Development of an Integrated Disposable Device for SARS-CoV-2 Nucleic Acid Extraction and Detection.

Objective: To develop a highly sensitive and rapid nucleic acid detection method for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods: We designed, developed, and manufactured an integrated disposable device for SARS-CoV-2 nucleic acid extraction and detection. The precision of the liquid transfer and temperature control was tested. A comparison between our device and a commercial kit for SARS-Cov-2 nucleic acid extraction was performed using real-time fluorescence reverse transcription polymerase chain reaction (RT-PCR). The entire process, from SARS-CoV-2 nucleic acid extraction to amplification, was evaluated. Results: The precision of the syringe transfer volume was 19.2 ± 1.9 µL (set value was 20), 32.2 ± 1.6 (set value was 30), and 57.2 ± 3.5 (set value was 60). Temperature control in the amplification tube was measured at 60.0 ± 0.0 °C (set value was 60) and 95.1 ± 0.2 °C (set value was 95) respectively. SARS-Cov-2 nucleic acid extraction yield through the device was 7.10 × 10 6 copies/mL, while a commercial kit yielded 2.98 × 10 6 copies/mL. The mean time to complete the entire assay, from SARS-CoV-2 nucleic acid extraction to amplification detection, was 36 min and 45 s. The detection limit for SARS-CoV-2 nucleic acid was 250 copies/mL. Conclusion: The integrated disposable devices may be used for SARS-CoV-2 Point-of-Care test (POCT).

Development of a Recombinase-aided Amplification Combined With Lateral Flow Dipstick Assay for the Rapid Detection of the African Swine Fever Virus.

OBJECTIVE: To establish a sensitive, simple and rapid detection method for African swine fever virus (ASFV) B646L gene. METHODS: A recombinase-aided amplification-lateral flow dipstick (RAA-LFD) assay was developed in this study. Recombinase-aided amplification (RAA) is used to amplify template DNA, and lateral flow dipstick (LFD) is used to interpret the results after the amplification is completed. The lower limits of detection and specificity of the RAA assay were verified using recombinant plasmid and pathogenic nucleic acid. In addition, 30 clinical samples were tested to evaluate the performance of the RAA assay. RESULTS: The RAA-LFD assay was completed within 15 min at 37 °C, including 10 min for nucleic acid amplification and 5 minutes for LFD reading results. The detection limit of this assay was found to be 200 copies per reaction. And there was no cross-reactivity with other swine viruses. CONCLUSION: A highly sensitive, specific, and simple RAA-LFD method was developed for the rapid detection of the ASFV.

A New Method for Ultra-sensitive P24 Antigen Assay Based on Near-infrared Fluorescent Microsphere Immunochromatography.

OBJECTIVE: To develop a rapid, highly sensitive quantitative method for detecting P24 antigen based on near-infrared fluorescent microsphere immunochromatography. METHODS: First, we prepared a lateral flow assay test strip, and labeled the detection antibody using a fluorescent microsphere. Second, we optimized the antibody labeling conditions. Third, we optimized the detection conditions. Fourth, we created a working curve. Fifth, we conducted a methodological assessment of the established fluorescent microsphere immunochromatography method. Sixty-six clinical samples were tested, and we compared the established fluorescent microsphere immunochromatography with the quantitative ELISA method. RESULTS: According to the working curve, the detection limit of the method is 3.4 pg/mL, and the detection range is 3.4 pg/mL to 10 ng/mL. The average intra-assay recovery was 99.6%, and the Coefficient of Variation (CV) was 5.4%-8.6%; the average inter-assay recovery was 97.3%, and the CV was 8.5%-11%. The detection rate of fluorescent microsphere immunochromatography was higher than ELISA method, and had a good correlation with ELISA. CONCLUSION: The P24 antigen quantitative detection method based on near-infrared fluorescent microsphere immunochromatography has the advantages of rapid detection, high sensitivity, and wide detection range; thus, it is suitable for early clinical diagnosis and continuous monitoring of AIDS.

Neutralizing Antibody Titer Test of Ebola Recombinant Protein Vaccine and Gene Vector Vaccine pVR-GP-FC.

