Simultaneous Quantification of Vancomycin, Linezolid and Voriconazole in Human Plasma by UHPLC-MS/MS: Application in Therapeutic Drug Monitoring.

OBJECTIVE: Individual differences challenge the treatment of vancomycin, linezolid and voriconazole in severe infections. This study aimed to build a simple and economical method for simultaneous determination of the three antibiotics in human plasma by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and provided a reference for therapeutic drug monitoring (TDM) of infected patients. METHODS: The plasma samples were precipitated by acetonitrile and detected and separated on a shim-pack GIST C18 column following the gradient elution within 5 min. Mass quantification was performed on multiple reaction monitoring mode under positive electrospray ionization. RESULTS: The linear ranges of vancomycin, linezolid and voriconazole were 1.00-100.00, 0.10-15.00 and 0.10-20.00 µg·mL-1, respectively, with good linearity (R2 > 0.99). The accuracy and precision, matrix effect, extraction recovery and stability were validated, and the results all meet the acceptance criteria of China Food and Drug Administration (CFDA) guidelines. CONCLUSION: The UHPLC-MS/MS method was established and validated for the simultaneous determination of vancomycin, linezolid and voriconazole in human plasma and successfully applied to routine TDM for individualized treatment.

Identification of cholest-4-ene-3,6-dione as a Novel Neuroprotectant in Ischemic Stroke and Its Lipidomics.

Purpose: Stroke is a leading cause of disability and death globally. However, there are few clinical drugs for stroke therapy. Novel and effective neuroprotectants are called on the way. Methods: In this study, 93 steroids from a constructed steroidal library were randomly numbered and blindly evaluated in an L-glutamate-induced HT-22 oxidative stress model. The neuroprotective effects of 5 candidates were further investigated in potassium deprivation-induced apoptosis of cerebellar granule neurons (CGNs), D-glutamate-induced excitotoxicity of CGNs, and cortical neuron (CN) models. Results: Interestingly, unblinding revealed that cholest-4-ene-3,6-dione (78), a cholesterol derivative, was first found to have comprehensive neuroprotective effects in all cell models. 78 administration also decreased the infarction volume and improved motor function in middle cerebral artery occlusion (MCAO) model rats. Additionally, 78 treatment decreased intercellular reactive oxygen species (ROS) and NO production in the HT-22 cell model. Finally, lipidomics and molecular docking results showed that 78 may exert its neuroprotective effects by increasing platelet-activating factor (PAF) analog 1-(9Z-pentadecenoyl)-glycero-3-phosphocholine production. Conclusion: This study indicates that 78, a novel neuroprotectant, is a promising therapeutic candidate with comprehensive neuroprotective effects for the treatment of ischemic stroke by decreasing ROS/reactive nitrogen species (RNS) levels and increasing 1-(9Z-pentadecenoyl)-glycero-3-phospho-choline production.

Simultaneous Quantitation of Anlotinib and Osimertinib by Isotope-Labeled UHPLC-MS/MS in Human Plasma: Application in NSCLC Patients.

Anlotinib and osimertinib are a class of tyrosine kinase inhibitors for the treatment of malignant tumor. The combination of anlotinib and osimertinib is currently used for treating non-small cell lung cancer (NSCLC) patients. This study aimed to develop a simple and rapid isotope-labeled UHPLC-MS/MS method for the simultaneous determination of anlotinib and osimertinib in human plasma. The analytes were extracted by protein precipitation with acetonitrile and were then separated on a Shim-pack GIST C18 column. The detection was performed on Shimadzu 8050 triple quadruple mass spectrometer in the positive electrospray ionization mode with multiple reaction monitoring. The precursor-to-product ion transitions were m/z 408.10→ 339.75, 500.25→ 72.20 and 413.50 â†’ 344.50 for anlotinib, osimertinib and D5-anlotinib, respectively. Validation is based on US Food and Drug Administration guidelines. The linearity ranges were 0.5-100 ng/mL for anlotinib and were 1-500 ng/mL for osimertinib with the correlation coefficients (r  2) ≥ 0.99. Accuracy and precision, matrix effect, extraction recovery and stability of anlotinib and osimertinib were acceptable after validation. The UHPLC-MS/MS method was successfully validated and was applied to monitor the concentration of anlotinib and osimertinib in NSCLC patients.

