Identification and characterization of endogenous biomarkers for hepatic vectorial transport (OATP1B3-P-gp) function using metabolomics with serum pharmacology.

The organic anion-transporting polypeptide 1B3 and P-glycoprotein (P-gp) provide efficient directional transport (OATP1B3-P-gp) from the blood to the bile that serves as a key determinant of hepatic disposition of the drug. Unfortunately, there is still a lack of effective means to evaluate the disposal ability mediated by transporters. The present study was designed to identify a suitable endogenous biomarker for the assessment of OATP1B3-P-gp function in the liver. We established stably transfected HEK293T-OATP1B3 and HEK293T-P-gp cell lines. Results showed that azelaic acid (AzA) was an endogenous substrate for OATP1B3 and P-gp using serum pharmacology combined with metabolomics. There is a good correlation between the serum concentration of AzA and probe drugs of rOATP1B3 and rP-gp when rats were treated with their inhibitors. Importantly, after 5-fluorouracil-induced rat liver injury, the relative mRNA level and expression of rOATP1B3 and rP-gp were markedly down-regulated in the liver, and the serum concentration of AzA was significantly increased. These observations suggest that AzA is an endogenous substrate of both OATP1B3 and P-gp, and may serve as a potential endogenous biomarker for the assessment of the function of OATP1B3-P-gp for the prediction of changes in the pharmacokinetics of drugs transported by OATP1B3-P-gp in liver disease states.

Fusidic Acid and Its Major Active Metabolite Penetration into Cerebrospinal Fluid for Assessing Treatment of Intracranial Infections.

Fusidic acid (FA) had excellent antimicrobial effects due to its unique mechanism of action. Since 1962, FA has been widely used in the systemic and topical treatment of staphylococcal infections and exhibits a well-characterized potency against methicillin-susceptible Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and methicillin-resistant coagulase-negative Staphylococci. In view of the spectrum of activity, no cross-resistance with other clinically used antibiotics, and potential penetration into brain tissue, FA was used to treat possible gra-positive bacteria in 3 patients with intracranial infections in the present report. FA and its active metabolite (3-keto FA) were measured in plasma and cerebrospinal fluid (CSF) to assess the treatment of FA, and the results indicated that 1,500 mg per day of FA was sufficient to achieve therapeutic concentrations in both plasma and CSF in intracranial infection patients, while the dosage did not experience unexpected regimen-related toxicity.

Kanglaite enhances the efficacy of cisplatin in suppression of hepatocellular carcinoma via inhibiting CKLF1 mediated NF-κB pathway and regulating transporter mediated drug efflux.

