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.

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.

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.

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.

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.

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.

Pre-treatment of oncolytic reovirus improves tumor accumulation and intratumoral distribution of PEG-liposomes.

PEGylated liposomes (PEG-liposomes) are a promising drug delivery vehicle for tumor targeting because of their efficient tumor disposition profiles via the enhanced permeability and retention (EPR) effect. However, tumor targeting of PEG-liposomes, particularly their delivery inside the tumors, is often disturbed by physical barriers in the tumor, including tumor cells themselves, extracellular matrices, and interstitial pressures. In this study, B16 melanoma tumor-bearing mice were injected intravenously with oncolytic reovirus before administration of PEG-liposomes to enhance PEG-liposomes' tumor disposition. Three days after reovirus administration, significant expression of reovirus sigma 3 protein, elevation of apoptosis-related gene expression, and activation of caspase 3 in the tumors were found. Apoptotic cells were found inside the tumors. These data indicated that reovirus efficiently replicated in the tumors and induced apoptosis of tumor cells. The tumor disposition levels of PEG-liposomes were approximately doubled by reovirus pre-administration, compared with a PBS-pretreated group. PEG-liposomes were widely distributed in the tumors of reovirus-pretreated mice, whereas in the PBS-pretreated group, PEG-liposomes were found mainly around or inside the blood vessels in the tumors. Pre-treatment with reovirus also improved the tumor accumulation of PEG-liposomes in human pancreatic BxPC-3 tumors. 3D imaging analysis of whole BxPC-3 tumors demonstrated that pretreatment with reovirus led to the enhancement of PEG-liposome accumulation inside the tumors. Combination treatment with reovirus and paclitaxel-loaded PEG-liposomes (PTX-PEG-liposomes) significantly suppressed B16 tumor growth. These results provide important information for clinical use of combination therapy of reovirus and nanoparticle-based drug delivery system (DDS).

Nanocrystal Preparation of Poorly Water-Soluble Drugs with Low Metal Contamination Using Optimized Bead-Milling Technology.

Nanocrystal preparation using bead milling is an important technology to enhance the solubility of poorly water-soluble drugs. However, there are safety concerns regarding the metal contaminants generated during bead milling. We have previously reported optimized bead-milling parameters that could minimize metal contamination and demonstrated comparable performance to NanoCrystal®, a world-leading contamination-free technology. This study aimed to investigate the applicability of optimized milling parameters for preparing nanocrystals of several poorly water-soluble drugs exhibiting various physicochemical properties. Using our optimized bead-milling parameters, we found that all the tested drugs could be ground into nanosized particles within 360 min. Notably, fenofibrate, which has a low melting point, could be ground into nanosized particles owing to the low level of heat generated during bead milling. Additionally, the concentration of metal contaminants in all the drugs prepared using the optimized milling parameters were approximately ten to twentyfold lower than those prepared without the optimized parameters and were comparable to those prepared using polycarbonate beads, known to minimize metal contamination during bead milling. Our results provide insights into the development of drug nanocrystals with low metal contamination using bead milling.

Analysis of absorption-enhancing mechanisms for combinatorial use of spermine with sodium taurocholate in Caco-2 cells.

Previously, we reported that the combined use of spermine (SPM) and sodium taurocholate (STC) (SPM-STC) significantly improves the oral absorption of rebamipide (BCS class IV) and pulmonary absorption of interferon-α without any harmful histopathological changes in the gastrointestinal tract and lungs, respectively. In the present study, we examined the effect of SPM-STC on the transport of fluorescein isothiocyanate-labeled dextrans (FDs) across Caco-2 cell monolayers and attempted to clarify the mechanisms underlying the transport enhancement caused by SPM-STC. SPM-STC were found to significantly enhance the transport of FDs, while the treatment with SPM-STC was not harmful, and the decrease in transepithelial electrical resistance was transient and reversible. The voltage-clamp study clearly indicated that the opening of the paracellular route could be mainly responsible for the enhanced transport of FD-4. As for the mechanisms, it was found that SPM-STC caused a significant increase in membrane fluidity, which would lead to the enhanced transport of small-molecule drugs such as rebamipide. Since SPM-STC increased intracellular Ca2+ via Ca2+ uptake through Ca2+ channels and Ca2+ release from the endoplasmic reticulum stimulated by the IP3 pathway, the subsequent possible activation of the MLCK signaling pathway would have led to the contraction of the actin-myosin ring. The rearrangement of tight junction-constituting proteins induced through the MAPK pathway has also been suggested as a possible mechanism for opening tight junctions. Claudin-4, a key protein constituting the tight junction, merged with F-actin along with the plasma membrane, was significantly decreased, which would be at least partial structural evidence for the tight-junction opening.

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.

Effects of particle size and release property of paclitaxel-loaded nanoparticles on their peritoneal retention and therapeutic efficacy against mouse malignant ascites.

