A Reliable and Effective UPLC-MS/MS Method for the Determination of Oprozomib in Rat Plasma.

Oprozomib, as a second-generation orally bioavailable protease inhibitor (PI), is undergoing clinical evaluation for the treatment of haematological malignancies. In relapsed refractory multiple myeloma (RRMM) patients, oprozomib has shown good efficacy as a single agent or combination therapy. In this experiment, our purpose was to validate a sensitive and rapid method for the determination of oprozomib concentration in rat plasma by ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The samples were treated with acetonitrile as the precipitant and separated by gradient elution using a Waters Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 µm). Using the selective reaction monitoring (SRM) method, the measurement was finished with the ion transitions of m/z 533.18 ⟶ 199.01 for oprozomib and m/z 493.03 ⟶ 112.03 for tepotinib (internal standard, IS), respectively. Meanwhile, acetonitrile and 0.1% formic acid aqueous solution were used as the mobile phase, and the flow rate was 0.3 mL/min. The lower limit of quantification (LLOQ) of the method was 1.0 ng/mL, and the linear relationship was good in the range of 1.0-100 ng/mL. In addition, the precision of four concentration levels was determined with the values of 3.1-7.3% and the accuracy was from -14.9% to 12.9%. Moreover, the recovery was determined to be from 85.1% to 96.1%, and the values of matrix effect were no more than 110.4%. The optimized UPLC-MS/MS method was also suitable for the pharmacokinetic study of rats after a single oral administration of 21 mg/kg oprozomib.

An Efficient UPLC-MS/MS Method Established to Detect Relugolix Concentration in Rat Plasma.

Relugolix, a gonadotropin-releasing hormone (GnRH) receptor antagonist, has been well studied in the treatment of endometriosis symptomatic. It is mainly metabolized by the CYP3A subfamily of P450 enzymes, while minorly metabolized by CYP2C8. Daidzein in different dose groups exhibited a certain induction on the mRNA expression level of CYP3A4 and resulted in the potent induction of CYP3A4. However, it is still unknown whether daidzein and relugolix interact. We developed an effective ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method to study the effect of daidzein on the pharmacokinetics of relugolix in rats after oral administration of 12 mg/kg relugolix in a single or mixed of 50 mg/kg daidzein. The results showed that the method had respectable linearity (r 2 > 0.999) on the scale of 0.7-1000 ng/mL. The intra-day precision was between 3.0% and 8.4% in this assay, and the inter-day was between 4.0% and 11.7%. The intra-day accuracy was from -4.3% to 6.1%, and the inter-day was 2.9% to 12.1%. Another three key indicators, including the stability, the recovery rate of extraction and the new technique's matrix effect, were perfectly in accord with the test verification rule in the biological medium by the United States Food and Drug Administration. Meanwhile, treatment with daidzein led to a decrease in Cmax and AUC0-t of relugolix by about 15.56% and 21.36%, respectively. Although there was no statistical difference in pharmacokinetic parameters, it reflected the induction trend of daidzein on relugolix metabolism for food-drug interaction. It would provide reference and improvement value for subsequent experiments.

Effect of quercetin on the pharmacokinetics of selexipag and its active metabolite in beagles.

CONTEXT: As an inhibitor cytochrome P450 family 2 subfamily C polypeptide 8 (CYP2C8), quercetin is a naturally occurring flavonoid with its glycosides consumed at least 100 mg per day in food. However, it is still unknown whether quercetin and selexipag interact. OBJECTIVE: The study investigated the effect of quercetin on the pharmacokinetics of selexipag and ACT-333679 in beagles. MATERIALS AND METHODS: The ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to investigate the pharmacokinetics of orally administered selexipag (2 mg/kg) with and without quercetin (2 mg/kg/day for 7 days) pre-treatment in beagles. The effect of quercetin on the pharmacokinetics of selexipag and its potential mechanism was studied through the pharmacokinetic parameters. RESULTS: The assay method was validated for selexipag and ACT-333679, and the lower limit of quantification for both was 1 ng/mL. The recovery and the matrix effect of selexipag were 84.5-91.58% and 94.98-99.67%, while for ACT-333679 were 81.21-93.90% and 93.17-99.23%. The UPLC-MS/MS method was sensitive, accurate and precise, and had been applied to the herb-drug interaction study of quercetin with selexipag and ACT-333679. Treatment with quercetin led to an increased in Cmax and AUC0-t of selexipag by about 43.08% and 26.92%, respectively. While the ACT-333679 was about 11.11% and 18.87%, respectively. DISCUSSION AND CONCLUSION: The study indicated that quercetin could inhibit the metabolism of selexipag and ACT-333679 when co-administration. Therefore, the clinical dose of selexipag should be used with caution when co-administered with foods high in quercetin.