OBJECTIVE: In previous studies, we immunized mice with Ebola recombinant protein vaccine and gene vector vaccine. Both stimulated high levels of humoral immunity. In this work, we constructed a pseudovirus containing Ebola membrane proteins to verify whether the two immunization strategies can induce neutralizing antibodies in mice. METHODS: A pseudovirus containing an Ebola virus membrane protein based on the HIV-1 viral gene sequence was constructed and evaluated using a known neutralizing antibody. The titer of the neutralizing antibody in the sera of mice immunized with the recombinant protein and the gene vector vaccine was examined using a neutralization test. RESULTS: Ebola pseudovirus was successfully prepared and applied for neutralizing antibody detection. Immunological experiments showed that recombinant protein GP-Fc and gene vaccine pVR-modGP-Fc had good immunogenicity. The titer of the bound antibody in the serum after 8 weeks of immunization in mice was more than 1:105, and the recombinant protein induced greater humoral immunity. The results of the neutralization test based on the Ebola pseudovirus system demonstrated that both vaccines induced production of protective antibodies, while the gene vaccine induced a higher titer of neutralizing antibodies. CONCLUSION: An Ebola pseudovirus detection system was successfully established and used to evaluate two Ebola vaccines. Both produced good immunogenicity. The findings lay the foundation for the development of new Ebola vaccines and screening for neutralizing monoclonal antibodies.

CD63 inhibits the cell migration and invasion ability of tongue squamous cell carcinoma.

The present study evaluated the expression and potential role of CD63 in the migration and invasion of tongue squamous cell carcinoma (TSCC) cells. Immunohistochemistry (IHC) was used to investigate the association between the expression level of CD63 protein and the histological differentiation of samples from 40 patients with TSCC and four normal tongue tissue specimens. RNA interference (RNAi) and gene transfection technology were used to alter the expression of CD63 in TCA8113 cells. The stable silencing and overexpression of CD63 in the TCA8113 cell line was used to assess the impact of the CD63 expression level on the migratory and invasive abilities of TCA8113 cells in a wound healing assay and a Transwell invasion assay. The effect of CD63 on the expression of matrix metalloproteinase (MMP)-2 and -9 were evaluated by western blot analysis. The results of IHC revealed a positive association between the CD63 expression level and the histopathological differentiation of TSCC and a negative association between the CD63 expression level and lymph node metastasis in TSCC. Western blotting revealed that the expressions of MMP-2 and MMP-9 were clearly upregulated in CD63-silenced TCA8113 cells but reduced in CD63-overexpressing TCA8113 cells, compared with the control. The wound-healing speed and the number of cells invading Matrigel-coated filters were negatively associated with the CD63 expression level. In summary, the results of the present study revealed that CD63 may be an inhibitor of TSCC malignancy and lymph node metastasis and may have applications in the prediction of prognosis and gene therapy for patients of TSCC.

CCND1-BCL2 Gene Network: A direct target of Amifostine in human acute megakaryocytic leukemia cells.

Amifostine, 2-(3-aminopropyl) aminoethyl phosphorothioate, is a broad-spectrum cytoprotective agent used to treat nuclear radiation and chemical weapon injuries. Recently, amifostine has been shown to have a profound biological influence on tumor cells. To examine the effects and mechanisms underlying the effects of amifostine on human acute megakaryocytic leukemia, we evaluated the efficacy of amifostine against Dami cells and observed a cell cycle arrest in G2 /M phase. Amifostine treatment also induced cell apoptosis of Dami cells which corresponds to formal studies. Through whole-genome microarray and bioinformatics analyses, we found that amifostine affected the gene expression of CCND1, BCL2, and CASP3 which revealed the mechanism amifostine acted on Dami cells. Thus, CCND1-BCL2 Gene Network is predicted to be a direct target of amifostine treating human acute megakaryocytic leukemia, which may provide a novel potential target for the therapy of several subtypes of human AML.

Effects of Jia-Wei-Xiao-Yao-San on the Peripheral and Lymphatic Pharmacokinetics of Paclitaxel in Rats.

