Simultaneous determination of sinomenine and its metabolites desmethyl-sinomenine and sinomenine N-oxide in rat plasma by liquid chromatography tandem mass spectrometry.

Sinomenine is an active ingredient extracted from herb medicine, which has been prescribed to treat rheumatoid arthritis in clinics. The present work was to develop a simple method to simultaneously determine sinomenine and its metabolites desmethyl sinomenine and sinomenine N-oxide in rat plasma by liquid chromatography tandem mass spectrometry. Precursor-to-product transitions for detection were m/z 330.2 > 239.1 for sinomenine, m/z 316.2 > 239.1 for desmethyl-sinomenine, m/z 346.2 > 314.1 for sinomenine N-oxide and m/z 286.2 > 153.2 for morphine (internal standard), respectively. During the validation and sample quantification, an excellent linear calibration range was observed for all the analytes with correlation coefficients more than 0.999 (r > 0.99). The extraction recovery was more than 85%. No significant matrix effect and carryover were observed. The precision was less than 6.45%, whereas accuracy ranged from -4.10% to 7.23%. The validated method has been successfully applied to the pharmacokinetic study of sinomenine, desmethyl sinomenine, and sinomenine N-oxide in rat plasma after oral administration of sinomenine at a single dose of 5 mg/kg. The results suggested that sinomenine was rapidly metabolized into its metabolite desmethyl sinomenine and sinomenine N-oxide.

A simple and sensitive liquid chromatography triple quadrupole mass spectrometry method for the determination of XL092 in monkey plasma and its application to pharmacokinetic study.

XL092 is a potent ATP-competitive inhibitor of multiple receptor tyrosine kinases that is undergoing clinical development for the treatment of lung cancer. In this study, an LC triple quadrupole mass spectrometry method was established to measure XL092 in monkey plasma. A Waters ACQUITY UPLC BEH C18 column was used for chromatographic separation. The mobile phase consisted of water containing 0.1% formic acid and acetonitrile with a gradient elution at the flow rate of 0.4 mL/min. Multiple reaction monitoring mode was used for quantitative analysis of XL092 in positive electrospray ionization. In the concentration range of 0.5-1000 ng/mL, XL092 showed excellent linearity in monkey plasma with a correlation coefficient greater than 0.995 (r > 0.995). The lowest limit of quantification was 0.5 ng/mL. The intra- and inter-day relative standard deviations were <9.99%, while the relative error ranged from -12.50% to 8.10%. The mean recovery was over 82.51%. XL092 was stable in monkey plasma after storage under certain conditions. The validated method was demonstrated to be selective, sensitive, and reliable, and has been successfully applied to the pharmacokinetic study of XL092 in monkey plasma. XL092 showed moderate short half-life (~3.81 h) and good oral bioavailability (80%).

Evidence for cytochrome P450 3A4-mediated metabolic activation of SCO-267.

SCO-267 is a potent G-protein-coupled receptor 40 agonist that is undergoing clinical development for the treatment of type 2 diabetes mellitus. The current work was undertaken to investigate the bioactivation potential of SCO-267 in vitro and in vivo. Three SCO-267-derived glutathione (GSH) conjugates (M1-M3) were found both in rat and human liver microsomal incubations supplemented with GSH and nicotinamide adenine dinucleotide phosphate. Two GSH conjugates (M1-M2) together with two N-acetyl-cysteine conjugates (M4-M5) were detected in the bile of rats receiving SCO-267 at 10 mg/kg. The identified conjugates suggested the generation of quinone-imine and ortho-quinone intermediates. CYP3A4 was demonstrated to primarily catalyze the bioactivation of SCO-267. In addition, SCO-267 concentration-, time-, and NADPH-dependently inactivated CYP3A in human liver microsomes using testosterone as a probe substrate, along with KI and kinact values of 4.91 µM and 0.036 min-1 , respectively. Ketoconazole (a competitive inhibitor of CYP3A) displayed no significant protective effect on SCO-267-induced CYP3A inactivation. However, inclusion of GSH showed significant protection. These findings revealed that SCO-267 undergoes a facile CYP3A4-catalyzed bioactivation with the generation of quinone-imine and ortho-quinone intermediates, which were assumed to be involved in SCO-267 induced CYP3A inactivation. These findings provide further insight into the bioactivation pathways involved in the generation of reactive, potentially toxic metabolites of SCO-267. Further studies are needed to evaluate the influence of SCO-267 metabolism on the safety of this drug in vivo.