Development of a Prognostic Five-Gene Signature for Diffuse Lower-Grade Glioma Patients.

Background: Diffuse lower-grade gliomas (LGGs) are infiltrative and heterogeneous neoplasms. Gene signature including multiple protein-coding genes (PCGs) is widely used as a tumor marker. This study aimed to construct a multi-PCG signature to predict survival for LGG patients. Methods: LGG data including PCG expression profiles and clinical information were downloaded from The Cancer Genome Atlas (TCGA) and the Chinese Glioma Genome Atlas (CGGA). Survival analysis, receiver operating characteristic (ROC) analysis, and random survival forest algorithm (RSFVH) were used to identify the prognostic PCG signature. Results: From the training (n = 524) and test (n = 431) datasets, a five-PCG signature which can classify LGG patients into low- or high-risk group with a significantly different overall survival (log rank P < 0.001) was screened out and validated. In terms of prognosis predictive performance, the five-PCG signature is stronger than other clinical variables and IDH mutation status. Moreover, the five-PCG signature could further divide radiotherapy patients into two different risk groups. GO and KEGG analysis found that PCGs in the prognostic five-PCG signature were mainly enriched in cell cycle, apoptosis, DNA replication pathways. Conclusions: The new five-PCG signature is a reliable prognostic marker for LGG patients and has a good prospect in clinical application.

Simultaneous quantification of vortioxetine, fluoxetine and their metabolites in rat plasma by UPLC-MS/MS.

In this study, a specific and quick ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was fully developed and validated for simultaneous measurement of the rat plasma levels of vortioxetine (VOR), Lu AA34443 (the major metabolite of VOR), fluoxetine and its metabolite norfluoxetine with diazepam as the internal standard (IS). After a simple protein precipitation with acetonitrile for sample preparation, the separation of the analytes were performed on an Acquity UPLC BEH C18 (2.1 × 50 mm, 1.7 µm) column, with acetonitrile and 0.1% formic acid in water as mobile phase by gradient elution. The detection was achieved on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring mode via an electrospray ionization source. Good linearity was observed in the calibration curve for each analyte. The data of precision, accuracy, matrix effect, recovery and stability all conformed to the bioanalytical method validation of acceptance criteria of US Food and Drug Administration recommendations. The newly developed UPLC-MS/MS method allowed simultaneous quantification of VOR, fluoxetine and their metabolites for the first time and was successfully applied to a pharmacokinetic study in rats.

Determination and Pharmacokinetic Profiles of Four Active Components From Scrophularia ningpoensis Hemsl. in Rats.

Acteoside, angoroside C, harpagoside, and cinnamic acid, which are the main bioactive ingredients of Scrophularia ningpoensis Hemsl., have wide clinical use with various biological effects. A new and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was established with taxifolin as the internal standard (IS) in this study and was successfully used to study the pharmacokinetic profiles of four active components from S. ningpoensis Hemsl. in rats after sublingual intravenous administration. After protein precipitation with acetonitrile, the mobile phase (consisting of acetonitrile and 0.1% formic acid) was used to separate the analytes on an Acquity UPLC BEH C18 chromatography column (2.1 × 50 mm, 1.7 µm) under gradient elution. The precursor-to-product ion transitions of 623.4 → 161.3 m/z for acteoside, 783.5 → 175.0 m/z for angoroside C, 493.3 → 345.2 m/z for harpagoside and 147.2 → 103.4 m/z for cinnamic acid were monitored by mass spectrometry with negative electrospray ionization in the multiple reaction monitoring (MRM) mode. The concentration range of 10-1,000 ng/ml could be detected by this method with a lower limit of quantification (LLOQ) of 10 ng/ml for each analyte. The intra- and inter-day precision (RSD%) of the method ranged from 2.6 to 9.9% and 2.7-11.5%, respectively. Meanwhile, the accuracy (RE%) was -9.6-10.7% in this developed method. The mean recoveries of four active components from S. ningpoensis Hemsl. were more than 76.7% with negligible matrix effects. The four active components from S. ningpoensis Hemsl. were stable under multiple storage and process conditions. A new, sensitive and simple analytical method had been established and was successfully applied to the pharmacokinetic profiles of four active components from S. ningpoensis Hemsl. in rats after sublingual intravenous administration.

