Evaluation of neurotoxicity of anticancer drugs using nematode Caenorhabditis elegans as a model organism.

It is well established that platinum-based drugs, including oxaliplatin (L-OHP) and cisplatin (CDDP), as well as microtubule inhibitors paclitaxel (PTX) and vincristine (VCR), are associated with chemotherapy-induced peripheral neuropathy (CIPN). In this study, we examined and compared the characteristics of neuropathies induced by L-OHP, CDDP, PTX, and VCR to evaluate whether Caenorhabditis elegans (C. elegans) could serve as a model organism for human CIPN. Worms were cultured on nematode growth medium plates, and L1 larvae synchronized by gel filtration were employed. We then performed bioassays and examined motility. In the motility test, exposure was performed for 2, 24, and 48 hr, and time-dependent effects were measured for each exposure time and 24 hr after terminating exposure. Herein, we observed that L-OHP and CDDP exerted concentration-dependent effects above a certain concentration, and PTX and VCR exerted concentration-dependent negative effects in the bioassay. Motility recovered in L-OHP-, PTX-, and VCR-treated worms on terminating exposure. However, CDDP exposure tended to reduce motility even 24 hr after terminating exposure. L-OHP exposure could decrease motility 2 hr after exposure, with a trend toward recovery 24 hr after terminating drug exposure. The findings of the present study revealed that C. elegans could exhibit neuropathy characteristics suggested to be similar to those observed in humans, indicating that this organism could be a suitable model to explore human CIPN.

Development of a useful single-reference HPLC method for therapeutic drug monitoring of phenytoin and carbamazepine in human plasma.

A single reference high-performance liquid chromatographic (SR-HPLC) method was developed and validated for the therapeutic drug monitoring (TDM) of phenytoin (PHT) and carbamazepine (CBZ) in plasma from patients. The analytical parameters evaluated were linearity, limit of quantification (LOQ), selectivity, accuracy, and stability according to the US Food and Drug Administration (FDA) guideline. The developed method shows good linearity (r2 > 0.999; LOQ-50 µg/mL), and LOQ values were 1.56 µg/mL for PHT and 0.40 µg/mL for CBZ at 254 nm. For the development of SR-HPLC method, we evaluated to improve the detection wavelength, stirred retention time, and stability for SR, and selected 5-(p-methylphenyl)-5-phenylhydantoin for PHT (relative molar sensitivity, RMS = 0.848) and 10-methoxyiminostilbene for CBZ (RMS = 0.263). The established differential definite quantities of PHT and CBZ in plasma samples were similar using the RMS and absolute calibration methods based on RSD < 5.10%. A preliminary application was performed using chemiluminescent immunoassay and SR-HPLC method, in which the detectable values of the correlation coefficient and the slope of the intercept were PHT: 0.964 and 0.992647, and CBZ: 0.969 and 1.072089, respectively. Based on these results, we propose that the SR-HPLC method with RMS would be offered to the useful and accurate TDM of various medicines in plasma/serum samples.

In vivo and in silico analyses of estrogenic potential of equine estrogens in medaka (Oryzias latipes).

Equine estrogens (EEs) are widely used in hormone replacement therapy pharmaceuticals for postmenopausal women. Previous studies have shown that EEs occur in the aquatic environment; however, the potential estrogenicity and risk of EEs in aquatic organisms, including fish, have yet to be studied in detail. Therefore, we evaluated the estrogenic potential of major EEs, namely equilin (Eq), 17α-dihydroequilin (17α-Eq), 17ß-dihydroequilin (17ß-Eq), equilenin (Eqn), 17α-dihydroequilenin (17α-Eqn), and 17ß-dihydroequilenin (17ß-Eqn), on medaka (Oryzias latipes) using in vivo and in silico assays. Quantitative real-time RT-PCR analyses revealed that expression levels of choriogenin L (ChgL) and choriogenin H (ChgH) in medaka embryos responded to various types and concentrations of EEs in a concentration-dependent manner, whereas transcription levels of vitellogenin 1 were not significantly affected by any of the EEs in the concentration range tested. The order of the in vivo estrogenic potencies of EEs was as follows: 17ß-Eq > Eq > 17ß-Eqn > Eqn > 17α-Eqn > 17α-Eq. Additionally, the 50% effective concentrations (EC50) of 17ß-Eq was lower than that of 17ß-estradiol. We also investigated the interaction potential of EEs with medaka estrogen receptor (ER) subtypes in silico using a three-dimensional model of the ligand-binding domain (LBD) for each ER and docking simulations. All six EEs were found to interact with the LBDs of ERα, ERß1, and ERß2. The order of the in silico interaction potentials of EEs with each ER LBD was as follows: 17ß-Eq > 17α-Eq > Eq > 17ß-Eqn > 17α-Eqn > Eqn. Furthermore, we identified the key amino acids that interact with EEs in each ER LBD; our findings suggest that amino acids and/or their hydrogen bonding may be responsible for the ligand-specific interactions with each ER. This study is the first to comprehensively analyze the estrogenic potential of EEs in medaka both in vivo and in silico.