Pharmacokinetics and bioavailability study of ginsenoside Rk1 in rat by liquid chromatography/electrospray ionization tandem mass spectrometry.

Ginsenoside Rk1 (Rk1) exhibited various potent biological activities. However, its pharmacokinetic profile in vivo remains unclear. In the present study, a simple and sensitive liquid chromatography tandem mass spectrometry method was developed and validated for determination of Rk1 in rat plasma and applied in a pharmacokinetic study. The sample was precipitated with acetonitrile and separated on a Zorbax Eclipse XDB C18 column (50 × 2.1 mm, 1.8 µm). The mobile phase was composed of 0.1% formic acid in water and acetonitrile at a flow rate of 0.4 mL/min. Rk1 and internal standard (ginsenoside Rg3) were quantitatively monitored with precursor-to-product ion transitions of m/z 765.4 → 441.5 and m/z 783.5 → 621.4, respectively. The assay was linear over the concentration range of 5-1000 ng/mL (r > 0.99) with the LLOQ of 5 ng/mL. Other parameters including intra- and inter-day precision and accuracy, extraction recovery and matrix effect were within the acceptable limits. The analyte was stable under the tested storage conditions. The validated method has been successfully applied to a pharmacokinetic study of Rk1 in rat plasma after intravenous (5 mg/kg) and oral (25 mg/kg, 50 mg/kg) administration. After oral administration, Rk1 could be detected in blood at 30 min and reached the highest concentration at 4.29~4.57 h. Our results demonstrated that Rk1 showed low clearance, moderate half-life (3.09-3.40 h) and low bioavailability (2.87-4.23%). The study will provide information for the further application of Rk1.

Evaluation of the pharmacokinetics, tissue distribution and excretion studies of YMR-65, a tubulin polymerization inhibitor with potential anticancer activity, in rats using UPLC-MS/MS.

1. YMR-65, 5-(5-bromo-1-methyl-1H-indol-3-yl)-3-(3-methoxyphenyl)-4, 5-dihydro-1H-pyrazole-1-carboxamide, is a new tubulin polymerization inhibitor with encouraging anticancer activity. 2. The validated ultra-performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) method was successfully applied to the pharmacokinetics, tissue distribution and excretion study of YMR-65 after oral and intravenous administration. The area under concentration-time curve (AUC0-∞) for YMR-65 were 151.67 ± 54.48 and 459.45 ± 49.23 ng/ml*h for oral and intravenous administration at the dosage of 1.5 mg/kg, respectively and the oral bioavailability was about 33.01%. Moreover, YMR-65 was extensively distributed in heart, liver, spleen, lung, kidney, stomach, intestine and testis and the highest were detected in heart, followed by stomach, intestine and liver. The majority of YMR-65 was excreted via feces and its accumulative excretion ratio during the period of 96 h was 19.83 ± 3.01%, but only 1.54 ± 0.37 and 0.215 ± 0.026% for urine within 96 h and bile within 10 h after intravenous administration, respectively, though the fecal and urine excretion were incomplete within 96 h. 3. In summary, this study defined the pharmacokinetic characteristics of YMR-65 in vivo and the important data can be a useful resource for further research and development.

Epigallocatechin-3-gallate decreases the transport and metabolism of simvastatin in rats.

