The effect of shikonin on the metabolism of lapatinib in vitro, and in vivo.

PURPOSE: The purpose of this study was to develop an assay for simultaneous determination of lapatinib and its metabolites (N-dealkylated lapatinib and O-dealkylated lapatinib) by ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), and to determine the interaction between shikonin and lapatinib in vitro, in vivo, in silico and its mechanism of action. METHODS: A new UPLC-MS/MS method for the determination of the concentrations of lapatinib and its metabolites was developed. In vivo, Sprague-Dawley (SD) rats were given lapatinib with or without shikonin. In vitro, to study the interaction mechanism, rat liver microsomes (RLMs), human liver microsomes (HLMs) and recombinant human CYP3A4.1 were used for determining enzyme kinetics. Lastly, we used in silico molecular docking to investigate the molecular mechanism of inhibition. RESULTS: The selectivity, precision, accuracy, stability, matrix effect and recovery of UPLC-MS/MS all met the requirements of quantitative analysis of biological samples. Administration of lapatinib combined with shikonin resulted in significantly increased pharmacokinetic parameters (AUC(0-t) and Cmax) of lapatinib, indicating that shikonin increased the exposure of lapatinib in rats. Moreover, in vitro kinetic measurements indicated that shikonin was a time-independent inhibitor, which inhibited the metabolism of lapatinib through a competitive mechanism in RLMs, while noncompetitive inhibition type in both HLMs and CYP3A4.1. Molecular docking analysis further verified the non-competitive inhibition of shikonin on lapatinib in CYP3A4.1. CONCLUSION: We developed an UPLC-MS/MS assay for simultaneous determination of lapatinib and its metabolites. It could be successfully applied to the study of pharmacokinetic interaction of shikonin on the inhibition of lapatinib metabolism in vivo and in vitro. In the end, further studies are needed to determine if such interactions are indeed valid in humans and if the interaction is clinically relevant.

Genetic variations of CYP3A4 on the metabolism of itraconazole in vitro.

Itraconazole is a triazole anti-infective drug that has been proven to prevent and treat a variety of fungal and viral infections and has been considered to be a potential therapeutic remedy for COVID-19 treatment. In this study, we aimed to completely evaluate the impacts of Cytochrome P450 3A4 (CYP3A4) variant proteins and drug interactions on the metabolism of itraconazole in recombinant insect microsomes, and to characterize the potential mechanism of substrate selectivity. Incubations with itraconazole (0.2-15 µM) in the presence/absence of lopinavir or darunavir were assessed by CYP3A4 variants, and the metabolite hydroxyitraconazole concentrations were measured by UPLC-MS/MS. Our data showed that when compared with CYP3A4.1, 4 variants (CYP3A4.9, .10, .28 and .34) displayed no significant differences, and 3 variants (CYP3A4.14, .15 and .19) exhibited increased intrinsic clearance (CLint), whereas the remaining 17 variant proteins showed decreased enzyme activities for the catalysis of itraconazole. Moreover, the inhibitory effects of lopinavir and darunavir on itraconazole metabolism varied in different degrees. Furthermore, different changed trend of the kinetic parameters in ten variants (CYP3A4.5, .9, .10, .16, .19, .24, .28, .29, .31, and .33) were observed, especially CYP3A4.5 and CYP3A4.16, and this may be related to the metabolic site-heme iron atom distance. In the present study, we functionally analyzed the effects of 25 CYP3A4 protein variants on itraconazole metabolism for the first time, and provided comprehensive data on itraconazole metabolism in vitro. This may help to better assess the metabolism and elimination of itraconazole in clinic to improve the safety and efficacy of its clinical treatment and also provide new possibilities for the treatment of COVID-19.

Effects of CYP2C19 inhibitors on mavacamten pharmacokinetics in rats based on UPLC-MS/MS.

