Magnetic/Acoustic Dual-Controlled Microrobot Overcoming Oto-Biological Barrier for On-Demand Multidrug Delivery against Hearing Loss.

Multidrug combination therapy in the inner ear faces diverse challenges due to the distinct physicochemical properties of drugs and the difficulties of overcoming the oto-biologic barrier. Although nanomedicine platforms offer potential solutions to multidrug delivery, the access of drugs to the inner ear remains limited. Micro/nanomachines, capable of delivering cargo actively, are promising tools for overcoming bio-barriers. Herein, a novel microrobot-based strategy to penetrate the round window membrane (RWM) is presented and multidrug in on-demand manner is delivered. The tube-type microrobot (TTMR) is constructed using the template-assisted layer-by-layer (LbL) assembly of chitosan/ferroferric oxide/silicon dioxide (CS/Fe3O4/SiO2) and loaded with anti-ototoxic drugs (curcumin, CUR and tanshinone IIA, TSA) and perfluorohexane (PFH). Fe3O4 provides magnetic actuation, while PFH ensures acoustic propulsion. Upon ultrasound stimulation, the vaporization of PFH enables a microshotgun-like behavior, propelling the drugs through barriers and driving them into the inner ear. Notably, the proportion of drugs entering the inner ear can be precisely controlled by varying the feeding ratios. Furthermore, in vivo studies demonstrate that the drug-loaded microrobot exhibits superior protective effects and excellent biosafety toward cisplatin (CDDP)-induced hearing loss. Overall, the microrobot-based strategy provides a promising direction for on-demand multidrug delivery for ear diseases.

Reversible opening of the blood-labyrinth barrier by low-pressure pulsed ultrasound and microbubbles for the treatment of inner ear diseases.

Systemic drug administration provides convenience and non-invasive benefits for preventing and treating inner ear diseases. However, the blood-labyrinth barrier (BLB) restricts the transport of drugs to inner ear tissues. Ultrasound can stimulate specific areas and penetrate tissues, with the potential to overcome physiological barriers. We present a novel strategy based on low-pressure pulsed ultrasound assisted by microbubbles (USMB) to transiently open the BLB and deliver therapeutics into the inner ear. A pulsed ultrasound device with adjustable pressure was established; the generated ultrasound was transmitted through the external auditory canal into the guinea pig's inner ear. We observed that the application of microbubbles allowed the use of safe and efficient ultrasound conditions to penetrate the BLB. We found that USMB-mediated BLB opening seemed to be associated with a reduced expression of the tight junction proteins zonula occludens-1 and occludin. Following intravenous administration, hydrophilic dexamethasone sodium phosphate (DSP), hydrophobic curcumin (CUR), as well as drug-loaded nanoparticles (Fe3O4@CUR NPs) could be efficiently delivered into the inner ear. We observed better drug accumulation in the perilymph of the inner ear, resulting in less drug (cisplatin)-induced ototoxicity. Furthermore, physiological, hematological, and histological studies showed that the modulation of the BLB by low-pressure USMB was a safe process without significant adverse effects. We conclude that USMB could become a promising strategy for the systematic delivery of therapeutics in the treatment of inner ear diseases.

Association of Valproic Acid and Its Main Metabolites' Plasma Concentrations with Clinical Outcomes among Epilepsy Patients: A 10-Year Retrospective Study Based on Therapeutic Drug Monitoring.

Valproic acid (VPA) is a first-line antiepileptic drug with broad efficacy. Due to significant individual differences in its metabolism, therapeutic drug monitoring is commonly used. However, the recommended therapeutic range (50-100 µg/mL) is inadequate for predicting clinical outcomes. Additionally, the relationship between VPA metabolites and clinical outcomes remains unclear. In this retrospective study, 485 Chinese Southern Han epilepsy patients receiving VPA monotherapy were analyzed after reaching steady-state levels. Plasma concentrations of VPA and its five main metabolites were determined by liquid chromatography-mass spectrometry (LC-MS). We assessed the relevance of the recommended therapeutic VPA range for clinical outcomes and explored the association between VPA/metabolites levels and treatment efficacy/adverse effects. Vitro experiments were conducted to assess 4-ene-VPA hepatotoxicity. The therapeutic range of VPA exhibited no significant correlation with clinical outcomes, and plasma concentrations of VPA failed to serve as predictive indicators for treatment response/adverse effects. Treatment responders had higher 2-PGA concentrations (median, 26.39 ng/mL versus 13.68 ng/mL), with a threshold of 36.5 ng/mL for optimal epilepsy treatment. Patients with abnormal liver function had a higher 4-ene-VPA median concentration (6.41 µg/mL versus 4.83 µg/mL), and the ratio of 4-ene-VPA to VPA better predicted VPA-induced hepatotoxicity (area under the curve, 0.718) than 4-ene-VPA concentration. Vitro experiments revealed that 4-ene-VPA was more hepatotoxic than VPA in HepaRG and L02 cell lines. Total plasma VPA concentration does not serve as a predictor of clinical outcomes. 2-PGA concentrations may be associated with efficacy, whereas the ratio of 4-ene-VPA to VPA may be considered a better biomarker (threshold 10.03%) for VPA-induced hepatotoxicity. SIGNIFICANCE STATEMENT: This was the first and largest observational cohort in China to explore the relationship between patients' parent and metabolites concentrations of VPA and clinical outcomes during the maintenance of VPA monotherapy in epileptic patients. This study provided feasible references of VPA for epilepsy clinical treatment with a larger sample of patients compared with previous studies for a more definitive conclusion based on real-world situations. We found two potential biomarkers in predicting efficacy and liver injury, respectively. This breakthrough has the potential to assist in the rational use of VPA.