1. This study aimed to investigate the potential impact of epigallocatechin-3-gallate (EGCG) on the pharmacokinetic behaviors of simvastatin and its metabolite simvastatin acid and explored the possible role of metabolizing enzymes and transporters of this food-drug interaction. 2. Female SD rats were intravenously administered with EGCG (5 mg/kg), ketoconazole (10 mg/kg) and rifampin (10 mg/kg), followed by intravenous administration of 2 mg/kg simvastatin. In vitro, the effects of EGCG on Cytochrome P450 enzymes (CYP450) and organic anion transporting polypeptides (OATPs) were studied using human hepatic microsomes and human embryonic kidney 293 (HEK293) cells overexpressing OATP1B1 or OATP1B3. The results showed that areas under concentration-time (AUC) curves of simvastatin and simvastatin acid increased by 2.21- and 1.4-fold while the clearance was reduced by 2.29- and 1.4-fold, respectively, when co-administered with EGCG. In vitro experiments suggested the inhibitory effect of EGCG on CYP enzymes (IC50: 18.37 ± 1.36 µM, 26.08 ± 1.51 µM for simvastatin and simvastatin acid, respectively). Simvastatin transport by OATP1B1 and OATP1B3 was also inhibited by EGCG (IC50: 8.68 ± 1.27 µM and 22.67 ± 1.42 µM, respectively). 3. The presently reported novel food-drug interaction between EGCG and simvastatin involves the inhibition of not only CYP450 enzymes but also OATPs by EGCG.

Investigation on pharmacokinetics, tissue distribution and excretion of 1-triacontanol in rats by gas chromatography-tandem mass spectrometry (GC-MS/MS).

1. 1-Triacontanol (TA) recently shows promising anti-tumor activity. The present study was aimed to develop a sensitive gas chromatography-tandem mass spectrometry method to explore the pharmacokinetic profiles, distribution and excretion of TA in Sprague-Dawley rats after oral administration of TA. Chromatography separation was performed on a HP-5MS column. 1-Octacosanal was used as the internal standard (IS). Quantification of TA and IS was carried out at m/z 495.6 → 97.0 and m/z 467.5 → 97.0, respectively, in positive electron ionization and multiple reaction monitoring mode. The pharmacokinetic parameters were calculated by non-compartmental analysis. 2. The area under concentration-time curve AUC0-6 h and AUC0-∞ for TA at 60 mg/kg were 87.737 ± 13.574 and 93.617 ± 17.62, respectively. The mean residence time was 3.25 ± 0.17 h. In addition, the elimination half-lives (t1/2) were (2.37 ± 1.23, 1.27 ± 0.49, 2.07 ± 0.93) h after single oral administration of 30, 60 and 120 mg/kg of TA. After oral administration, TA was extensively distributed in stomach and intestine. The majority of TA excreted via feces, and its accumulative excretion ratio during the period of 72 h was 26.68 ± 7.14%, but only 0.0023 ± 0.0015% and 0.0027 ± 0.0006% for urines and bile, respectively. The absolute bioavailability (F, %) of TA was about 2.0%.

Trace quantification of 1-triacontanol in beagle plasma by GC-MS/MS and its application to a pharmacokinetic study.

1-Triacontanol (TA), a member of long chain fatty alcohol, has recently been received great attention owing to its antitumor activity. In this study, an accurate, sensitive and selective gas chromatography-tandem mass spectrometry method was developed and validated for the quantification of TA in beagle plasma using 1-octacosanal as the internal standard (IS) for the first time. With temperature programming, chromatographic separation was carried out on an HP-5MS column, using helium as carrier gas and argon as collision gas, both at a flow rate of 1 mL/min. TA was analyzed using positive ion electrospray ionization in multiple-reaction monitoring mode, with the precursor to product ion transitions of m/z 495.6 → 97.0 and m/z 467.5 → 97.0 for TA and the IS, respectively. The lower limit of quantitation, linearity, intra- and interday precision, accuracy, stability, extraction recovery and matrix effect of TA were within the acceptable limits. The validated method was successfully applied to a pharmacokinetic study of TA in beagles.