CONTEXT: CYP2C19 is an important member of the human cytochrome P450 2C (CYP2C) family. Mavacamten is a novel treatment of patients with symptomatic obstructive hypertrophic cardiomyopathy (HCM) which was metabolized mainly by CYP2C19. OBJECTIVE: In this study, we firstly reported and validated a quantitative analysis method of mavacamten in rat plasma based on ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), which was applied to the drug-drug interaction (DDI) study between mavacamten and CYP2C19 inhibitors (fluvoxamine, fluoxetine and fluconazole) in rats. MATERIALS AND METHODS: Vericiguat was used as the internal standard (IS), and the analyte and IS were measured with electrospray ion (ESI) source in positive ion mode on a XEVO TQ-S triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode. RESULTS: In the scope of 1.0-100 ng/mL, this assay had excellent linearity. Both intra-day and inter-day accuracy of the analyte ranged from -2.4% to 9.1%, while the precision was ≤4.2%. Matrix effect, recovery, and stability were evaluated and validated to meet the requirements for the guidelines of bioanalytical assay. When compared with the control group, AUC0→∞ of mavacamten in fluconazole, fluoxetine and fluvoxamine were increased by 125.5%, 110.7% and 43.6%, respectively, which demonstrated that CYP2C19 inhibitors could inhibit mavacamten metabolism. CONCLUSIONS: The results showed that CYP2C19 inhibitors could significantly improve the bioavailability of mavacamten in rats, which indicated that we should pay more attention to the patient's condition to prevent the occurrence of side effects when used mavacamten in combination with CYP2C19 inhibitors.

Ultrasound-induced destruction of heparin-loaded microbubbles attenuates L-arginine-induced acute pancreatitis.

PURPOSE: Acute pancreatitis (AP) involves sudden inflammation caused by abnormal activation of pancreatic enzymes. The mechanisms underlying AP include oxidative stress, high levels of inflammatory mediators and inflammatory cell infiltration. Heparin, a key therapeutic drug, exerts anti-inflammatory, antioxidative, and anticoagulative effects. However, safe and effective drug delivery remains an obstacle. This study is the first to investigate the therapeutic effects of heparin-loaded microbubbles (HPMB) combined with ultrasound (UHPMB) and the role of heparin in acoustic cavitation. METHODS: The characteristics of the microbubbles, including particle size, concentration, release, stability, and development, were studied. Heparin concentration in the HPMB was measured, and heparin-induced anticoagulation was evaluated. Drug safety was explored using hemolysis and cell viability assessments. The ability of HPMB to alleviate oxidative stress and inflammation were investigated in vitro. L-arginine induces AP in vivo. UHPMB was used for AP treatment. Serum amylase levels were measured and pancreatic architecture and pathological features were evaluated to determine AP severity. In vivo efficacy was evaluated, and the underlying mechanism of heparin action during acoustic cavitation was explored. RESULTS: HPMB was spherical and presented as an emulsion-like solution without aggregation. HPMB was visible and stable and effectively released the drug under ultrasound (US). HPMB and UHPMB led to lower AP severity than in the untreated group. US-targeted microbubble destruction (UTMD) enhanced the therapeutic effect by decreasing oxidative stress and inflammation in AP models without injuring vital organs. UHPMB regulated VEGF/Flt-1 and SOD-1 expression. HPMB can also mitigate oxidative stress and inflammation in H2O2-pretreated cells. CONCLUSION: UHPMB exhibits a strong ability not only to selectively target pancreatic lesions and release heparin but also to provide efficient protection by inhibiting oxidative stress and inflammation.

Evaluation of the inhibitory effect of quercetin on the pharmacokinetics of tucatinib in rats by a novel UPLC-MS/MS assay.

CONTEXT: Tucatinib (CYP2C8 substrate) and quercetin (CYP2C8 inhibitor) are two common drugs for the treatment of cancer. However, the effect of quercetin on the metabolism of tucatinib remains unknown. OBJECTIVE: We validated a sensitive method to quantify tucatinib levels in rat plasma based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), which was successfully employed to explore the effect of quercetin on tucatinib pharmacokinetics in rats. MATERIALS AND METHODS: An Acquity UPLC BEH C18 column was applied to achieve the separation of tucatinib and internal standard (IS) talazoparib after protein precipitation with acetonitrile. Then, we used this assay to investigate the effect of different doses of quercetin (25, 50 and 100 mg/kg) on the exposure of orally administered tucatinib (30 mg/kg) in 24 Sprague-Dawley (SD) rats, which were randomly divided into three quercetin pre-treated groups and one control group (n = 6). RESULTS: Our developed assay was verified in all aspects of bioanalytical method validation, involving lower limit of quantification (LLOQ), selectivity, accuracy and precision, calibration curve, extraction recovery, matrix effect and stability. After pre-treatment with 100 mg/kg quercetin, AUC0→t, AUC0→∞ and Cmax of tucatinib were remarkably increased by 75.4%, 75.8% and 59.1% (p < 0.05), respectively, while CLz/F was decreased significantly by 47.3% (p < 0.05) when compared with oral administration of 30 mg/kg tucatinib alone. This change is dose-dependent. CONCLUSIONS: This study will help better understand the pharmacokinetic properties of tucatinib with concurrent use with quercetin, and more clinical verifications were inspired to confirm whether this interaction has clinical significance in humans.