ETHNOPHARMACOLOGICAL RELEVANCE: Kanglaite (KLT) is an active extract of the Coix lacryma-jobi seed, which can benefit Qi and nourish Yin, and disperse the accumulation of evils. It is used as a biphasic broad-spectrum anti-cancer drug, and shows synergistic effects with radiotherapy and chemotherapy. However, the mechanism of KLT combined with cisplatin (CDDP) against hepatocellular carcinoma (HCC) has not been elucidated. AIM OF THE STUDY: The aim of present study was to investigate the potential synergistic effects of KLT and CDDP on HepG2 cells, discussing the possible mechanisms from the perspective of CKLF1 and NF-κB mediated inflammatory response and chemoresistance, and the involvement of drug efflux transporters. MATERIALS AND METHODS: CDDP injured HepG2 cells were used to investigate the effects of KLT on chemotherapeutics treated HCC. Effects of KLT pretreatment on CDDP injured HepG2 cells were determined by MTT, wound healing assay, and transwell assay. Expression of chemokine-like factor 1 (CKLF1) and activation of nuclear factor κB (NF-κB) were examined by qPCR, western blot, and immunofluorescence staining. Furthermore, to study the role of CKLF1 in KLT mediated effects on this CDDP injured HCC cell model, HepG2 cells overexpressed with CKLF1 gene were used. Cell viability and NF-κB activation were investigated. Moreover, TNF-α and IL-1ß levels were measured by Elisa analysis and western blot to evaluate the inflammatory response. Additionally, ATP-binding cassette (ABC) drug efflux transporters, MDR1, MRP2, and BCRP were also determined in present study. RESULTS: KLT pretreatment followed by CDDP treatment was found to show synergistic effects, which showed by decreased cell viability, migration and invasion ability of HepG2 cells. Expression of CKLF1 enhanced significantly in CDDP treated HepG2 cells, and KLT decreased this elevation obviously. Furthermore, CDDP activated NF-κΒ and promoted translocation of NF-κB toward the nucleus. KLT inhibited the activation of NF-κΒ, which sensitized cancer cells. Overexpression of CKLF1 reversed the effects of KLT on CDDP injured HepG2 cells, which exhibited by increased cell viability and enhanced activation of NF-κΒ. CDDP induced NF-κΒ activation could also lead to excessive inflammatory response, and KLT can suppress the aggravating inflammation which may be beneficial for tumor progression. Furthermore, we found that ABC drug efflux transporters MDR1, MRP2, and BCRP in CDDP treated HepG2 cells were decreased when pretreated with KLT. CONCLUSIONS: KLT pretreatment may increase the effects of CDDP on HepG2 cells, by exhibiting cooperative effects on suppression of HepG2 cells. The mechanisms may partly by inhibiting CKLF1 mediated NF-κB pathway, which may contribute to inflammation of tumor microenvironment and chemoresistance of CDDP. Inhibition of transporter-mediated drug efflux is also involved in KLT mediated sensitization effects of CDDP.

Determination of Total and Unbound Meropenem, Imipenem/Cilastatin, and Cefoperazone/Sulbactam in Human Plasma: Application for Therapeutic Drug Monitoring in Critically Ill Patients.

BACKGROUND: Critically ill patients show several pathophysiological alterations that can complicate antibiotic dosing. Hence, there is a strong rationale to individualize anti-infective dosing in these patients by using therapeutic drug monitoring (TDM). The current study aimed to develop and validate a liquid chromatography-tandem mass spectrometry method for the simultaneous determination of total and unbound plasma concentrations of 3 commonly used antibiotics (meropenem, imipenem/cilastatin, and cefoperazone/sulbactam) in the treatment of infections in critically ill patients in China, which could be suitable for routine TDM in hospital laboratories. METHODS: The unbound drug was separated from the bound drug by ultrafiltration. Simple protein precipitation was used for sample preparation. Meropenem, imipenem/cilastatin, cefoperazone/sulbactam, and their corresponding internal standards were then resolved using the Waters CORTECS C18 column. All the compounds were detected using electrospray ionization in the positive/negative ion-switching mode. RESULTS: The calibration curves were linear for all compounds, with correlation coefficients (R) above 0.99 for total concentrations in human plasma and unbound concentrations in the plasma ultrafiltrate. For both total and unbound drugs, the relative errors and intra-assay/interassay relative standard deviations were below 15%. The limit of quantification was 0.05 mcg/mL for both total plasma concentrations and plasma ultrafiltrate concentrations of all compounds. CONCLUSIONS: The method was simple, rapid, and reliable and is currently being used to provide a TDM service to enhance the efficacious use of the 3 antibiotics.

Multicomponent determination of traditional Chinese medicine preparation yin-zhi-huang injection by LC-MS/MS for screening of its potential bioactive candidates using HepaRG cells.

Yin-zhi-huang (YZH) injection is an injectable multiherbal prescription derived from the ancient Chinese medicine formula of Yin-chen-hao-tang, which is widely used in the clinic for the treatment of jaundice and chronic liver diseases. To date, the systematic study of the components in this multiherbal prescription still lacks suitable analytical methods that are able to simultaneously detect a broad array of components at low concentrations. In this study, a new liquid chromatography-tandem mass spectrometry method using dynamic multiple reaction monitoring mode was developed to determine multiple peaks in traditional Chinese medicine preparation YZH injection. This simple, selective and sensitive method enabled the quantification of 22 components with standard materials with a lower limit of quantification of 1.46-12.5 ng/mL in cell lysates. This method was successfully applied to celluar uptake and binding investigation of components in YZH injection. The results indicated that this strategy might be a useful approach for rapidly screening of the potential bioactive candidates from YZH injection, and the discovered candidates could be used to investigate the pharmacodynamics in further studies.