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.

Impacts of Clinical Pharmacist Intervention on the Secondary Prevention of Coronary Heart Disease: A Randomized Controlled Clinical Study.

Coronary heart disease (CHD) is one of the leading causes of morbidity and mortality worldwide, and more efforts should be made to reduce the risk of cardiovascular events. This study aimed to investigate the impact of clinical pharmacist intervention on the prognosis of acute coronary syndrome (ACS) in Chinese patients with CHD. Two hundred and forty patients who had ACS were recruited. Participants were randomly assigned to the intervention group (n = 120) or the control group (n = 120). The intervention group received a medication assessment and education by the clinical pharmacist at discharge and telephone follow-ups at 1 week and 1 and 3 months after discharge. The control group received usual care. The primary outcomes of this study were the proportion of patients who had major adverse cardiovascular events (MACEs), including mortality, nonfatal myocardial infarction (MI), stroke, and unplanned cardiac-related rehospitalizations within 6 and 12 months after hospital discharge. Secondary outcome was self-reported medication adherence to evidence-based medications for CHD (antiplatelets, statins, ß-blockers, and angiotensin-converting enzyme inhibitors or angiotensin receptor blockers). Of 240 enrolled patients, 238 (98.3%) completed 6-month follow-up, and 235 (97.9%) completed 12-month follow-up. There were no significant differences between intervention and control groups in the percentages of patients who incurred MACEs within the 6-month follow-up (3.3% vs 7.6%, respectively, P = 0.145) or 12-month follow-up (10.9% vs 12.1%, respectively, P = 0.783). Significant improvements were found in the prescribing rates of ß-blockers and all four classes of medications at discharge in the intervention group compared with the control group (P = 0.001 and P = 0.009, respectively). There was no significant difference between the intervention and control groups in the use of all four classes of medications at the 6-month follow-up (48.3% vs 45.8%, respectively, P = 0.691) and 12-month follow-up (47.9% vs 46.6%, respectively, P = 0.836). The use of ß-blockers was nonsignificantly higher in the intervention group than in the control group at the 6-month follow-up (74.2% vs. 64.4%, P = 0.103) and 12-month follow-up (74.8% vs 63.8%, P = 0.068). Clinical pharmacist intervention had no significant effects on reduction in cardiovascular events among patients with CHD. Further studies with larger sample sizes and longer time frames for both intervention and follow-up are needed to validate the role of the clinical pharmacist in the morbidity and mortality of CHD. Clinical Trial Registration: chictr.org.cn, identifier ChiCTR-IOR-16007716.

Simultaneous Determination of Rivaroxaban and Enalapril in Rat Plasma by UPLC-MS/MS and Its Application to A Pharmacokinetic Interaction Study.

BACKGROUND AND OBJECTIVES: There have been no animal experiments and clinical studies on the pharmacokinetic interaction between rivaroxaban and enalapril. To investigate whether a potential pharmacokinetic interaction is present between rivaroxaban and enalapril, a rapid and sensitive Ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to determine the concentration of rivaroxaban and enalapril in rat plasma and was then applied to a pharmacokinetic interaction study. METHODS: The analytes were separated on an Acquity UPLC BEH C18 chromatography column (2.1 × 50 mm, 1.7 µm) with acetonitrile and 0.1% formic acid as the mobile phase with gradient elution. The mass spectrometer was operated in multiple reaction monitoring mode to monitor the precursor-to-product ion transitions of 436.1 → 145.1 m/z for rivaroxaban, 377.3 → 234.2 m/z for enalapril and 285.2 → 193.1 m/z for diazepam (IS). RESULTS: The method was validated over the concentration range of 1.0-200 ng/mL for rivaroxaban and 0.5-100 ng/mL for enalapril. The intra- and inter-day precision and accuracy of the quality control (QC) samples exhibited relative standard deviations (RSD) < 9.4% and the accuracy values ranged from - 8.3 to 9.6%. After co-administration of rivaroxaban and enalapril, the maximum plasma concentration (Cmax) and area under the systemic drug concentration-time curve from time 0 to infinity (AUC0-∞) of rivaroxaban were significantly increased by 19.6% (p < 0.05) and 21.3% (p < 0.05), respectively. On the contrary, the plasma clearance rate (CL/F) of rivaroxaban and enalapril was significantly decreased by 17.8% (p < 0.05) and 23.8% (p < 0.05), respectively. CONCLUSIONS: The UPLC-MS/MS method was successfully applied to simultaneous determination of rivaroxaban and enalapril in rat plasma and applied to study the pharmacokinetic interaction between rivaroxaban and enalapril. The co-administration of rivaroxaban and enalapril resulted in a significant drug interaction in rats.