Functional assessment of the effects of CYP3A4 variants on acalabrutinib metabolism in vitro.

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.

Simultaneous quantification and pharmacokinetic investigation of selexipag and its main metabolite ACT-333679 in rat plasma by UPLC-MS/MS method.

In the present study, an accurate, simple and fast bioanalytical method based on ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) technique for simultaneous quantification of plasma selexipag and its main metabolite ACT-333679 concentrations in rats was optimized and established. The purpose of chromatographic separation of selexipag, ACT-333679 and the internal standard (IS, diazepam) was accomplished using an Acquity BEH C18 (2.1 mm × 50 mm, 1.7 µm) column. The mobile phase was consisted of acetonitrile (solution A) and 0.1 % formic acid in water (solution B) in a linear gradient elution procedure with a flow rate of 0.40 mL/min. The measurement of the analytes and IS was explored using a XEVO TQ-S triple quadrupole tandem mass spectrometer, which was comprised with electrospray ionization (ESI) source in positive ion mode. Selected multiple reaction monitoring (MRM) mode was employed to detect the parent-to-daughter ion transitions as follows: m/z 497.4 → 302.2 for selexipag, m/z 420.1 → 378.2 for ACT-333679, and m/z 285.0 → 154.0 for diazepam (IS), respectively. The new UPLC-MS/MS method showed good linearity respectively at the calibration curve range of 0.05-50 ng/mL for selexipag, and 0.05-250 ng/mL for ACT-333679. The intra- and inter-day of accuracy and precision were all within the acceptable limits in the bioanalytical method, and the results of recovery and matrix effect were also met the requirements. The newly developed UPLC-MS/MS assay was forward successfully used to describe the pharmacokinetic profiles of selexipag and ACT-333679 in rats after oral treatment with 6.0 mg/kg selexipag.

Inhibitory effects of voriconazole, itraconazole and fluconazole on the pharmacokinetic profiles of ivosidenib in rats by UHPLC-MS/MS.

Ivosidenib, as an oral mutant isocitrate dehydrogenase 1 (mIDH1) inhibitor, was awarded approval in the USA for the targeted therapy of relapsed or refractory acute myeloid leukemia (AML) in adult patients, who also had a susceptible enzyme to mIDH1. The aim of our present study was to develop and validate an accurate and fast assay based on the ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) technique for the quantification of ivosidenib in plasma and to investigate the possible effects of different CYP3A4 inhibitors (voriconazole, itraconazole and fluconazole) on ivosidenib metabolism in rats. After the fast protein crash with acetonitrile, chromatographic separation of ivosidenib and erlotinib (used as the internal standard in this experiment, IS) was accomplished using an Acquity BEH C18 (2.1 mm × 50 mm, 1.7 µm) column, and detection of the analyte was also performed using a Xevo TQ-S triple quadrupole tandem mass spectrometer in the positive ion electrospray ionization (ESI) interface. The assay showed enough linearity over a 0.5-6000 ng/mL calibration range. The application of the validated bioanalytical method based on the UHPLC-MS/MS technique was further successfully exhibited in an animal study of the drug-drug interaction between ivosidenib (50 mg/kg) and voriconazole (20 mg/kg)/itraconazole (20 mg/kg)/fluconazole (20 mg/kg) in rats. Voriconazole, itraconazole and fluconazole increased the exposure of ivosidenib in plasma by different degrees and also had a potential inhibitory effect on the metabolism of ivosidenib. Thus, a dose reduction or interruption of ivosidenib may be important to guide the practice of clinical medicine.

