CONCLUSIONS: Our in vivo and in vitro results demonstrated that there is a drug-drug interaction between ticagrelor and rivaroxaban in rats. Further studies need to be carried out to verify whether similar interactions truly apply in humans and whether these interactions have clinical significance.

Pharmacokinetics and bioavailability of gelsenicine in mice by UPLC-MS/MS.

Gelsenicine is an indole alkaloid isolated from Gelsemium elegans Benth. In recent years, the role of G. elegans Benth preparations in anti-tumor, analgesic, dilatation and dermatological treatment has attracted attention, and it has been applied clinically, but it is easy to cause poisoning with its use. An UPLC-MS/MS method was established to determine the gelsenicine in mouse blood, and the pharmacokinetics of gelsenicine after intravenous (0.1 mg/kg) and intragastric (0.5 and 1 mg/kg) administration was studied. Deltalin was used as internal standard; a UPLC BEH C18 column was used for chromatographic separation. The mobile phase consisted of acetonitrile and 10 mmol/L ammonium acetate (0.1% formic acid) with a gradient elution flow rate of 0.4 mL/min. Multiple reaction monitoring mode was used for quantitative analysis of gelsenicine in electrospray ionization positive interface. Proteins from mouse blood were removed by acetonitrile precipitation. A validation of this method was performed in accordance with the US Food and Drug Administration guidelines. In the concentration range of 0.05-100 ng/mL, the gelsenicine in the mouse blood was linear (r > 0.995), and the lower limit of quantification was 0.05 ng/mL. In the mouse blood, the intra-day precision RSD was <12%, the inter-day precision RSD was <15%, the accuracy ranged from 89.8 to 112.3%, the average recovery was >76.8%, and the matrix effect was between 103.7 and 108.4%, which meet the pharmacokinetic research requirements of gelsenicine. The UPLC-MS/MS method is sensitive, rapid and selective, and has been successfully applied to the pharmacokinetic study of gelsenicine in mice. The absolute bioavailability of gelsenicine is 1.13%.

[Analysis of clinical manifestation and genetic mutation in a child with X-linked chondrodysplasia punctata 2].

OBJECTIVE: To analyze clinical manifestations and genetic mutation in a child with severe short stature and other malformations. METHODS: The child has undergone history taking and physical examination. Genome DNA was extracted from peripheral blood samples of the proband and her family members. Candidate genes were captured with Agilent SureSelect and sequenced on an Illumina platform. Suspected mutation was verified by Sanger sequencing. RESULTS: The patient, a six-year-and-10-month old girl, presented with non-symmetrical short stature, dysmorphism, abnormalities of limbs and spine, amblyopia of left eye, and cataract of right eye, in addition with frequent respiratory infection and micturition. Laboratory testing suggested 25-hydroxy vitamin D deficiency (18.9 ng/mL). Spine X-ray showed multiple malformations with centrums. Her mother also featured short stature (138 cm). Her aunt had short stature (130 cm) and limb-length discrepancy. Her little brother was 2.5 years old, and his height was 81 cm (-3.4 SD). Exome sequencing revealed a heterozygous mutation c.184C to T (p.Arg62Trp) in the proband and her mother. The same mutation was not found in her father and brother. CONCLUSION: The patient was diagnosed with X-linked chondrodysplasia punctata 2. Mutation of the EBP gene probably underlied the disease in this family.

Pharmacokinetic study of ardisiacrispin A in rat plasma after intravenous administration by UPLC-MS/MS.

In this work, a sensitive and selective UPLC-MS/MS method for determination of ardisiacrispin A in rat plasma was developed. Cyasterone used as an internal standard (IS) and protein precipitation by acetonitrile-methanol (9:1, v/v) was used to prepare samples. Chromatographic separation was achieved on a UPLC BEH C18 column (2.1 × 100 mm, 1.7 µm) with 0.1% formic acid and acetonitrile as the mobile phase with gradient elution. An electrospray ionization source was applied and operated in positive ion mode; multiple reaction monitoring mode was used for quantification using target fragment ions m/z 1083.5 → 407.1 for ardisiacrispin A and m/z 521.3 → 485.2 for IS. Calibration plots were linear throughout the range 5-2000 ng/mL for ardisiacrispin A in rat plasma. Mean recoveries of ardisiacrispin A in rat plasma ranged from 80.4 to 92.6%. The values of RSD of intra- and inter-day precision were both <11%. The accuracy of the method was between 97.3 and 105.6%. The method was successfully applied to pharmacokinetic study of ardisiacrispin A after intravenous administration in rats.