Paclitaxel is effective against breast cancer. The herbal medicine, Jia-Wei-Xiao-Yao-San (JWXYS), is the most frequent prescription used to relieve the symptoms of breast cancer treatments. The aim of the study was to investigate the herb-drug interaction effects of a herbal medicine on the distribution of paclitaxel to lymph. A validated ultraperformance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) method was used to determine the paclitaxel levels in rat plasma and lymph after intravenous infusion of paclitaxel alone with or without 7 days of JWXYS pretreatment. The pharmacokinetic results indicate that paclitaxel concentrations in plasma exceeded those in lymph by approximately 3.6-fold. The biodistribution of paclitaxel from plasma to lymph was 39 ± 5%; however, this increased to 45 ± 4% with JWXYS pretreatment. With JWXYS pretreatment, the AUC and C max of paclitaxel in plasma were significantly reduced by approximately 1.5-fold, compared to paclitaxel alone. Additionally, JWXYS decreased the AUC and C max of paclitaxel in lymph. However, the lymph absorption rate of paclitaxel with or without JWXYS pretreatment was not significantly changed (27 ± 3 and 30 ± 2%, resp.). Our findings demonstrate that when paclitaxel is prescribed concurrently with herbal medicine, monitoring of the blood pharmacokinetics of paclitaxel is recommended.

Pharmacokinetics of Maleic Acid as a Food Adulterant Determined by Microdialysis in Rat Blood and Kidney Cortex.

Maleic acid has been shown to be used as a food adulterant in the production of modified starch by the Taiwan Food and Drug Administration. Due to the potential toxicity of maleic acid to the kidneys, this study aimed to develop an analytical method to investigate the pharmacokinetics of maleic acid in rat blood and kidney cortex. Multiple microdialysis probes were simultaneously inserted into the jugular vein and the kidney cortex for sampling after maleic acid administration (10 or 30 mg/kg, i.v., respectively). The pharmacokinetic results demonstrated that maleic acid produced a linear pharmacokinetic phenomenon within the doses of 10 and 30 mg/kg. The area under concentration versus time curve (AUC) of the maleic acid in kidney cortex was 5-fold higher than that in the blood after maleic acid administration (10 and 30 mg/kg, i.v., respectively), indicating that greater accumulation of maleic acid occurred in the rat kidney.

The Drug-Drug Effects of Rhein on the Pharmacokinetics and Pharmacodynamics of Clozapine in Rat Brain Extracellular Fluid by In Vivo Microdialysis.

Clozapine, an atypical antipsychotic agent, is highly effective in treatment-resistant schizophrenia; however, its major side effect is constipation. Instead of laxatives, rhein is a pharmacologically active component found in Rheum palmatum L., a medicinal herbal remedy for constipation. The purpose of this study is to determine whether rhein impacts the pharmacokinetics (PK) and pharmacodynamics (PD) of clozapine in brain when used to relieve clozapine-induced constipation. Here, we have investigated not only the PK of clozapine in blood but also the effects of rhein on the PK of clozapine in blood and in brain extracellular fluid together with the PD effects on neurotransmitters in extracellular fluid. The concentrations of clozapine and norclozapine in biologic samples were measured by ultra-performance liquid chromatography-tandem mass spectrometry. The drug-drug effects of rhein on extracellular neurotransmitter efflux in the rat medial prefrontal cortex (mPFC) produced by clozapine were assayed by high-performance liquid chromatography-electrochemical detection. The results demonstrate that the clozapine PK was nonlinear. Pretreatment with rhein for 7 days increased the total blood concentration of clozapine, but significantly reduced the unbound clozapine concentrations in the mPFC by approximately 3-fold. Furthermore, 7 days of rhein pretreatment thoroughly abolished the efflux of dopamine and its metabolite (3,4-dihydroxyphenylacetic acid) and altered the profile of homovanillic acid, another metabolite of dopamine, in the mPFC. In conclusion, rhein was found to substantially decrease clozapine and norclozapine concentrations in the mPFC dialysate, and this is accompanied by lower concentrations in the neurotransmitters in the same biophase. These findings suggest that a detailed clinical study for drug-drug interactions is recommended.