Development of a LC-MS/MS method for simultaneous determination of metoprolol and its metabolites, α-hydroxymetoprolol and O-desmethylmetoprolol, in rat plasma: application to the herb-drug interaction study of metoprolol and breviscapine.

A simple, specific and sensitive LC-MS/MS method was developed and validated for the simultaneous determination of metoprolol (MET), α-hydroxymetoprolol (HMT) and O-desmethylmetoprolol (DMT) in rat plasma. The plasma samples were prepared by protein precipitation, then the separation of the analytes was performed on an Agilent HC-C18 column (4.6 × 250 mm, 5 µm) at a flow rate of 1.0 mL/min, and post-column splitting (1:4) was used to give optimal interface flow rates (0.2 mL/min) for MS detection; the total run time was 8.5 min. Mass spectrometric detection was achieved using a triple-quadrupole mass spectrometer equipped with an electrospray source interface in positive ionization mode. The method was fully validated in terms of selectivity, linearity, accuracy, precision, stability, matrix effect and recovery over a concentration range of 3.42-7000 ng/mL for MET, 2.05-4200 ng/mL for HMT and 1.95-4000 ng/mL for DMT. The analytical method was successfully applied to herb-drug interaction study of MET and breviscapine after administration of breviscapine (12.5 mg/kg) and MET (40 mg/kg). The results suggested that breviscapine have negligible effect on pharmacokinetics of MET in rats; the information may be beneficial for the application of breviscapine in combination with MET in clinical therapy.

[Study of pharmacokinetics of digoxin in ovariectomized rats model].

This study aims to investigate the change of plasma concentration of digoxin (DIG) in rats with ovariectomy. Twelve female SD rats were randomly assigned into ovariectomized group and sham group (n = 6). All rats plasma was collected after a single dose of 2 mg x kg(-1) DIG administrated orally, serum DIG concentration was determined by LC-MS/MS. The level of P-gp in the intestinal was analyzed by Western blotting. Pharmacokinetic calculations were performed on each individual using DAS 2.0 practical pharmacokinetic software. Compared with the sham group, C(max) of ovariectomized group decreased significantly (P < 0.01). There was no significant difference of AUC(0-t), and the level of P-gp was elevated in ovariectomized group. It was found that C(max) of DIG was significantly reduced after ovariectomy, and the change was associated with the decreased level of estrogen, which contributes to the increased level of P-gp.

[Simultaneous determination of repaglinide and pravastatin sodium in rat plasma by LC-ms/MS and its application on pharmacokinetic interactions study].

The study aims to establish a method for simultaneous determination of repaglinide and pravastatin sodium in rat plasma by LC-MS/MS and to study its pharmacokinetic interactions. Eighteen male SD rats were divided into repaglinide group, pravastatin sodium group and co-administration group. Blood samples were collected at different times after oral administration. Repaglinide and pravastatin sodium in rat plasma were separated by Agilent HC-C18 with the mobile phase consisting of methanol-0.1% formic acid (80 : 20). Detection and quantification were performed by using ESI-MS. The detector was operated in selected Reaction-monitoring mode at m/z 453.3-->230.1 for repaglinide, m/z 447.2-->327.4 for pravastatin sodium and m/z 285.1-->192.9 for diazepam as the internal standard. The calibration curve obtained was linear (R2>0.99) over the concentration range of 9.77-10,000 ng.mL-1 for repaglinide and 4.88-625 ng.mL-1 for pravastatin sodium. Compared with the single administration group, Cmax and AUC0-6h of repaglinide increased significantly (P<0.05) and tmax of pravastatin sodium prolonged (P<0.05) in co-administration group. The method is found to be simple, sensitive and accurate for determining the concentration of repaglinide and pravastatin sodium in rat plasma. There exists pharmacokinetic interactions in the co-administration of repaglinide and pravastatin sodium.

