Advances in Cyclodextrins and Their Derivatives in Nano-Delivery Systems.

The diversity of cyclodextrins and their derivatives is increasing with continuous research. In addition to monomolecular cyclodextrins with different branched chains, cyclodextrin-based polymers have emerged. The aim of this review is to summarize these innovations, with a special focus on the study of applications of cyclodextrins and their derivatives in nano-delivery systems. The areas covered include nanospheres, nano-sponges, nanogels, cyclodextrin metal-organic frameworks, liposomes, and emulsions, providing a comprehensive and in-depth understanding of the design and development of nano-delivery systems.

Effects of the aquatic pollutant sulfamethoxazole on the innate immunity and antioxidant capacity of the mud crab Scylla paramamosain.

Sulfamethoxazole (SMZ) is commonly used in aquaculture to treat bacterial infections, but its long-term residual properties in natural water can pose a direct threat to aquatic animals. This study is to investigate the effects of continuous exposure to SMZ on mud crabs (Scylla paramamosain) at four different concentrations (0, 10, 100, and 1000 ng/L) that reflect the range found in natural aquatic environments. The results confirmed that SMZ exposure reduced the expression levels of genes related to the innate immunity in mud crabs, including JAK, Astakine, TLR, and Crustin. It also stimulated oxidative stress, caused the production of reactive oxygen species and lower activities of antioxidant enzymes such as peroxidase, superoxide dismutase, catalase, and glutathione. SMZ exposure damaged the DNA of crab hemocytes and hepatopancreas tissue, and reduced the phagocytosis, ultimately leading to a decreased survival rates of mud crabs infected with Vibrio alginolyticus. These findings demonstrate that SMZ exposure has immunotoxic effects on mud crabs' innate immunity and reduces the ability to resist pathogen infections.

Effects of geniposide on innate immunity and antiviral activity of Scyllaparamamosain.

In this study, we examined the impact of geniposide on the innate immunity of the mud crab Scylla paramamosain, specifically in relation to WSSV infection. Through the use of in vitro cell culture experiments, we assessed the effects of geniposide on various parameters of hemocyte activity in S. paramamosain. Our findings revealed that high doses of geniposide inhibited hemocyte growth, with an optimal dose of 100 mg/kg determined. Additionally, we observed that geniposide increased the total hemocyte counts in S. paramamosain following WSSV infection. Geniposide also enhanced the enzymatic activities in hemolymph following treatment. The enzymes affected by geniposide encompassed ACP (acid phosphatase), POD (phenol oxidase catalase), PO (phenoloxidase), SOD (superoxide dismutase), CAT (catalase), and LZM (lysozyme). Furthermore, the activities of ACP, POD, PO, and LZM were also observed to increase subsequent to infection with WSSV. Notably, geniposide was found to enhance the phagocytosis of V. alginolyticus within the hemocytes. Geniposide can reduce hemocyte apoptosis rates after treatment, as well as hemocytes infected with WSSV. Furthermore, geniposide treatment significantly up-regulated the expression level of Myosin, but expression levels of Astakine, C-type lectin (CTL), STAT, JAK, proPO, minichromosome maintenance protein (MCM7), caspase-3 and crustin were down-regulated in the hemocytes. Additionally, geniposide treatment inhibited WSSV replication in hemocytes of S. paramamosain, and enhanced the survival rates of mud crabs following WSSV infection. These experimental results provide evidence that geniposide can improve the immune response by regulating humoral immunity and cellular immunity, and enhance pathogen resistance in S. paramamosain.

Effects of environmental pollutant benzop[α]yrene on the innate immunity of Scylla paramamosain and its mechanism.

Benzo[α]pyrene (BaP), a polycyclic aromatic hydrocarbon, is present in the aquatic environment and may be harmful to aquatic animals. We exposed the mud crab Scylla paramamosain to BaP for 7 days, the of superoxide dismutase (SOD), catalase (CAT), phenoloxidase (PO), lysozyme (LZM), glutathione (GSH), glutathione-S-transferase (GST), and acid phosphatase (ACP) activities in the hemolymph of mud crab were reduced. Additionally, the reactive oxygen species content was increased in mud crabs after exposed to BaP. When BaP concentration was increased, the total hemocyte count (THC), the survival rate of hemocytes and their proliferation were decreased. Histopathology analysis revealed damaged hepatopancreas cells, which indicating that BaP exposure is cytotoxic to crab hemocytes. However, the degree of DNA damage did not worsen with increasing BaP concentration. The expression levels of p53, MCM7, Caspase-3, and Myosin were changed with increasing concentration of BaP, which indicated that BaP exposure may affect apoptosis and phagocytosis in mud crabs. As BaP concentration was increased, the apoptosis rate of hemocytes was increased and the phagocytosis was decreased. These results confirmed that BaP exposure inhibited the innate immune response of mud crabs. A possible explanation for this effect is that BaP reduces the antioxidant enzyme activity and increases the reactive oxygen species content in mud crabs, thereby oxidizing and damaging hemocytes, which stimulates phagocytosis and apoptosis and negatively affects the innate immunity of S. paramamosain.

