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Yu Been Shin,1,* Ju-Yeon Choi,2,* Dae Hwan Shin,1 Jeong-Won Lee2,3 1College of Pharmacy, Chungbuk National University, Cheongju, 28160, Republic of Korea; 2Research Institute for Future Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea; 3Department of Obstetrics and Gynecology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea*These authors contributed equally to this workCorrespondence: Dae Hwan Shin, College of Pharmacy, Chungbuk National University, Osongsaengmyeong 1-ro, Osong-eup, Heungdeok-gu, Cheongju, 28160, Republic of Korea, Tel +82 43 261 2820, Fax +82 43 268 2732, Email dshin@chungbuk.ac.kr Jeong-Won Lee, Department of Obstetrics and Gynecology, Samsung Medical Center, Sungkyunkwan University School of Medicine, 81, Irwon-ro, Gangnam-gu, Seoul, 06351, South Korea, Tel +82-2-3410-1382, Fax +82-2-3410-0630, Email garden.lee@samsung.comPurpose: We aimed to inhibit ovarian cancer (OC) development by interfering with microtubule polymerization and inhibiting mTOR signaling. To achieve this, previously developed micelles containing fenbendazole and rapamycin were applied.Methods: Herein, we prepared micelles for drug delivery using fenbendazole and rapamycin at a 1:2 molar ratio and methoxy poly(ethylene glycol)-b-poly(caprolactone)(mPEG-b-PCL) via freeze-drying. We revealed their long-term storage capacity of up to 120 days. Furthermore, a cytotoxicity test was performed on the OC cell line HeyA8, and an orthotopic model was established for evaluating in vivo antitumor efficacy.Results: Fenbendazole/rapamycin-loaded mPEG-b-PCL micelle (M-FR) had an average particle size of 37.2 ± 1.10 nm, a zeta potential of − 0.07 ± 0.09 mV, and a polydispersity index of 0.20 ± 0.02. Additionally, the average encapsulation efficiency of fenbendazole was 75.7 ± 4.61% and that of rapamycin was 98.0 ± 1.97%. In the clonogenic assay, M-FR was 6.9 times more effective than that free fenbendazole/rapamycin. The in vitro drug release profile showed slower release in the combination formulation than in the single formulation.Conclusion: There was no toxicity, and tumor growth was suppressed substantially by our formulation compared with that seen with the control. The findings of our study lay a foundation for using fenbendazole and rapamycin for OC treatment.Keywords: ovarian cancer treatment, combination therapy, drug delivery, nanoformulation

Hee Ji Shin,1,* Min Jeong Jo,1,* Ik Sup Jin,1 Chun-Woong Park,1 Jin-Seok Kim,2 Dae Hwan Shin1 1College of Pharmacy, Chungbuk National University, Cheongju, 28160, Republic of Korea; 2Drug Information Research Institute (DIRI), College of Pharmacy, Sookmyung Women’s University, Seoul, 04310, Republic of Korea*These authors contributed equally to this workCorrespondence: Dae Hwan ShinCollege of Pharmacy, Chungbuk National University, Osongsaengmyeong 1-ro, Osong-eup, Heungdeok-gu, Cheongju, 28160, Republic of KoreaTel +82 43 261 2820Fax +82 43 268 2732Email dshin@chungbuk.ac.krPurpose: We aimed to develop a nanocarrier formulation incorporating fenbendazole (FEN) and rapamycin (RAPA) with strong efficacy against A549 cancer cells. As FEN and RAPA are poorly soluble in water, it is difficult to apply them clinically in vivo. Therefore, we attempted to resolve this problem by encapsulating these drugs in polymeric micelles.Methods: We evaluated drug synergy using the combination index (CI) values of various molar ratios of FEN and RAPA. We formed and tested micelles composed of different polymers. Moreover, we conducted cytotoxicity, stability, release, pharmacokinetic, and biodistribution studies to investigate the antitumor effects of FEN/RAPA-loaded mPEG-b-PCL micelles.Results: We selected mPEG-b-PCL-containing FEN and RAPA at a molar ratio of 1:2 because these particles were consistent in size and had high encapsulation efficiency (EE, %) and drug loading (DL, %) capacity. The in vitro cytotoxicity was assessed for various FEN, RAPA, and combined FEN/RAPA formulations. After long-term exposures, both the solutions and the micelles had similar efficacy against A549 cancer cells. The in vivo pharmacokinetic study revealed that FEN/RAPA-loaded mPEG-b-PCL micelles had a relatively higher area under the plasma concentration–time curve from 0 to 2 h (AUC0– 2 h) and 0 to 8 h (AUC0– 8 h) and plasma concentration at time zero (Co) than that of the FEN/RAPA solution. The in vivo biodistribution assay revealed that the IV injection of FEN/RAPA-loaded mPEG-b-PCL micelles resulted in lower pulmonary FEN concentration than the IV injection of the FEN/RAPA solution.Conclusion: When FEN and RAPA had a 1:2 molar ratio, they showed synergism. Additionally, using data from in vitro cytotoxicity, synergism between a 1:2 molar ratio of FEN and RAPA was observed in the micelle formulation. The FEN/RAPA-loaded mPEG-b-PCL micelle had enhanced bioavailability than the FEN/RAPA solution.Keywords: combination therapy, drug interaction, cytotoxicity, biodistribution, bioavailability

As an active pharmaceutical ingredient, dapagliflozin propanediol monohydrate (D-PD) has been used in the solvated form consisting of dapagliflozin compounded with (S)-propylene glycol and monohydrate at a 1:1:1 ratio. However, dapagliflozin propanediol loses the solvent's reduced lattice structure at slightly higher temperatures. Due to its sensitive solid-state stability, the temperature and humidity are strictly controlled during the production and storage of dapagliflozin. Thus, crystalline molecular complexes containing pharmaceutical salts, solvates, monohydrates, and cocrystals have recently been developed as alternative strategies. This study investigated the dapagliflozin free base (D-FB), D-PD, and dapagliflozin l-proline cocrystals (D-LP). Their solid-state behavior was also evaluated in stress stability studies. The compounds were analyzed using scanning electron microscopy (SEM), powder X-ray diffraction (PXRD), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), Fourier-transform infrared (FT-IR) spectroscopy, dynamic vapor sorption (DVS), and powder rheology testing. In addition, Carr's index, the Hausner ratio, contact angle, and intrinsic dissolution rate were calculated. Dapagliflozin exhibited distinct physical properties depending upon the differences in solid form and also showed significant differences in solid-state behavior in the stress stability test. In conclusion, D-LP was superior to D-FB or D-PD in physicochemical and mechanical properties.