Potential mechanisms underlying embryonic developmental toxicity caused by benzo[a]pyrene in Japanese medaka (Oryzias latipes).

Polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (BaP), are widely distributed in air, water, and sediments; however, limited data are available regarding their potential adverse effects on the early life stages of fish. In this study, we evaluated the embryonic teratogenicity and developmental toxicity of BaP in Japanese medaka (Oryzias latipes) using a nanosecond pulsed electric field (nsPEF) technique and predicted their molecular mechanisms via transcriptome analysis. The gas chromatography/mass spectrometry analyses revealed that the BaP was efficiently incorporated into the embryos by nsPEF treatment. The embryos incorporating BaP presented typical teratogenic and developmental effects, such as cardiovascular abnormalities, developmental abnormalities, and curvature of backbone. DNA microarray analysis revealed several unique upregulated genes, such as those involved in cardiovascular diseases, various cellular processes, and neural development. Furthermore, the gene set enrichment and network analyses found several genes and hub proteins involved in the developmental effects of BaP on the embryos. These findings suggest a potential mechanism of teratogenicity and developmental toxicity caused by exposure to BaP. The nsPEF and transcriptome analyses in combination can be effective for evaluating the potential effects of chemical substances on medaka embryos.

Occurrence and seasonal variation of equine estrogens, equilin and equilenin, in the river water of Japan: Implication with endocrine-disrupting potentials to Japanese medaka (Oryzias latipes).

In this study, we determined the concentration of equine estrogens, such as equilin (Eq) and equilenin (Eqn), in the river water collected from nine research stations in Hokkaido, Japan. The LC-MS/MS analysis revealed that Eq concentrations were 2.7 ±â€¯6.7, 0.22 ±â€¯0.12, and 1.2 ±â€¯0.64 ng/L in Sep 2015, Feb 2016, and Jul 2016, respectively. Eqn had concentration levels similar to those of Eq. Comparison of the concentrations at nine research stations showed that seasonal variation was observed in the detected Eq and Eqn concentration levels. This study was the first to show the occurrences and seasonal variation of Eq and Eqn in the river water of Japan. We further investigated the reproductive and transgenerational effects of Eq in Japanese medaka (Oryzias latipes) exposed to 10, 100, and 1000 ng/L for 21 days and assessed the transcriptional profiles of the estrogen-responsive genes in the livers of both sexes. The reproduction assay demonstrated that 1000 ng/L of Eq adversely affected the reproduction (i.e. fecundity) in the F0 generation and that the hatching of F1 generation fertilized eggs was reduced in the 100 and 1000 ng/L treatment groups. Our qRT-PCR assay revealed that the mRNA expression levels of hepatic vitellogenin 1 and 2, choriogenin L and H, and estrogen receptor α were significantly up-regulated in males exposed to 100 and/or 1000 ng/L of Eq. In contrast, the transcriptional levels of several genes, such as pregnane X receptor and cytochrome P450 3A, were down-regulated in the livers of males after the 21-d exposure. These results suggest that Eq has endocrine-disrupting potential such as reproductive and transgenerational effects by the modulation of hepatic estrogen-responsive genes expression on medaka.