Safety of romiplostim and eltrombopag for children with immune thrombocytopenia: a pharmacovigilance study of the FDA adverse event reporting system database.

BACKGROUND: Romiplostim and eltrombopag are thrombopoietin receptor agonists (TPORAs) that have been approved by the FDA on 22 August 2008 and 20 November 2008 for pediatric immune thrombocytopenia (ITP). However, postmarketing pharmacovigilance of TPORAs in children still attracts much attention. We aimed to evaluate the safety of the TPORAs romiplostim and eltrombopag using data from the Adverse Event Reporting System database of FDA (FAERS). RESEARCH DESIGN AND METHODS: We conducted a disproportionality analysis and analyzed data from the FAERS database to characterize the key features of adverse events (AEs) associated with TPO-RAs approved for children under 18 years of age. RESULTS: Since their approval in the market in 2008, 250 and 298 reports of romiplostim and eltrombopag use in children have been published in the FAERS database, respectively. The most frequent AE associated with romiplostim and eltrombopag was epistaxis. Neutralizing antibodies and vitreous opacities showed the strongest signals for romiplostim and eltrombopag, respectively. CONCLUSIONS: The labeled AEs for romiplostim and eltrombopag in children were analyzed. Unlabeled AEs may reflect the potential of new clinical individuals. Early recognition and management of AEs that appear in children treated with romiplostim and eltrombopag are of key importance in clinical practice.

A novel DNA damage repair-related signature for predicting prognositc and treatment response in non-small lung cancer.

DNA damage repair (DDR) is essential for maintaining genome integrity and modulating cancer risk, progression, and therapeutic response. DDR defects are common among non-small lung cancer (NSCLC), resulting in new challenge and promise for NSCLC treatment. Thus, a thorough understanding of the molecular characteristics of DDR in NSCLC is helpful for NSCLC treatment and management. Here, we systematically analyzed the relationship between DDR alterations and NSCLC prognosis, and successfully established and validated a six-DDR gene prognostic model via LASSO Cox regression analysis based on the expression of prognostic related DDR genes, CDC25C, NEIL3, H2AFX, NBN, XRCC5, RAD1. According to this model, NSCLC patients were classified into high-risk subtype and low-risk subtype, each of which has significant differences between the two subtypes in clinical features, molecular features, immune cell components, gene mutations, DDR pathway activation status and clinical outcomes. The high-risk patients was characterized with worse prognosis, lower proportion and number of DDR mutations, unique immune profile and responsive to immunetherapy. And the low-risk patients tend to have superior survival, while being less responsive to immunotherapy and more sensitive to treatment with DNA-damaging chemotherapy drugs. Overall, this molecular classification based on DDR expression profile enables hierarchical management of patients and personalized clinical treatment, and provides potential therapeutic targets for NSCLC.

Preliminary evidence from a multicenter prospective observational study of the safety and efficacy of chloroquine for the treatment of COVID-19.

Effective therapies are urgently needed for the SARS-CoV-2 pandemic. Chloroquine has been proved to have antiviral effect against coronavirus in vitro. In this study, we aimed to assess the efficacy and safety of chloroquine with different doses in COVID-19. In this multicenter prospective observational study, we enrolled patients older than 18 years old with confirmed SARS-CoV-2 infection excluding critical cases from 12 hospitals in Guangdong and Hubei Provinces. Eligible patients received chloroquine phosphate 500 mg, orally, once (half dose) or twice (full dose) daily. Patients treated with non-chloroquine therapy were included as historical controls. The primary endpoint is the time to undetectable viral RNA. Secondary outcomes include the proportion of patients with undetectable viral RNA by day 10 and 14, hospitalization time, duration of fever, and adverse events. A total of 197 patients completed chloroquine treatment, and 176 patients were included as historical controls. The median time to achieve an undetectable viral RNA was shorter in chloroquine than in non-chloroquine (absolute difference in medians -6.0 days; 95% CI -6.0 to -4.0). The duration of fever is shorter in chloroquine (geometric mean ratio 0.6; 95% CI 0.5 to 0.8). No serious adverse events were observed in the chloroquine group. Patients treated with half dose experienced lower rate of adverse events than with full dose. Although randomized trials are needed for further evaluation, this study provides evidence for safety and efficacy of chloroquine in COVID-19 and suggests that chloroquine can be a cost-effective therapy for combating the COVID-19 pandemic.