Airborne particulate matter has been designated as a class 1 carcinogen by the World Health Organization. Nitrate is a toxic substance that accounts for a large proportion of particulate matter, and nitrate toxicity has long been reported. In this study, we aimed to optimize the adsorption and removal of particulate matter containing nitrate for effective elimination by the lungs. To this end, particles were designed to optimize the inhalation and removal efficiencies. These particles were prepared as chitosan-based particles containing N-acetylcysteine by using emulsion diffusion methods. Chitosan adsorbs nitrate, while N-acetylcysteine dissolves mucus. This removal mechanism has been found to occur in various in vitro models that mimic respiratory environments and in vivo models. In particular, the removal of exogenous substances, such as particulate matter, by the motility of respiratory cilia through mucolytic effect was investigated. This new approach for the adsorption and elimination of toxic substances entering the lungs represents an alternative defense mechanism against exposure to nitrates from air pollution.

Animals have been used for the testing of new drugs and nanoparticles to determine their safety and effectiveness before application in humans. Numerous drugs and nanoparticles have been developed through animal tests, and development in medical nanotechnology is progressing rapidly. However, a good experiment requires many animals. The primary measures to reduce animal experiments include reducing the number of animals used in each experiment and replacing laboratory animals with inanimate, cellular, or inferior animals to reduce sacrifices. Developing nanoparticles using simulations rather than experiments may be another alternative. Instead of animals, human cells or animal-derived organs, tissues, or cells could be used in experiments. These methods allow rapid toxicity and efficacy testing at low cost. However, the disadvantage of these methods is that they cannot accurately replicate the complex interrelationships between human organs, biological reactions to specific routes of administration, or toxicity of substances resulting from metabolic processes. To overcome these shortcomings of in vitro tests, new technologies such as organoids and organ-on-chips are progressing. These can be used to quickly examine the efficacy of newly developed drugs and nanoparticles and can be useful in developing patient-tailored agents. These technologies offer some promise but it is still difficult to entirely replace animal experiments. However, the scope of methods to replace experimental animals is widening, suggesting the potential to refine, reduce, and ultimately replace animal testing.

Insufficient efficacy of current single drug therapy of cancers have led to the advancement of combination drug-loaded formulations. Specifically, polymeric micelles have been focused on as efficient injectable vehicles for the delivery of several anticancer drugs simultaneously to cancer cells. These nano delivery systems have evolved with advancements in the area of nanotechnology. The current review presents a summary of the past events that have led to the procession of nanomicelles and novel nanotechnologies for combinatorial drug delivery. It also focuses on the advantages, disadvantages, and considerations for the design of nanotechnologies for combinatorial drug delivery systems. The opportunities and challenges of nanotechnologies in drug delivery to overcome current disadvantages are also discussed. Furthermore, we have added findings regarding the trends and perspectives regarding nanotechnologies for combinatorial anticancer drug delivery.

Orbitides are plant-derived small cyclic peptides with a wide range of biological activities. Phytochemical investigation of the whole plants of Dianthus chinensis was performed with the aim to discover new bioactive orbitides. Five undescribed proline-containing orbitides, dianthiamides A–E (1–5), were isolated from a methanolic extract of Dianthus chinensis. Their structures were elucidated by extensive analysis of 1D and 2D NMR and HRESI–TOF–MS as well as ESI–MS/MS fragmentation data. The absolute configuration of the amino acid residues of compounds 1–5 was determined by Marfey’s method. All compounds were tested for their cytotoxic activity, and dianthiamide A (1) exhibited weak activity against A549 cell line with IC50 value of 47.9 μM.

Coronavirus disease 2019 (COVID-19), caused by a new strain of coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is spreading rapidly worldwide. Nafamostat mesylate (NFM) suppresses transmembrane serine protease 2 and SARS-CoV-2 S protein-mediated fusion. In this study, pharmacokinetics and lung distribution of NFM, administered via intravenous and intratracheal routes, were determined using high performance liquid chromatography analysis of blood plasma, lung lumen using bronchoalveolar lavage fluid, and lung tissue. Intratracheal administration had higher drug delivery and longer residual time in the lung lumen and tissue, which are the main sites of action, than intravenous administration. We confirmed the effect of lecithin as a stabilizer through an ex vivo stability test. Lecithin acts as an inhibitor of carboxylesterase and delays NFM decomposition. We prepared inhalable microparticles with NFM, lecithin, and mannitol via the co-spray method. The formulation prepared using an NFM:lecithin:mannitol ratio of 1:1:100 had a small particle size and excellent aerodynamic performance. Spray dried microparticles containing NFM, lecithin, and mannitol (1:1:100) had the longest residual time in the lung tissue. In conclusion, NFM-inhalable microparticles were prepared and confirmed to be delivered into the respiratory tract, such as lung lumen and lung tissue, through in vitro and in vivo evaluations.

Three new naphthalene derivatives, namely mukungalenes A – C (1-3), together with three known ones, syriacusin B (4), syriacusin A (5) and parvifloral (6), were isolated from the roots of Hibiscus syriacus. The structures were determined on the basis of combined spectroscopic analysis and comparison with literature data. The isolated compounds dose-dependently inhibited the proliferation of A549 lung cancer cells. Among the isolated compounds (1∼6), compound 2 showed the most potent anti-proliferative activity on A549 cells with an IC50 value of 0.26 μM.