Development of a novel and quick UPLC-MS/MS method for the pharmacokinetic analysis of duvelisib in beagle dogs.

Duvelisib, a new oral phosphoinositide-3-kinase (PI3K)-δ and PI3K-γ inhibitor, was recently approved in the USA as the therapeutic drug for patients with the diseases of relapsed or refractory chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL). In the present study of our research, a quick and simple bioanalytical method based on ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) technique was fully explored and established for the quantification of plasma duvelisib concentrations from beagle dog in which gilteritinib was used as the internal standard (IS). After a simple and quick protein precipitation treated with acetonitrile, the chromatographic separation of the analyte was carried out on an Acquity BEH C18 column (2.1 mm × 50 mm, 1.7 µm) conducted in a gradient elution procedure where acetonitrile (solvent A) and 0.1 % formic acid in water (solvent B) consisted as the mobile phase. The measurements of the analyte and IS were explored using a XEVO TQS triple quadrupole tandem mass spectrometer, which was comprised with electrospray ionization (ESI) source in positive ion mode. Selected reaction monitoring (SRM) mode was employed to detect the parent-to-daughter ion transitions as follows: m/z 416.88 → 281.88 for duvelisib, and m/z 553.09 → 436.01 for IS, respectively. The assay was successfully established in the calibration range from 0.5 to 3000 ng/mL for duvelisib, where the lower limit of quantification (LLOQ) was set at 0.5 ng/mL. The precisions of intra-day and inter-day for duvelisib were all below 12.6 %, and the accuracies were from -2.5% to 14.1%. Both matrix effect and mean recovery of the analyte and IS were all acceptable, and the analyte was stable during the assay and storage in dog plasma samples. The novel established bioanalytical method based on UPLC-MS/MS technique was effectively employed to the investigation of the pharmacokinetic profile of duvelisib in beagle dogs following a 1.34 mg/kg single dose of oral administration.

UPLC-MS/MS method for the determination of talazoparib in rat plasma and its pharmacokinetic study.

In the present study, an accurate and sensitive ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the determination of plasma talazoparib concentration in rats was developed and established. The purpose of chromatographic separation of talazoparib and the internal standard (bosutinib) was achieved on an Acquity BEH C18 (2.1 mm × 50 mm, 1.7 µm) column with a flow rate of 0.40 mL/min, using a gradient elution with acetonitrile and 0.1% formic acid in water as the mobile phase. The detection was performed on a XEVO TQ-S triple quadrupole tandem mass spectrometer coupled with electrospray ionization interface under positive-ion multiple reaction monitoring (MRM) mode with the precursor-to-product ion transitions of m/z 381.3 → 285.2 for talazoparib and m/z 530.2 → 141.2 for bosutinib (IS), respectively. The method was linear over the range of 0.5-200 ng/mL for talazoparib. The accuracies and precisions of intra- and inter-day were all within the acceptance limits, and no matrix effect was observed in this method. The validated method was further employed to a pharmacokinetic study of talazoparib after oral treatment with 0.2 mg/kg talazoparib to rats.

Validated UPLC-MS/MS method for quantification of fruquintinib in rat plasma and its application to pharmacokinetic study.

A new, simple, and sensitive ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for quantification of fruquintinib was established to assess the pharmacokinetics of fruquintinib in the rat. The internal standard working solution was added to the plasma sample for extraction before analysis. The Acquity UPLC BEH C18 chromatography column (2.1 mm ×50 mm, 1.7 µm) was used to separated analytes under gradient elution using acetonitrile and 0.1% formic acid as the mobile phase. Positive multiple reaction monitoring modes were chosen to detect fruquintinib and diazepam (IS). The precursor-to-product ion transitions were 394.2 → 363.2 for fruquintinib and m/z 285 → 154 for IS. The current method was linear over the concentration range of 1.0-1000 ng/mL for fruquintinib with a correlation coefficient of 0.9992 or better. The matrix effect of fruquintinib and IS was acceptable under the current method. The intra- and interday precision (RSD%) and accuracy (RE%) were within 11.9% and ±13.7%, respectively. The recovery, stability, and sensitivity were validated according to the United States Food and Drug Administration (FDA) regulations for bioanalytical method validation. The analytical method had been validated and applied to a pharmacokinetic study of fruquintinib in rat.