Gene expression analyses of vitellogenin, choriogenin and estrogen receptor subtypes in the livers of male medaka (Oryzias latipes) exposed to equine estrogens.

In the present study, we investigated transcriptional profiles of estrogen-responsive genes, such as vitellogenins (Vtg1 and Vtg2), choriogenins (ChgL and ChgH) and estrogen receptor subtypes (ERα, ERß1, and ERß2), in the liver of male medaka fish (Oryzias latipes) that were exposed to six equine estrogens (1-300 ng l(-1) ) for 3 days. Our quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses revealed that the expression levels of hepatic Vtg, Chg and ERα genes in male medaka responded to various types and concentrations of equine estrogens. The estrogenic potentials of the tested chemicals were in the order of equilin > 17ß-estradiol > equilenin > 17ß-dihydroequilin > 17ß-dihydroequilenin > 17α-dihydroequilin > 17α-dihydroequilenin, showing the higher estrogenic potential of equilin than that of 17ß-estradiol. Our results also showed that the estrogenicities of 17ß-dihydroequilin and 17ß-dihydroequilenin were more potent than that of 17α-dihydroequilin and 17α-dihydroequilenin. Furthermore, in gene expression analyses of hepatic ER subtypes, observations were made to note that 17ß-estradiol and equilin induced ERα transcription in male medaka, and the ERα transcription level had significantly positive correlations with the expression of Vtg and Chg genes. In contrast, in the same 17ß-estradiol and equilin treatment groups, it was shown that the transcription levels of hepatic ERß1 and/or ERß2 had significantly negative correlations with the expression of Vtg and Chg genes. These results suggested some potential involvement of the ER subtypes in the regulation of Vtg and Chg gene expressions in the liver. This is the first report describing the comprehensive analyses of in vivo estrogenicity of the equine estrogens in male medaka. Copyright © 2016 John Wiley & Sons, Ltd.

Safe and effective delivery of small interfering RNA with polymer- and liposomes-based complexes.

We developed binary and ternary complexes based on polymers and liposomes for safe and effective delivery of small interfering RNA (siRNA). Anti-luciferase siRNA was used as a model of nucleic acid medicine. The binary complexes of siRNA were prepared with cationic polymers and cationic liposomes such as polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, poly-L-arginine (PLA), trimethyl[2,3-(dioleoxy)-propyl]ammonium chloride (DOTMA), and cholesteryl 3ß-N-(dimetylaminnoethyl)carbamate hydrochloride (DC-Chol). The ternary complexes were constructed by the addition of γ-polyglutamic acid (γ-PGA) to the binary complexes. The complexes were approximately 54-153 nm in particle size. The binary complexes showed a cationic surface charge although an anionic surface charge was observed in the ternary complexes. The polymer-based complexes did not show a silencing effect in the mouse colon carcinoma cell line expressing luciferase regularly (Colon26/Luc cells). The binary complexes based on liposomes and their ternary complexes coated by γ-PGA showed a significant silencing effect. The binary complexes showed significant cytotoxicity although the ternary complexes coated by γ-PGA did not show significant cytotoxicity. The ternary complexes coated by γ-PGA suppressed luciferase activity in the tumor after their direct injection into the tumors of mice bearing Colon26/Luc cells. Thus, we have newly identified safe and efficient ternary complexes of siRNA for clinical use.

[Susceptibility of spore-forming butyric acid bacteria to antimicrobial agents].

Antimicrobial agents occasionally cause certain adverse effects, such as diarrhea and loose stool, by altering the composition of the intestinal flora. Antibiotic-resistant lactic acid bacteria are used to prevent these adverse effects. Although these bacteria are not resistant to several recently introduced antimicrobial agents, bacterial preparations are still sometimes prescribed concomitantly with these antimicrobial agents. In this study, we investigated whether the administration of the spore-forming butyric acid bacteria Clostridium butyricum improves the adverse clinical effects by preventing diarrhea. Inhibition of C. butyricum growth was observed with 17 of the 20 antimicrobial agents used. However, dilution of 11 of these 17 agents resulted in the regrowth of C. butyricum. These results suggest that C. butyricum may survive exposure to several antibiotic agents by forming spores. Further, a decrease in the antimicrobial agent concentration in the gastrointestinal tract permits the vegetative growth of C. butyricum, which functions as a probiotic.