Decreased Penetration Mechanism of Ranitidine Due to Application of Sodium Sulfobutyl Ether-ß-Cyclodextrin.

Permeability has an important effect on drug absorption. In this study, the effect of different concentrations of sodium sulfobutyl ether-ß-cyclodextrin (SBE-ß-CD) on the absorption of ranitidine was investigated to examine the mechanism of permeability changes. The results of a parallel artificial membrane permeability assay (PAMPA) showed that increasing the concentration of sodium sulfobutyl ether-ß-cyclodextrin, 0, 0.12% (w/v), 0.36% (w/v) and 3.6% (w/v), respectively, caused the apparent permeability coefficient of ranitidine to decrease to 4.62 × 10-5, 4.5 × 10-5, 3.61 × 10-5 and 1.08 × 10-5 in Caco-2 cells, respectively. The same results were obtained from an oral pharmacokinetic study in rats. Further studies indicated that SBE-ß-CD significantly increased the zeta potential of ranitidine. SBE-ß-CD interacted with ranitidine charges to form a complex that reduced ranitidine permeability, and SBE-ß-CD should be chosen with caution for drugs with poor permeability.

Effects of Hizikia fusiforme polysaccharides on innate immunity and disease resistance of the mud crab Scylla paramamosain.

In this study, we extracted the polysaccharides from Hizikia fusiforme (HFPs) and evaluated their effects on the immune response of the mud crab Scylla paramamosain. Compositional analysis revealed that HFPs were composed mainly of mannuronic acid (49.05%) and fucose (22.29%) as sulfated polysaccharides, and the sugar chain structure was ß-type. These results indicated that HFPs have potential antioxidant and immunostimulation activity in vivo or in vitro assays. Through this research, we found that HFPs inhibited viral replication in white spot syndrome virus (WSSV)-infected crabs and promoted phagocytosis of Vibrio alginolyticus by hemocytes. Quantitative PCR results showed that HFPs up-regulated the expression levels of astakine, crustin, myosin, MCM7, STAT, TLR, JAK, CAP, and p53 in crab hemocytes. HFPs also promoted the activities of superoxide dismutase and acid phosphatase and the hemolymph antioxidant activities of crabs. HFPs maintained peroxidase activity after WSSV challenge, thereby providing protection against oxidative damage caused by the virus. HFPs also promoted apoptosis of hemocytes after WSSV infection. In addition, HFPs significantly enhanced the survival rate of WSSV-infected crabs. All results confirmed that HFPs improved the innate immunity of S. paramamosain by enhancing the expression of antimicrobial peptides, antioxidant enzyme activity, phagocytosis, and apoptosis. Therefore, HFPs have potential for use as therapeutic or preventive agents to regulate the innate immunity of mud crabs and protect them against microbial infection.

Preparation and Characterization of Furosemide Solid Dispersion with Enhanced Solubility and Bioavailability.

Furosemide (FMD), as a potent circulating diuretic, is commonly used for the treatment of hypertension and edema arising from cardiac, renal, and hepatic failure. However, the low solubility of furosemide restricts its dissolution and bioavailability. In this study, Polyvinylpyrrolidone K30 (PVP-K30), mesoporous (Syloid 244FP, Syloid XDP 3050), and non-mesoporous (Aeroperl 300, Aerosil 200) silica were chosen as combined carrier to develop novel amorphous solid dispersions of furosemide, and then its dissolution and bioavailability were evaluated. Characterization study included XRD, DSC, TGA, SEM, FT-IR, and molecular docking. We found that FMD:PVP-K30:244FP achieved its best performance in terms of dissolution at the ratio of 1:1:1 when PVP-K30 and mesoporous silica Syloid 244FP (244FP) were chosen as combined carrier. SEM, DSC, and XRD studies indicated that furosemide existed in an amorphous form in the solid dispersion. FT-IR and molecular docking analysis showed that there might be an intermolecular interaction between FMD and the carrier. Moreover, the in vivo pharmacokinetics study revealed that the bioavailability of solid dispersion in rats had significant improvement. In particular, Cmax and AUClast were greatly increased by 2.69- and 2.08-fold in the solid dispersion (FMD-PVP-K30-244FP) group, respectively, and the relative bioavailability was 208.00%. In conclusion, the solid dispersion (FMD-PVP-K30-244FP) can significantly improve the solubility and oral bioavailability of furosemide. Mesoporous silica can be used as an excellent carrier material for furosemide, which can provide new ideas and methods for improving the stability of solid dispersion and further improving the dissolution of insoluble drugs. Graphical Abstract.