Determination of four omega-3 polyunsaturated fatty acids by UPLC-MS/MS in plasma of hyperlipidemic and normolipidemic subjects.

BACKGROUND: Omega-3 polyunsaturated fatty acids (PUFAs), including alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosapentaenoic acid (DPA), play critical roles in numerous biochemical reactions. Our aim is to develop a rapid and sensitive method for simultaneous determination of ALA, EPA, DHA and DPA in the plasma of hyperlipidemic and normolipidemic subjects. METHODS: An ultra-high-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method of ALA, EPA, DHA, and DPA was developed with chlorzoxazone as the internal standard (IS). The analytes were separated on an Acquity BEH C18 column (2.1 mm × 100 mm, 1.7 µm) with gradient elution by acetonitrile and 0.1% ammonia water. ALA, EPA, DHA, DPA, and IS were determined by negative electrospray ionization (ESI-) with multiple reaction monitoring (MRM) at m/z 277.42/259.05, 301.20/257.00, 327.30/283.40, 329.24/285.32, and 168.03/132.02. A total of 80 normolipidemic subjects and 83 hyperlipidemic subjects, who underwent testing for plasma lipids, liver and kidney functions, and blood routine blood test (BRT), were enrolled. RESULTS: There was good linearity for ALA within 1-10 µg/mL, and EPA, DHA and DPA were within 0.125-10 µg/mL. The relative standard deviation (RSD) of precision was below 15%. The concentrations of ALA, EPA, DHA and DPA were 3.47 ±â€¯2.58, 0.41 ±â€¯0.26, 2.93 ±â€¯1.39 and 0.25 ±â€¯0.21 µg/mL, respectively, in normolipidemic subjects, increasing to 4.14 ±â€¯3.71, 0.57 ±â€¯0.46, 3.43 ±â€¯2.13, 0.27 ±â€¯0.25 µg/mL, respectively in hyperlipidemic subjects. Among them, only the EPA concentration was significantly different between two groups. There was a high correlation between ALA, EPA, DHA and DPA. CONCLUSION: We developed a rapid and sensitive method for simultaneously determination of ALA, EPA, DHA and DPA in hyperlipidemic and normolipidemic subjects. In hyperlipidemic and normolipidemic subjects, concentrations of ALA were highest, followed by DHA, EPA and DPA; there were high degrees of correlation between each value.

Drug-drug interaction study of imatinib and voriconazole in vitro and in vivo.

Background: In clinical practice, common problem polypharmacy could result in the increased risks of drug-drug interactions (DDIs). Co-administered imatinib (IMA) and voriconazole (VOR) as one treatment protocol in cancer patients with fungal infections are common.Purpose: The aim of the present study was to assess the potential DDIs associated with the concurrent use of IMA and VOR in rat liver microsomes (RLMs) and in rats.Methods and results: The concentration levels of IMA, VOR, and their metabolites N-desmethyl IMA (CGP74588) and N-oxide voriconazole (N-oxide VOR) were determined by ultra performance liquid chromatography-tandem mass spectrometry. In vitro study of RLMs, VOR inhibited the IMA metabolism with the half-maximal inhibitory concentration (IC50) of 105.20 µM, while IC50 for IMA against VOR was 61.30 µM. After co-administered IMA and VOR in rats, the C max of IMA was increased significantly, while the AUC0→t, AUC0→∞, and C max of CGP74588 were decreased significantly. In addition, similar results were also found that the main pharmacokinetic parameters (AUC0→t, AUC0→∞, MRT0→∞, T max, and C max) of VOR were increased significantly, while the AUC0→t, AUC0→∞, and C max of N-oxide VOR were decreased significantly. Incorporation of all the results indicated that both drugs had a inhibitory effect on each other's metabolism in vitro and in vivo.Conclusion: Thus, it is of great value to monitor the concomitant use of IMA and VOR in the clinic to reduce the risks of unexpected clinical outcomes.

Quantitative bioanalytical assay for the human epidermal growth factor receptor (HER) inhibitor dacomitinib in rat plasma by UPLC-MS/MS.