Synthesis and biological evaluation of allylated mono-carbonyl analogues of curcumin (MACs) as anti-cancer agents for cholangiocarcinoma.

A series of new allylated mono-carbonyl curcumin analogues (MACs) were designed and synthesized. In vitro cytotoxic activities of allylated MACs 6a-h together with previously reported analogues 4a-i and 7a-e, were tested against human cholangiocarcinoma cell lines including HUCCA, QBC-939 and RBE. Of all the compounds tested, 6c exhibited potent in vitro antiproliferative activity against the three tested cancer cell lines with IC50 values of 8.7, 9.3 and 8.9µM, respectively. Cell cycle analysis showed that 6c inhibited cell proliferation due to G2/M arrest. Furthermore, mechanistic studies revealed that 6c dose-dependently increased the level of Bax and inhibited the expression of Bcl-2, to induce cancer cell apoptosis. Taken together, this work provides a novel series of anti-cancer candidates for the treatment of cholangiocarcinoma.

Pharmacokinetic study of dendrobine in rat plasma by ultra-performance liquid chromatography tandem mass spectrometry.

Dendrobine, considered as the major active alkaloid compound, has been used for the quality control and discrimination of Dendrobium which is documented in the Chinese Pharmacopoeia. In this work, a sensitive and simple ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for determination of dendrobine in rat plasma is developed. After addition of caulophyline as an internal standard (IS), protein precipitation by acetonitrile-methanol (9:1, v/v) was used to prepare samples. Chromatographic separation was achieved on a UPLC BEH C18 (2.1 ×100 mm, 1.7 µm) column with acetonitrile and 0.1% formic acid as the mobile phase with gradient elution. An electrospray ionization source was applied and operated in positive ion mode; multiple reaction monitoring mode was used for quantification using target fragment ions m/z 264.2 → 70.0 for dendrobine and m/z 205.1 → 58.0 for IS. Calibration plots were linear throughout the range 2-1000 ng/mL for dendrobine in rat plasma. The RSDs of intra-day and inter-day precision were both <13%. The accuracy of the method was between 95.4 and 103.9%. The method was successfully applied to pharmacokinetic study of dendrobine after intravenous administration. Copyright © 2015 John Wiley & Sons, Ltd.

Pharmacokinetic interaction study combining lapatinib with vorinostat in rats.

BACKGROUND: Because lapatinib and vorinostat [suberoylanilide hydroxamic acid (SAHA)] are both new anticancer drugs, interactions between SAHA and lapatinib remain unclear. This study examines pharmacokinetic interactions in simultaneous oral administration of SAHA and lapatinib to rats. METHODS: Twenty-four rats were divided randomly into 3 groups: a lapatinib group (lapatinib 25 mg/kg, n = 8), a SAHA group (SAHA 25 mg/kg, n = 8), and a coadministration group (SAHA 25 mg/kg and lapatinib 25 mg/kg, n = 8). Using ultrahigh-performance liquid chromatography-tandem mass spectrometry, the concentrations of lapatinib and SAHA were determined in the plasma of the test rats. RESULTS: Statistically significant pharmacokinetic differences appeared for lapatinib levels between the lapatinib and the coadministration group. When lapatinib was coadministered with SAHA, the AUC0-t decreased from 39,816.6 to 23,712.8 ng/ml ∙ h (p < 0.05), while the mean residence time (MRT)0-t increased from 7.0 to 10.3 h (p < 0.05) and t1/2 increased from 3.5 to 6.4 h (p < 0.05). Between the SAHA levels for the SAHA group and those for the coadministration group, there appeared to be no statistically significant differences. CONCLUSION: The resulting data indicate that, when administered together, lapatinib does not influence the pharmacokinetic profile of SAHA in rats, while, in contrast, SAHA influences the pharmacokinetic profile of lapatinib.

Determination and validation of hupehenine in rat plasma by UPLC-MS/MS and its application to pharmacokinetic study.

