3-deazaneplanocin A, a histone methyltransferase inhibitor, improved the chemoresistance induced under hypoxia in melanoma cells.

One of common characteristics of solid tumors is low O2 level, so-called hypoxia, which plays a critical role in chemoresistance. Epigenetic mechanism such as DNA methylation and histone modification is involved in cancer development and progression. There is ample evidence that epigenetic drugs reversed acquired chemoresistance in cancer cells under normal O2 level, normoxia. However, it remains unknown whether epigenetic drugs improve acquired chemoresistance under hypoxia. The aim of our study was to investigate whether epigenetic drugs can improve the chemoresistance induced under hypoxia in cancer cells. In murine melanoma B16-BL6 (B16) cells, the culture under hypoxia, 1%O2 caused the elevated expression of hypoxia-inducible factor-1α (HIF-1α) and its target genes. The chemoresistance to 7-ethyl-10-hydroxycamptothecin (SN-38, the active metabolite of irinotecan) was also acquired under hypoxia in B16 cells. In addition, as epigenetic mechanisms, the protein expression of the enhancer of zeste homolog 2 (EZH2), histone methyltransferase and its target histone H3 trimethylation at lysine 27 (H3K27Me3) level increased under hypoxia. The induction of H3K27Me3 under hypoxia was suppressed by EZH2 siRNA and 3-deazaneplanocin A (DZNep), an EZH2 inhibitor. Furthermore, both EZH2 siRNA and DZNep significantly reduced the cell viability after SN-38 treatment and improved the chemoresistance to SN-38 under hypoxia. These results indicated that the chemoresistance to SN-38 under hypoxia would arise from epigenetic mechanism, H3K27Me3 elevation due to EZH2 induction. In conclusion, a histone methyltransferase EZH2 inhibitor, DZNep was capable of tackling acquired chemoresistance via the suppression of histone methylation induced under hypoxic tumor microenvironment.

Preparation and Evaluation of Paclitaxel-Loaded PEGylated Niosomes Composed of Sorbitan Esters.

Niosomes are non-ionic surfactant (NIS)-based bilayer vesicles and, like liposomes, have great potential as drug-delivery systems. Our previous study revealed that polyethylene glycol (PEG) niosomes using different sorbitan ester (Span) surfactants (sorbitan monoester, Span 20, 40, 60, 80; sorbitan triester, Span 65) distributed within tumors similarly to PEG liposomes. The aim of this study was to encapsulate efficiently an anti-cancer drug, paclitaxel (PTX) into Span PEG niosomes, and evaluate PTX release profiles and anti-tumor efficacy of PTX-loaded Span PEG niosomes. Niosome sizes ranged between 100-150 nm, and the PTX encapsulation efficiency was more than 50%. All niosomes examined, in the presence of serum, yielded sustained PTX-release profiles. PTX release at 24 and 48 h from Span 80 PEG niosomes was significantly the highest among the other Span PEG niosomes examined. In C26 tumor-bearing mice, PTX-loaded Span 40 PEG niosomes (the lowest PTX release in vitro) suppressed tumor growth while PTX-loaded Span 80 PEG niosomes (the highest PTX release in vitro) did not. Thus, we succeeded in the control of PTX release from Span PEG niosomes by modifying the component of niosomes, and it influenced the effects of drugs loaded into niosomes. This demonstrates that the excellent NIS physicochemical properties of Spans make them an ideal candidate for anti-cancer drug-carrier niosomes.

Suppression of Phagocytic Activity Leads to the Efficient Surface Modification of Macrophages with Liposomes for Developing a Biomimetic Drug Delivery System.