The impact of drug transporters on adverse drug reaction.

In this review, we have highlighted the adverse drug reaction mediated by transporters from two aspects: (1) competitive interactions between drug and drug/metabolite/endogenous substance mediated by transporters; (2) the expression/function change of transporter due to physiologic factors, disease, and drugs induction. It indicated that transporters exhibited a broad substrate specificity with a degree of overlap, which could change the pharmacokinetics of drugs and cause toxicity due to competition interactions among substrates. In addition, the expression and function of transporters were regulated by physiological conditions, pathological conditions, and drugs induction, which could cause adverse drug reaction and interindividual differences. Furthermore, one substrate was always medicated by several transporters and often subjected to metabolism by CYP enzymes, so we should be more aware of the increased plasma concentration of drugs caused by drug transporters as well as drug metabolizing enzymes synergistically, especially for drugs with narrow therapeutic window. In addition, the weightiness for one transporter to induce drugs plasma/tissue concentration change could be different in different condition. On the whole, transporters were corresponding with systemic/organs exposure of drug/metabolites/endogenous compounds. So understanding the expression and function in drug transporters will result in better strategies for optimal dosage regimen and reduce the risk for drug adverse reaction as well as adverse drug-drug interactions.

Effects of pyridoxine on the intestinal absorption and pharmacokinetics of isoniazid in rats.

Pyridoxine is always simultaneously administered orally with isoniazid for tuberculosis patients in the clinic to prevent or treat the nervous system side effects induced by isoniazid. So the aim of this research was to investigate the effects of pyridoxine on the intestinal absorption and pharmacokinetics of isoniazid. The intestinal absorption of isoniazid with or without pyridoxine was investigated by the rat single-pass intestinal perfusion model in situ, and a high-performance liquid chromatographic method was applied to study the pharmacokinetics of isoniazid with or without pyridoxine. The results suggested that the intestinal apparent permeability (P app) and intestinal absorption rate constant (K a) for isoniazid (30 µg/ml) were decreased by 43.7 and 36.4 %, respectively, by co-perfused pyridoxine (40 µg/ml). In vivo, the effect of pyridoxine on isoniazid pharmacokinetic correlated with the doses of pyridoxine. The blood concentrations of isoniazid at the absorption phase were affected by co-administered pyridoxine, but the AUC and C max of isoniazid were not greatly affected by pyridoxine as expected from the inhibition by pyridoxine of the intestinal absorption of isoniazid, which could be caused by its rapid absorption phase. Therefore, although the intestinal absorption of isoniazid could be significantly inhibited by pyridoxine, the pharmacokinetics of isoniazid oral administration was not greatly affected by the decreased intestinal absorption of isoniazid due to its rapid absorption.

Pharmacokinetic variation of ofloxacin based on gender-related difference in the expression of multidrug resistance-associated protein (Abcc2/Mrp2) in rat kidney.

The present study aimed to investigate the pharmacokinetic variation of ofloxacin based on gender-related difference in the expression of multidrug resistance-associated protein (Abcc2/Mrp2) in rat kidney. The concentrations of ofloxacin in rat plasma and urine were determined after tail vein administration (30 mg x kg(-1)) by high-performance liquid chromatography (HPLC) method. Expression of Mrp2 in kidney of male and female rats was qualitatively and quantitatively detected by immunohistochemistry and flow cytometry, separately. The results showed that AUC value of ofloxacin was lower in male rats than that in female rats and the total amount of ofloxacin excreted in the urine was higher in male rats than that in female rats. And the expression of Mrp2 in male rat kidney was higher than that in female rats. All results suggested that gender-related differences in pharmacokinetics of ofloxacin may be attributed to the differences in the expression of Mrp2 in kidney of male and female rats.