Simultaneous determination of curcumin, tetrahydrocurcumin, quercetin, and paeoniflorin by UHPLC-MS/MS in rat plasma and its application to a pharmacokinetic study.

Curcumin (CUR) is a bioactive compound present in many composite prescriptions of traditional Chinese medicine together with quercetin (QR) and paeoniflorin (PF). Little is known about the influence of QR and PF on the absorption and metabolism of CUR when the three compounds are orally co-administered. In this study, a rapid, sensitive, and reliable ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the simultaneous determination of CUR, tetrahydrocurcumin (THC), QR, and PF in rat plasma by using tinidazole as the internal standard (IS). A liquid-liquid extraction method with ethyl acetate was used to pre-treat the plasma samples. Chromatographic separation was conducted on a C18 column with isocratic elution using acetonitrile and 0.1% formic acid water solution (80:20, v/v) as the mobile phase at the flow rate of 0.3 mL/min. A TSQ Quantum Access Max API mass spectrometer equipped with electrospray ionisation (ESI) source in selection reaction monitoring (SRM) mode was employed to determine transitions of m/z 369.0 → 176.9, 373.1 → 137.0, 303.0 → 228.9, 478.9 → 120.9, 248.1 → 121.0 for CUR, THC, QR, PF, and IS, respectively. The selectivity, precision, accuracy, extraction recovery, matrix effect, and stability of the method were validated. This developed and validated method was successfully applied in the pharmacokinetic study of CUR, THC, QR, and PF in rats. The effects of QR and PF on the pharmacokinetics of CUR and its metabolite, THC, were evaluated in the plasma of Sprague-Dawley rats that were orally co-administered CUR, QR, and PF. The results showed that the combined use of QR, PF, and CUR has a possible influence on the metabolism and excretion of CUR. Our work provides a fundamental method for the rapid simultaneous determination of CUR, THC, QR, and PF in rat plasma. Furthermore, this study will provide a basic method for the analysis of pharmacokinetic interaction of CUR, QR, and PF and offer a scientific basis for a possible combination therapy with the three compounds.

Effects of SLCO1B1 and GATM gene variants on rosuvastatin-induced myopathy are unrelated to high plasma exposure of rosuvastatin and its metabolites.

Myotoxicity is a significant factor contributing to the poor adherence and reduced effectiveness in the treatment of statins. Genetic variations and high drug plasma exposure are considered as critique causes for statin-induced myopathy (SIM). This study aims to explore the sequential influences of rosuvastatin (RST) pharmacokinetic and myopathy-related single-nucleotide polymorphisms (SNPs) on the plasma exposure to RST and its metabolites: rosuvastatin lactone (RSTL) and N-desmethyl rosuvastatin (DM-RST), and further on RST-induced myopathy. A total of 758 Chinese patients with coronary artery disease were enrolled and followed up SIM incidents for 2 years. The plasma concentrations of RST and its metabolites were determined through a validated ultra-performance liquid chromatography mass spectrometry method. Nine SNPs in six genes were genotyped by using the Sequenom MassArray iPlex platform. Results revealed that ABCG2 rs2231142 variations were highly associated with the plasma concentrations of RST, RSTL, and DM-RST (Padj < 0.01, FDR < 0.05). CYP2C9 rs1057910 significantly affected the DM-RST concentration (Padj < 0.01, FDR < 0.05). SLCO1B1 rs4149056 variant allele was significantly associated with high SIM risk (OR: 1.741, 95% CI: 1.180-2.568, P = 0.0052, FDR = 0.0468). Glycine amidinotransferase (GATM) rs9806699 was marginally associated with SIM incidents (OR: 0.617, 95% CI: 0.406-0.939, P = 0.0240, FDR = 0.0960). The plasma concentrations of RST and its metabolites were not significantly different between the SIM (n = 51) and control groups (n = 707) (all P > 0.05). In conclusion, SLCO1B1 and GATM genetic variants are potential biomarkers for predicting RST-induced myopathy, and their effects on SIM are unrelated to the high plasma exposure of RST and its metabolites.