Pharmacokinetics of quercetin-loaded nanodroplets with ultrasound activation and their use for bioimaging.

Bubble formulations have both diagnostic and therapeutic applications. However, research on nanobubbles/nanodroplets remains in the initial stages. In this study, a nanodroplet formulation was prepared and loaded with a novel class of chemotherapeutic drug, ie, quercetin, to observe its pharmacokinetic properties and ultrasonic bioimaging of specific sites, namely the abdominal vein and bladder. Four parallel groups were designed to investigate the effects of ultrasound and nanodroplets on the pharmacokinetics of quercetin. These groups were quercetin alone, quercetin triggered with ultrasound, quercetin-encapsulated in nanodroplets, and quercetin encapsulated in nanodroplets triggered with ultrasound. Spherical vesicles with a mean diameter of 280 nm were formed, and quercetin was completely encapsulated within. In vivo ultrasonic imaging confirmed that the nanodroplets could be treated by ultrasound. The results indicate that the initial 5-minute serum concentration, area under the concentration-time curve, elimination half-life, and clearance of quercetin were significantly enhanced by nanodroplets with or without ultrasound.

Local pelvic irradiation modulates Pharmacokinetics of 5-Fluorouracil in the plasma but not in the Lymphatic System.

BACKGROUND: 5-fluorouracil (5-FU) is employed to enhance radiotherapy (RT) effect. Here, we evaluated the influence of whole-pelvic irradiation on the pharmacokinetics (PK) of 5-FU in plasma and lymphatic system of rats as the experimental model. METHODS: RT with 2 Gy was delivered to the whole pelvis of Sprague-Dawley rats. 5-FU at 100 mg/kg was intravenously infused 24 hours after radiation. The pharmacokinetics of 5-FU in plasma and lymphatic system were calculated. RESULTS: RT at 2 Gy reduced the area under the plasma concentration vs. time curve and mean residence time of 5-FU by 21.5% and 31.5%, respectively compared with those of non-RT controls. By contrast, RT at 2 Gy increased drug clearances of 5-FU by 28.2% when compared with those of non-RT controls. There was no significant difference in T1/2, Cmax and Vss in plasma between both groups. Intriguingly, 5-Fu could be detected in the lymphatic system. In addition, the AUC in 5-FU without and with RT was 3.3-fold and 4.9-fold greater for lymph than for plasma, respectively. Compared with the non-RT group, the RT group showed increase in distribution of 5-FU in the lymphatic system (p = 0.001). CONCLUSIONS: The local whole pelvic RT at 2 Gy could modulate systemic PK of 5-FU in plasma of rats and intravenous 5-FU passing into the lymphatic system was proved. The metabolism of 5-FU might be modulated by RT but the distribution of 5-FU from blood circulation to the lymphatic system might not be changed. The RT-PK phenomena in plasma provide references for adjustment of drug administration. Chemotherapy drugs entering the lymphatic system is worthy of further investigation.

Pelvic irradiation modulates the pharmacokinetics of cisplatin in the plasma and lymphatic system.

BACKGROUND: Cisplatin (CDDP) is employed to enhance radiotherapy's (RT) effect for various cancers. However, the effects of local RT on chemotherapeutics in the plasma and lymphatic system remain unclear. Here, we evaluated the influence of pelvic irradiation on the pharmacokinetics (PK) of CDDP using rats as an experimental model. METHODS AND MATERIALS: RT with 2 Gy and 0.5 Gy were delivered to the whole pelvis of Sprague-Dawley rats. CDDP at 5 mg/kg and 10 mg/kg was intravenously infused 24 hours after radiation for the plasma and lymphatic system, respectively. The pharmacokinetics of CDDP in the plasma and lymphatic system were calculated. RESULTS: Compared with sham-irradiated controls, the whole pelvic irradiation increased the area under the concentration versus time curve (AUC) of CDDP (5 mg/kg) in the plasma by 80% at 0.5 Gy and 87% at 2 Gy, respectively. In contrast, the AUC of CDDP decreased in bile by 13% at both dose levels. Intriguingly, RT could also increase the AUC of CDDP (10 mg/kg) in the lymphatic fluid by 87% at 2 Gy. In addition, the AUC in CDDP without and with RT was 2.8-fold and 3.4-fold greater for the lymph system than for the plasma, respectively. CONCLUSIONS: A local pelvic RT could modulate the systemic PK of CDDP in both the plasma and lymphatic fluids of the rats. The RT-PK phenomena are worth further investigation.