Macrophages selectively infiltrate the lesion sites of several diseases, including cancers, and, thus, have attracted attention as a biomimetic drug delivery carrier. To achieve the efficient drug loading of macrophages with minimal cytotoxicity, drugs are preferably encapsulated into nanoparticles, such as liposomes, and modified on the surface of macrophages rather than being incorporated into cells. However, liposomes are rapidly taken up by macrophages after binding to the cell surface because of their strong phagocytic activity. To overcome this, we herein attempted to modify the surface of macrophages with liposomes by suppressing their phagocytic activity using a pretreatment with anionic liposomes. We confirmed that 1,2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DSPG)- and cholesterol-rich anionic liposomes were efficiently taken up by RAW264.7 murine macrophage-like cells. Furthermore, the cellular uptake of anionic liposomes by RAW264.7 cells was higher in the absence of fetal bovine serum (FBS) than in its presence. Moreover, the viability of RAW264.7 cells was maintained above 90% when cells were incubated with anionic liposomes for 3 h, whereas viability was markedly decreased after a 24-h incubation. Based on these results, we pretreated RAW264.7 cells by an incubation with DSPG- and cholesterol-rich liposomes for 3 h in the absence of FBS. This pretreatment significantly inhibited the internalization of other liposomes, which subsequently bound to the cell surface. Therefore, we succeeded in modifying the surface of macrophages with liposomes, and liposome-modified macrophages have potential as a biomimetic active drug delivery carrier.

Improvement of resistance to oxaliplatin by vorinostat in human colorectal cancer cells through inhibition of Nrf2 nuclear translocation.

Vorinostat (suberoylanilide hydroxamic acid: SAHA), a histone deacetylase inhibitor, has potential benefit of improving the resistance to conventional other anti-cancer drugs. This study was aimed to clarify whether SAHA improves the resistance to oxaliplatin (L-OHP), a platinum-based anticancer drug using L-OHP-resistant HCT116 cells (HCT116/OxR), established from colorectal cancer (CRC) cell line HCT116. HCT116/OxR cells showed cross-resistance to other platinum-based drugs. Pre-treatment with SAHA improved the sensitivity of both L-OHP and its metabolite in HCT116/OxR cells, but not in parental HCT116 cells. However, pre-treatment with SAHA did not affect the sensitivity of other platinum-based drugs. These results indicated that SAHA specifically improved the sensitivity of L-OHP in HCT116/OxR cells. Focusing on NF-E2 p45-related factor 2-Kelch-like ECH-associated protein 1 pathway (Nrf2-Keap1) pathway, which is activated by oxidative stress such as the treatment with anti-cancer drugs, mechanisms behind these observations were elucidated. In HCT116/OxR cells transfected with Nrf2 siRNA, the improving effects on L-OHP resistance by SAHA were abolished, suggesting that Nrf2-Keap1 pathway was involved in L-OHP-resistance. In addition, L-OHP metabolite significantly induced the expression of the nuclear protein Nrf2 and its target gene mRNA expression in HCT116/OxR cells. Pre-treatment with SAHA suppressed these changes observed in HCT116/OxR cells. In conclusion, this study demonstrated that SAHA improved L-OHP resistance by inhibiting Nrf2-Keap1 activation via Nrf2 nuclear translocation by L-OHP metabolite.

Effect of Excessive Serotonin on Pharmacokinetics of Cephalexin after Oral Administration: Studies with Serotonin-Excessive Model Rats.

PURPOSE: Serotonin (5-HT) is important for gastrointestinal functions, but its role in drug absorption remains to be clarified. Therefore, the pharmacokinetics and oral absorption of cephalexin (CEX) were examined under 5-HT-excessive condition to understand the role of 5-HT. METHODS: 5-HT-excessive rats were prepared by multiple intraperitoneal dosing of 5-HT and clorgyline, an inhibitor for 5-HT metabolism, and utilized to examine the pharmacokinetics, absorption behavior and the intestinal permeability for CEX. RESULTS: Higher levels of 5-HT in brain, plasma and small intestines were recognized in 5-HT-excessive rats, where the oral bioavailability of CEX was significantly enhanced. The intestinal mucosal transport via passive diffusion of CEX was significantly increased, while its transport via PEPT1 was markedly decreased specifically in the jejunal segment, which was supported by the decrease in PEPT1 expression on brush border membrane (BBM) of intestinal epithelial cells. Since no change in antipyrine permeability and significant increase in FITC dextran-4 permeability were observed in 5-HT-excessive rats, the enhanced permeability for CEX would be attributed to the opening of tight junction, which was supported by the significant decrease in transmucosal electrical resistance. In 5-HT-excessive rats, furthermore, total body clearance of CEX tended to be larger and the decrease in PEPT2 expression on BBM in kidneys was suggested to be one of the reasons for it. CONCLUSIONS: 5-HT-excessive condition enhanced the oral bioavailability of CEX in rats, which would be attributed to the enhanced permeability across the intestinal mucosa via passive diffusion through the paracellular route even though the transport via PEPT1 was decreased.