Acute toxicity of pharmaceutical and personal care products on freshwater crustacean (Thamnocephalus platyurus) and fish (Oryzias latipes).

Pharmaceutical and personal care products (PPCPs) enter aquatic environments via sewage treatment facilities and their potentially toxic effects on biota, particularly aquatic organisms, are of considerable concern. In this study, we investigated the acute toxicity of selected PPCPs on a freshwater crustacean (Thamnocephalus platyurus) and a fish species (Oryzias latipes). The 24-hr median lethal concentration (LC(50)) values of ibuprofen, mefenamic acid, indometacin, carbamazepine, propranolol, ifenprodil, clarithromycin and triclosan for T. platyurus were estimated to be 19.59, 3.95, 16.14, > 100, 10.31, 4.43, 94.23 and 0.47 mg/l respectively. Conversely, the 96-hr LC(50) values for these PPCPs were estimated at > 100, 8.04, 81.92, 45.87, 11.40, 8.71, > 100 and 0.60 mg/l for O. latipes, respectively. The toxic sensitivity of T. platyurus to these PPCPs, except for carbamazepine, was therefore higher than for O. latipes. No acute toxicity effects were associated with PPCPs, such as atenolol, disopyramide, famotidine, fluconazole, erythromycin and levofloxacin, in the two aquatic organisms at the concentrations tested in this study (> 100 mg/l). These findings may help us to understand the potential biological effects and risks associated with PPCP exposure in aquatic organisms. Further long-term studies are required to fully assess the growth and reproduction of these compounds on aquatic biota.

Influence of murine hepatitis induced by D-(+)-galactosamine hydrochloride and lipopolysaccharide on gene expression of polyethylenimine/plasmid DNA polyplex.

We investigated the influence of murine hepatitis induced by D-(+)-galactosamine and lipopolysaccharide (D-GalN/LPS) on polyethylenimine (PEI)-mediated plasmid DNA (pDNA) delivery. pDNA encoding firefly luciferase was used as the model reporter gene. PEI was used as the non-viral vector because of its high gene expression and low toxicity. The activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mice indicated the highest peaks at 12 h after D-GalN/LPS injection, then the activities of serum ALT and AST rapidly decreased. We determined luciferase activity in various organs of D-GalN/LPS-treated mice and control mice after an intravenous administration of PEI/pDNA complexes. High transgene expression was observed in the liver, spleen, and lung of both mice. Compared to the control mice, a significant increase of transgene expression was observed in the liver of D-GalN/LPS-treated mice after D-GalN/LPS injection. The transgene expression in the spleen and lung decreased at 6 and 12 h after D-GalN/LPS injection. In conclusion, we found that murine hepatitis induced by D-GalN/LPS injection can influence PEI-mediated pDNA delivery and its influence was different from that induced by CCl(4) injection which was reported previously. These results demonstrated the necessity of considering the timing and dose of gene therapy according to the disease and its stage.

Hybrid vector including polyethylenimine and cationic lipid, DOTMA, for gene delivery.

We developed polyethylenimine (PEI) lipopolyplexes with N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethlylammonium chloride (DOTMA) and pDNA to investigate their usefulness for in vitro and in vivo gene delivery. The charge ratio of the complex to pDNA was calculated with molar values of nitrogen of PEI, and nitrogen of DOTMA to phosphate of pDNA. The polyplexes were prepared at charge ratio 2 (polyplex 2P) and 8 (polyplex 8P). DOTMA solution was added to polyplex 2P to prepare lipopolyplexes at charge ratio 1 (lipopolyplex 2P-1D), 2 (lipopolyplex 2P-2D), and 4 (lipopolyplex 2P-4D). The particle size of the complex was significantly reduced by the addition of DOTMA and settled to 74-114nm, indicating pDNA compaction. The addition of DOTMA to polyplex 2P decreased pDNA dissociation from the complex and degradation in serum. The addition of DOTMA to polyplex 2P remarkably increased gene expression in HepG2 cells in the absence or presence of FBS. These lipopolyplexes showed little cytotoxicity in the presence of FBS. After intravenous injection of the lipopolyplexes into mice, high-gene expression in the liver, spleen, and lung was observed with lipopolyplex 2P-2D, lipopolyplex 2P-4D, and polyplex 8P. In particular, lipopolyplex 2P-4D showed the highest gene expression.