Introduction: In this study, a dual release bi-layer tablet containing Fesoterodine fumarate (Fst) 5 mg and Mirabegron (Mrb) 50 mg was prepared to investigate the different release behavior of each drug in bilayer tablet. The bilayer tablet was prepared based on monolayer-tablet formulation of each drug. Methods: The optimized bi-layer tablet showed an in vitro dissolution profile similar to commercial reference tablets Toviaz and Betmiga, based on a satisfactory similarity factor. Drug-release kinetics of each drug in the bilayer tablet were evaluated based on dissolution profiles. Drug-release behavior was evaluated by observing the surface of each layer by scanning electron microscopy and measuring the changes in weight and volume of the tablet during dissolution. Drug transfer between each layer was also investigated by Fourier -transform infrared spectroscopic imaging by observing the cross-section of the bilayer tablet cut vertically during dissolution. Results: The release of Fst was well suited for the Higuchi model, and the release of Mrb was well suited for the Hixson-crowell model. Compared with dissolution rate of each monolayer tablet, that of Fst in the bilayer tablet was slightly reduced (5%), but the dissolution rate of Mrb in bilayer tablet was dramatically decreased (20%). Also, a drug-release study confirmed that polymer swelling was dominant in Fst layer compared with polymer erosion, and degradation was dominant in MRB layer. Fourier-transform infrared imaging and 3-D image reconstruction showed that drug transfer in the bilayer tablet correlates with the results of drug-release behavior. Conclusion: These findings are expected to provide scientific insights in the development of a dual-release bilayer drug-delivery system for Fst and Mrb.

CPD409 is a novel sodium channel blocker for pain control in chemotherapy-induced peripheral neuropathy with a very poor water solubility of less than 10 μg/mL. The aim of this study is to improve the aqueous solubility of CPD409 by using inclusion complexes with β-cyclodextrin (β-CD) and 2-hydroxypropyl-β-cyclodextrin (HP-β-CD). The inclusion complexes were prepared by two different methods; common solvent evaporation (CSE) and freeze-drying (FD) method. The phase solubility study indicated that HP-β-CD showed 3-times higher formation constant with CPD409 than β-CD. Inclusion complexes at a molar ratio of 1:2 increased the solubility of CPD409 by 130 times when compared with pure CPD409. The physicochemical characterization of the prepared formulation confirmed the formation of CPD409/HP-β-CD inclusion complexes. In dissolution study, inclusion complexes at a molar ratio of 1:2 released 70% of CPD409 but CSE method showed more rapid dissolution profile than FD method. Pharmacokinetic study also showed an increased bioavailability in 1:2 formulation compared to pure CPD409. In conclusion, inclusion complexes of CSE and FD method at a molar ratio of 1:2 showed increased solubility, dissolution rate, and bioavailability of CPD409. Therefore, complexation of CPD409 with HP-β-CD using CSE and FD method at a molar ratio 1:2 can be promising strategy for effective oral delivery.

Itraconazole, which has been widely used as an antifungal-agent, is revisited as an anticancer drug but its low solubility still remains a major hurdle. Inclusion complex was used to enhance the solubility of itraconazole, followed by encapsulating into liposome for glioblastoma. Itraconazole-inclusion complex was well formed at 1:1, 1:2 and 1:3 molar ratios of itraconazole: hydroxypropyl-β-cyclodextrin (HP-β-CD) as determined by differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD) and Fourier transform infrared spectroscopy (FT-IR) analyses. Itraconazole-HP-β-CD inclusion complex was then encapsulated in liposome and its size was 120.5 ± 53.1 nm in diameter with 50% encapsulation efficiency. Stem cell-like property, as determined by the population ratio of CD90+/CD133+, was decreased from 3.38 to 1.46% when the U87-MG-TL cells were treated with 100 µM itraconazole/HP-β-CD-loaded liposome. Anti-proliferative effect of itraconazole/HP-β-CD-loaded liposome on U87-MG-TL cells was slightly better than that of free itraconazole (IC50 of 17 µM vs. 26 µM). Moreover, anti-proliferative effect of Itraconazole/HP-β-CD-loaded liposome on U87-MG-TL cells was higher than that of free itraconazole when the cells were co-treated with temozolomide (IC50 of 1.58 mM vs. 2.35 mM). Therefore, itraconazole/HP-β-CD-loaded liposomal formulation could serve as a promising strategy for targeting the glioblastoma multiforme.

Poly(ethylene glycol)-block-poly( d , l -lactic acid) (PEG-b-PLA) micelles affect drug solubilization, and a paclitaxel (PTX) loaded-PEG-b-PLA micelle (PTX-PM) is approved for cancer treatment due to injection safety and dose escalation (Genexol-PM®) compared to Taxol®. However, PTX-PM is unstable in blood, has rapid clearance, and causes dose-limiting toxicity. We have synthesized a prodrug for PTX (7-OH), using oligo(lactic acid) as a novel pro-moiety (o(LA)8-PTX) specifically for PEG-b-PLA micelles, gaining higher loading and slower release of o(LA)8-PTX over PTX. Notably, o(LA)8-PTX prodrug converts into PTX by a backbiting reaction in vitro, without requiring esterases. We hypothesize that o(LA)8-PTX-loaded PEG-b-PLA micelles (o(LA)8-PTX-PM) has a lower Cmax and higher plasma AUC than PTX-PM for improved therapeutic effectiveness. In Sprague-Dawley rats at 10 mg/kg, compared to o(LA)8-PTX-PM (10% w/w loading) and PTX-PM (10%), o(LA)8-PTX-PM (50% w/w loading) produces a 2- and 3-fold higher plasma AUC0–24 of PTX, lactic acid-PTX, and o(LA)2-PTX (o(LA)0-2-PTX), respectively. For o(LA)8-PTX-PM at 10 and 50% w/w loading, PTX and lactic acid-PTX are major bioactive metabolites, respectively. Fast prodrug conversion of o(LA)8-PTX in vivo versus in vitro (by backbiting) suggests that o(LA)8 is a good substrate for esterases. At 60 mg/kg (qwx3), o(LA)8-PTX-PM (50%) has higher antitumor activity than o(LA)8-PTX-PM (10%) and PTX-PM (10%) in a syngeneic 4T1-luc breast tumor model based on measurements of tumor volume, 4T1-luc breast tumor bioluminescence, and survival. Importantly, intravenous administration of o(LA)8-PTX-PM is well tolerated by BALB/c mice. In summary, oligo(lactic acid)8-PTX is more compatible than PTX with PEG-b-PLA micelles, more stable, and may expand the role of PEG-b-PLA micelles from “solubilizer” into “nanocarrier” for PTX as a next-generation taxane for cancer.