Ethosomes, binary ethosomes and transfersomes of terbinafine hydrochloride: a comparative study.

The aim of this study was to compare the skin permeation of ethosomes, binary ethosomes and transfersomes of Terbinafine Hydrochloride (TH) under non-occlusive conditions. These lipid vesicles were prepared and characterized for shape, size, zeta-potential and entrapment efficiency. Franz diffusion cells and confocal laser scanning microscopy (CLSM) were used for the percutaneous absorption studies. The quantity of drug in the skin from ethosomes, binary ethosomes (the weight ratio of ethanol to propylene glycol 7:3, ethanol-PG = 7:3, w/w), and transfersomes was 1.26, 1.51 (p <0.05), 1.56 (p <0.01) times higher than that of TH from traditional liposomes (control). The skin deposition of the applied dose (DD%) of TH from ethosomes, binary ethosomes, and transfersomes was 3.34 (p < 0.05), 9.88 (p < 0.01), 2.52 times higher than that of TH from control. The results of CLSM experiments showed that penetration depth and fluorescence intensity of Rhodamine B from binary ethosomes was much greater than that from ethosomes and transfersomes. These results indicated the binary ethosomes (ethanol-PG = 7:3, w/w) most effectively permitted drug penetration through skin; transfersomes made drug easiest to accumulate in the skin. Ethosomes improved drug delivery with greater improvement in skin permeation than improvement in skin deposition.

Sympodiomycopsis kandeliae sp. nov., a basidiomycetous anamorphic fungus from mangroves, and reclassification of Sympodiomycopsis lanaiensis as Jaminaea lanaiensis comb. nov.

Three ustilaginomycetous anamorphic strains were isolated from flowers of Kandelia candel in mangrove forests of Taiwan. Phylogenetic analyses based on the combined sequences of internal transcribed spacer 1 (ITS1)-5.8S-ITS2 and the D1/D2 domain of the large-subunit (LSU) rDNA indicated that the closest recognized species was Sympodiomycopsis paphiopedili. The results of a DNA-DNA hybridization experiment and the physiological characteristics showed that the three strains represent a novel species within the genus Sympodiomycopsis. The name Sympodiomycopsis kandeliae sp. nov. is proposed, with FIRDI 007(T) (=BCRC 23165(T) =CBS 11676(T)) as the type strain. In addition, based on phenotypic characteristics and the phylogenetic analyses of the combined sequences of the ITS region and D1/D2 domain of the LSU rDNA, Sympodiomycopsis lanaiensis was clustered with the genus Jaminaea. A new combination, Jaminaea lanaiensis comb. nov. (type strain LM418(T) =DSM 18755(T) =ATCC MYA-4092(T) =NRRL Y-48466(T) =CBS 10858(T) =BCRC 23177(T)), is proposed.

Synergistic penetration of ethosomes and lipophilic prodrug on the transdermal delivery of acyclovir.

The aim of this study was to investigate the lipophilic prodrug as a means of promoting acyclovir (ACV) that exhibited biphasic insolubility into the ethosomes for optimum skin delivery. Acyclovir Palmitate (ACV-C(16)) was synthesized as the lipophilic prodrug of ACV. The ethosomal system and the liposomal system bearing ACV or ACV-C(16) were prepared, respectively. The systems were characterized for shape, zeta potential value, particle size, and entrapment efficiency. Franz diffusion cells and confocal laser scanning microscopy were used for the percutaneous absorption studies. The results showed that the entrapment efficiency of ACV-C(16) ethosomes (87.75%) were much higher than that of ACV ethosomes (39.13%). The quantity of drug in the skin from ACV-C(16) ethosomes at the end of the 24 h transdermal experiment (622.89 microg/cm(2)) was 5.30 and 3.43 times higher than that from ACV-C(16) hydroalcoholic solution and ACV ethosomes, respectively. This study indicated that the binary combination of the lipophilic prodrug ACV-C(16) and the ethosomes synergistically enhanced ACV absorption into the skin.