Electrophysiological characterization of tight junctional pathway of rabbit cornea treated with ophthalmic ingredients.

The purpose of this study was to investigate the continuous and real-time influence of ophthalmic ingredients on rabbit cornea by monitoring electrophysiological characteristics. The tight junctional permeabilities of FITC-dextran 4,400 (FD-4) was also determined through the cornea in the presence of ophthalmic ingredients. Intact cornea showed approximately one k-ohmxcm(2) of transepithelial electrical resistance (TEER) and extremely low permeability of FD-4. The ophthalmic ingredients used in the present study were benzalkonium chloride (BK; 0.002%, 0.01%, 0.05%), ethylenediaminetetraacetic acid (EDTA; 0.5%), capric acid (C10; 0.25%), saponin (SP; 0.1%), taurocholic acid (TA; 1.0%) and sodium dodecyl sulfate (SDS; 0.01%). They were previously reported to be effective on corneal penetrations of various drugs at those concentrations without severe toxicity. These ingredients decreased TEER and increased corneal permeability of FD-4. BK reduced TEER in a concentration-dependent manner. There was a significant correlation (gamma=0.860) between the permeability coefficient (Papp) of FD-4 and conductance (Gm), which is the reciprocal value of TEER. It was also indicated that Papp and Gm have a relationship with the corneal cytotoxicity of the ingredients. In conclusion, an electrophysiological method using isolated cornea was very useful to determine the continuous and real-time influence of ophthalmic ingredients on the cornea. In this method, electrophysiological conductance must be able to predict corneal tight junction permeability and the corneal cytotoxicity of ingredients.

[Investigation for proper use of probiotics in Nagasaki University Hospital of Medicine and Dentistry].

Antimicrobial agents sometimes cause the adverse effects of diarrhea and loose stool. Antibiotic-resistant lactic acid bacteria are used to prevent these adverse effects. The bacteria are not resistant to several antimicrobial agents, although the bacterium preparations are sometimes prescribed the antimicrobial agents concomitantly. Therefore this paper reports that the minimal inhibitory concentration of three new antimicrobial agents against antibiotic-resistant lactic acid bacteria were determined using a microdilution method with cation-adjusted Mueller-Hinton broth. Furthermore, we investigated antimicrobial agents that are prescribed concomitantly with antibiotic-resistant lactic acid bacterium preparations or a clostridium butyricum preparation. The bacteria were susceptible to the three new antimicrobial agents. Approximately 50% of the bacterium preparations were prescribed alone, and 30% were prescribed concomitantly with antimicrobial agents that show antimicrobial activity against the bacteria. Consequently, we suggest that pharmacists need to confirm prescriptions and to provide more drug information on antibiotic-resistant lactic acid bacterium preparations.

Transport of timolol and tilisolol in rabbit corneal epithelium.

The purpose of this study is to characterize the transport of tilisolol and timolol through the corneal epithelium, which is believed to be a tight barrier of ocular drug absorption. Cultured normal rabbit corneal epithelial cells (RCEC) were used to investigate drug transport. Primary RCEC were seeded on a filter membrane of Transwell-COL insert coated with fibronectin and grown in Dulbecco's modified Eagle's medium/nutrient mixture F-12 with various supplements. Beta-blocker permeability through the RCEC layer was measured to assess the transcellular permeability coefficient (P(transcell)) in the absence or presence of inhibitors. The transcellular permeability of tilisolol was dependent on drug concentration although timolol showed no concentration dependency. Tilisolol flux from the apical to the basal side was larger than in the opposite direction although timolol showed no direction dependency. The transcellular permeability of tilisolol from the apical to the basal side was inhibited by sodium azide, tetraethylammonium, quinidine, taurocholic acid, guanidine and carnitine. Tilisolol had an active mechanism in uptake to the corneal epithelium, probably by the organic cation transporter family, although timolol predominantly permeated via passive diffusion. This RCEC system was useful to characterize the ocular permeation mechanism of drugs.