Fluvastatin (FLUVA), which is a common anti-hypercholesterolemia drug, exhibits potential anticancer activity as it suppresses the proliferation, angiogenesis, and metastasis of breast cancer cells via inhibiting 3-hydroxy-methyl glutaryl-coenzyme A (HMG-CoA) reductase. In this study, hyaluronan-conjugated FLUVA-encapsulating liposomes (HA-L-FLUVA) were evaluated for their anticancer efficacy in vitro and in vivo. The particle size, zeta potential, and encapsulation efficiency of HA-L-FLUVA were 158.36 &plusmn, 1.78 nm, &minus, 24.85 &plusmn, 6.26 mV, and 35%, respectively. Growth inhibition of breast cancer stem cells (BCSCs) by HA-L-FLUVA was more effective than that by free FLUVA. The half maximal inhibitory concentration (IC50) values of FLUVA, L-FLVUA, and HA-L-FLUVA were 0.16, 0.17, and 0.09 &mu, M, respectively. The in vivo anticancer effect of HA-L-FLUVA in combination with doxorubicin (DOX) was more effective than that of free FLUVA, free DOX, and HA-L-FLUVA. The longest survival of mice was achieved by treatment with FLUVA (15 mg/kg) and HA-L-FLUVA (15 mg/kg) + DOX (3 mg/kg), followed by HA-L-FLUVA (15 mg/kg), Dulbecco&rsquo, s phosphate buffered saline, and DOX (3 mg/kg). No more than 10% body weight loss was observed in the mice injected with FLUVA, indicating that the drug was not toxic. Taken together, these results indicate that HA-L-FLUVA could serve as an effective anticancer drug by inhibiting the growth of both breast cancer cells and cancer stem cells.

Fenbendazole (FEN), a broad-spectrum benzimidazole anthelmintic, suppresses cancer cell growth through various mechanisms but has low solubility and achieves low blood concentrations, which leads to low bioavailability. Solubilizing agents are required to prepare poorly soluble drugs for injections, however, these are toxic. To overcome this problem, we designed and fabricated low-toxicity Soluplus&reg, polymeric micelles encapsulating FEN and conducted toxicity assays in vitro and in vivo. FEN-loaded Soluplus&reg, micelles had an average particle size of 68.3 &plusmn, 0.6 nm, a zeta potential of &minus, 2.3 &plusmn, 0.2 mV, a drug loading of 0.8 &plusmn, 0.03%, and an encapsulation efficiency of 85.3 &plusmn, 2.9%. MTT and clonogenic assays were performed on A549 cells treated with free FEN and FEN-loaded Soluplus&reg, micelles. The in vitro drug release profile showed that the micelles released FEN more gradually than the solution. Pharmacokinetic studies revealed lower total clearance and volume of distribution and higher area under the curve and plasma concentration at time zero of FEN-loaded Soluplus&reg, micelles than of the FEN solution. The in vivo toxicity assay revealed that FEN-loaded Soluplus&reg, micelle induced no severe toxicity. Therefore, we propose that preclinical and clinical safety and efficacy trials on FEN-loaded Soluplus&reg, micelles would be worthwhile.

Fingolimod has been evaluated for use as an anticancer agent. However, many steps are required to synthesize fingolimod because of its intricate structure. A fingolimod analogue, N-(4-(2-((4-methoxybenzyl)amino)ethyl)phenyl)heptanamide (MPH), also has anti-cancer effects and is easier to synthesize but is poorly soluble in water. To compensate for its poor water solubility, MPH-loaded polymeric micelles were prepared by thin film hydration method using various polymers and the physicochemical properties of the MPH-loaded micelles such as particle size, drug-loading (DL, %), and encapsulation efficiency (EE, %) were evaluated. A storage stability test was conducted to select the final formulation and the release profile of the MPH-loaded micelles was confirmed by in vitro release assay. MPH-loaded mPEG-b-PLA micelles were selected for further testing based on their stability and physicochemical properties; they were stable for stable for 14 days at 4 °C and 25 °C and for 7 days at 37 °C. They showed anti-cancer efficacy against both A549 and U87 cancer cells. Encapsulation of MPH in polymeric micelles did not decrease the in vitro cytotoxicity of MPH. The findings of this study lay the groundwork for future formulations that enable the effective and stable delivery of poorly water-soluble agents.

The conventional treatment of pulmonary arterial hypertension (PAH) with oral bosentan hydrate has limitations related to the lack of pulmonary selectivity. In this study, we verified the hypothesis of the feasibility of dry powder inhalation of bosentan as an alternative to oral bosentan hydrate for the treatment of PAH. Inhalable bosentan microparticles with the capability of delivery to the peripheral region of the lungs and enhanced bioavailability have been formulated for PAH. The bosentan microparticles were prepared by the co-spray-drying method with bosentan hydrate and mannitol at different weight ratios. The bosentan microparticles were then characterized for their physicochemical properties, in vitro dissolution behavior, and in vitro aerodynamic performance. The in vivo pharmacokinetics and pathological characteristics were evaluated in a monocrotaline-induced rat model of PAH after intratracheal powder administration of bosentan microparticles, in comparison to orally administered bosentan hydrate. The highest performance bosentan microparticles, named SDBM 1:1, had irregular and porous shape. These microparticles had not only the significantly highest aerosol performance (MMAD of 1.91 μm and FPF of 51.68%) in the formulations, but also significantly increased dissolution rate, compared with the raw bosentan hydrate. This treatment to the lungs was also safe, as evidenced by the cytotoxicity assay. Intratracheally administered SDBM 1:1 elicited a significantly higher Cmax and AUC0-t that were over 10 times higher, compared with those of the raw bosentan hydrate administered orally in the same dose. It also exhibited ameliorative effects on monocrotaline-induced pulmonary arterial remodeling, and right ventricular hypertrophy. The survival rate of the group administrated SDBM1:1 intratracheally was 0.92 at the end of study (Positive control and orally administrated groups were 0.58 and 0.38, respectively). In conclusion, SDBM 1:1 showed promising in vitro and in vivo results with the dry powder inhalation. The inhaled bosentan microparticles can be considered as a potential alternative to oral bosentan hydrate for the treatment of PAH.