Epimedium sagittatum inhibits TLR4/MD-2 mediated NF-κB signaling pathway with anti-inflammatory activity.

BACKGROUND: Epimedium sagittatum (Sieb.et Zucc.) Maxim., Ying-Yang-Huo in Chinese has been used as a traditional Chinese medicine and is deemed to "reinforce the kidney Yang". Previous studies showed that E. sagittatum could modulate the immune system and treat some chronic disease such as rheumatic arthritis, cardiovascular diseases and osteoporosis. The aim of this study is to evaluate the anti-inflammatory effects of ethyl acetate extracts (YYHs) of E. sagittatum and its mechanisms of action. METHODS: In order to explore the composition of YYHs, YYHs was analyzed using high performance liquid chromatography-mass spectrometry-mass spectrometry (HPLC-MS/MS) and in comparison with reference standards. Anti-inflammatory model was established in LPS-induced RAW264.7 cells. The levels of nitric oxide (NO) were measured with the Griess reagent. Production of tumor necrosis factor-alpha (TNF-α) and interleukin-2 (IL-2) were measured by enzyme-linked immunosorbent assays (ELISA). In addition, expression of p-p65 protein and TLR4/MD-2 complex was detected by western blots and flow cytometric, respectively. Nuclear factor kappa B (NF-κB) nuclear translocation was observed by fluorescence microscope. RESULTS: A total of eight compounds were identified, of which icariside II was the most abundant compound. YYHs (12.5-50 µg/mL) had no obvious cytotoxic effect on cells, and remarkably inhibited LPS-induced production of NO, TNF-α and IL-2 with a dose-dependent manner. Additionally, YYHs up-regulated expression of p-p65 and TLR4/MD-2 complex. Further research showed that YYHs significantly suppressed NF-κB p65 nuclear translocation. CONCLUSION: In brief, YYHs contributed to the inhibition of LPS-induced inflammatory response through the TLR4/MD-2-mediated NF-κB pathway and may be a potential choice to combat inflammation diseases. It includes a schema of pathways at the end of the paper.

Polymorphisms of peroxisome proliferator-activated receptor γ (PPARγ) and cluster of differentiation 36 (CD36) associated with valproate-induced obesity in epileptic patients.

RATIONALE: Valproate (VPA) is a choice for the treatment of primary generalized epilepsies and partial epilepsies. Unfortunately, weight gain or obesity is one of the most frequent adverse effects of VPA treatment. Genetic factors were shown to be involved in the effect. OBJECTIVE: The aim of this study was to investigate the association of selected single nucleotide polymorphisms (SNPs) of cluster of differentiation 36 (CD36) and peroxisome proliferator-activated receptor γ (PPARγ) with VPA-induced weight gain and obesity in epileptic patients. METHODS: A total of 225 Chinese Han epilepsy patients receiving VPA treatment were recruited in the study. Height and weight for the calculation of body mass index (BMI) were measured at the initiation of VPA therapy and in the follow-up examination. A BMI of 25 kg/m2 or higher was defined as obesity on the basis of the World Health Organization (WHO) criteria for Asian populations. Four SNPs in CD36 (rs1194197, rs7807607) and PPARγ (rs10865710, rs2920502) were genotyped using the Sequenom® MassArray iPlex platform. RESULTS: About 19.6% of epileptic patients receiving VPA therapy were found to become obese. After covariate analysis of age, gender, sex, height, initial BMI, and VPA dosage, the CD36 rs1194197 C allele and rs7807607 T allele (OR, 0.31; 95%CI, 0.13-0.72; P = 0.009 and OR, 0.38; 95%CI; 0.18-0.83; P = 0.02, respectively) were identified as protective factors for VPA-induced obesity. The PPARγ rs10865710 C allele carriers were found to be less likely to suffer from VPA-induced obesity compared with GG genotype carriers (OR, 0.04; 95%CI, 0.01-0.12; P < 0.001). After a Bonferroni correction for multiple comparisons, the genotypic associations of CD36 rs1194197 and PPARγ rs10865710 and the allelic association of CD36 rs7807607 with obesity remained statistically significant. CONCLUSIONS: Our data first indicated that CD36 and PPARγ polymorphisms may be associated with VPA-induced obesity and weight gain, suggesting that CD36 and PPARγ may have potential value in predicting VPA-induced obesity in Chinese Han epileptic patients.