Partial hepatectomy enhances polyethylenimine-mediated plasmid DNA delivery.

Polyethylenimine (PEI) is widely used for non-viral transfection in vitro and in vivo. Hepatectomy is an interesting and considerable factor modifying PEI-mediated gene expression. We investigated the gene expression in mice over time following partial hepatectomy after an intravenous injection of PEI/plasmid DNA (pDNA) complex. pDNA encoding firefly luciferase was used as the model reporter gene. The hepatectomized liver was rapidly regenerated until 72 h. After 168 h, the liver weight of hepatectomized mice was similar to that of control mice. Slight liver function impairment was only observed at 1-24 h after hepatectomy in alanine aminotransferase and aspartate aminotransferase levels. Luciferase activity in the liver of partial hepatectomized mice at 48 h after partial hepatectomy increased by 70 times compared with that of control mice; however, luciferase activities did not significantly differ between hepatectomized mice and control mice in the spleen, lung, kidney, and heart. Among the lobes, luciferase activity by gram of tissue was not significantly different, indicating that gene expression enhancement by partial hepatectomy occurred equally throughout the liver. In conclusion, our findings demonstrate that liver resection is an influencing factor on PEI-mediated gene delivery in mice. These results indicate the necessity of considering cell division in PEI-mediated pDNA delivery.

PEGylated liposomes loading palmitoyl prednisolone for prolonged blood concentration of prednisolone.

We investigated the pharmacokinetic behavior of palmitoyl prednisolone (Pal-PLS) and its liposomes with L-alpha-distearoylphosphatidylcholine (DSPC) and cholesterol (Chol) with or without L-alpha-distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG 2000) after their intravenous administration in rats. Pal-PLS rapidly disappeared from the systemic circulation and prednisolone (PLS) was regenerated after the administration of DSPC/Chol liposomes. PEGylated liposomes including DSPE-PEG 2000, however, successfully maintained high blood concentrations of Pal-PLS and PLS. The blood profiles of drugs after the administration of liposomal Pal-PLS were analyzed according to a two-compartment model. The larger content of DSPE-PEG 2000 in DSPC/Chol liposomes showed a lower first order elimination rate constant from the central compartment (K(el)) and clearance (CL). The area under the concentration-time curve (AUC) of Pal-PLS and PLS in PEGylated liposomes was larger than DSPC/Chol liposomes. The mean resident time (MRT) of Pal-PLS and PLS was also prolonged by PEGylated liposomes. Although DSPC/Chol liposomes showed a high distribution of Pal-PLS in the liver and spleen, PEGylated liposomes significantly decreased the liver distribution of Pal-PLS. The biliary and urinary excretions of drugs for 240 min after drug administration were less than 1% of the administrated dose in any formulations. In conclusion, PEGylated liposomes, including Pal-PLS, are useful for maintain the PLS concentration in the blood after intravenous administration.

Influence of disease stage on polyethylenimine-mediated plasmid DNA delivery in murine hepatitis.

In order to determine the influence of hepatic disease-stage on polyethylenimine-mediated gene delivery, we investigated branched and linear polyethylenimine (B-PEI, L-PEI)-mediated plasmid DNA delivery with time in murine hepatitis induced by a subcutaneous injection of tetrachloro carbon (CCl(4)). Plasmid DNA (pDNA) encoding firefly luciferase was used as the model reporter gene. We determined luciferase activity in various organs of CCl(4)-treated mice and control mice after an intravenous administration of B-PEI and L-PEI/pDNA complexes. Both B-PEI and L-PEI/pDNA complexes showed significantly lower gene expression in the liver, spleen, and lung at the stage of severe hepatitis (18 h after CCl(4) injection), whereas the complexes induced gene expression in the liver at the liver regeneration stage (48 h after CCl(4) injection). Significant differences in gene expressions between CCl(4)-treated mice and control mice vanished in most organs at the hepatitis subsidence stage (168 h after CCl(4) injection), indicating that the influence of hepatitis induced by CCl(4) was reversible with PEI-mediated gene delivery. Our findings demonstrated that murine hepatitis induced by CCl(4) could influence polyethylenimine-mediated plasmid DNA delivery according to the disease stage. These results indicate the necessity of considering the timing and dose of gene therapy according to the disease stage.