Investigation on pharmacokinetics, tissue distribution and excretion of a novel platinum anticancer agent in rats by inductively coupled plasma mass spectrometry (ICP-MS).

1. DN604 is a new platinum agent with encouraging anticancer activity. The present study was to explore the pharmacokinetic profiles, distribution and excretion of platinum in Sprague-Dawley rats after intravenous administration of DN604. A sensitive and selective inductively coupled plasma mass spectrometry (ICP-MS) method was established for determination of platinum in biological specimens. The pharmacokinetic parameters were calculated by a non-compartmental method. 2. The area under concentration-time curve AUC0-t and AUC0-∞ for platinum originating from DN604 at 10 mg/kg were 25.15 ± 1.29 and 28.72 ± 1.04 µg/hml, respectively. The mean residence time MRT was 36.59 ± 6.65 h. The volume of distribution Vz was 11.42 ± 2.49 l/kg and clearance CL was 0.18 ± 0.01 l/h/kg. In addition, the elimination half-life T1/2z was 44.83 ± 9.75 h. After intravenous administration of DN604, platinum was extensively distributed in most of tested tissues except brain. The majority of platinum excreted via urine, and its accumulative excretion ratio during the period of 120 h was 63.5% ± 7.7% for urine, but only 6.94% ± 0.11% for feces. 3. The satisfactory half-life, wide distribution and high excretion made this novel platinum agent worthy of further research and development.

Effect of triacontanol on the pharmacokinetics of docetaxel in rats associated with induction of cytochrome P450 3A1/2.

Triacontanol was confirmed to have a potential anti-cancer effect, the aim was to assess whether the co-administration of triacontanol alters the exposure of docetaxel via inducing hepatic CYP3A1/2 activity. The concentration of docetaxel in rats pretreated with triacontanol for seven successive days was determined, and the expression levels of CYP3A protein and mRNA were analyzed by the western blot and real time polymerase chain reaction (RT-PCR) technique, respectively. 2. The concentrations of docetaxel in rats pretreated with triacontanol were decreased, with 61.5%, 61.9% decrease in AUC0-24h and 65.7%, 54.9% reduction in Cmax (120 and 180 mg kg(-1), respectively) compared with the control. Hepatic clearance of docetaxel was enhanced in vitro and in vivo at dosage of 120 and 180 mg kg(-1), and CYP3A activity was up-regulated by measuring the formation rate of 1-hydroxymidazolam. Triacontanol preferentially induced protein expression level of CYP3A2 in a dose-dependent manner and of CYP 3A1 at dosage of 120 and 180 mg kg(-1). The mRNA expression of CYP3A1 was moderately different with the western blot results, but the trends appeared similar. CYP3A2 mRNA level was not markedly affected by triacontanol. 3. The significant triacontanol-docetaxel interaction was largely due to the induction of CYP3A1/2, which brought useful information in the clinical therapy when the combination is administered in human.

Pharmacokinetics, tissue distribution, excretion and plasma protein binding studies of wogonin in rats.

Wogonin is a natural anticancer candidate. The purpose of this study was to explore the pharmacokinetic profiles, tissue distribution, excretion and plasma protein binding of wogonin in Sprague-Dawley rats. A rapid, sensitive, and specific LC-MS/MS method has been developed for the determination of wogonin in different rat biological samples. After i.v. dosing of wogonin at different levels (10, 20 and 40 mg/kg) the elimination half-life was approximately 14 min, the AUC0-∞ increased in a dose disproportional manner from 112.13 mg/L·min for 10 mg/kg to 758.19 mg/L·min for 40 mg/kg, indicating a non linear pharmacokinetic profile. After i.g. dosing at 100 mg/kg, plasma levels of wogonin peaked at 28 min with a Cmax value of 300 ng/mL and a very low oral bioavailability (1.10%). Following i.v. single dose (20 mg/kg), wogonin was detected in all examined tissues (including testis) with the highest levels in kidney and liver. Approximately 21% of the administered dose was excreted as unchanged drug (mainly via non-biliairy fecal route (16.33%). Equilibrium dialysis was used to evaluate plasma protein binding of wogonin at three concentrations (0.1, 0.5 and 2 µg/mL). Results indicated a very high protein binding degree (over 90%), reducing substantially the free fraction of the compound.