Dacomitinib is a highly selective irreversible small-molecule inhibitor of the human epidermal growth factor receptor (HER) family of tyrosine kinases. A simple and quick bioanalytical method was completely developed and validated for the assay and pharmacokinetic investigation of dacomitinib in rat plasma using ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Proteins in 0.1 mL plasma samples were prepared by precipitant acetonitrile containing ibrutinib as the internal standard (IS). Separation of the analyte from plasma samples was carried out on an Acquity UPLC BEH C18 column using acetonitrile and 0.1% formic acid in water as mobile phase for gradient elution. The total run time and the elution time of dacomitinib were 3.0 min and 1.07 min, respectively. Positive-ion electrospray ionization (ESI) and multiple reaction monitoring (MRM) on a triple quadrupole tandem mass spectrometer were used for detection at the transitions of m/z 470.1 → 124.1 for dacomitinib and m/z 441.2 → 84.3 for ibrutinib (IS), respectively. In the range of 1-150 ng/mL, the calibration curve of dacomitinib was linear with a lower limit of quantitation (LLOQ) of 1 ng/mL. Mean recovery of dacomitinib in plasma was in the range of 76.9-84.1%. The inter- and intra-day precision (RSD) was in the scope of 1.7-8.7% and the accuracy (RE) ranged from -6.1 to 8.5%. Stability studies under different conditions were indicated to be stable. A pharmacokinetic study after oral administration of 40 mg/kg dacomitinib in rats illustrated the applicability of the new presented determination of dacomitinib.

UPLC-MS/MS method for the quantification of ertugliflozin and sitagliptin in rat plasma.

In the present study, an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) approach was designed to concurrently measure the levels of ertugliflozin and sitagliptin in rat plasma with diazepam as the internal standard (IS). Acetonitrile-based protein precipitation was applied for sample preparation, then analytes (ertugliflozin and sitagliptin) were subjected to gradient elution chromatography with a mobile phase composed of acetonitrile (A) and 0.1% formic acid in water (B). Ertugliflozin was monitored by m/z 437.2 → 329.0 transition for quantification and m/z 437.2 → 207.5 transition for qualification, and sitagliptin was determined by m/z 408.2 → 235.0 transition for quantification and m/z 408.2 → 174.0 transition for qualification by multiple reaction monitoring (MRM) in positive ion electrospray ionization (ESI) source. When the concentration of ertugliflozin ranged from 1 to 1000 ng/mL and sitagliptin ranged from 2 to 2500 ng/mL, the method exhibited good linearity. For both ertugliflozin and sitagliptin, the intra- and inter-day precision were determined with the values of 1.6-10.9% and 0.8-13.3%, respectively; and the accuracy ranged from -5.7% to 14.6%. Matrix effect, extraction recovery, and stability data were in line with the stipulated FDA guidelines for validating a bioanalytical method. The validity of the designed method was confirmed through the pharmacokinetic experiments.

Functional Characterization of 22 CYP3A4 Protein Variants to Metabolize Ibrutinib In Vitro.

Cytochrome P450 3A4 (CYP3A4) is quantitatively the most important P450 enzyme in adults. It is suggested that CYP3A4 genetic polymorphisms may influence the rate of the metabolism and elimination of CYP3A4 substrates in human beings. Ibrutinib is an anticancer drug and primarily metabolized by CYP3A4. The aim of this study was to systematically investigate the effects of 22 CYP3A4 protein variants on the metabolism of ibrutinib in vitro. When compared with wild-type CYP3A4.1, two variants (CYP3A4.17 and CYP3A4.24) had no detectable enzyme activity; five variants (CYP3A4.10, .11, .18, .23 and .33) exhibited no significant differences; another five variants (CYP3A4.3, .4, .9, .19 and .34) showed increased intrinsic clearance values, while the remaining nine variants (CYP3A4.2, .5, .14, .15, .16, .28, .29, .31 and .32) displayed decreased enzymatic activities in different degrees. As the first study of 22 CYP3A4 protein variants in ibrutinib metabolism, these comprehensive data may help in the clinical assessment of the metabolism and elimination of ibrutinib and also offer a reference to the personalized treatment of ibrutinib in clinic.

Genetic Variants Associated with Gestational Hypertriglyceridemia and Pancreatitis.