CD133+ cells in glioblastoma multiforme (GBM) display glioma stem cell (GSC) properties and have been considered to be the culprit in tumor recurrence, justifying the exploration of potential therapeutic modalities targeting CD133+ cells. Such strategies require a drug-delivery vehicle, and increasingly, the unique properties of PEGylated liposomes are being exploited for this purpose. More advanced liposomal delivery studies have suggested conjugation of CD133 antibodies as a suitable method for targeting GSCs. For targeting studies, GSCs separated from U87 GBM cells using a magnetic bead separation method were challenged with liposomes encapsulating gemcitabine (GEM) conjugated with a CD133 monoclonal antibody (PEG-lipo-CD133-GEM). An in vitro study showed that conjugation of CD133 antibody significantly enhanced the cytotoxicity of GEM through endocytosis of CD133 surface markers overexpressed on GSCs. The anti-tumor effect of PEG-lipo-CD133-GEM was 15 times higher than that of free GEM, presumably reflecting the specific targeting of the CD133 surface marker by PEG-lipo-CD133-GEM and the enhanced stability and cytotoxicity through the PEGylated liposome formulation in xenograft models. Moreover, monitoring of body weight changes showed that the use of PEGylated liposomes significantly reduced the toxicity of GEM. Taken together, our studies demonstrate that PEG-lipo-CD133-GEM shows promise for the treatment of gliomas in vitro and in xenograft models.

Background A larger optical zone for photorefractive keratectomy may improve optical quality and stability. However, there is need for limiting ablation diameter in that a larger ablation diameter requires greater ablation depth, and minimizing ablation depth may reduce adverse effects on postoperative wound healing, haze and keratoectasia. In this study, we compared the changes in clinical outcomes and the degree of regression between a 6.0 mm optical zone and 6.5 mm optical zone following PRK. Methods The records of 95 eyes that had undergone PRK with a 6.0 OZ (n = 40) and a 6.5 OZ (n = 55) were retrospectively reviewed. We compared data including the spherical equivalent of manifest refraction (SE of MR), simulated K (Sim K), thinnest corneal thickness, change in thinnest corneal thickness (the initial value divided by corrected diopter [ΔTCT/CD]), Q value, corneal higher order aberrations (HOAs) and spherical aberration (SA) pre-operation, at 3 and 6 months postoperative and at the last follow-up visit (Mean; 20.71 ± 10.52, 17.47 ± 6.57 months in the 6.0 and 6.5 OZ group, respectively). Results There were no significant differences in the SE of MR, Sim K and UDVA between the 6.0 OZ group and the 6.5 OZ group over 1 year of follow-up after PRK, and the 6.0 OZ group required less ΔTCT/CD than the 6.5 OZ group. The 6.5 OZ group showed better results in terms of post-operative HOAs of RMS, SA and Q value. When comparing that pattern of change in Sim K, there was no significant difference between the 6.0 OZ group and the 6.5 OZ group. Conclusions The clinical refractive outcomes and regression after PRK using Mel 90 excimer laser with a 6.0 OZ were comparable to those with a 6.5 OZ.

Rationale: Chitinase 3-like 1 (Chi3L1) protein is up-regulated in various diseases including solid cancers. According to Genome-Wide Association Study (GWAS)/Online Mendelian Inheritance in Man (OMIM)/Differentially Expressed Gene (DEG) analyses, Chi3L1 is associated with 38 cancers, and more highly associated with cancer compared to other oncogenes such as EGFR, TNFα, etc. However, the mechanisms and pathways by which Chi3L1 is associated with cancer are not clear. In current study, we investigated the role of Chi3L1 in lung metastasis. Methods: We performed the differentially expressed gene analysis to explore the genes which are associated with Chi3L1 using the web-based platform from Biomart. We investigated the metastases in lung tissues of C57BL/6 mice injected with B16F10 melanoma following treatment with Ad-shChi3L1. We also investigated the expression of USF1 and Chi3L1 in Chi3L1 KD mice lung tissues by Western blotting and IHC. We also analyzed lung cancer cells metastases induced by Chi3L1 using migration and cell proliferation assay in human lung cancer cell lines. The involvement of miR-125a-3p in Chi3L1 regulation was determined by miRNA qPCR and luciferase reporter assay. Results: We showed that melanoma metastasis in lung tissues was significantly reduced in Chi3L1 knock-down mice, accompanied by down-regulation of MMP-9, MMP-13, VEGF, and PCNA in Chi3L1 knock-down mice lung tissue, as well as in human lung cancer cell lines. We also found that USF1 was conversely expressed against Chi3L1. USF1 was increased by knock-down of Chi3L1 in mice lung tissues, as well as in human lung cancer cell lines. In addition, knock-down of USF1 increased Chi3L1 levels in addition to augmenting metastasis cell migration and proliferation in mice model, as well as in human cancer cell lines. Moreover, in human lung tumor tissues, the expression of Chi3L1 was increased but USF1 was decreased in a stage-dependent manner. Finally, Chi3L1 expression was strongly regulated by the indirect translational suppressing activity of USF1 through induction of miR-125a-3p, a target of Chi3L1. Conclusion: Metastases in mice lung tissues and human lung cancer cell lines were decreased by KD of Chi3L1. USF1 bound to the Chi3L1 promoter, however, Chi3L1 expression was decreased by USF1, despite USF1 enhancing the transcriptional activity of Chi3L1. We found that USF1 induced miR-125a-3p levels which suppressed Chi3L1 expression. Ultimately, our results suggest that lung metastasis is suppressed by knock-down of Chi3L1 through miR-125a-3p-mediated up-regulation of USF1.