Yi-Qi-Huo-Xue decoction alleviates intracerebral hemorrhage injury through inhibiting neuronal autophagy of ipsilateral cortex via BDNF/TrkB pathway.

BACKGROUND: Yi-Qi-Huo-Xue Decoction (YQHXD), a traditional Chinese medicine formula, has demonstrated efficacy in the clinical treatment of intracerebral hemorrhage (ICH) for over a decade. Nevertheless, the precise pharmacotherapeutic compounds of YQHXD capable of penetrating into cerebral tissue and the pharmacological underpinnings of YQHXD remain ambiguous. METHODS: The active components of YQHXD in rat brains was analyzed by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. The potential targets, pathways and biological progresses of YQHXD ameliorating ICH induced injury was predicted by network pharmacology. Moreover, collagenase-induced ICH rat model, primary cortex neurons exposed to hemin and molecular docking were applied to validate the molecular mechanisms of YQHXD. RESULTS: Eleven active components of YQHXD were identified within the brains. Employing the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, our investigation concentrated on the roles of autophagy and the BDNF/TrkB signaling pathway in the pharmacological context. The pharmacological results revealed that YQHXD alleviated neurological dysfunction, brain water content, brain swelling, and pathological injury caused by ICH. Meanwhile, YQHXD inhibited autophagy influx and autophagosome in vivo, and regulated cortex neuronal autophagy and TrkB/BDNF pathway both in vivo and in vitro. Subsequently, N-acetyl serotonin (NAS), a selective TrkB agonist, was employed to corroborate the significance of the BDNF/TrkB pathway in this process. The combination of NAS and YQHXD did not further enhance the protective efficacy of YQHXD in ICH rats. Additionally, outcomes of molecular docking analysis revealed that nine compounds of YQHXD exhibited potential regulatory effects on TrkB. CONCLUSIONS: Ipsilateral neuronal autophagy and BDNF/TrkB pathway were activated 72 h after ICH. YQHXD effectively resisted injury induced by ICH, which was related with suppression of ipsilateral neuronal autophagy via BDNF/TrkB pathway. This study provides novel insights into the therapeutic mechanisms of traditional Chinese medicine in the context of ICH treatment.

A class of novel tubulin polymerization inhibitors exert effective anti-tumor activity via mitotic catastrophe.

In current work, a class of novel 4,5-dihydro-1H-pyrazole-1-carboxylate derivatives (E01-E28) were designed, synthesized and evaluated. Among them, the most potent compound E24 exhibited comparable activity against a panel of cancer cells (GI50 ranging 0.05-0.98 µM) and tubulin polymerization inhibition (IC50 = 1.49 µM) with reference drug CA-4(P) (GI50 ranging 0.019-0.32 µM, IC50 = 2.18 µM). The following assays indicated that compound E24 disturbed the dynamics of tubulin catastrophe and rescue, which triggered G2/M arrest, leading to ROS accumulation, cleavage of PARP and apoptosis. Molecular dynamics simulation validated that compound E24 could tightly bind into tubulin heterodimers with ß Lys 254 and ß Cys 241 of tubulin in the docking pose. Metabolic stability and pharmacokinetics parameters were also determined. The half time (t1/2) displayed species differences in three microsomes. The plasma elimination half-life (t1/2), peak plasma concentration (Cmax), mean retention time (MRT), the area under the curve (AUC0-∞) and distribution volume (Vz) of E24 after intravenous administration were 0.90 ± 0.22 h, 594.50 ± 97.23 ng/mL, 1.09 ± 0.22 h, 413.67 ± 105.64 ng/mL*h and 5.03 ± 1.82 L/kg, respectively. In HeLa-xenografts, compound E24 exhibited obvious antitumor efficacy via the suppression of tumor growth without weight loss of body or organ. In brief, compound E24 might be a hopeful candidate with excellent properties for oncotherapy as tubulin polymerization inhibitor.