Increased Oxidative Damage of RNA in Early-Stage Nephropathy in db/db Mice.

To evaluate RNA oxidation in the early stage of diabetic nephropathy, we applied an accurate method based on isotope dilution high-performance liquid chromatography-triple quadruple mass spectrometry to analyze the oxidatively generated guanine nucleosides in renal tissue and urine from db/db mice of different ages. We further investigated the relationship between these oxidative stress markers, microalbumin excretion, and histological changes. We found that the levels of 8-oxo-7,8-dihydroguanosine (8-oxoGuo) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) were increased in the urine and renal tissue of db/db mice and db/db mice with early symptoms of diabetic nephropathy suffered from more extensive oxidative damage than lean littermate control db/m mice. Importantly, in contrast to the findings in db/m mice, the 8-oxoGuo levels in the urine and renal tissue of db/db mice were higher than those of 8-oxodGuo at four weeks. These results indicate that RNA oxidation is more apparent than DNA oxidation in the early stage of diabetic nephropathy. RNA oxidation may provide new insight into the pathogenesis of diabetic nephropathy, and urinary 8-oxoGuo may represent a novel, noninvasive, and easily detected biomarker of diabetic kidney diseases if further study could clarify its source and confirm these results in a large population study.

Analysis of the oxidative damage of DNA, RNA, and their metabolites induced by hyperglycemia and related nephropathy in Sprague Dawley rats.

We used a sensitive and accurate method based on isotope dilution high-performance liquid chromatography-triple quadrupole mass spectrometry (ID-LC-MS/MS) to determine the levels of 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxo-dGsn) and 8-oxo-7,8-dihydroguanosin (8-oxo-Gsn) in various tissue specimens, plasma, and urine of hyperglycemic Sprague Dawley rats induced by streptozotocin (STZ). The oxidative DNA and RNA damages were observed in various organs and the amounts of 8-oxo-dGsn and 8-oxo-Gsn derived from DNA and RNA were increased with hyperglycemic status. In contrast to the results of the nucleic acid samples derived from tissues, the levels of 8-oxo-Gsn in urine and plasma were significantly higher compared with that of 8-oxo-dGsn, which most likely reflected the RNA damage that occurs more frequently compared with DNA damage. For the oxidative stress induced by hyperglycemia, 8-oxo-Gsn in urine may be a sensitive biomarker on the basis of the results in urine, plasma, and tissues. In addition, high levels of urinary 8-oxo-Gsn were observed before diabetic microvascular complications. Based on that the 8-oxo-dGsn was associated with diabetic nephropathy and RNA was more vulnerable to oxidative stress compared with DNA. We also propose that 8-oxo-Gsn is correlated with diabetic nephropathy and that 8-oxo-Gsn in urine could be a useful and sensitive marker of diabetic nephropathy.

Characterization of a novel CYP2C9 mutation (1009C>A) detected in a warfarin-sensitive patient.

Warfarin is the most frequently prescribed anticoagulant for the long-term treatment in the clinic. Recent studies have shown that polymorphic alleles within the CYP2C9, VKORC1, and CYP4F2 genes are related to the warfarin dosage requirement. In this study, a novel non-synonymous mutation (1009C>A) in CYP2C9 was detected in a warfarin-hypersensitive patient, while the other two candidate genes were both found to be homozygous for the wild-type alleles. The newly identified point mutation results in an amino acid substitution at position 337 of the CYP2C9 protein (P337T) and has been designated as the novel allele CYP2C9*58. When expressed in insect cell microsomes, the relative intrinsic clearance values of the CYP2C9.58 variant for tolbutamide and losartan were quite similar to those of the typical defective variant CYP2C9.3, whereas the clearance value of CYP2C9.58 for diclofenac was slightly higher than that of another typical defective variant CYP2C9.2. These data suggested that when compared with wild-type CYP2C9.1, the enzymatic activity of the novel allelic variant has been greatly reduced by the 1009C>A mutation. If patients carrying this allele take drugs metabolized by CYP2C9, their metabolic rate might be slower than that of wild-type allele carriers and thus much more attention should be paid to their clinical care.