Glioblastoma stem cells (GSCs), which are identified as subpopulation of CD133+/ALDH1+, are known to show resistance to the most of chemotherapy and radiation therapy, leading to the recurrence of tumor in glioblastoma multiforme (GBM) patients. Also, delivery of temozolomide (TMZ), a mainline treatment of GBM, to the GBM site is hampered by various barriers including the blood-brain barrier (BBB). A dual-targeting immunoliposome encapsulating TMZ (Dual-LP-TMZ) was developed by using angiopep-2 (An2) and anti-CD133 monoclonal antibody (CD133 mAb) for BBB transcytosis and specific delivery to GSCs, respectively. The size, zeta potential and drug encapsulation efficiency of Dual-LP-TMZ were 203.4nm in diameter, -1.6mV and 99.2%, respectively. The in vitro cytotoxicity of Dual-LP-TMZ against U87MG GSCs was increased by 425- and 181-folds when compared with that of free TMZ and non-targeted TMZ liposome (LP-TMZ) (10.3μM vs. 4380μM and 1869μM in IC50, respectively). Apoptosis and anti-migration ability of Dual-LP-TMZ in U87MG GSCs were also significantly enhanced comparing with those of free TMZ or LP-TMZ. In vivo study clearly showed a significant reduction in tumor size after intravenous administrations of Dual-LP-TMZ to the orthotopically-implanted brain tumor mice when compared with free TMZ or LP-TMZ. Increased life span (ILS) and median survival time (MST) of tumor-bearing mice were also increased when treated with Dual-LP-TMZ (211.2% in ILS and 49.2days in MST) than with free TMZ (0% in ILS and 23.3day in MST). These data indicate that conjugation of both An2 peptide and CD133 mAb to TMZ-encapsulating liposome is very effective in delivering the TMZ to GSCs via BBB, suggesting a potential use of Dual-LP-TMZ as a therapeutic modality for GBM.

Epothilones are microtubule inhibitors that are promising alternatives to paclitaxel due to enhanced anticancer efficacy. While epothilones are slightly more water soluble than paclitaxel and more active against paclitaxel-resistant cells, they still require formulation with Cremophor EL and/or cosolvents and drug resistance still limits therapeutic efficacy. In this report, we showed that the combinational treatment of epothilone B (EpoB), 17-N-allylamino-17-demethoxygeldanamycin (17-AAG, Hsp90 inhibitor), and rapamycin (mTOR inhibitor) displays strong anticancer activity in vitro and in vivo. To address the poor water solubility of this 3 drug-combination, they were co-loaded into poly(ethylene glycol)-block-poly(D,L-lactic acid) (PEG-b-PLA) micelles, and the 3-in-1 loaded PEG-b-PLA micelle (m-EAR) was characterized in terms of drug loading efficiency, particle size, release kinetics. The m-EAR achieved high levels of all three drugs in water; formed micelles with hydrodynamic diameters at ca. 30 nm and released the drugs in a sustained manner in vitro at rates slower than individually loaded PEG-b-PLA micelles. In A549-derived xenograft mice, m-EAR (2.0, 15.0, and 7.5 mg/kg) caused tumor regression after four weekly injections, whereas EpoB alone (2.0 mg/kg) was the same as control. No severe changes in body weight relative to PBS control were observed, attesting to the safety of m-EAR. Collectively, these results suggest that m-EAR provides a simple, but effective and safe EpoB-based combination nanomedicine for cancer therapy.

Alpinumisoflavone, a major compound in unripe Cudrania tricuspidata fruit is reported to exhibit numerous beneficial pharmacological activities, such as osteoprotective, antibacterial, estrogenic, anti-metastatic, atheroprotective, antioxidant, and anticancer effects. Despite its medicinal value, alpinumisoflavone is poorly soluble in water, which makes it difficult to formulate and administer intravenously (i.v.). To overcome these limitations, we used methoxy poly(ethylene glycol)-b-poly(d,l-lactide) (mPEG-b-PLA) polymeric micelles to solubilize alpinumisoflavone and increase its bioavailability, and evaluated their toxicity in vivo. Alpinumisoflavone-loaded polymeric micelles were prepared using thin-film hydration method, and their physicochemical properties were characterized for drug release, particle size, drug-loading (DL, %), and encapsulation efficiency (EE, %). The in vitro drug release profile was determined and the release rate of alpinumisoflavone from mPEG-b-PLA micelles was slower than that from drug solution, and sustained. Pharmacokinetic studies showed decreased total clearance and volume of distribution of alpinumisoflavone, whereas area under the curve (AUC) and bioavailability were significantly increased by incorporation in mPEG-b-PLA micelles. In vivo toxicity assay revealed that alpinumisoflavone-loaded mPEG-b-PLA micelles had no severe toxicity. In conclusion, we prepared an intravenous (i.v.) injectable alpinumisoflavone formulation, which was solubilized using mPEG-b-PLA micelles, and determined their physicochemical properties, pharmacokinetics, and toxicity profiles.

Hair is one of the key samples for judging drug abuse in the field of forensic science. However, few studies have examined synthetic cannabinoids and their metabolites in human hair. Synthetic cannabinoids are a class of chemicals that bind to cannabinoid receptors, but they differ structurally from the cannabinoids found in cannabis. They have been sold sprayed on dried, shredded plant material under brand names such as "Spice" since the 2000s. In South Korea, synthetic cannabinoids have been widely distributed since 2009 and many types detected up to now. Unlike traditional drugs such as methamphetamine and cannabis, the abuse trends of synthetic cannabinoids were variable by regions and changed according to the times. If new types of synthetic cannabinoids become popular which has been altered in some structures, it becomes difficult to identify using exist analytical method. Therefore, it is important to develop a new analytical method for synthetic cannabinoids currently being abused in society. In this study, we developed simultaneous analytical methods for the detection of 18 synthetic cannabinoids and 41 of their metabolites in authentic human hair samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Selectivity, linearity, limits of detection (LODs), limits of quantification (LOQs), precision, accuracy, matrix effect, recovery, and process efficiency were evaluated, and all results were acceptable. Additionally, the distribution of synthetic cannabinoids in the head hair of Korean drug abusers from 2016 to 2018 was investigated. Hair samples from 43 individuals suspected of synthetic cannabinoid use were provided by law enforcement agencies. The drugs detected most prevalently in the head hair of Korean drug abusers were AB-CHMINACA and JWH-210.