Malignant ascites accounts for abdominal pain, dyspnea and anorexia, all of which decrease quality of life in cancer patients. Intraperitoneal chemotherapy is a useful method for managing malignant ascites and nanoparticulate drug delivery system makes it more effective by increasing peritoneal retention of anti-cancer drugs. In this study, we prepared paclitaxel-loaded emulsions and liposomes with different particle sizes and drug release properties, and evaluated their peritoneal retention and therapeutic efficacy in Ehrlich's ascites carcinoma (EAC)-bearing mice. Liposomes with the size of 100 nm were rapidly absorbed from peritoneal cavity into blood after intraperitoneal injection into EAC-bearing mice, whereas 1000-nm liposomes were highly retained in peritoneal cavity. Accordingly, 1000 nm liposomes significantly prolonged survival time of EAC-bearing mice but did not inhibit the ascites accumulation because of too poor paclitaxel release. On the other hand, although peritoneal retention of emulsions themselves was similar irrespective of their sizes, 270-nm emulsions showed the higher PTX retention in ascites than other emulsions, and resulted in significantly prolonged survival time and lower accumulation of ascites in EAC-bearing mice. These results indicate that not only particle size but drug release property is one of key determinants of the biodisposition and therapeutic efficacy of intraperitoneally injected nanoparticulate PTX against malignant ascites.

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 Doxorubicin Release Rate From Polyethylene Glycol-Modified Liposome on Anti-tumor Activity in B16-BL6 Tumor-Bearing Mice.

To investigate the effect of doxorubicin (DOX) release rates from polyethylene glycol (PEG)-liposomes on the anti-tumor activity, several in-vitro and in-vivo studies were performed by utilizing three types of DOX-PEG-liposomes showing the slow (L-Slow), middle (L-Mid) and fast (L-Fast) release rates of DOX. L-Mid provided the highest anti-tumor activity in B16-BL6 tumor-bearing mice, although the largest amount of DOX distribution into the tumor tissue was observed in L-Slow-administered mice and the lowest was in L-Fast-administered mice. To elucidate the reason for this discrepancy, DOX distribution into cancer cells constituting the tumor tissue was determined and the highest DOX distribution into cancer cells was observed in L-Mid-administered mice. These results clearly indicate that the adequate drug release rate from liposome should make it possible to deliver the substantial amounts of drugs into cancer cells, leading to the actual anti-tumor activity.

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.

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.

Possible Regulation of P-glycoprotein Function by Adrenergic Agonists in a Vascular-luminal Perfused Preparation of Small Intestine.

Although the functions of small intestine are largely regulated by enteric nervous system (ENS), an independent intrinsic innervation, as well as central nervous system (CNS), the neural regulation of drug absorption from the small intestine still remains to be clarified. To obtain some information on it, the effect of adrenergic agonists on P-glycoprotein (P-gp) function was investigated by utilizing a vascular-luminal perfused rat small intestine. Adrenaline significantly decreased the secretion of rhodamine-123 (R-123) into the intestinal lumen, but dibutyryl cAMP (DBcAMP) significantly enhanced R-123 secretion. The inhibition study with quinidine clearly indicated that the decrease in secretory clearance of R-123 by adrenaline or the increase by DBcAMP would be attributed to the decrease or increase in P-gp activity, respectively. Expression levels of P-gp in whole mucosal homogenates were not changed at all by any chemicals examined, but those on brush border membrane (BBM) of intestinal epithelial cells were significantly decreased or increased by adrenaline or DBcAMP, respectively. Furthermore, changes in P-gp activity caused by adrenergic agonists and DBcAMP were significantly correlated with changes in expression level of P-gp in BBM, suggesting that the trafficking of P-gp from cytosolic pool to BBM would be regulated by adrenergic agonists and DBcAMP.

Sequential administration of PEG-Span 80 niosome enhances anti-tumor effect of doxorubicin-containing PEG liposome.

To improve the anti-tumor effect of polyethylene glycol-modified liposome containing doxorubicin (DOX-PEG liposome), the effect of sequential administration of PEG-Span 80 niosome was investigated for Colon-26 cancer cells (C26)-bearing mice. The concept of the current study is as follows: Since both particulates would be accumulated in the tumor tissue due to the enhanced permeability and retention (EPR) effect, PEG-Span 80 niosome, mainly composed of synthetic surfactant (Span 80), would interact with DOX-PEG liposome and be a trigger to induce the release of DOX from the liposome within the tumor tissue, leading to the improvement of anti-tumor effect of DOX-PEG liposome. To find out an adequate liposome for this strategy, several PEG liposomes with different compositions were examined in terms of drug release enhancement and it was found that PEG-Span80 niosome could significantly enhance the release of calcein and DOX from a PEG liposome composed of 90% hydrogenated soybean phosphatidylcholine (HSPC) and 10% cholesterol. The sequential administration of PEG-Span 80 niosome at 24 or 48 h after dosing of DOX-PEG liposome provided a higher anti-tumor effect than the single dose of DOX-PEG liposome in the C26-bearing mice. Particularly, the 24 h-later dosing of PEG-Span 80 niosome has been found to be more effective than the 48 h-later dosing. It was also confirmed that the coexistence of PEG-Span 80 niosome with DOX-PEG liposome in 50% serum or in 50% supernatant of tumor tissue homogenate significantly increased DOX release from PEG liposome, suggesting that DOX release from DOX-PEG liposome within tumor tissue would be enhanced via the interaction with PEG-Span 80 niosome. This strategy would lead to the safer and more inexpensive chemotherapy, since it could make it possible to provide the better anti-tumor effect by utilizing the lower dose of DOX.