Combination of azacytidine and curcumin is a potential alternative in decitabine-resistant colorectal cancer cells with attenuated deoxycytidine kinase.

Decitabine (DAC), a DNA methyltransferase (DNMT) inhibitor is a novel anti-cancer drug regulating epigenetic mechanisms. Similar to conventional anti-cancer drugs, drug resistance to DAC also has been reported, resulting in tumor recurrence. Our previous study using colorectal cancer HCT116 cells found the decrease in deoxycytidine kinase (dCK) (activation enzyme of DAC) and the increase in cytidine deaminase (inactivation enzyme of DAC) in acquired DAC-resistant HCT116 (HCT116/DAC) cells. The aim of our study was to clarify the involvement of dCK and CDA in DAC resistance. In order to tackle DAC resistance, it was also examined whether other DNMT inhibitors such as azacytidine (AC) and polyphenols are effective in DAC-resistant cancer cells. When dCK siRNA was transfected into HCT116 cells, IC50 value of DAC increased by about 74-fold and reached that of HCT116/DAC cells with attenuated dCK. dCK siRNA to HCT116 cells also abolished DNA demethylation effects of DAC. In contrast, CDA siRNA to HCT116 cells did not influence the efficacy of DAC. In addition, CDA siRNA to HCT116/DAC cells with increased CDA did not restore the compromised effects of DAC. These results suggested that attenuated dCK but not increased CDA mainly contributed to DAC resistance. Regarding dCK in HCT116/DAC cells, a point mutation with amino acid substitution was observed while the product size and expression of mRNA coding region did not change, suggesting that dCK protein was decreased by post-transcriptional regulation. AC and polyphenols showed no cross-resistance in HCT116/DAC cells. AC but not polyphenols exerted DNA demethylation effect. Among polyphenols, curcumin (Cur) showed the most synergistic cytotoxicity in combination with AC while DNA demethylation effect of AC was partly maintained. Taken together, combination of AC and Cur would be a promising alternative to tackle DAC resistance mainly due to attenuated dCK.

Novel Platform for Predicting Drug Effects in Patients with Acromegaly: Translational Exposure-Response Evaluation of Growth Hormone-Inhibitory Effect of Octreotide after Growth Hormone-Releasing Hormone Stimulation.

Acromegaly is a chronic systemic disease characterized by facial and peripheral changes caused by soft tissue overgrowth and is associated with multiple comorbidities. Despite available surgical and medical therapies, suitable treatments for acromegaly are still lacking. Efficient drug development requires an understanding of the exposure-response (E-R) relationship based on nonclinical and early clinical studies. We aimed to establish a platform to facilitate the development of novel drugs to treat acromegaly. We evaluated the E-R relationship of the growth hormone (GH)-inhibitory effect of the somatostatin analog octreotide under growth hormone-releasing hormone + arginine stimulation in healthy participants and compared the results with historical data for patients with acromegaly. This randomized five-way crossover study included two placebo and three active-treatment periods with different doses of octreotide acetate. GH secretion in the two placebo periods was comparable, which confirmed the reproducibility of the response with no carryover effect. GH secretion was inhibited by low-, medium-, and high-dose octreotide acetate in a dose-dependent manner. We also examined the E-R relationship in monkeys as a preclinical drug evaluation study and in rats as a more convenient and simple system for screening candidate drugs. The E-R relationships and EC50 values were similar among animals, healthy participants, and patients with acromegaly, which suggests that GH stimulation studies in early research and development allowed simulation of the drug response in patients with acromegaly. SIGNIFICANCE STATEMENT: This study demonstrated similar exposure-response relationships in terms of the growth hormone-inhibitory effect of octreotide after growth hormone-releasing hormone stimulation among healthy participants, monkeys, and rats. The research methods and analyses utilized in this study will be useful for simulating the dosages and therapeutic effects of drugs for acromegaly and will facilitate the research and development of novel therapeutic agents with similar modes of action.