Differential expression of microRNAs in mud crab Scylla paramamosain in response to white spot syndrome virus (WSSV) infection.

Till date numerous microRNAs (miRNAs) have been discovered from various organisms, including fish, shellfish and crustaceans. The miRNAs are known to regulate immune functions in crustaceans, but little is known about the role of miRNAs against viral infection in crab. We performed small RNA sequencing to characterize the differentially expressed miRNAs in WSSV infected Scylla paramamosain, in comparison to that in uninfected crab, at 2 h and 12 h post infection. In total, 24 host miRNAs were up-regulated and 25 host miRNAs were down-regulated in response to WSSV infection at 2 h post infection. And 27 host miRNAs were up-regulated and 30 host miRNAs were down-regulated in response to WSSV infection at 12 h post infection. Further, the gene ontology analysis revealed that many signaling pathways were mediated by these miRNAs. The integral component of membrane is the most important biological process and endocytosis pathway is the most important pathway, which indicates that endocytosis is very important for WSSV infection. This study is one important attempt at characterizing crab miRNAs that response to WSSV infection, and will help unravel the miRNA pathways involved in antiviral immunity of crab.

Amplification of tumor antigen presentation by NLGplatin to improve chemoimmunotherapy.

Oxaliplatin is a chemotherapeutic agent widely used in cancer treatment whereas its immunosuppressive effect hinders the progress of immunotherapy. Here we have synthesized a new compound NLGplatin constructed by combining oxaliplatin (OXA) and indoleamine 2,3-dioxygenase (IDO) inhibitor NLG919. The NLGplatin acquires chemotherapeutic properties of OXA and can activate the immune system, and also retains the ability to inhibit IDO enzyme activity without affecting the proliferation of immune cells. This difunctional drug has a great potential to achieve effective cancer chemoimmunotherapy.

Differentially expressed genes of the shrimp Marsupenaeus japonicus in response to infection by white spot syndrome virus or Vibrio alginolyticus.

This is the first study to use a next-generation high-throughput DNA sequencing technique, the Illumina Hiseq2000 method, to analyze the transcriptome from hemocytes of Marsupenaeus japonicus. A total of 80,929,652 Illumina reads, including 79,525,942 high quality reads were obtained in this study. From these, 40,231 unigenes with a mean length of 1557 bp were assembled using Trinity de novo software and 28,746 cDNA were matched in the NCBI database. Then we compared the transcriptome changes after white spot syndrome virus (WSSV) or Vibrio alginolyticus infection. A total of 19,872 putative proteins were classified functionally into 25 molecular families in the cluster of orthologous groups. KEGG pathway analysis identified that the metabolic pathway possessed more unigenes (1358 unigenes), followed by biosynthesis of secondary metabolites, Huntington's disease and RNA transport. Important immune functions like apoptosis, phagocytosis, and lysosomes were in response to WSSV and V. alginolyticus early infection. Only 26 transcripts were significantly up-regulated or down-regulated after WSSV infection showed compared with V. alginolyticus. Crustin-like protein, endonuclease-reverse transcriptase, putative nuclease HARBI1-like, diphthamide biosynthesis protein 7 and hormone receptor 3 were involved in the immune response to WSSV or V. alginolyticus infection. These transcriptome datasets accelerate our understanding of the innate immune mechanisms in M. japonicus and other crustaceans.

Antibiotics and chemical disease-control agents reduce innate disease resistance in crayfish.