In this work, a sensitive and selective ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for determination of hupehenine in rat plasma was developed and validated. After addition of imperialine as an internal standard (IS), protein precipitation by acetonitrile-methanol (9:1, v/v) was used to prepare samples. Chromatographic separation was achieved on a UPLC BEH C18 column (2.1 × 100 mm, 1.7 µm) with 0.1% formic acid and acetonitrile as the mobile phase with gradient elution. An electrospray ionization source was applied and operated in positive ion mode; multiple reaction monitoring mode was used for quantification using target fragment ions m/z 416.3 → 98.0 for hupehenine, and m/z 430.3 → 138.2 for IS. Calibration plots were linear throughout the range 2-2000 ng/mL for hupehenine in rat plasma. Mean recoveries of hupehenine in rat plasma ranged from 92.5 to 97.3%. Relative standard deviations of intra-day and inter-day precision were both <6%. The accuracy of the method was between 92.7 and 107.4%. The method was successfully applied to a pharmacokinetic study of hupehenine after either oral or intravenous administration. For the first time, the bioavailability of hupehenine was reported as 13.4%.

Simultaneous determination of bupropion, metroprolol, midazolam, phenacetin, omeprazole and tolbutamide in rat plasma by UPLC-MS/MS and its application to cytochrome P450 activity study in rats.

A specific ultra-performance liquid chromatography tandem mass spectrometry method is described for the simultaneous determination of bupropion, metroprolol, midazolam, phenacetin, omeprazole and tolbutamide in rat plasma with diazepam as internal standard, which are the six probe drugs of the six cytochrome P450 isoforms CYP2B6, CYP2D6, CYP3A4, CYP1A2, CYP2C19 and CYP2C9. Plasma samples were protein precipitated with acetonitrile. The chromatographic separation was achieved using a UPLC® BEH C18 column (2.1 × 100 mm, 1.7 µm). The mobile phase consisted of acetonitrile and water (containing 0.1% formic acid) with gradient elution. The triple quadrupole mass spectrometric detection was operated by multiple reaction monitoring in positive electrospray ionization. The precisions were <13%, and the accuracy ranged from 93.3 to 110.4%. The extraction efficiency was >90.5%, and the matrix effects ranged from 84.3 to 114.2%. The calibration curves in plasma were linear in the range of 2-2000 ng/mL, with correlation coefficient (r(2) ) >0.995. The method was successfully applied to pharmacokinetic studies of the six probe drugs of the six CYP450 isoforms and used to evaluate the effects of erlotinib on the activities of CYP2B6, CYP2D6, CYP3A4, CYP1A2, CYP2C19 and CYP2C9 in rats. Erlotinib may inhibit the activity of CYP2B6 and CYP3A4, and may induce CYP2C9 of rats.

Pharmacokinetic interaction study of combining imatinib with dasatinib in rats by UPLC-MS/MS.

This study examined whether oral administration of dasatinib to the rats with imatinib led to any pharmacokinetic interactions. Twenty-four rats were divided randomly into three groups, imatinib group (imatinib 25 mg/kg, n = 8), dasatinib group (dasatinib 15 mg/kg, n = 8) and co-administration group (dasatinib 15 mg/kg and imatinib 25 mg/kg, n = 8). The concentration of imatinib and dasatinib in rat plasma was determined by a sensitive and simple UPLC-MS/MS method. There was statistical pharmacokinetics difference for imatinib in the imatinib group and co-administration group, when co-oral administration imatinib with dasatinib, MRT(0-t) increased (p < 0.01). There was statistical pharmacokinetics difference for dasatinib in the dasatinib group and co-administration group, when co-oral administration dasatinib with imatinib, Cmax and AUC increased (p < 0.01), CL and V decreased (p < 0.01). These data indicate dasatinib could slightly influence the pharmacokinetic profile of imatinib in rats, and imatinib could influence the pharmacokinetic profile of dasatinib in rats, which might cause drug-drug interactions when using imatinib with dasatinib.

[Effects of ribavirin aerosol on viral exclusion of patients with hand-foot-mouth disease].