Development of Novel Bead Milling Technology with Less Metal Contamination by pH Optimization of the Suspension Medium.

To develop novel contamination-less bead milling technology without impairing grinding efficiency, we investigated the effect of the formulation properties on the grinding efficiency and the metal contamination generated during the grinding process. Among the various formulations tested, the combination of polyvinylpyrrolidone and sodium dodecyl sulfate was found to be suitable for efficiently pulverizing phenytoin. However, this stabilization system included a relatively strong acid, which raised the concern of possible corrosion of the zirconia beads. An evaluation of the process clearly demonstrated that acidic pH promoted bead dissolution, suggesting that this could be suppressed by controlling the pH of the suspension. Among the various pH values tested, the metal contamination generated during the grinding process could be significantly reduced in the optimized pH range without significant differences in the particle size of the phenytoin suspension after pulverization. In addition, the contamination reduction by pH optimization in the presence of physical contact among the beads was approximately 10-times larger than that without bead contact, suggesting that pH optimization could suppress not only bead dissolution but also the wear caused by bead collisions during the grinding process. These findings show that pH optimization is a simple but effective approach to reducing metal contamination during the grinding process.

Pharmacokinetic-Pharmacodynamic-Efficacy Modeling of ONO-7579, a Novel Pan-Tropomyosin Receptor Kinase Inhibitor, in a Murine Xenograft Tumor Model.

The orally available and novel small molecule ONO-7579 (N-{2-[4-(2-amino-5-chloropyridin-3-yl)phenoxy]pyrimidin-5-yl}-N'-[2-(methanesulfonyl)-5-(trifluoromethyl)phenyl]urea) is a highly potent and selective pan-tropomyosin receptor kinase (TRK) inhibitor. The objective of the present study was to characterize the pharmacokinetic (PK), pharmacodynamic (PD), and antitumor efficacy relationships of ONO-7579 in mice xenografted with a human colorectal cancer cell line, KM12 (harboring the tropomyosin 3 (TPM3) -neurotrophic tyrosine receptor kinase 1 fusion gene), via a PK/PD modeling approach. Plasma and tumor concentrations of ONO-7579, tumor levels of phosphorylated TPM3-TRKA (pTRKA), and tumor volumes in the murine model were measured with a single or multiple dose of ONO-7579 (0.06-0.60 mg/kg) administered once daily. The PK/PD/efficacy models were developed in a sequential manner. Changes in plasma concentrations of ONO-7579 were described with an oral one-compartment model. Tumor concentrations of ONO-7579 were higher than plasma concentrations, and changes in ONO-7579 tumor concentrations were described with an additional tumor compartment that had no influence on plasma concentrations. pTRKA in tumors was described with a direct Emax model, and the tumor ONO-7579 concentration causing 50% of the maximum effect was estimated to be 17.6 ng/g. In addition, a pTRKA-driven tumor growth inhibition model indicated that ONO-7579 started to sharply increase the antitumor effect at pTRKA inhibition rates >60% and required >91.5% to reduce tumors. In conclusion, the developed PK/PD/efficacy models revealed a "switch-like" relationship between pTRKA inhibition rate and antitumor effect in a murine KM12 xenograft model, demonstrating that pTRKA in tumors could serve as an effective biomarker for scheduling the dose regimen in early-stage clinical studies. SIGNIFICANCE STATEMENT: In recent years, clinical development of TRK inhibitors in patients with neurotrophic tyrosine receptor kinase fusion-positive solid tumors has been accelerated. This research found that phosphorylated TRKA was a useful biomarker for explaining the antitumor efficacy of TRK inhibitors using a pharmacokinetic/pharmacodynamic modeling approach in xenograft mice. This finding suggests a rational dosing regimen in early-stage clinical studies for ONO-7579 (N-{2-[4-(2-amino-5-chloropyridin-3-yl)phenoxy]pyrimidin-5-yl}-N'-[2-(methanesulfonyl)-5-(trifluoromethyl)phenyl]urea), a novel pan-TRK inhibitor.