Differential Functions of Two Metalloproteases, Mrmep1 and Mrmep2, in Growth, Sporulation, Cell Wall Integrity, and Virulence in the Filamentous Fungus Metarhizium robertsii.

The Metarhizium genus of filamentous entomopathogenic fungi plays a pivotal role in regulating insect populations. Metalloproteases (MEPs) are a widely distributed and diverse family of hydrolytic enzymes that are important toxicity factors in the interactions between fungi and their hosts. Herein, we characterized two MEPs, Mrmep1 and Mrmep2, in Metarhizium robertsii using gene deletion. Growth rates of the resulting ΔMrmep1 and ΔMrmep2 mutants decreased by 16.2 and 16.5%, respectively, relative to the wild-type (WT) strain. Both mutants were less sensitive to cell wall-perturbing agents, sodium dodecyl sulfate and Congo red than the WT strain, whereas did not show any obvious changes in fungal sensitivity to ultraviolet B irradiation or heat stress. The conidial yield of ΔMrmep1, ΔMrmep2, and ΔMrmep1ΔMrmep2 mutants decreased by 56.0, 23, and 53%, respectively. Insect bioassay revealed that median lethal time values against Galleria mellonella increased by 25.5% (ΔMrmep1), 19% (ΔMrmep2), and 28.8% (ΔMrmep1ΔMrmep2) compared with the WT strain at a concentration of 1 × 107 conidia mL-1, suggesting attenuated fungal virulence in the ΔMrmep1, ΔMrmep2, and ΔMrmep1ΔMrmep2 strains. During fungal infection, transcription levels of Mrmep1 was 1.6-fold higher than Mrmep2 at 36 h post inoculation. Additionally, transcription levels of gallerimycin gene were 1.2-fold, 2.18-fold, and 2.5-fold higher in insects infected with the ΔMrmep1, ΔMrmep2, or ΔMrmep1ΔMrmep2 mutant than those infected with the WT strain, respectively. Our findings suggest that Mrmep1 and Mrmep2 are differentially contributed to the growth, sporulation, cell wall integrity, and virulence of M. robertsii.

Determination of oroxylin A and oroxylin A 7-O-d-glucuronide in HepG2 cell lysate and subcellular fractions with SPE-UPLC-MS/MS: Cellular pharmacokinetic study to indicate anti-cancer mechanisms.

Targeting therapy of anti-cancer drugs has been gaining increasing attention. Cell pharmacokinetics have been used for in vitro disposition evaluation, as well as drug-drug interaction for anti-cancer drugs, revealing their fate after entering tumor. Flavonoid compound oroxylin A (OA) possesses strong anti-cancer effects especially on the liver and breast cancer. However, despite the low bioavailability, the disposition of OA and its active metabolite in the target cancer cells remained unclear. In current study, a highly sensitive and selective solid phase extraction (SPE)-UPLC-MS/MS method was developed and validated to simultaneously quantify the concentrations of OA and its major active metabolite oroxylin A 7-O-d-glucuronide (OG) in HepG2 cell lysate and multiple subcellular organelle fractions. The proposed method appeared to be suitable for the analysis with desirable linearity(R2 > 0.99). The relative standard deviations (RSDs) of intra- and inter-assay precision and accuracy were less than 9.9% and -7.7%, 8.4% and 11% for OA and OG in cell lysate respectively. The intra- precision and accuracy was less than 9.5% and -11.3%, 9.4% and 12.3% for OA and OG in subcellular organelles respectively. The range of absolute recovery of this method in the cell lysate was from 73.1% ±â€¯1.4% to 87.9% ±â€¯6.7%. The RSDs of matrix effects of the quality control (QC) samples were below 15%. The uptake and distribution experiments demonstrated a time-dependent transport characteristic in HepG2 cell lines. Furthermore, both OA and OG were mainly distributed into nuclei after taken up by the tumor cells. In addition, OG was also distributed into mitochondria, which indicates another potential target of OG. The present study, for the first time, reports the in vitro cell pharmacokinetics profiles of OA and OG in tumor cell lines in vitro.