The aquaculture industry has developed rapidly in recent years, and in China Crayfish Procambarus clarkii represent an important aquaculture fishery. However, bacterial and viral diseases are becoming an increasingly serious threat, causing considerable economic losses. Farmers use a large number of drugs and chemicals to destroy pathogenic microorganisms and to purify aquaculture water. The purpose of this study was to assess the effects of such drugs on crayfish immune systems. Five of the most commonly used fishery drugs and water treatment chemicals were analyzed: norfloxacin, calcium hypochlorite, quick lime, povidone iodine and copper sulfate. Crayfish immune activity tests revealed that total hemocytes counts, as well as the activities of phenoloxidase and superoxide dismutase, decreased following exposure to all five treatments. These treatments, especially calcium hypochlorite and norfloxacin, significantly enhanced hemocyte apoptosis in crayfish, regardless of disease status. Calcium hypochlorite, in particular, led to a significant decrease in the survival rates of crayfish infected with white spot syndrome virus or Vibrio alginolyticus. Our results indicate that water treatment and disease control compounds commonly used in aquaculture can reduce the innate immunity and therefore disease resistance of crayfish.

Regulating the Golgi apparatus by co-delivery of a COX-2 inhibitor and Brefeldin A for suppression of tumor metastasis.

Finding a cure for breast cancer currently remains a medical challenge in due to the failure of common treatment methods to inhibit invasion and metastasis of cancer cells, which eventually leads to recurrence of breast cancer. Many secreted proteins are overexpressed and play crucial roles in tumorigenesis and development. The Golgi apparatus is a key protein processing and secretion factory in which metastasis-associated proteins are modified, transported and secreted; thus, regulating the Golgi apparatus of tumor cells is a viable strategy to inhibit tumor metastasis. Herein, celecoxib (CLX) and Brefeldin A (BFA) were encapsulated into the biocompatible polymer PLGA-PEG to form nanoparticles that act on the Golgi apparatus to treat metastatic breast cancer; CLX is a specific COX-2 inhibitor which accumulates in the Golgi apparatus, and BFA is a protein transport inhibitor fusing the Golgi apparatus into endoplasmic reticulum. The optimized CLX and BFA co-loaded nanoparticles (CBNPs) possessed good physicochemical properties. CBNPs efficiently damaged the Golgi apparatus within 30 min and showed enhanced cytotoxicity of CLX and BFA toward murine metastatic breast cancer 4T1 cells. The migration and invasion abilities of the cells were dramatically suppressed by the CBNPs. Further, the expression and secretion of metastasis-associated proteins such as matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) were remarkably decreased. Our findings showed that co-delivering CLX and BFA to regulate the Golgi apparatus may be an efficient strategy to inhibit breast cancer growth and suppress tumor cell metastasis.

[Development of an aptamer/fluorescence dye PicoGreen-based method for detection of fumonisin B1].

Fumonisin B1 (FB1) is a carcinogenic mycotoxin found in commodities such as corn and corn-originated products. An aptamer-based method for detection of FB1 was developed using the fluorescent dye PicoGreen, which can recognize and bind double-stranded DNA. A peak fluorescence of PicoGreen was obtained in 15 min in the presence of FB1 aptamer, which formed a double-stranded hybridizer DNA with its complementary strand. The excitation and emission wavelengths for PicoGreen detection were 480 nm and 520 nm, respectively. The sensitivity of this aptamer/PicoGreen-based method was 0.1 µg/L. This method showed a good linearity for FB1 concentration ranging from 0.1 to 1 µg/L. The entire detection procedure for FB1 could be completed within 40 min. No cross reactions were observed with any other mycotoxins against aflatoxin B1, ochratoxin A, citrinin and zearalenone, demonstrating high specificity towards FB1 aptamer. Agreement between commercial, antibody-based enzyme-linked immunosorbent assay (ELISA) kit and aptamer method was excellent with a kappa value of 0.857. Taken together, this aptamer/PicoGreen-based method is more cost-effective, time-saving and useful than ELISA for detection of FB1.

Upregulation of COX-2 in the lung cancer promotes overexpression of multidrug resistance protein 4 (MRP4) via PGE2-dependent pathway.

It is apparent that lung cancer is associated with inflammation, with accompanying hallmark elevations of cyclooxygenase 2 (COX-2) and prostaglandin E2 (PGE2) levels. However, the effects of these changes on MRP efflux transporters have not been thoroughly investigated before. Here, we report that upregulation of COX-2 can induce overexpression of MRP4 in both A549 non-small-cell lung cancer cell lines and mouse lung cancer models. In A549 cells, phorbol 12-myristate 13-acetate (PMA) treatment induced upregulation of COX-2 and MRP4 together, but not other MRP transporters. Transient overexpression of human COX-2 cDNA also specifically increased COX-2 and MRP4. Moreover, COX inhibitor treatment and COX-2-specific siRNA significantly inhibited the upregulation of MRP4. Additionally, PMA-treatment increased extracellular PGE2 levels, likely due to increased MRP4 function. Likewise, COX-2-specific siRNA reduced extracellular PGE2 levels. Furthermore, COX-2 upregulation resulted in an increase in mPGES-1, an enzyme responsible for PGE2 production. Finally, metastasized lung cancer model mice exhibited increased expression levels of COX-2 and MRP4, as well as mPGES-1. In conclusion, the present study suggests that overexpression of MRP4 in lung cancer may be attributable to COX-2 upregulation via a PGE2-dependent pathway.