Silymarin in liposomes and ethosomes: pharmacokinetics and tissue distribution in free-moving rats by high-performance liquid chromatography-tandem mass spectrometry.

The aim of this study was to prepare silymarin formulations (silymarin entrapped in liposomes and ethosomes, formulations referred to as LSM and ESM, respectively) to improve oral bioavailability of silymarin and evaluate its tissue distribution by liquid chromatography with tandem mass spectrometry (LC-MS/MS) in free-moving rats. Silibinin is the major active constituent of silymarin, which is the main component to be analyzed. A rapid, sensitive, and repeatable LC-MS/MS method was developed and validated in terms of precision, accuracy, and extraction recovery. Furthermore, the established method was applied to study the pharmacokinetics and tissue distribution of silymarin in rats. The size, ζ potential, and drug release of the formulations were characterized. These results showed that the LSM and ESM encapsulated formulations of silymarin may provide more efficient tissue distribution and increased oral bioavailability, thus improving its therapeutic bioactive properties in the body.

Comparative pharmacokinetics of rhein in normal and loperamide-induced constipated rats and microarray analysis of drug-metabolizing genes.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhein is a pharmacological active component found in Rheum palmatum L. that is the major herb of the San-Huang-Xie-Xin-Tang (SHXXT), a medicinal herbal product used as a remedy for constipation. Here we have investigated the comparative pharmacokinetics of rhein in normal and constipated rats. Microarray analysis was used to explore whether drug-metabolizing genes will be altered after SHXXT treatment. MATERIALS AND METHODS: The comparative pharmacokinetics of rhein in normal and loperamide-induced constipated rats was studied by liquid chromatography with electrospray ionization tandem mass spectrometry (LC-MS/MS). Gene expression profiling in drug-metabolizing genes after SHXXT treatment was investigated by microarray analysis and real-time polymerase chain reaction (RT-PCR). RESULTS: A validated LC-MS/MS method was applied to investigate the comparative pharmacokinetics of rhein in normal and loperamide-induced constipated rats. The pharmacokinetic results demonstrate that the loperamide-induced constipation reduced the absorption of rhein. Cmax significantly reduced by 2.5-fold, the AUC decreased by 27.8%; however, the elimination half-life (t1/2) was prolonged by 1.6-fold. Tmax and mean residence time (MRT) were significantly prolonged by 2.8-fold, and 1.7-fold, respectively. The volume of distribution (Vss) increased by 2.2-fold. The data of microarray analysis on gene expression indicate that five drug-metabolizing genes, including Cyp7a1, Cyp2c6, Ces2e, Atp1b1, and Slc7a2 were significantly altered by the SHXXT (0.5 g/kg) treatment. CONCLUSION: The loperamide-induced constipation reduced the absorption of rhein. Since among the 25,338 genes analyzed, there were five genes significantly altered by SHXXT treatment. Thus, information on minor drug-metabolizing genes altered by SHXXT treatment indicates that SHXXT is relatively safe for clinical application.

Development of a microdialysis system to monitor lamivudine in blood and liver for the pharmacokinetic application in herbal drug interaction and the gene expression in rats.