Impact of a pharmacist-led education and follow-up service on anticoagulation control and safety outcomes at a tertiary hospital in China: a randomised controlled trial.

OBJECTIVES: This study was designed to evaluate the impact of a pharmacist-led anticoagulation service on international normalised ratio (INR) control and other outcomes among patients receiving warfarin therapy at a tertiary hospital in Zhuhai, China. METHODS: In this randomised controlled trial, adult patients who were newly initiated on warfarin with intended treatment duration of at least 3 months were recruited. Participants were randomly allocated to receive the pharmacist-led education and follow-up service (PEFS) or usual care (UC). Anticoagulation control was calculated as the proportions of time within the target INR range (TTR) and time within the expanded target range (TER). KEY FINDINGS: A total of 152 participants (77 in the PEFS group and 75 in the UC group) were included. Within 180 days after hospital discharge, the PEFS group spent more TER than the UC group (54.4% versus 42.0%; P = 0.024), whereas the difference in TTR did not reach statistical significance (35.9% versus 29.5%; P = 0.203). No major bleeding events were observed, and the cumulative incidences of major thromboembolic events (6.5% versus 9.3%) and mortality (1.3% versus 1.3%) were similar between the two groups (P> 0.05). At 30 days postdischarge, the PEFS group had better warfarin knowledge by answering 57.5% of questions correctly, compared with the UC group (43.0%) (P = 0.003). CONCLUSIONS: The PEFS markedly enhanced anticoagulation control and warfarin knowledge but there was room for improvement. The expansion of pharmacists' clinical role and the development of more effective education and follow-up strategies are warranted to optimise anticoagulation management services in China.

[PREPARATION AND BIOCOMPATIBILITY OF IN SITU CROSSLINKING HYALURONIC ACID HYDROGEL].

OBJECTIVE: To fabricate in situ crosslinking hyaluronic acid hydrogel and evaluate its biocompatibility in vitro. METHODS: The acrylic acid chloride and polyethylene glycol were added to prepare crosslinking agent polyethylene glycol acrylate (PEGDA), and the molecular structure of PEGDA was analyzed by Flourier transformation infrared spectroscopy and 1H nuclear magnetic resonance spectroscopy. Hyaluronic acid hydrogel was chemically modified to prepare hyaluronic acid thiolation (HA-SH). And the degree of HA-SH was analyzed qualitatively and quantitatively by Ellman method. HA-SH solution in concentrations (W/V) of 0.5%, 1.0%, and 1.5% and PEGDA solution in concentrations (W/V) of 2%, 4%, and 6% were prepared with PBS. The two solutions were mixed in different ratios, and in situ crosslinking hyaluronic acid hydrogel was obtained; the crosslinking time was recorded. The cellular toxicity of in situ crosslinking hyaluronic acid hydrogel (1.5% HA-SH and 4% PEGDA mixed) was tested by L929 cells. Meanwhile, the biocompatibility of hydrogel was tested by co-cultured with human bone mesenchymal stem cells (hBMSCs). RESULTS: Flourier transformation infrared spectroscopy showed that most hydroxyl groups were replaced by acrylate groups; 1H nuclear magnetic resonance spectroscopy showed 3 characteristic peaks of hydrogen representing acrylate and olefinic bond at 5-7 ppm. The thiolation yield of HA-SH was 65.4%. In situ crosslinking time of hyaluronic acid hydrogel was 2 to 70 minutes in the PEGDA concentrations of 2%-6% and HA-SH concentrations of 0.5%-1.5%. The hyaluronic acid hydrogel appeared to be transparent. The toxicity grade of leaching solution of hydrogel was grade 1. hBMSCs grew well and distributed evenly in hydrogel with a very high viability. CONCLUSIONS: In situ crosslinking hyaluronic acid hydrogel has low cytotoxicity, good biocompatibility, and controllable crosslinking time, so it could be used as a potential tissue engineered scaffold or repairing material for tissue regeneration.