Correlation of fibroblast growth factor 21 serum levels with metabolic parameters in Japanese subjects.

OBJECTS: Since serum level of fibroblast growth factor 21 (FGF21) has been implicated as a potential biomarker for the early detection of the metabolic syndrome and type 2 diabetes, we examined how FGF21 serum levels are correlated with metabolic parameters in Japanese subjects. METHODS: FGF21 levels were analyzed by enzyme-linked immunosorbent assays. Spearman's correlation and multiple stepwise regression analyses were used to examine the relationship between serum FGF21 and other factors. A Mann-Whitney U test was performed between the normal and high groups for triglycerides and systolic blood pressure (BP) respectively. RESULTS: By univariate correlation analysis, serum FGF21 levels were significantly associated with triglyceride levels, systolic BP, diastolic BP, pulse pressure, body mass index (BMI), age, fasting plasma glucose (FPG) levels, and total cholesterol levels. Multiple regression analysis (adjusted for age, gender, and BMI) showed that serum FGF21 levels were independently and significantly associated with triglyceride levels and systolic BP. Serum FGF21 levels were significantly higher in subjects with high triglyceride levels and high systolic BP compared with those who had normal triglyceride levels and normal systolic BP respectively. CONCLUSIONS: This study found that FGF21 levels might be a biomarker for some metabolic disorders associated with metabolic syndrome.

Development of a LC-MS method for quantification of FK-3000 and its application to in vivo pharmacokinetic study in drug development.

FK-3000 can inhibit proliferation of carcinomas and arrest the growth of carcinoma cells through cytotoxic (apoptosis induction) and cytostatic (cell cycle arrest) effects. A rapid and sensitive assay was developed and validated using liquid chromatography-mass spectrometry (LC-MS) for FK-3000 in rat plasma. FK-3000 was extracted with ethyl acetate from rat plasma samples, and the residue containing the FK-3000 was dried in a gentle stream of nitrogen and reconstituted with acetonitrile. The FK-3000 was quantified using high-performance liquid chromatography (HPLC; Waters Alliance 2695) with a reversed phase Gemini column (3 mm × 150 mm, 5 µm; Phenomenex, USA) and a Waters Micromass ZQ detector. FK-3000 and phenazine, an internal standard (IS), were analyzed by selected ion monitoring (SIM) at m/z transitions of 418.45 and 256, respectively. A lower limit of quantification (LLOQ) of 10 ng/mL was observed, with a linear dynamic range from 10 to 10,000 ng/mL (R>0.999). The accuracy, precision, recovery, matrix effects, and stability of the assay were deemed acceptable according to the FDA guidance for industry (bioanalytical method validation). The FK-3000 concentration was measured in plasma samples up to 6 h following FK-3000 administration at an oral dose of 20 mg/kg. The findings indicate that the assay method is suitable for routine pharmacokinetic (PK) studies of FK-3000 in rats.

Quantification of oltipraz using liquid chromatography-tandem mass spectrometry and its application to a pharmacokinetic study in rat plasma.

An assay method for the determination of oltipraz, a candidate drug for the treatment of liver fibrosis and liver cirrhosis, was developed in rat plasma using a fast-flow protein precipitation (FF-PPT) method coupled with LC-MS/MS for quantification to reduce the labor and to improve the speed of analysis. The applicability of the assay to pharmacokinetic studies was also evaluated. Oltipraz and ethyl-oltipraz, an internal standard (IS), were analyzed by multiple reaction monitoring (MRM) at m/z transitions of 227→193 and 241→174, respectively. A lower limit of quantification (LLOQ) of 20 ng/mL was observed, with a linear dynamic range from 20 to 4000 ng/mL (R>0.997). The accuracy, precision, dilution, recovery, and stability of the assay were deemed acceptable according to FDA guidelines. Oltipraz concentrations were measured successfully in plasma samples up to 12h post-dose in rats that had received an oral dose of 60 mg/kg. The findings indicate that the assay method is rapid and sensitive to oltipraz, showing applicability for pharmacokinetics (PK) studies of oltipraz in other small animals, including rats.