A validated LC-MS/MS method for the simultaneous determination of thalidomide and its two metabolites in human plasma: Application to a pharmacokinetic assay.

An accurate and sensitive LC-MS/MS method for determining thalidomide, 5-hydroxy thalidomide and 5'-hydroxy thalidomide in human plasma was developed and validated using umbelliferone as an internal standard. The analytes were extracted from plasma (100 µL) by liquid-liquid extraction with ethyl acetate and then separated on a BETASIL C18 column (4.6 × 150 mm, 5 µm) with mobile phase composed of methanol-water containing 0.1% formic acid (70:30, v/v) in isocratic mode at a flow rate of 0.5 mL/min. The detection was performed using an API triple quadrupole mass spectrometer in atmospheric pressure chemical ionization mode. The precursor-to-product ion transitions m/z 259.1 → 186.1 for thalidomide, m/z 273.2 → 161.3 for 5-hydroxy thalidomide, m/z 273.2 → 146.1 for 5'-hydroxy thalidomide and m/z 163.1 → 107.1 for umbelliferone (internal standard, IS) were used for quantification. The calibration curves were obtained in the concentrations of 10.0-2000.0 ng/mL for thalidomide, 0.2-50.0 ng/mL for 5-hydroxy thalidomide and 1.0-200.0 ng/mL for 5'-hydroxy thalidomide. The method was validated with respect to linear, within- and between-batch precision and accuracy, extraction recovery, matrix effect and stability. Then it was successfully applied to estimate the concentration of thalidomide, 5-hydroxy thalidomide and 5'-hydroxy thalidomide in plasma samples collected from Crohn's disease patients after a single oral administration of thalidomide 100 mg.

A rapid and simple HPLC-MS/MS method for the simultaneous quantification of valproic acid and its five metabolites in human plasma and application to study pharmacokinetic interaction in Chinese epilepsy patients.

Valproic acid(VPA) is a classic drug that used to treat epilepsy in monotherapy or combination with other anticonvulsant drugs such as lamotrigine (LTG). Although it was reported that VPA could increase lamotrigine trough concentration in clinical practice, there was no report about the effect of LTG on the trough concentration of VPA and its main metabolites, such as 4-ene-VPA, 3-OH-VPA, 4-OH-VPA, 3-keto-VPA, 2-PGA which are related to the therapeutic and toxic effects of VPA. In this study, a simple and rapid method for the simultaneous determination of VPA and its five metabolites in human plasma using HPLC-MS/MS was developed for the first time. Benzoic acid was used as an internal standard (IS). Separation was performed on a Hypersil GOLD C18 column by isocratic elution using acetonitrile: 10mM ammonium acetate solution (90:10, v/v) as mobile phase at a flow rate of 0.3mL/min. A triple quadrupole mass spectrometer operating in the negative ion-switching, electron spray ionization mode with selection reaction monitoring (SRM) was employed to determine transitions of m/z 143.183→143.183, 157.033→113.165, 173.017→129.074, 159.058→101.082, 140.870→140. 870, 159.049→123.076, 121.035→77.136 for VPA, 2-PGA, 3-keto-VPA, 3-OH-VPA, 4-ene-VPA, 4-OH-VPA and IS, respectively. The method also afforded satisfactory results in terms of sensitivity, specificity, precision (intra- and inter-batch), accuracy, recovery, matrix effect and stability. This method was successfully applied to evaluate the effect of LTG on the trough concentration of VPA, 2-PGA, 3-keto-VPA, 3-OH-VPA, 4-ene-VPA, 4-OH-VPA in Chinese epilepsy patients. The result showed that there was no significant difference in the concentration of VPA and its five metabolites between patients in VPA monotherapy and patients in therapy combining VPA with LTG.

A rapid and sensitive UHPLC-MS/MS method for quantification of 83b1 in plasma and its application to bioavailability study in rats.