Severe hypertriglyceridemia is a well-known cause of pancreatitis. Usually, there is a moderate increase in plasma triglyceride level during pregnancy. Additionally, certain pre-existing genetic traits may render a pregnant woman susceptible to development of severe hypertriglyceridemia and pancreatitis, especially in the third trimester. To elucidate the underlying mechanism of gestational hypertriglyceridemic pancreatitis, we undertook DNA mutation analysis of the lipoprotein lipase (LPL), apolipoprotein C2 (APOC2), apolipoprotein A5 (APOA5), lipase maturation factor 1 (LMF1), and glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1) genes in five unrelated pregnant Chinese women with severe hypertriglyceridemia and pancreatitis. DNA sequencing showed that three out of five patients had the same homozygous variation, p.G185C, in APOA5 gene. One patient had a compound heterozygous mutation, p.A98T and p.L279V, in LPL gene. Another patient had a compound heterozygous mutation, p.A98T & p.C14F in LPL and GPIHBP1 gene, respectively. No mutations were seen in APOC2 or LMF1 genes. All patients were diagnosed with partial LPL deficiency in non-pregnant state. As revealed in our study, genetic variants appear to play an important role in the development of severe gestational hypertriglyceridemia, and, p.G185C mutation in APOA5 gene appears to be the most common variant implicated in the Chinese population. Antenatal screening for mutations in susceptible women, combined with subsequent interventions may be invaluable in the prevention of potentially life threatening gestational hypertriglyceridemia-induced pancreatitis.

A novel lipoprotein lipase gene missense mutation in Chinese patients with severe hypertriglyceridemia and pancreatitis.

BACKGROUND: Alterations or mutations in the lipoprotein lipase (LPL) gene contribute to severe hypertriglyceridemia (HTG). This study reported on two patients in a Chinese family with LPL gene mutations and severe HTG and acute pancreatitis. METHODS: Two patients with other five family members were included in this study for DNA-sequences of hyperlipidemia-related genes (such as LPL, APOC2, APOA5, LMF1, and GPIHBP1) and 43 healthy individuals and 70 HTG subjects were included for the screening of LPL gene mutations. RESULTS: Both patients were found to have a compound heterozygote for a novel LPL gene mutation (L279V) and a known mutation (A98T). Furthermore, one HTG subject out of 70 was found to carry this novel LPL L279V mutation. CONCLUSIONS: The data from this study showed that compound heterozygote mutations of A98T and L279V inactivate lipoprotein lipase enzymatic activity and contribute to severe HTG and acute pancreatitis in two Chinese patients. Further study will investigate how these LPL gene mutations genetically inactivate the LPL enzyme.

Intestinal and peripheral fibrinogen-like protein 2 expression in inflammatory bowel disease.

BACKGROUND: Fibrinogen-like protein 2 (FGL2), a new member of the fibrinogen-like family, has recently been identified as a novel immunosuppressive molecule. AIM: The purpose of this work was to investigate intestinal and peripheral expression of FGL2 in patients with inflammatory bowel disease (IBD), mainly ulcerative colitis (UC) and Crohn's disease (CD). METHODS: FGL2 expression in mucosal biopsies from three groups (UC group (n = 61), CD group (n = 54), and controls group (n = 35)) was detected by immunohistochemistry. Concentrations of FGL2 in plasma from 50 UC patients, 45 CD patients, and 30 controls were analyzed by enzyme-linked immunosorbent assay. Western blot of FGL2 protein and real-time fluorescent quantitative PCR of FGL2 mRNA expression by peripheral mononuclear cells was performed. Correlations of FGL2 expression with disease type, activity, and location, and with measured laboratory data, including C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR), were examined. RESULTS: Intestinal and peripheral FGL2 protein data showed that FGL2 expression was significantly up-regulated in both UC and CD patients compared with controls (P < 0.001). Expression of FGL2 was higher in UC and CD patients with active disease than in those with inactive disease (P < 0.001). Moreover, FGL2 mRNA expression was significantly higher in patients with active disease than in those with inactive disease (P < 0.050). Expression of FGL2 protein was correlated with disease activity indices, CRP levels, and ESR levels. CONCLUSION: Expression of FGL2 was up-regulated in IBD patients with active disease. Measurement of FGL2 may be used as a helpful biomarker for understanding immunopathogenesis and for assessment of IBD.