Inhibitory effects of ketoconazole and rifampin on OAT1 and OATP1B1 transport activities: considerations on drug-drug interactions.

Ketoconazole and rifampin are the most widely used compounds examined in recent drug-drug interaction (DDI) studies, and they have multiple roles in modulating drug metabolizing enzymes and transporters. To determine the underlying mechanisms of DDI, this study was performed to investigate the inhibitory effects of ketoconazole and rifampin on the functions of OAT1 and OATP1B1, and to evaluate the potential of ketoconazole and rifampin for DDI with substrate drugs for these transporters in a clinical setting. Ketoconazole inhibited OATP1B1-mediated transport activity, while rifampin inhibited OAT1 and OATP1B1. Inhibition by rifampin and ketoconazole of the uptake of olmesartan, a substrate for OAT1 and OATP1B1, was evaluated in oocytes overexpressing these transporters. The K(i) values for rifampin on OAT1 and OATP1B1-mediated olmesartan uptake were 62.2 and 4.42 µM, respectively, and the K(i) value for ketoconazole on OATP1B1-mediated olmesartan uptake was 66.1 µM. As measured plasma concentrations of rifampin and ketoconazole were 7.29 and 6.4-13.3 µM, respectively, the likelihood of an OATP1B1-mediated drug-drug interaction between rifampin and olmesartan is thought to be possible, whereas OAT1 or OATP1B1-mediated DDI between rifampin or ketoconazole and olmesartan appears unlikely in the clinical setting.

Sitagliptin attenuates metformin-mediated AMPK phosphorylation through inhibition of organic cation transporters.

To assess potential interactions between sitagliptin and metformin, we sought to characterize the in vitro inhibitory potency of sitagliptin on the uptake of MPP(+) and metformin, representative substrates for OCTs, and to evaluate the pharmacological pathways that may be affected by the combination of metformin and sitagliptin. Among the OATs and OCTs screened, OAT3-mediated salicylate uptake and OCT1- and OCT2-mediated MPP(+) uptake were inhibited by sitagliptin. The K(i) values of sitagliptin for OCT1- and OCT2-mediated metformin uptake were 34.9 and 40.8 µM, respectively. As OCT1 is the gate protein for metformin action in the liver, we investigated whether sitagliptin-mediated OCT1 inhibition affected metformin-induced activation of AMPK signalling. Treatment with sitagliptin in MDCK-OCT1 and HepG2 cells resulted in a reduced level of phosphorylated AMPK, with K(i) values of 38.8 and 43.3 µM, respectively. These results suggest that the inhibitory potential of sitagliptin on OCT1 may attenuate the first step of metformin action, that is, the phosphorylation of AMPK. Nevertheless, the likelihood of a drug-drug interaction between sitagliptin and metformin is believed to be remote in usual clinical setting.

Increased affinity to canalicular P-gp via formation of lipophilic ion-pair complexes with endogenous bile salts is associated with mw threshold in hepatobiliary excretion of quaternary ammonium compounds.

OBJECTIVES: We intended to elucidate the mechanism of the molecular weight (Mw) threshold (i.e., 200 +/- 50) for appreciable hepatobiliary excretion of quaternary ammonium compounds (QACs) in rats. METHODS: We measured the effect of ion-pair complexation of QACs with taurodeoxycholate (TDC), an endogenous anionic bile salt, on the apparent partition coefficients (APC) of QACs between n-octanol and phosphate buffer, and the inhibition of organic cation transporter1 (OCT1)- and P-glycoprotein (P-gp)-mediated transport of representative substrates. RESULTS: By measuring the APC, we demonstrated that there is a Mw threshold of 200 +/- 50 in the ion-pair complexation of QACs with an endogenous bile salt, TDC. We also demonstrated, by measuring the inhibition of relevant transports, that a Mw threshold of 200 +/- 50 exists for the binding of QACs to canalicular P-gp, but not for sinusoidal OCT1. The Mw threshold values for ion-pair formation and P-gp binding were identical and consistent with the reported Mw threshold value for appreciable biliary excretion of QACs in rats. CONCLUSIONS: Mw-dependent binding of QACs to canalicular P-gp contributes in part to the mechanism of the Mw threshold of 200 +/- 50. The formation of lipophilic ion-pair complexes with bile salts, followed by stronger binding to canalicular P-gp, appears to accelerate biliary excretion of QACs with a high Mw.