OBJECTIVE: To explore the roles of ribavirin aerosol in the prevention and treatment of hand-foot-mouth disease (HFMD). METHODS: For this prospective, multicenter, randomized, double-blind and placebo-controlled trial, a total of 300 HFMD outpatients from 3 class 3A hospitals from July 2011 to June 2013 were divided into treatment (ribavirin aerosol) and control (placebo) groups (n = 150 each). The age range was 6 months to 6 years. The proportion of male and female was 1.5: 1. Temperature, herpes of mouth and skin rash were observed before and after treatments. Before treatment and 6-7 days after, their specimens of throat swab were collected and the levels of EV71 and CA16 detected with reverse transcription (RT)-PCR. The software SAS 9.2 was used for statistic data analyses. RESULTS: Before treatment, no significant statistical difference existed in parameters between treatment and control groups (all P > 0.05). After treatment, the degree of herpes of mouth and papulovesicle of skin in treatment group was better than that in control group (100.0% (147/147) vs 83.9% (120/143), χ(2) = 109.21, P < 0.01); (100.0% (147/147) vs 95.9% (139/145) , χ(2) = 6.38, P < 0.05) . The virus negative conversion rates had significant inter-group difference (80.0% (102/110) vs 41.8% (41/98) , χ(2) = 37.06, P < 0.01). The temperature, compliance and differences were not significant (all P > 0.05). The effective rate of comprehensive efficacy in treatment group was higher than that in control group (93.9% (122/130) vs 52.0% (64/123), χ(2) = 111.08, P < 0.01). No obvious adverse drug reaction was observed. CONCLUSIONS: Ribavirin aerosol has multiple advantages of lower dose, quicker onset, higher local concentration, better clinical efficacy and fewer side-effects. Its therapeutic effect for local lesion is better than that for systemic lesion. Thus it may shorten the duration of oropharyngeal and skin lesions and lower the number and time of viral release.

The Impact of Baohuoside I on the Metabolism of Tofacitinib in Rats.

Purpose: To study the potential drug-drug interactions between tofacitinib and baohuoside I and to provide the scientific basis for rational use of them in clinical practice. Methods: A total of eighteen Sprague-Dawley rats were randomly divided into three groups: control group, single-dose group (receiving a single dose of 20 mg/kg of baohuoside I), and multi-dose group (receiving multiple doses of baohuoside I for 7 days). On the seventh day, each rat was orally administered with 10 mg/kg of tofacitinib 30 minutes after giving baohuoside I or vehicle. Blood samples were collected and determined using UPLC-MS/MS. In vitro effects of baohuoside I on tofacitinib was investigated in rat liver microsomes (RLMs), as well as the underlying mechanism of inhibition. The semi-inhibitory concentration value (IC50) of baohuoside I was subsequently determined and its inhibitory mechanism against tofacitinib was analyzed. Furthermore, the interactions between baohuoside I, tofacitinib and CYP3A4 were explored using Pymol molecular docking simulation. Results: The administration of baohuoside I orally has been observed to enhance the area under the concentration-time curve (AUC) of tofacitinib and decrease the clearance (CL). The observed disparity between the single-dose and multi-dose groups was statistically significant. Furthermore, our findings suggest that the impact of baohuoside I on tofacitinib metabolism may be a mixture of non-competitive and competitive inhibition. Baohuoside I exhibit an interaction with arginine (ARG) at position 106 of the CYP3A4 enzyme through hydrogen bonding, positioning itself closer to the site of action compared to tofacitinib. Conclusion: Our study has demonstrated the presence of drug-drug interactions between baohuoside I and tofacitinib, which may arise upon pre-administration of tofacitinib. Altogether, our data indicated that an interaction existed between tofacitinib and baohuoside I and additional cares might be taken when they were co-administrated in clinic.

Effect of P. corylifolia on the pharmacokinetic profile of tofacitinib and the underlying mechanism.