Shock absorbing function study on denucleated intervertebral disc with or without hydrogel injection through static and dynamic biomechanical tests in vitro.

Hydrogel injection has been recently proposed as a novel therapy for disc degenerative diseases, with the potential to restore the spine motion and the intervertebral disc height. However, it remains unknown whether the new technique could also maintain the shock absorbing property of the treated intervertebral disc. In this study, 18 porcine lumbar bone-disc-bone specimens were collected and randomly divided into three groups: the normal with intact intervertebral discs, the mimic for the injection of disulfide cross-linked hyaluronan hydrogels following discectomy, and the control disc with discectomy only. In the static compression test, specimens in the mimic group exhibited displacements similar to those in the normal discs, whereas the control group showed a significantly larger displacement range in the first two steps (P < 0.05). With the frequency increasing, all specimens generally displayed an increasing storage modulus, decreasing loss modulus, and tanδ. At any frequency point, the control group exhibited the largest value in all the three parameters among three groups while the normal group was the lowest, with the mimic group being mostly close to the normal group. Therefore, the hydrogel injection into the intervertebral discs greatly restored their shock absorbing function, suggesting that the technique could serve as an effective approach to maintaining biomechanical properties of the degenerative intervertebral disc.

Electrostatic self-assembly of multilayer copolymeric membranes on the surface of porous tantalum implants for sustained release of doxorubicin.

Many studies in recent years have focused on surface engineering of implant materials in order to improve their biocompatibility and other performance. Porous tantalum implants have increasingly been used in implant surgeries, due to their biocompatibility, physical stability, and good mechanical strength. In this study we functionalized the porous tantalum implant for sustained drug delivery capability via electrostatic self-assembly of polyelectrolytes of hyaluronic acid, methylated collagen, and terpolymer on the surface of a porous tantalum implant. The anticancer drug doxorubicin was encapsulated into the multilayer copolymer membranes on the porous tantalum implants. Results showed the sustained released of doxorubicin from the functionalized porous tantalum implants for up to 1 month. The drug release solutions in 1 month all had inhibitory effects on the proliferation of chondrosarcoma cell line SW1353. These results suggest that this functionalized implant could be used in reconstructive surgery for the treatment of bone tumor as a local, sustained drug delivery system.

High performance liquid chromatography-tandem mass spectrometric determination of rupatadine in human plasma and its pharmacokinetics.

A simple, rapid, sensitive and selective liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the quantification of rupatadine in human plasma using estazolam as internal standard (IS). Following liquid-liquid extraction, the analytes were separated using a mobile phase of methanol-ammonium acetate (pH 2.2; 5mM) (50:50, v/v) on a reverse phase C18 column and analyzed by a triple-quadrupole mass spectrometer in the positive ion and multiple reaction monitoring (MRM) mode, m/z 416-->309 for rupatadine and m/z 295-->267 for the IS. The assay exhibited a linear dynamic range of 0.1-100 ng/ml for rupatadine in human plasma. The lower limit of quantification (LLOQ) was 0.1 ng/ml with a relative standard deviation of less than 20%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The validated LC-MS/MS method has been successfully applied to study the pharmacokinetics of rupatadine in healthy volunteers.

Sensitive and selective liquid chromatography-tandem mass spectrometry method for the quantification of aniracetam in human plasma.