Great attentions have been drawn by quinoline for its broad bioactivity as anti-fungal, anti-bacterial and anti-tumor activities. Compared with cisplatin, 83b1, a quinoline derivative, showed equal activity in anti-tumor and lower cyctotoxicity in normal cell. In this study, a simple, rapid and sensitive method for determination of 83b1 in rat plasma using UHPLC-MS/MS was developed for the first time. Loratadine was used as an internal standard (IS). Separation was performed on an Xterra MS C18 column by isocratic elution using acetonitrile: water solution with 1‰ formic acid (90:10, v/v) as mobile phase at a flow rate of 0.3mL/min. A triple quadrupole mass spectrometer operating in the positive ion-switching electron spray ionization mode with selection reaction monitoring (SRM) was employed to determine 83b1 and IS transitions of m/z 321.82→147.84, 382.71→258.76 for 83b1 and Loratadine, respectively. The values of specificity, linearity and lower limit of quantification, intra- and inter- day precision and accuracy, extraction recovery, matrix effect and stability for this method satisfied the acceptable limits. The lower limit of quantification was 0.5ng/mL with a linear range of 0.5-1500ng/mL. The validated method was employed to study the bioavailability of 83b1 in rat by dosing with intravenous injection (1mg/kg) and gavage (10mg/kg), and the oral bioavailability of 83b1 in rat was calculated as 20.9±8.8%.

Determination of a novel Aurora-A (AurA) kinase AKI603 by UPLC-MS/MS and its application to a bioavailability study in rat.

A simple, sensitive and accurate ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the determination of AKI603 in rat plasma has been firstly developed and validated. After a simple liquid-liquid extraction (LLE) with ethyl acetate, the analytes were separated on C18 column (2.1×100mm, 1.9µm, Thermo) by gradient elution with mobile phase of water (A) (containing 5mM ammonium acetate and 0.1% formic acid) and methanol (B) with a flow rate of 0.3mLmin(-1) and then analyzed by mass spectrometry in the positive multiple reactions monitoring (MRM) mode. The mass transitions monitored were m/z 410.0→352.9, m/z 457.1→367.9 for AKI603 and internal standard (Ly-7z), respectively. The developed method was validated for specificity, linearity and lower limit of quantification, intra- and inter-day precision and accuracy, extraction recovery, matrix effect and stability whose values satisfied the acceptable limits. The calibration curves for AKI603 was linear in concentration ranges of 0.025-5000ngmL(-1). The method has been successfully used to the bioavailability study of AKI603 administered to rats intravenously (2.5mg/kg) or orally (25mg/kg). The oral bioavailability of AKI603 in rats was calculated as 28.7±9.7%.

Synthesis of Deuterated Benzopyran Derivatives as Selective COX-2 Inhibitors with Improved Pharmacokinetic Properties.

We designed a series of specifically deuterated benzopyran analogues as new COX-2 inhibitors with the aim of improving their pharmacokinetic properties. As expected, the deuterated compounds retained potency and selectivity for COX-2. The new molecules possess improved pharmacokinetic profiles in rats compared to their nondeuterated congeners. Most importantly, the new compounds showed pharmacodynamic efficacy in several murine models of inflammation and pain. The benzopyran derivatives were separated into their enantiomers, and the activity was found to reside with the S-isomers. To streamline the synthesis of the desired S-isomers, an organocatalytic asymmetric domino oxa-Michael/aldol condensation reaction was developed for their preparation.

Validation of visualized transgenic zebrafish as a high throughput model to assay bradycardia related cardio toxicity risk candidates.

Drug-induced QT prolongation usually leads to torsade de pointes (TdP), thus for drugs in the early phase of development this risk should be evaluated. In the present study, we demonstrated a visualized transgenic zebrafish as an in vivo high-throughput model to assay the risk of drug-induced QT prolongation. Zebrafish larvae 48 h post-fertilization expressing green fluorescent protein in myocardium were incubated with compounds reported to induce QT prolongation or block the human ether-a-go-go-related gene (hERG) K⁺ current. The compounds sotalol, indapaminde, erythromycin, ofoxacin, levofloxacin, sparfloxacin and roxithromycin were additionally administrated by microinjection into the larvae yolk sac. The ventricle heart rate was recorded using the automatic monitoring system after incubation or microinjection. As a result, 14 out of 16 compounds inducing dog QT prolongation caused bradycardia in zebrafish. A similar result was observed with 21 out of 26 compounds which block hERG current. Among the 30 compounds which induced human QT prolongation, 25 caused bradycardia in this model. Thus, the risk of compounds causing bradycardia in this transgenic zebrafish correlated with that causing QT prolongation and hERG K⁺ current blockage in established models. The tendency that high logP values lead to high risk of QT prolongation in this model was indicated, and non-sensitivity of this model to antibacterial agents was revealed. These data suggest application of this transgenic zebrafish as a high-throughput model to screen QT prolongation-related cardio toxicity of the drug candidates.