The aim of study is to develop a novel multiple microdialysis technique coupled to a validated chromatographic system for the measurement of protein-unbound form lamivudine and investigation of its herb-drug interaction in rat blood and liver. Furthermore, gene expression changes of drug metabolizing enzymes in rat were evaluated by microarray analysis after being treated with a traditional Chinese herbal formulation, Long-Dan-Xie-Gan-Tang (LDXGT). The analyte was separated by a reverse-phase C18 column using the mobile phase comprising methanol and 10mM KH2PO4 (15:85, v/v, adjusted to pH 6.0 with NaOH) with the flow rate of 0.8mL/min, and the UV wavelength was set at 270nm. The processes of method validation followed Food and Drug Administration (FDA) guidelines. The pharmacokinetic data demonstrated that the area under the concentration-time curve (AUC) of the lamivudine alone and the LDXGT pretreated group were 532±37.6 and 550±44.2minµg/mL in rat blood after lamivudine administration (10mg/kg, i.v.) and 682±196 and 642±153minµg/mL in rat liver, respectively. The herb-drug pharmacokinetic interaction showed that with either lamivudine alone or in combination with pretreated with LDXGT, the pharmacokinetic parameters were not significantly changed except the apparent volume of distribution (Vd) at a high dose of lamivudine (30mg/kg). In addition, microarray analysis showed that among 70 altered genes (selection criteria: |Fold change|≧2 and p<0.05), only 11 genes were involved in drug metabolism and indicated that a relatively small portion of drug metabolizing genes in liver were altered at the genome level after the therapeutic dose of LDXGT treatment. In conclusion, these studies provide constructive information to interpret the herb-drug interactions between lamivudine and a popular Chinese herbal formulation.

Determination of bioactive components in Chinese herbal formulae and pharmacokinetics of rhein in rats by UPLC-MS/MS.

Rhein (4,5-dihydroxy-9,10-dioxoanthracene-2-carboxylic acid, cassic acid) is a pharmacological active component found in Rheum palmatum L. the major herb of San-Huang-Xie-Xin-Tang (SHXXT), a medicinal herbal product used as a remedy for constipation. Here we have determined multiple bioactive components in SHXXT and investigated the comparative pharmacokinetics of rhein in rats. A sensitive and specific method combining liquid chromatography with electrospray ionization tandem mass spectrometry has been developed and validated to simultaneously quantify six active compounds in the pharmaceutical herbal product SHXXT to further study their pharmacokinetics in rats. Multiple reaction monitoring (MRM) was employed for quantification with switching electrospray ion source polarity between positive and negative modes in a single run. There were no significant matrix effects in the quantitative analysis and the mean recovery for rhein in rat plasma was 91.6%±3.4%. The pharmacokinetic data of rhein demonstrate that the herbal formulae or the single herbal extract provide significantly higher absorption rate than the pure compound. This phenomenon suggests that the other herbal ingredients of SHXXT and rhubarb extract significantly enhance the absorption of rhein in rats. In conclusion, the herbal formulae (SHXXT) are more efficient than the single herb (rhubarb) or the pure compound (rhein) in rhein absorption.

Chemical and Physical Methods to Analyze a Multicomponent Traditional Chinese Herbal Prescription Using LC-MS/MS, Electron Microscope, and Congo Red Staining.

This study develops several chemical and physical methods to evaluate the quality of a traditional Chinese formulation, Jia-Wei-Xiao-Yao-San. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with electrospray ionization was used to measure the herbal biomarkers of saikosaponin A, saikosaponin D, ferulic acid, and paeoniflorin from this herbal formula. A scanning electron microscope (SEM) and light microscopy photographs with Congo red staining were used to identify the cellulose fibers if raw herbal powder had been added to the herbal pharmaceutical product. Moreover, water solubility and crude fiber content examination were used to inspect for potential herbal additives to the herbal pharmaceutical products. The results demonstrate that the contents of the herbal ingredients of saikosaponin A, saikosaponin D, ferulic acid, and paeoniflorin were around 0.351 ± 0.017, 0.136 ± 0.010, 0.140 ± 0.005, and 2.281 ± 0.406 mg/g, respectively, for this herbal pharmaceutical product. The physical examination data demonstrate that the raw herbal powder had rough, irregular, lumpy, filamentous, and elongated shapes, as well as strong Congo red staining. In addition, water solubility and crude fiber content were not consistent in the herbal pharmaceutical products.

Determination and pharmacokinetics of di-(2-ethylhexyl) phthalate in rats by ultra performance liquid chromatography with tandem mass spectrometry.