Optimization of Analytical Conditions for Hydrophilic Nucleic Acids Using Mixed-Mode and Reversed-Phase Pentabromobenzyl Columns.

The aim of this study was to investigate appropriate analytical conditions for hydrophilic nucleosides and nucleotides (monophosphates and triphosphates) by HPLC methods using a mixed-mode AX-C18 column with anion-exchange and hydrophobic interactions by quaternary ammonium and C18, respectively, and a reversed-phase pentabromobenzyl (PBr) column with dispersion force and hydrophobic interactions by PBr group. The higher compound polarity led to stronger retention on AX-C18 (triphosphates > monophosphates > nucleosides). AX-C18 demonstrated feasible retention of nucleotides via anion-exchange interaction by increasing the salt and methanol concentrations. In contrast, on PBr, the lower compound polarity led to stronger retention. On PBr, feasible retention of both nucleosides and nucleotides was obtained via dispersion interactions with purine and pyrimidine rings by increasing the methanol concentration. Regarding the pH of phosphate buffer used as the mobile phase, pH 7.0 should be used in measuring nucleoside triphosphates on AX-C18, whereas pH 2.5 is better suited for measuring nucleotides on PBr. In terms of selectivity to highly hydrophilic nucleotides, the mixed-mode AX-C18 column had an advantage over the reverse-phase PBr column. In contrast, PBr column was more versatile than the AX-C18 column. Taken together, HPLC analyses of nucleosides and nucleotides should be carried out by optimizing the interactions between the stationary phase and nucleic acids.

Decitabine exerted synergistic effects with oxaliplatin in colorectal cancer cells with intrinsic resistance to decitabine.

Epigenetic modifiers such as decitabine (DAC), a DNA methyltransferase inhibitor, have the potential benefit of combination therapy with conventional anti-cancer drugs. This study was aimed to clarify whether DAC at the low concentration without cytotoxic effects exerts synergistic effects with conventional anti-cancer drugs in human colorectal cancer cell line, HT29 cells low-sensitive to DAC. DAC at very low concentration below its IC20 synergistically enhanced cytotoxicity of oxaliplatin (L-OHP), and the enhancement was the most remarkable for L-OHP among several anti-cancer drugs tested. DAC was found to be metabolized and incorporated into DNA in HT29 cells. Combination of L-OHP with DAC did not increase the protein expression of γH2A.X, the earliest indicator of DNA damage, in HT29 cells. On the other hand, although DAC alone did not produce reactive oxygen species (ROS), DAC significantly enhanced ROS production by L-OHP in HT29 cells. Furthermore, enhanced cytotoxicity of L-OHP by DAC was cancelled with the presence of N-acetyl-l-cysteine, a ROS scavenger. The mRNA expression of ROS-generating enzymes was significantly increased by combination in HT29 cells. Taken together, indirect enhancement of ROS production by DAC at the low concentration with negligible cytotoxicity should be at least involved in synergistic effects with L-OHP in HT29 cells with intrinsic resistance to DAC.

Quantitative Estimation of the Effect of Nasal Mucociliary Function on in Vivo Absorption of Norfloxacin after Intranasal Administration to Rats.