Quantification of triacontanol and its PEGylated prodrug in rat plasma by GC-MS/MS: Application to a pre-clinical pharmacokinetic study.

PEGylation techniques have been increasingly employed in drug delivery system and chemical modification of compounds with low aqueous solubility. Triacontanol (TA) is a natural product with several pharmacological activities, but its low aqueous solubility significantly limited its application. PEGylated triacontanol (PEG-TA) was designed as the prodrug to improve the aqueous solubility and pharmacokinetic properties of TA. On the basis of salting-out assisted liquid-liquid extraction (SALLE) and saponification sample preparation procedure, a reliable gas chromatography tandem mass spectrometric (GC-MS/MS) method was developed and validated for the quantification of PEG-TA and its metabolite TA in rat plasma after separation and transformation. Acetonitrile-methanol (9:1, v/v) and ammonium acetate (10 M) were utilized to separate PEG-TA and TA (including conjugated TA with fatty acid). Saponification facilitated the complete conversion of PEG-TA into TA, so PEG-TA could be indirectly quantified. The results revealed that the GC-MS/MS method had excellent selectivity, accuracy and linearity. Calibration curves were linear (R2>0.99) within the range of 20.0-1000.0 ng/mL for TA and 100.0-10,000.0 ng/mL for PEG-TA. The intra- and inter-day precision of quality control samples were within 15%, and their accuracy values varied from 93.54% to 113.38%. This analytical method has been successfully applied to pharmacokinetic study of PEG-TA. This study can facilitate the further exploration and quantification of PEGylated prodrugs.

Pharmacodynamic and pharmacokinetic characteristics of YMR-65, a tubulin inhibitor, in tumor-bearing mice.

YMR-65​, 5-(5-bromo-1-methyl-1H-indol-3-yl)-3-(3-methoxyphenyl)-4, 5-dihydro-1H-pyrazole-1-carboxamide, is a potential tubulin inhibitor exhibiting good anticancer activity. In our study, we illustrated the biological activities in HepG2 cells and the pharmacodynamic and pharmacokinetic profiles were evaluated in murine H22 hepatoma-bearing mice. Molecular docking assay and colchicine competition assay indicated that YMR-65 could bind tightly to the colchicine binding site of tubulin. Further investigation demonstrated that YMR-65 arrested cells in the G2/M phase of cell cycle and induced apoptosis in HepG2 cells. Compared with control group, the tumor growth inhibition determined by final relative volume of tumor/the initial tumor volume were 32.57%, 24.00% and 34.95%, respectively, for YMR-65 (10 mg/kg), YMR-65 (20 mg/kg) and CA4P (10 m/kg) groups. Besides there were no obvious body change or tissue damage (enhanced by histopathology study). YMR-65 administration at 10 and 20 mg/kg in H22 tumor-bearing mice resulted in 1.87- and 1.80-fold longer half time (t1/2) and 0.36- and 0.78-fold lower area under concentration-time curve (AUC0-∞) in plasma in contrast with normal mice at 10 mg/kg. Furthermore, YMR-65 showed a wide distribution to various tissues or tumor and the highest distribution index (the ratio of AUCtissue or tumor/AUCplasma) was found in tumor, which implied that it might accumulate in tumor after administration. In brief, our results indicated that YMR-65 was a promising candidate with high antitumor efficacy and low tissue damage.

Hydrophilic interaction liquid chromatography-tandem mass spectrometry method for the determination of intact oxaliplatin in cells: validated and applied in colon cancer HCT-116 cell line.