This work aimed to explore the mechanisms underlying the interaction of the active furanocoumarins in P. corylifolia on tofacitinib both in vivo and in vitro. The concentration of tofacitinib and its metabolite M8 was determined using UPLC-MS/MS. The peak area ratio of M8 to tofacitinib was calculated to compare the inhibitory ability of furanocoumarin contained in the traditional Chinese medicine P. corylifolia in rat liver microsomes (RLMs), human liver microsomes (HLMs) and recombinant human CYP3A4 (rCYP3A4). We found that bergapten and isopsoralen exhibited more significant inhibitory activity in RLMs than other furanocoumarins. Bergapten and isopsoralen were selected to investigate tofacitinib drug interactions in vitro and in vivo. Thirty rats were randomly allocated into 5 groups (n = 6): control (0.5% CMC-Na), low-dose bergapten (20 mg/kg), high-dose bergapten (50 mg/kg), low-dose isopsoralen (20 mg/kg) and ketoconazole. 10 mg/kg of tofacitinib was orally intervented to each rat and the concentration level of tofacitinib in the rats were determined by UPLC-MS/MS. More imporrantly, the results showed that bergapten and isopsoralen significantly inhibited the metabolism of tofacitinib metabolism. The AUC(0-t), AUC(0-∞), MRT(0-t), MRT(0-∞) and Cmax of tofacitinib increased in varying degrees compared with the control group (all p < 0.05), but CLz/F decreased in varying degrees (p < 0.05) in the different dose bergapten group and isopsoralen group. Bergapten, isopsoralen and tofacitinib exhibit similar binding capacities with CYP3A4 by AutoDock 4.2 software, confirming that they compete for tofacitinib metabolism. P. corylifolia may considerably impact the metabolism of tofacitinib, which can provide essential information for the accurate therapeutic application of tofacitinib.

Dabrafenib alleviates hepatotoxicity caused by lenvatinib via inhibiting the death receptor signaling pathway.

Lenvatinib is a multi-target inhibitor that exerts anti-tumor effects by inhibiting angiogenesis and is now commonly used as a first-line treatment for hepatocellular carcinoma. However, with the widespread use of lenvatinib, the problem of serious and fatal hepatotoxicity has become increasingly prominent. Currently, the mechanism behind this toxicity is not yet understood, and as a result, there is a lack of safe and effective intervention strategies with minimal side effects. Here, we established the model of lenvatinib-induced liver injury in vivo and in vitro and found that lenvatinib caused hepatotoxicity by inducing apoptosis. Further mechanistic studies in cellular models revealed that lenvatinib upregulated death receptor signaling pathway, which activated the downstream effector Caspase-8, and ultimately led to apoptosis. Meanwhile, lenvatinib-induced apoptosis was associated with ROS generation and DNA damage. In addition, after screening marketed drugs and natural products in combination with cellular modeling, we identified a potential co-administered drug, dabrafenib, which could alleviate lenvatinib-induced hepatotoxicity. Further mechanistic studies revealed that dabrafenib attenuated lenvatinib-induced hepatotoxicity by inhibiting the activation of the death receptor signaling pathway. Subsequently, cancer cell proliferation assays confirmed that dabrafenib did not antagonize the antitumor effects of lenvatinib. In conclusion, our results validate that apoptosis caused by the death receptor signaling pathway is the key cause of lenvatinib-induced hepatotoxicity, and dabrafenib alleviates lenvatinib-induced hepatotoxicity by inhibiting this pathway.

Characterization of six missense mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene in Chinese children with hypophosphatasia.

AIMS: Hypophosphatasia, a rare inherited disease characterized by defective mineralization of bone and teeth, is caused by various mutations in the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP) gene. Our aim was to determine the mutations on TNSALP gene in three Chinese children diagnosed as having hypophosphatasia. METHODS: Genomic DNA was extracted from whole blood samples of patients and their parents. The TNSALP coding regions were then sequenced. Plasmids expressing wild-type or various mutants were built and in vitro studies were performed in order to determine whether these amino acid replacements could affect the TNSALP enzymatic activity. RESULTS: Six missense mutations were identified from three independent pedigrees. Of the six missense mutations, four were novel and two had been previously reported. The Y28D, A111T and T389N mutants displayed only negligible ALP activity in vitro compared to the wild-type (WT) TNSALP. The defect was mainly due to the significantly decreased protein expression in the 66 KD immature forms and the nearly undetectable protein expression in the 80 KD mature forms. Moreover, all three mutants had a dominant negative effect on the WT protein when co-transfected with TNSALP (WT). M219V and R136L mutants both exhibited partial enzymatic activities which were consistent with reduced protein expression in both forms of TNSALP which further exhibited moderate dominant-negative effect. In addition, Y388H mutant showed weak ALP activity. Western blot analysis indicated that the extreme reduction in signal from the mature forms of TNSALP could be the main cause of decreased enzymatic activity, since a strong signal was observed in the immature forms. CONCLUSION: Six missense mutations were identified in three Chinese hypophosphatasia pedigrees with subnormal serum ALP activity. Our results show that the low activity of serum ALP in the three patients is due mainly to a defect in the protein expression of the mutants. This may be the underling molecular mechanism for hypophosphatasia in these patients.