Nasal drug delivery has attracted significant attention as an alternative route to deliver drugs having poor bioavailability. Large-molecule drugs, such as peptides and central nervous system drugs, would benefit from intranasal delivery. Drug absorption after intranasal application depends on the nasal retention of the drug, which is determined by the nasal mucociliary clearance. Mucociliary clearance (MC) is an important determinant of the rate and extent of nasal drug absorption. The aim of the present study was to clarify the effect of the changes in MC on in vivo drug absorption after nasal application, and to justify the pharmacokinetic model to which the MC parameter was introduced, to enable prediction of bioavailability after intranasal administration. The pharmacokinetics of norfloxacin (NFX) after intranasal administration were evaluated following the modification of nasal MC by pretreatment with the MC inhibitors propranolol and atropine and the MC enhancers terbutaline and acetylcholine chloride. From the relationship between nasal MC and bioavailability after nasal application, prediction of drug absorption was attempted on the basis of our pharmacokinetic model. Propranolol and atropine enhanced the bioavailability of NFX by 90 and 40%, respectively, while the bioavailability decreased by 30% following terbutaline and 40% following acetylcholine chloride. As a result of changes in the MC function, nasal drug absorption was changed depending on the nasal residence time of the drug. On the basis of our pharmacokinetic model, the nasal drug absorption can be precisely predicted, even when the MC is changed. This prediction system allows the quantitative evaluation of changes in drug absorption due to changes in nasal MC and is expected to contribute greatly to the development of nasal formulations.

Nanoparticle-Based Photodynamic Therapy: Current Status and Future Application to Improve Outcomes of Cancer Treatment.

Photodynamic therapy (PDT) is an emerging cancer treatment that uses photosensitizers (PS), along with light to activate them, resulting in oxidation of various biological components in cancer tissues. However, since most potential PS are solubilized and given as aqueous solution, PS is non-specifically distributed in the body, leading to the induction of various side effects in normal tissues that are exposed to daylight such as skin and eyes. To overcome the problem associated with non-specific in vivo disposition of PS, various approaches have been applied to develop safer dosage forms for PS with more efficient tumor delivery and lower disposition to normal tissues. Passive drug targeting to tumors with nanoparticulate formulations has been recognized as one of the potentially useful approaches to improve the poor tissue specificity of conventional cancer chemotherapy and this approach should also be applicable for more efficient tumor delivery of PS. In this review article, several issues concerning the efficacy of PDT using nanoparticle-based formulations are discussed and our recent attempts to temporally enhance the vascular permeability within tumors with photodynamic treatment for the better therapeutic outcome of nanoparticle-based therapy are introduced.

Improvement of Oral Bioavailability of N-251, a Novel Antimalarial Drug, by Increasing Lymphatic Transport with Long-Chain Fatty Acid-Based Self-Nanoemulsifying Drug Delivery System.

PURPOSE: The objective of this study was to improve the absorption behavior of N-251, a novel antimalarial drug, by preparing an appropriate self-nanoemulsifying drug delivery system (SNEDDS). METHODS: Two different types of SNEDDS formulations, medium-chain fatty acid-based SNEDDS (MC-SNEDDS) and long-chain fatty acid-based SNEDDS (LC-SNEDDS), were prepared based on pseudo-ternary phase diagram, and examined for their in vivo oral absorption behavior in rats. RESULTS: Oral dosing of MC-SNEDDS formulations significantly improved the bioavailability (BA) of N-251 compared with N-251 powders. However, its high hepatic extraction limited the BA of N-251 to only 0.49 for MC-SNEDDS B, the best formulation of MC-SNEDDS. LC-SNEDDS formulations, especially LC-SNEDDS F provided the highest BA, 0.65, and successfully attenuated the inter-individual difference in the absorption behavior. Furthermore, it was confirmed that lymphatic transport of N-251 for LC-SNEDDS F was significantly increased up to around 3.19 times larger than that for MC-SNEDDS B. Simulation study suggested that 20 to 39% of N-251 uptaken by the small intestine would be delivered to lymphatic system after oral administration of LC-SNEDDS F. CONCLUSIONS: SNEDDS formulations significantly improved the absorption behavior of N-251 and long-chain fatty acid-based lipid further improved it by avoiding the hepatic first-pass elimination.

Uptake Pathway of Styrene Maleic Acid Copolymer-Coated Lipid Emulsions Under Acidic Tumor Microenvironment.