miR-33a-5p in small extracellular vesicles as non-invasive biomarker for oxaliplatin sensitivity in human colorectal cancer cells.

microRNAs (miRNAs) contained in small extracellular vesicles (sEVs) are candidates for non-invasive biomarkers. Oxaliplatin (L-OHP) has been approved for advanced colorectal cancer (CRC) chemotherapy. However, the response to L-OHP differs among CRC patients. In addition, CRC cells often acquire the resistance to L-OHP. This study aimed at the prediction of L-OHP sensitivity by measuring extracellular miRNAs levels. Firstly, we compared intracellular miRNAs expressions in L-OHP-sensitive CRC cells (SW620 and HCT116 cells) with those in acquired and intrinsic L-OHP-resistant cells. In microarray and real-time RT-PCR analyses, the intracellular miR-33a-5p, miR-210-3p, and miR-224-5p expressions were lower in acquired and intrinsic L-OHP-resistant CRC cells than sensitive cells. Furthermore, in SW620 cells, L-OHP sensitivity was decreased by miR-33a-5p inhibitor. On the other hand, miR-210-3p or miR-224-5p inhibitor did not affect L-OHP sensitivity in SW620 cells. Secondly, the amount of miR-33a-5p, miR-210-3p, and miR-224-5p in sEVs was compared. The amount of miR-33a-5p and miR-210-3p in sEVs secreted from acquired and intrinsic L-OHP-resistant cells tended to be small. miR-224-5p was not detected in sEVs secreted from three types of CRC cells examined. To the best of our knowledge, this is the first study demonstrating that miR-33a-5p and/or miR-210-3p in sEVs would be candidates for biomarkers of L-OHP sensitivity. In particular, miR-33a-5p is a promising candidate because it would be directly involved in L-OHP sensitivity.

Improvement of lipid solubility and oral bioavailability of a poorly water- and poorly lipid-soluble drug, rebamipide, by utilizing its counter ion and SNEDDS preparation.

Among drugs in development and/or in market, there are poorly water-soluble and poorly lipid-soluble compounds. Rebamipide, classified into BCS class IV, is one of those drugs which provide very low bioavailability and/or the difficulty of formulation for oral administration. Because of its low solubility in available lipoidal excipients, it was impossible to prepare an adequate SNEDDS formulation of rebamipide. Then, we tried to increase the solubility of rebamipide in lipoidal excipients for preparing a more practical SNEDDS formulation by making the complex with its counter ion, tetrabutylphosphonium hydroxide (TBPOH) or NaOH. Rebamipide concentration in ethanol was proportionally increased with the increment of TBPOH or NaOH added, indicating that the formation of complex with a counter ion should contribute to the solubilization of rebamipide in ethanol. Both Rebamipide-TBPOH complex (Reb-TBPOH) and Rebamipide-NaOH complex (Reb-NaOH) obtained by lyophilization showed no endothermic peak in DSC and no diffraction peak in XRPD, suggesting that the solid state of both complexes should be amorphous. Reb-TBPOH maintained the dissolution of rebamipide in SNEDDS vehicle (Capryol 90:Cremophor EL:Transcutol P = 4:3:3) at 20 mg/g at least for 28 days, while Reb-NaOH did it at 10 mg/g. In vitro dissolution study showed that Reb-TBPOH SNEDDS and Reb-NaOH SNEDDS containing rebamipide at 10 mg/g maintained the complete dissolution of rebamipide in FaSSIF (intestinal luminal condition). In the gastric luminal condition (pH3.9 acetate buffer), the high concentration, close to the complete dissolution, was transiently observed and quickly decreased to one-sixth of the maximum, but it was still around 70 times higher than that of the crystalline powder. The additional utilization of Eudragit EPO for SNEDDS preparations of both complexes successfully maintained the high concentrations of rebamipide in the gastric luminal condition. In vivo oral absorption studies clearly indicated that SNEDDS preparations utilizing Reb-counter ion complex successfully improved rebamipide absorption.