Inhibitory effect of Schisandrin on the pharmacokinetics of poziotinib in vivo and in vitro by UPLC-MS/MS.

BACKGROUND: As a pan-HER tyrosine kinase inhibitor with a promising application prospect, poziotinib is likely to be coadministered with Schisandrins in clinical treatment due to its anticancer activities. METHODS: Eighteen Sprague-Dawley rats were randomly divided into three groups: Schisandrin A group and Schisandrin B group (20 mg/kg daily for 1 week), and control group (vehicle). On day 8, poziotinib (2 mg/kg) was administered by oral gavage 30 min later. An in vitro study was developed to identify the possible mechanisms of Schisandrins on poziotinib metabolism. All analytes were detected by UPLC/MS-MS, and molecular docking was performed by AutoDock Tools. RESULTS: When rats were preadministered with Schisandrin A, AUC(0-∞) and Cmax of poziotinib were obviously increased by 0.79- and 1.17-fold, whereas the Vz/F and CLz/F values were dramatically decreased. The results in Schisandrin B group presented similarly. Both Schisandrin A and Schisandrin B were mixed inhibitors of poziotinib in RLMs, and Schisandrin B showed stronger inhibitory activity with IC50 values of 2.55 µM for M1 and 6.97 µM for M2. Molecular docking analysis demonstrated that Schisandrin A and Schisandrin B exhibited a strong binding ability towards CYP2D6 as compared to CYP3A4. CONCLUSION: All results provided the direct evidence of the pharmacokinetic drug-drug interactions (DDIs) between Schisandrin and poziotinib. Thus, particular attention should be paid when poziotinib is taken together with Schisandrins in clinical practice.

Effects of bergapten on the pharmacokinetics of macitentan in rats both in vitro and in vivo.

Macitentan was approved by the United States Food and Drug Administration (FDA) in 2013 for the treatment of pulmonary arterial hypertension (PAH). Bergapten is a furanocoumarin that is abundant in Umbelliferae and Rutaceae plants and is widely used in many Chinese medicine prescriptions. Considering the possible combination of these two compounds, this study is aimed to investigate the effects of bergapten on the pharmacokinetics of macitentan both in vitro and in vivo. Rat liver microsomes (RLMs), human liver microsomes (HLMs), and recombinant human CYP3A4 (rCYP3A4) were used to investigate the inhibitory effects and mechanisms of bergapten on macitentan in vitro. In addition, pharmacokinetic parameters were also studied in vivo. Rats were randomly divided into two groups (six rats per group), with or without bergapten (10 mg/kg), and pretreated for 7 days. An oral dose of 20 mg/kg macitentan was administered to each group 30 min after bergapten or 0.5% CMC-Na administration on day 7. Blood was collected from the tail veins, and the plasma concentrations of macitentan and its metabolites were assessed by ultra-performance liquid chromatography - tandem mass spectrometer (UPLC-MS/MS). Finally, we analyzed the binding force of the enzyme and two small ligands by in silico molecular docking to verify the inhibitory effects of bergapten on macitentan. The in vitro results revealed that the IC50 values for RLMs, HLMs, and rCYP3A4 were 3.84, 17.82 and 12.81 µM, respectively. In vivo pharmacokinetic experiments showed that the AUC(0-t), AUC(0-∞), and Cmax of macitentan in the experimental group (20,263.67 µg/L*h, 20,378.31 µg/L*h and 2,999.69 µg/L, respectively) increased significantly compared with the control group (7,873.97 µg/L*h, 7,897.83 µg/L*h and 1,339.44 µg/L, respectively), while the CLz/F (1.07 L/h/kg) of macitentan and the metabolite-parent ratio (MR) displayed a significant decrease. Bergapten competitively inhibited macitentan metabolism in vitro and altered its pharmacokinetic characteristics in vivo. Further molecular docking analysis was also consistent with the experimental results. This study provides a reference for the combined use of bergapten and macitentan in clinical practice.