Background: To compare the changes in clinical outcomes and the degree of regression between a 6.0 mm optical zone (6.0 OZ) and 6.5 mm optical zone (6.5 OZ) following photorefractive keratectomy (PRK). Methods: The records of 95 eyes that had undergone PRK with a 6.0 OZ (n = 40) and a 6.5 OZ (n = 55) were retrospectively reviewed. We compared data including the spherical equivalent of manifest refraction (SE of MR), simulated K (Sim K), thinnest corneal thickness, change in thinnest corneal thickness (the initial value divided by corrected diopter [ΔTCT/CD]), Q value, corneal higher order aberrations (HOAs) and spherical aberration (SA) pre-operation, at 3 and 6 months postoperative and at the last follow-up visit (Mean; 20.71 ± 10.52, 17.47 ± 6.57 months in the 6.0 and 6.5 OZ group, respectively). Results: There were no significant differences in the SE of MR, Sim K and UCVA between the 6.0 OZ group and the 6.5 OZ group over 1 year of follow-up after PRK, and the 6.0 OZ group required less ΔTCT/CD than the 6.5 OZ group. The 6.5 OZ group showed better results in terms of post-operative HOAs of RMS, SA and Q value. When comparing that pattern of change in Sim K, there was no significant difference between the 6.0 OZ group and the 6.5 OZ group. Conclusions: The clinical refractive outcomes and regression after PRK using Mel 90 excimer laser with a 6.0 OZ were comparable to those with a 6.5 OZ.

To investigate the risk factors for development of spontaneous consecutive exotropia (ScXT) among patients with refractive and nonrefractive accommodative esotropia (AET). Retrospective. Patients who were diagnosed with AET were reviewed from January, 2000 to December, 2016. The patients who developed ScXT after well corrected hyperopia were defined as exodeviation group (n = 51), and the patients who did not show exodeviation and were well controlled with eyeglasses were defined as the control group (n = 117). The changes in cycloplegic refraction, mean angle deviation at initial visit, time till the first correction of esodeviation, presence of amblyopia and accompanying strabismus were compared between the two groups. The mean interval from the first visit to correction of esodeviation under 8 PD in the exodeviation group was shorter than of the control group (P = 0.008). Patients in the exodeviation group showed more dissociated vertical deviation (DVD) (P = 0.015) and faster reduction in hyperopia per year (more hyperopic eye: P = 0.006; less hyperopic eye: P = 0.034) than the patients in the control group. Exodeviation was found mean 42.31 ± 41.13 months after hyperopia correction. There were no differences in angle deviation at initial visit, and presence of amblyopia. ScXT can be found in AET with faster reduction in hyperopia per year, accompanied by DVD, or in eyes with esodeviation corrected in relatively shorter time. It can be noted even in patients with good alignment over a long-term, so long-term follow-up is recommended.

Introduction: In this study, a dual release bi-layer tablet containing Fesoterodine fumarate (Fst) 5 mg and Mirabegron (Mrb) 50 mg was prepared to investigate the different release behavior of each drug in bilayer tablet. The bilayer tablet was prepared based on monolayer-tablet formulation of each drug. Methods: The optimized bi-layer tablet showed an in vitro dissolution profile similar to commercial reference tablets Toviaz and Betmiga, based on a satisfactory similarity factor. Drug-release kinetics of each drug in the bilayer tablet were evaluated based on dissolution profiles. Drug-release behavior was evaluated by observing the surface of each layer by scanning electron microscopy and measuring the changes in weight and volume of the tablet during dissolution. Drug transfer between each layer was also investigated by Fourier -transform infrared spectroscopic imaging by observing the cross-section of the bilayer tablet cut vertically during dissolution. Results: The release of Fst was well suited for the Higuchi model, and the release of Mrb was well suited for the Hixson-crowell model. Compared with dissolution rate of each monolayer tablet, that of Fst in the bilayer tablet was slightly reduced (5%), but the dissolution rate of Mrb in bilayer tablet was dramatically decreased (20%). Also, a drug-release study confirmed that polymer swelling was dominant in Fst layer compared with polymer erosion, and degradation was dominant in MRB layer. Fourier-transform infrared imaging and 3-D image reconstruction showed that drug transfer in the bilayer tablet correlates with the results of drug-release behavior. Conclusion: These findings are expected to provide scientific insights in the development of a dual-release bilayer drug-delivery system for Fst and Mrb.

Lacosamide (LCM) is a third-generation antiepileptic drug. Selective action of the drug on voltage-gated sodium channels reduces side effects. Oral administration of LCM shows good pharmacokinetic profile. However, the bitter taste of LCM is a barrier to the development of oral formulations. In this study, we aimed to prepare encapsulated LCM microparticles (MPs) for masking its bitter taste. Encapsulated LCM MPs were prepared with Eudragit&reg, E100 (E100), a pH-dependent polymer, by spray drying. Three formulations comprising different ratios of LCM and E100 (3:1, 1:1, and 1:3) were prepared. Physicochemical tests showed that LCM was in an amorphous state in the prepared formulations, and they were not miscible. LCM-E100 (1:3) had a rough surface due to surface enrichment of LCM. Increased E100 ratio in LCM-E100 MPs resulted in better taste-making effectiveness: LCM-E100 (1:1) and LCM-E100 (1:3) showed good taste-masking effectiveness, while LCM-E100 (3:1) could not mask the bitter taste of LCM. Dissolution results of the prepared formulations showed good correlation with taste-masking effectiveness. Nevertheless, high E100 ratio reduced the stability of the prepared formulations. Especially the difference in initial dissolution profile observed for LCM-E100 (1:3) indicated rapid reduction in taste-masking effectiveness and surface recrystallization. Therefore, LCM-E100 formulation in the ratio of 1:1 was selected as the best formulation with good taste-masking effectiveness and stability.