Transport of acebutolol through rabbit corneal epithelium.

The purpose of this study is to characterize transport of acebutolol through the corneal epithelium. Cultured normal rabbit corneal epithelial cells (RCEC) were used to investigate the drug transport. Primary RCEC were seeded on a filter membrane of Transwell-COL insert coated with fibronectin and were grown in Dulbecco's modified Eagle's medium/nutrient mixture F-12 with various supplements. Measurements of acebutolol permeability through RCEC layer were carried out to assess transcellular permeability coefficient (P(transcell)) in the absence or presence of inhibitors. Paracellular permeability coefficient (P(paracell)) was calculated by permeability coefficient of hydrophilic drugs (P(cell)). The transcellular permeability of acebutolol from apical side to basal side (A-to-B) showed concentration-dependency. The acebutolol flux in the A-to-B direction was smaller than that of opposite direction. Sodium azide, verapamil, and cyclosporin A enhanced the transcellular permeability of acebutolol in the A-to-B direction. Acebutolol permeability through an excised rabbit cornea was also increased by verapamil. Thus, it was suggested that acebutolol was actively secreted via P-glycoprotein in a corneal epithelium.

Prolonged blood concentration of prednisolone after intravenous injection of liposomal palmitoyl prednisolone.

We compared the pharmacokinetic behavior of drugs after an intravenous administration of prednisolone (PLS), palmitoyl prednisolone (Pal-PLS), and liposomal Pal-PLS in rats. Pal-PLS showed higher lipophilicity and higher binding to plasma protein than PLS, and PLS regeneration in rat blood and liver homogenates. After the intravenous administration of Pal-PLS solution in polyethylene glycol (PEG) 400 to rats, Pal-PLS disappeared from the blood in a two-phase mode and PLS was rapidly regenerated. Pal-PLS showed a significantly higher accumulation than PLS in the liver and lung. The administration of Pal-PLS incorporated into egg yolk phosphatidylcholine (EggPC)/cholesterol (Chol) liposomes enhanced Pal-PLS concentrations in the blood, liver, and lung compared to that of Pal-PLS solution in PEG 400, suggesting the rapid removal of liposomes by the mononuclear phagocytic system. Pal-PLS incorporated into PEGylated liposomes constituted with EggPC/Chol/1% L-alpha-distearoylphosphatidylethanolamine (DSPE)-PEG 2000 and EggPC/Chol/10% DSPE-PEG 2000 decreased the initial distribution of Pal-PLS, and successfully maintained the blood concentrations of Pal-PLS and PLS. Thus, we could change the pharmacokinetics of PLS by introducing the palmitoyl function into the molecule and its liposomal formulation including PEGylation. This is the first study to evaluate liposomal PLS constituted with a lipophilic derivative and PEG lipids.

In vitro activity of lauric acid or myristylamine in combination with six antimicrobial agents against methicillin-resistant Staphylococcus aureus (MRSA).

The objective of this study was to investigate the in vitro activities of lauric acid and myristylamine in combination with six antimicrobial agents against methicillin-resistant Staphylococcus aureus (MRSA). The combination effect of lipids and antimicrobial agents was evaluated by the checkerboard method to obtain a fractional inhibitory concentration (FIC) index. The effects of lauric acid + gentamicin (GM) and lauric acid + imipenem (IPM) combinations were synergistic against the clinical isolates in 12 combinations. An antagonistic FIC index was observed only with the myristylamine + GM combination. We investigated in detail the antimicrobial activity for two combinations that showed a synergistic effect. The cytotoxicity of lauric acid was not enhanced by the addition of GM and IPM. In time-kill studies, lauric acid + GM and lauric acid + IPM combinations at one-eighth of the minimum inhibitory concentration produced a bacteriostatic effect.