The purpose of this study was to elucidate and compare styrene maleic acid copolymer (SMA)-coated lipid emulsions (SMA emulsions) uptake pathway in vascular endothelial cells and surrounding cancer cells under not only neutral but also acidic pH, which is often observed in tumor microenvironment. DiI-labeled SMA emulsions were prepared using 1-palmitoyl-2-oleoyl-sn­glycero-3-phosphocholine and triolein. In murine melanoma B16-BL6 (B16) cells and human umbilical vein endothelial cells (HUVEC), DiI-labeled SMA emulsions uptake under near-neutral (pH 7.4) and acidic (pH 6.0) conditions was determined by fluorescent analysis. SMA emulsions were taken up more efficiently into HUVEC than B16 cells under acidic condition in a temperature-dependent manner. Uptake study using endocytosis inhibitors showed that SMA emulsions were taken up by macropinocytosis and clathrin-mediated endocytosis in B16 cells. In HUVEC, however, they were taken up by clathrin- and caveolae-independent, but dynamin-dependent pathway. SMA emulsions would be internalized efficiently into vascular endothelial cells as well as cancer cells under acidic microenvironment via different endocytosis pathways. SMA emulsions could be a promising drug delivery carrier for anti-angiogenic drugs.

Possible Regulation of P-Glycoprotein Function by Adrenergic Agonists II: Study with Isolated Rat Jejunal Sheets and Caco-2 Cell monolayers.

To clarify the regulation of drug absorption by the enteric nervous system, we investigated how adrenergic agonists (adrenaline (ADR), clonidine (CLO), dobutamine (DOB)) and dibutyryl cAMP (DBcAMP) affected P-glycoprotein (P-gp) function by utilizing isolated rat jejunal sheets and Caco-2 cell monolayers. ADR and CLO significantly decreased the secretory transport (Papptotal) of rhodamine-123 and tended to decrease the transport via P-gp (PappP-gp) and passive transport (Papppassive). In contrast, DBcAMP significantly increased and DOB tended to increase Papptotal and both tended to increase PappP-gpand Papppassive. Changes in P-gp expression on brush border membrane by adrenergic agonists and DBcAMP were significantly correlated with PappP-gp, while P-gp expression was not changed in whole cell homogenates, suggesting that the trafficking of P-gp would be responsible for its functional changes. Papppassive was inversely correlated with transmucosal or transepithelial electrical resistance, indicating that adrenergic agonists affected the paracellular permeability. Adrenergic agonists also changed cAMP levels, which were significantly correlated with PappP-gp. Furthermore, protein kinase A (PKA) or PKC inhibitor significantly decreased PappP-gp in Caco-2 cell monolayers, suggesting that they would partly contribute to the changes in P-gp activity. In conclusion, adrenergic agonists regulated P-gp function and paracellular permeability, which would be caused via adrenoceptor stimulation.

Preparation of stable and monodisperse paclitaxel-loaded bovine serum albumin nanoparticles via intermolecular disulfide crosslinking.

Paclitaxel (PTX) is one of the most used anti-cancer drugs worldwide. Due to its insolubility in water, the clinically available liquid formulation of PTX contains Cremophor EL that is responsible for severe hypersensitivity. Albumin-based nanoparticles have emerged as a promising carrier for anti-cancer drugs because albumin nanoparticles have high capacity to not only load lipophilic drugs without solubilizer but also accumulate in tumor by both passive and active mechanisms. In this study, we attempted to prepare solvent-free formulation of PTX-loaded bovine serum albumin (BSA) nanoparticles with high stability, and the in vitro stability in serum were comparatively assessed between our PTX-loaded BSA nanoparticles and clinically used nanoparticulate albumin-bound PTX (Abraxane®). PTX-loaded BSA nanoparticles were prepared by intermolecular disulfide crosslinking. When BSA molecules were used without denaturation by guanidinium, the obtained BSA nanoparticles showed broad size distribution. On the other hand, the nanoparticles composed of denatured BSA by guanidinium had a uniform size around 100 nm. The PTX encapsulation efficiency of BSA nanoparticles were approximately 30-40 %. In addition, in vitro gel filtration analysis and dialysis study demonstrated that PTX-loaded BSA nanoparticles had higher colloidal stability and sustained PTX release property than Abraxane® in serum. These results suggest that BSA nanoparticles is a promising drug carrier for improving therapeutic efficacy of PTX and reducing its adverse effects.