Purpose&mdash, This study was conducted to develop an automated detection algorithm for screening fundus abnormalities, including age-related macular degeneration (AMD), diabetic retinopathy (DR), epiretinal membrane (ERM), retinal vascular occlusion (RVO), and suspected glaucoma among health screening program participants. Methods&mdash, The development dataset consisted of 43,221 retinal fundus photographs (from 25,564 participants, mean age 53.38 &plusmn, 10.97 years, female 39.0%) from a health screening program and patients of the Kangbuk Samsung Hospital Ophthalmology Department from 2006 to 2017. We evaluated our screening algorithm on independent validation datasets. Five separate one-versus-rest (OVR) classification algorithms based on deep convolutional neural networks (CNNs) were trained to detect AMD, ERM, DR, RVO, and suspected glaucoma. The ground truth for both development and validation datasets was graded at least two times by three ophthalmologists. The area under the receiver operating characteristic curve (AUC), sensitivity, and specificity were calculated for each disease, as well as their macro-averages. Results&mdash, For the internal validation dataset, the average sensitivity was 0.9098 (95% confidence interval (CI), 0.8660&ndash, 0.9536), the average specificity was 0.9079 (95% CI, 0.8576&ndash, 0.9582), and the overall accuracy was 0.9092 (95% CI, 0.8769&ndash, 0.9415). For the external validation dataset consisting of 1698 images, the average of the AUCs was 0.9025 (95% CI, 0.8671&ndash, 0.9379). Conclusions&mdash, Our algorithm had high sensitivity and specificity for detecting major fundus abnormalities. Our study will facilitate expansion of the applications of deep learning-based computer-aided diagnostic decision support tools in actual clinical settings. Further research is needed to improved generalization for this algorithm.

Docetaxel (DTX), a taxane-based anticancer drug, and osthol (OTH), a coumarin-derivative compound, have shown anticancer effects against different types of cancers through various mechanisms. However, these drugs have low solubility in water and low oral bioavailability, and thus their clinical application is difficult. To overcome these problems, we encapsulated DTX and OTH in methoxy poly(ethylene glycol)-b-poly(caprolactone) (mPEG-b-PCL) and conducted studies in vitro and in vivo. We selected a 1:4 ratio as the optimal ratio of DTX and OTH, through combination index analysis in A549 cancer cells, and prepared micelles to evaluate the encapsulation efficiency, drug loading, particle size, and zeta potential. The in vitro drug-release profile showed that DTX/OTH-loaded mPEG-b-PCL micelles could slowly release DTX and OTH. In the clonogenic assay, DTX/OTH-loaded mPEG-b-PCL micelles showed 3.7 times higher inhibitory effect than the DTX/OTH solution. Pharmacokinetic studies demonstrated that micelles in combination with DTX and OTH exhibited increased area under curve and decreased clearance values, as ​​compared with single micelles.

To overcome cancer, various chemotherapeutic studies are in progress; among these, studies on nano-formulated combinatorial drugs (NFCDs) are being actively pursued. NFCDs function via a fusion technology that includes a drug delivery system using nanoparticles as a carrier and a combinatorial drug therapy using two or more drugs. It not only includes the advantages of these two technologies, such as ensuring stability of drugs, selectively transporting drugs to cancer cells, and synergistic effects of two or more drugs, but also has the additional benefit of enabling the spatiotemporal and controlled release of drugs. This spatial and temporal drug release from NFCDs depends on the application of nanotechnology and the composition of the combination drug. In this review, recent advances and challenges in the control of spatiotemporal drug release from NFCDs are provided. To this end, the types of combinatorial drug release for various NFCDs are classified in terms of time and space, and the detailed programming techniques used for this are described. In addition, the advantages of the time and space differences in drug release in terms of anticancer efficacy are introduced in depth.

Amphiphilic block copolymers (ABCs) assemble into a spherical nanoscopic supramolecular core/shell nanostructure termed a polymeric micelle that has been widely researched as an injectable nanocarrier for poorly water-soluble anticancer agents. The aim of this review article is to update progress in the field of drug delivery towards clinical trials, highlighting advances in polymeric micelles used for drug solubilization, reduced off-target toxicity and tumor targeting by the enhanced permeability and retention (EPR) effect. Polymeric micelles vary in stability in blood and drug release rate, and accordingly play different but key roles in drug delivery. For intravenous (IV) infusion, polymeric micelles that disassemble in blood and rapidly release poorly water-soluble anticancer agent such as paclitaxel have been used for drug solubilization, safety and the distinct possibility of toxicity reduction relative to existing solubilizing agents, e.g., Cremophor EL. Stable polymeric micelles are long-circulating in blood and reduce distribution to non-target tissue, lowering off-target toxicity. Further, they participate in the EPR effect in murine tumor models. In summary, polymeric micelles act as injectable nanocarriers for poorly water-soluble anticancer agents, achieving reduced toxicity and targeting tumors by the EPR effect.

Fungizone® (AmB-SD), amphotericin B solubilized by sodium deoxycholate, contains a highly aggregated form of the antifungal agent that causes dose-limiting renal toxicity. With the aim of reducing the formulation's toxicity by co-delivering monomeric amphotericin B (AmB) and sodium supplementation, we deaggregated AmB-SD with FDA-approved excipient PEG-DSPE in 0.9% NaCl-USP. Herein, we describe a reformulated AmB-SD with PEG-DSPE micelles that results in a less toxic drug with maintained antifungal activity.We compared the aggregation state and particle size of AmB-SD alone or combined with PEG-DSPE micelles. In vitro hemolytic activity and in vivo renal toxicity were measured to determine the toxicity of different formulations. In vitro antifungal assays were performed to determine differences in efficacy among formulations.PEG-DSPE micelles in saline deaggregated AmB-SD. Deaggregated AmB-SD exhibited significantly reduced in vitro and in vivo toxicity. In vitro antifungal studies showed no difference in minimum inhibitory and fungicidal concentrations of AmB-SD combined with PEG-DSPE relative to the drug alone.Reformulation of AmB-SD with PEG-DSPE micelles in saline facilitates co-delivery of monomeric AmB and sodium supplementation, potentially reducing the dose-limiting nephrotoxicity of AmB-SD. Ease of preparation and commercially available components lead us to acknowledge its potential for clinical use.