Di-(2-ethylhexyl) phthalate (DEHP) is used to increase the flexibility of plastics for industrial products. However, the illegal use of the plasticizer DEHP in food and drinks has been reported in Taiwan in 2011. In order to assess the exact extent of the absorption of DEHP via the oral route, the aim of this study is to develop a reliable and validated ultra performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) method to evaluate the oral bioavailability of DEHP in rats. The optimal chromatographic separation of DEHP and butyl benzyl phthalate (BBP; used as internal standard) were achieved on a C18 column. The mobile phase was consisted of 5 mM ammonium acetate-methanol (11:89, v/v) with a flow rate of 0.25 mL/min. The monitoring ion transitions were m/z 391.4 → 149.0 for DEHP and m/z 313.3 → 149.0 for BBP. The mean matrix effects of DEHP at low, medium and high concentrations were 94.5 ± 5.7% and 100.1 ± 2.3% in plasma and feces homogenate samples, respectively. In conclusion, the validated UPLC-MS/MS method is suitable for analyzing the rat plasma sample of DEHP and the oral bioavailability of DEHP was about 7% in rats.

Head and neck irradiation modulates pharmacokinetics of 5-fluorouracil and cisplatin.

BACKGROUND: 5-fluorouracil (5-FU) and cisplatin (CDDP) are used to enhance radiotherapy (RT) effect for head and neck (HN) cancers. However, the effect of local RT on systemic chemotherapeutics remains unclear. Here, we evaluated the influence of HN irradiation on the pharmacokinetics (PK) of 5-FU and CDDP in rats as experimental model. METHODS: The radiation dose distributions of HN cancer patients were determined for the low dose areas, which are generously deposited around the target volume. Two Gy and 0.5 Gy RT were selected. Single-fraction radiation was delivered to the HN of Sprague-Dawley rats. 5-FU at 100 mg/kg or CDDP at 5 mg/kg was intravenously infused 24 hours after radiation. RESULTS: Radiation at 2 Gy reduced the area under the plasma concentration vs. time curve (AUC) of 5-FU and CDDP by 16% and 29% compared to non-irradiated controls, respectively. This was accompanied by incremental total plasma clearance values. Intriguingly, low dose radiation at 0.5 Gy resulted in a similar pharmacokinetic profile, with a 17% and 33% reduction in the AUC of 5-FU and CDDP, respectively. The changes in AUC of bile, which increases with RT, were opposite to AUC of plasma for both drugs. CONCLUSIONS: The local HN RT could modulate systemic PK of 5-FU and CDDP in rats. This unexpected RT-PK phenomena may provide a reference for adjustment of drug administration and is worthy of further investigation. TRIAL REGISTRATION: ClinicalTrials.gov ID NCT01755585 and NCT01609114.

Pharmacokinetics of di-isononyl phthalate in freely moving rats by UPLC-MS/MS.

Di-isononyl phthalate (DINP) is a general-purpose plasticizer for polyvinyl chloride. However, this industrial chemical plasticizer used as a clouding agent has recently contaminated food and beverages that had been inspected by Taiwan Food and Drug Administration. This study develops a sensitive and specific method combining ultra-performance liquid chromatography with electrospray ionization tandem mass spectrometry (UPLC-MS/MS) to investigate the pharmacokinetics of DINP in freely moving rats. Multiple reaction monitoring (MRM) was used to monitor the transition of the protonated molecule m/z of 419 [M+H](+) to the product ion 149 for DINP. The analyte was analyzed by UPLC-MS/MS with C18 column (100×2.1mm, 1.7 µm) which was equilibrated and eluted with an isocratic mixture of acetonitrile-ammonium acetate water solution (90:10, v/v) at a flow rate of 0.3 mL/min. Linear calibration curves were obtained for DINP concentration ranges of 0.05-2.5 µg/mL in plasma and feces. The feces were homogenized mechanically using 50% acetonitrile as the medium. The pharmacokinetic curve demonstrates that the disposition of DINP in rat plasma was fitted well by the two-compartment model after DINP administration (10 mg/kg, i.v.). The elimination half-life of DINP was 364±146 min and 150±58 min for intravenous (10 mg/kg) and oral (100 mg/kg) administration, respectively. The pharmacokinetic data indicate that the oral bioavailability of DINP in freely moving rats was about 1.19%. The total DINP excretion up to 48 h was 13.64±3.99% in feces.