A rapid, sensitive and selective LC-MS/MS method was developed and validated for the quantification of aniracetam in human plasma using estazolam as internal standard (IS). Following liquid-liquid extraction, the analytes were separated using a mobile phase of methanol-water (60:40, v/v) on a reverse phase C18 column and analyzed by a triple-quadrupole mass spectrometer in the selected reaction monitoring (SRM) mode using the respective [M+H]+ ions, m/z 220-->135 for aniracetam and m/z 295-->205 for the IS. The assay exhibited a linear dynamic range of 0.2-100 ng/mL for aniracetam in human plasma. The lower limit of quantification (LLOQ) was 0.2 ng/mL with a relative standard deviation of less than 15%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The validated LC-MS/MS method has been successfully applied to study the pharmacokinetics of aniracetam in healthy male Chinese volunteers.

High-performance liquid chromatography-electrospray ionization-mass spectrometric determination of emedastine difumarate in human plasma and its pharmacokinetics.

A selective and sensitive method employing high-performance liquid chromatography (HPLC)-electrospray ionization (ESI)-mass spectrometry is developed and validated for the determination of emedastine difumarate in human plasma. With naphazoline hydrochloride as the internal standard, emedastine difumarate is extracted from plasma with ethyl acetate. The organic layer is evaporated, and the residue is redissolved in the mobile phase. An aliquot of 10 microL is chromatographically analyzed on a prepacked Phenomenex Luna 5u CN 100A (150 x 2.0-mm i.d.) column, using a mobile phase comprised of methanol-water (20 mM CH(3)COONH(4), pH 4.0) (80:20, v/v). Standard curves are linear (r(2) = 0.9990) over the concentration range of 0.05-30 ng/mL and had good accuracy and precision. The within- and between-batch precisions did not exceed 15% for the relative standard deviation. The lower limit of detection is 0.01 ng/mL. The validated HPLC-ESI-MS method is successfully used to study emedastine difumarate pharmacokinetics in 12 healthy volunteers.

High-performance liquid chromatography-electrospray ionization mass spectrometry determination of mitiglinide in human plasma and its pharmacokinetics.

A selective and sensitive method employing high-performance liquid chromatography-electrospray ionization mass spectrometry was developed and validated for the determination of mitiglinide in human plasma.With gliclazide as the internal standard, mitiglinide was extracted from plasma with n-hexane: = 80 : 20 (v/v). The organic layer was evaporated and the residue was redissolved in methanol: water (10 mM CH3COONH4, pH = 3.0) = 65 : 35 (v/v). An aliquot of 10 microl was chromatographically analyzed on a prepacked Shimadzu VP-ODS (5 microm, 150 x 2.0 mm i.d.) using the mobile phase comprising methanol: water (10 mM CH3COONH4) = 65 : 35 (v/v) by means of selected-ion monitoring mode mass spectrometry. Standard curves were linear (r2 = 0.9972) over the concentration range of 2.84-11 300 pmol/ml and had good accuracy and precision. The within- and between-batch precisions of the method were within 15% of standard deviation. The lower limit of detection was 1.42 pmol/ml. The validated HPLC/ESI-MS method has been successfully applied in the pharmacokinetics of mitiglinide in 12 healthy Chinese volunteers.

High-performance liquid chromatography electrospray ionization mass spectrometry determination of tulobuterol in rabbit's plasma.

A sensitive and specific liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) method has been developed and validated for the identification and quantification of tulobuterol in rabbits' plasma. After the addition of clenbuterol-HCl, the internal standard (IS) and 1.0 M sodium hydroxide solution, plasma samples were extracted using a solvent mixture comprised of 5% isopropanol in n-hexane. The compounds were separated on a prepacked Lichrospher CN (5 microm, 150 mm x 2.0 mm) column using a mixture of methanol-water (10 mM CH3COONH4, pH 4.0) as mobile phase. A Shimadzu LCMS-2010A mass spectrometer connected to a Shimadzu high performance liquid chromatograph (HPLC) was used to develop and validate the method. The method has shown to be sensitive and specific by testing six different blank plasma batches. Linearity was established for the range of concentrations 0.50-40.0 ng/mL with a coefficient of determination (r) of 0.9998. The intra-day precision was better than 15%. The lower limit of quantification (LLOQ) was identifiable and reproducible at 0.50 ng/mL. The proposed method enables the unambiguous identification and quantification of tulobuterol for pharmacokinetic, bioavailability or bioequivalence studies.