Pseudozyma pruni sp. nov., a novel ustilaginomycetous anamorphic fungus from flowers in Taiwan.

Two ustilaginomycetous anamorphic strains were isolated from flowers in Taiwan. Phylogenetic analysis based on the combined rRNA gene sequence of internal transcribed spacer 1 (ITS1)-5.8S-ITS2 and large-subunit D1/D2 domains indicated that the closest recognized species was Pseudozyma fusiformata. The results of DNA-DNA hybridization and physiological characteristics showed that the two strains represent a novel species within the genus Pseudozyma. The name Pseudozyma pruni sp. nov. is proposed, with FIRDI 005(T) (=BCRC 34227(T) =CBS 10937(T)) as the type strain.

Determination of tanshinone I in rat plasma by high-performance liquid chromatography and its application to pharmacokinetic studies.

This paper describes a rapid and sensitive high-performance liquid chromatographic (HPLC) method for the determination of the concentration of tanshinone I in rat plasma, and applies the method to pharmacokinetic study. The plasma is deproteinized with acetonitrile containing an internal standard (estradiolbenzoate). The HPLC assay is carried out using a Cosmosil C18 column. The mobile phase is acetonitrile, 0.05 mol/L(-1) ammonium acetate buffer with 1% acetic acid (66:34, v/v). The flow rate is 1.0 mL/min. The detection wavelength is set at 263 nm. The assay accuracy is better than 92%, and the precision of tanshinone I at low to high concentrations is better than 9% and 11% for intra-day and inter-day assays, respectively. The recovery of the method exceeds 88.3% for tanshinone I. The assay shows good linearity (r = 0.9998) over a relatively wide concentration range from 0.05 to 10.0 microg/mL. The method is used to determine the concentration-time profiles of tanshinone I in plasma following an intravenous injection of tanshinone I solution, and the pharmacokinetic parameters of tanshinone I are calculated for the first time by the Drug and Statistics 1.0 program. This assay is successfully applied to the determination of tanshinone I in rat plasma, and the developed method is applied to pharmacokinetic studies for the first time.

[Effects of Yuquan pills on pharmacokinetics of metformin hydrochloride in diabetic rats].

OBJECTIVE: To study effects of Yuquan pills on the pharmacokinetics process of metformin hydrochloride in diabetic rats. METHOD: After administration Yuquan pills 7 day to the diabetic rats, the metformin hydrochloride was orally administrated, then the blood samples were collected at different time. The concentrations of metformin hydrochloride in plasma were determined by HPLC method and the pharmacokinetic parameters were calculated. RESULT: The pharmacokinetic parameter Cmax of the controlling group and the testing group were respectively, 18.95, 21.76 mg x L(-1); t1/2 were 1,069.8, 1,767.4 min, respectively; CL/F were 0.013, 0.008 L x min(-1) x kg(-1); AUC were 10,042.1, 10,712.2 mg z L(-1) x min(-1) respectively. CONCLUSION: The pharmacokinetics process of metformin hydrochloride in diabetic rats fits one-compartment model. Yuquan pills has a significant effect on the pharmacokinetics of metformin hydrochloride in diabetic rats.

Polyphasic approach to the taxonomy of the Rhizopus stolonifer group.

Phylogenetic analysis of Rhizopus strains based on the D1/D2 region of LSU rDNA sequences yielded a phylogram with four well-supported clades. The R. microsporus clade concurs with classification obtained by traditional methods. The R. oryzae group was found to include species of the genus Amylomyces. The traditional R. stolonifer group was divided into two well-supported clades in the phylogram, with one clade comprising R. stolonifer var. stolonifer, R. sexualis var. sexualis, and R. sexualis var. americanus and the other clade comprising taxa with recurved sporangiophores; R. reflexus, R. stolonifer var. lyococcus, and R. circinans, identifying recurved sporangiophores as an important taxonomic character. The molecular data supported the recognition of this clade at the species level: R. lyococcus (basionym: Sporotrichum lyococcum).