Oxaliplatin is a platinum compound that is frequently prescribed for the chemotherapeutic treatment of colorectal cancer. In tumor cells, cellular uptake is the first step of oxaliplatin action. Cellular accumulation of oxaliplatin is considered to play an important role in anti-cancer efficacy. However, limited information about cellular accumulation of intact oxaliplatin is available. In this study, a sensitive hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) approach for the quantification of oxaliplatin in cells was developed and validated. The method allowed for a rapid and simple determination of intact oxaliplatin in cell lysate. The retention time of oxaliplatin was 3.04 min, which was achieved by applying a chromatographic gradient elution of 5 min. The lower limit of quantification (LLOQ) was 2 ng/mL and the analytical range of oxaliplatin was linear between 2-200 ng/mL. The intra-day precision and inter-day precision (RSD (relative standard deviation)) ranged from 0.52 to 7.89%, and the accuracy (RE (relative error)) was within ±â€¯4.5%. Matrix effects and recovery were acceptable. The method was successfully used for the determination of intact oxaliplatin uptake by HCT-116 colon cancer cells. Thus, our findings may prospectively support a celluar pharmacokinetic study and low concentration measurement of intact oxaliplatin in the clinic.

Coloaded Nanoparticles of Paclitaxel and Piperlongumine for Enhancing Synergistic Antitumor Activities and Reducing Toxicity.

The purpose of this study was to develop a nanocarrier system for codelivery of paclitaxel (PTX) and piperlongumine (PL) and investigate the therapeutic potential of improving efficacy and reducing toxicity. PTX and PL were formulated into poly lactic-co-glycolic acid and D-α-tocopheryl polyethylene glycol succinate via organic solvent evaporation method. The average diameter was 117.1 ± 1.9 nm, and the zeta potential was -43.25 ± 2.76 mV. PL facilitated the cellular uptake of PTX, and the increased cytotoxicity was similarly displayed. The formulation with the PTX/PL concentration ratio at 1:200 showed the best antitumor activity, the IC50 of PTX were 5.10 ± 0.08 nM in HepG2 cells, and 3.79 ± 1.01 nM in Michigan Cancer Foundation-7 cells. Correspondingly, the combination index was 0.79 and 0.76. Furthermore, intracellular uptake of PTX toward HepG2 cells in coencapsulated nanoparticles was significantly more than free solution. In addition, the antitumor effect of PTX/PL-PTNPs in the HepG2 xenograft tumor model suggested that the nanoparticles showed a higher antitumor efficacy with reduced toxicity to other tissues compared with free PTX. In summary, the results indicated that PTX/PL-PTNPs processed well characteristics and enhanced its therapeutic efficacy; thus, this delivery system could be clinically effective for treatment of cancers.

Co-delivery of docetaxel and palmitoyl ascorbate by liposome for enhanced synergistic antitumor efficacy.

Palmitoyl ascorbate (PA) as an antioxidant has the potential for the treatment of cancer. In the present study, a nanocarrier system was developed for co-delivery of docetaxel (DOC) with palmitoyl ascorbate and the therapeutic efficacy of a combination drug regimen was investigated. For this purpose, different ratios of docetaxel and palmitoyl ascorbate were co-encapsulated in a liposome and they all showed high encapsulation efficiency. The average diameters of the liposomes ranged from 140 to 170 nm. Negative zeta potential values were observed for all systems, ranged from -40 mV to -56 mV. Studies on drug release and cellular uptake of the co-delivery system demonstrated that both drugs were effectively taken up by the cells and released slowly. Moreover, the liposome loading drugs with DOC/PA concentration ratio of 1:200 showed the highest anti-tumor activity to three different types of tumor cells. The higher in vivo therapeutic efficacy with lower systemic toxicity of the DOC-PA200-LPs was also verified by the H22 tumor bearing mice model. Our results showed that such co-loaded delivery systems could serve as a promising therapeutic approach to improve clinical outcomes against hepatic carcinoma.