Nanoparticle-based passive drug targeting to tumors: considerations and implications for optimization.

There are many potential barriers to the effective delivery of small-molecule drugs to solid tumors. Most small-molecule chemotherapeutic drugs have a large volume of distribution upon intravenous administration, which is often associated with a narrow therapeutic index due to their high level of toxicity in healthy tissues. Nanoparticle-based therapeutics for tumor targeting have emerged as one of the promising approaches to overcome the lack of tissue specificity of conventional chemotherapeutic drugs. Various different concepts have been envisioned for nanoparticle-mediated drug targeting. Among them, the passive drug targeting strategy has been the most widely investigated, and numerous preclinical studies have provided insights into the validity of the strategy. This review article briefly introduces our recent findings related to the passive drug targeting strategy including its application in anti-angiogenic therapy, along with considerations to be taken into account and implications for the rational design of a passive drug targeting strategy.

In vitro evaluation of the ciliary beat frequency of the rat nasal epithelium using a high-speed digital imaging system.

Mucociliary clearance (MC) is an important factor in determining nasal drug absorption and the ciliary beat of ciliated epithelial cells of the nasal mucosa is the driving force of MC. However, the relationship between MC and ciliary beat frequency (CBF) is still ambiguous. The purpose of this study was to establish an evaluation method of CBF as an index of mucociliary function and examine the relationship between MC and CBF. A sequence of images of ciliary beating of an excised rat nasal septum was captured using a high-speed digital video camera. CBF (beats per second, Hz) was determined from periodic changes in the contrast value of a specific location in a sequence of images. CBF under control conditions was 8.49±0.38 Hz, which is similar to values reported for cultured human nasal epithelial cells and rat tracheal cells. ß-Adrenergic and cholinergic antagonists decreased CBF, while ß-adrenergic agonists and acetylcholine increased CBF. These results were similar with those observed for MC in our previous study. It was found that CBFs were significantly and linearly correlated with MC, indicating that MC is directly regulated by CBF and that this evaluation system allows the quantitative determination of nasal mucociliary function.

Efficient Liposome Loading onto Surface of Mesenchymal Stem Cells via Electrostatic Interactions for Tumor-Targeted Drug Delivery.

Mesenchymal stem cells (MSCs) have a tumor-homing capacity; therefore, MSCs are a promising drug delivery carrier for cancer therapy. To maintain the viability and activity of MSCs, anti-cancer drugs are preferably loaded on the surface of MSCs, rather than directly introduced into MSCs. In this study, we attempted to load liposomes on the surface of MSCs by using the magnetic anionic liposome/atelocollagen complexes that we previously developed and assessed the characters of liposome-loaded MSCs as drug carriers. We observed that large-sized magnetic anionic liposome/atelocollagen complexes were abundantly associated with MSCs via electrostatic interactions under a magnetic field, and its cellular internalization was lower than that of the small-sized complexes. Moreover, the complexes with higher atelocollagen concentrations showed lower cellular internalization than the complexes with lower atelocollagen concentrations. Based on these results, we succeeded in the efficient loading of liposomes on the surface of MSCs by using large-sized magnetic anionic liposomes complexed with a high concentration of atelocollagen. The constructed liposome-loaded MSCs showed a comparable proliferation rate and differentiation potential to non-loaded MSCs. Furthermore, the liposome-loaded MSCs efficiently adhered to vascular endothelial cells and migrated toward the conditioned medium from cancer cells in vitro and solid tumor tissue in vivo. These findings suggest that liposome-loaded MSCs could serve as an efficient cell-based drug carrier for tumor-targeted delivery.