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Jun Soo Park,1 Min Seop Kim,1 Min Yeong Joung,1 Hyun Jin Park,1 Myoung-Jin Ho,1 Jun Hyuk Choi,1 Jae Hee Seo,1 Woo Heon Song,1 Young Wook Choi,2 Sangkil Lee,3 Yong Seok Choi,1 Myung Joo Kang1 1College of Pharmacy, Dankook University, Cheonan, Republic of Korea; 2College of Pharmacy, Chung-Ang University, Seoul, Republic of Korea; 3College of Pharmacy, Keimyung University, Daegu, Republic of KoreaCorrespondence: Myung Joo Kang, College of Pharmacy, Dankook University, Dongnam-gu, Cheonan, 31116, Republic of Korea, Tel +82 41 550 1446, Fax +82 41 550 7899, Email kangmj@dankook.ac.krBackground: Montelukast (MTK), a representative leukotriene receptor antagonist, is currently being investigated as a potential candidate for treating Alzheimer’s disease. For potent and effective dosing in elderly patients, a parenteral prolonged delivery system is favored, with improved medication adherence with reduced dosage frequency.Purpose: This study aimed to design a nanocrystalline suspension (NS)-based MTK prolonged delivery system and evaluate its pharmacokinetics profile and local tolerability following subcutaneous administration.Methods: To decelerate the dissolution rate, the amorphous MTK raw material was transformed into a crystalline state using a solvent-mediated transformation method and subsequently formulated into NS using a bead-milling technique. The MTK NSs were characterized by morphology, particle size, crystallinity, and in vitro dissolution profiles. The pharmacokinetic profile and local tolerability at the injection site following subcutaneous injection of MTK suspension were evaluated in rats.Results: Microscopic and physical characterization revealed that the amorphous MTK powder was lucratively transformed into a crystalline form in acidic media (pH 4). MTK crystalline suspensions with different diameters (200 nm, 500 nm, and 3 μm) were uniformly prepared using bead-milling technology, employing polysorbate 80 as suspending agent. Prepared crystalline suspensions exhibited analogous crystallinity (melting point, 150°C) and size-dependent in vitro dissolution profiles. MTK NSs with particle sizes of 200 nm and 500 nm provided a protracted pharmacokinetic profile for up to 4 weeks in rats, with a higher maximum drug concentration in plasma than the 3 μm-sized injectable suspensions. Histopathological examination revealed that MTK NS caused chronic granulomatous inflammation at the injection site, which resolved after 4 weeks.Conclusion: The MTK parenteral NS delivery system is expected to be a valuable tool for treating Alzheimer’s disease with extended dose intervals.Graphical Abstract: Keywords: montelukast, parenteral prolonged release delivery, crystallinity, nanocrystalline suspension, bead-milling, pharmacokinetics, local tolerability

Eun A Kim, Jun Soo Park, Min Seop Kim, Min Young Jeong, Hyun Jin Park, Jun Hyuk Choi, Jae Hee Seo, Yong Seok Choi, Myung Joo Kang College of Pharmacy, Dankook University, Cheonan, Chungnam, 330-714, KoreaCorrespondence: Myung Joo KangCollege of Pharmacy, Dankook University, 119 Dandae-ro, Dongnam-gu, Cheonan, 330-714, KoreaTel +82 41 550 1446Fax +82 41 550 7899. Email kangmj@dankook.ac.krBackground: The titrated extract of Centella asiatica (CA) has received much attention as a cosmeceutical ingredient owing to its anti-wrinkle effect. However, due to the low solubility and high molecular weight of pharmacologically active constituents, including asiatic acid (AA), madecassic acid (MA), and asiaticoside (AS), it is challenging to fabricate high-payload topical preparations of CA with satisfactory skin absorption profiles.Purpose: This study aimed to design a high-payload topical preparation of CA using nanocrystallization technique and to evaluate its skin absorption profile and local tolerability.Methods: High-payload nanocrystal suspensions (NSs) were prepared using lab-scale bead-milling technology, by adjusting the type and amount of suspending agent, CA content, type of vehicle, and milling speed. CA-loaded NSs were characterized in terms of morphology, particle size, crystallinity, and in vitro dissolution pattern. Skin absorption of CA nanocrystals was evaluated using a vertical Franz diffusion cell mounted with porcine skin. In vivo skin irritation following topical application of high-payload NS was assessed in normal rats.Results: The optimized NS system, composed of 10% (w/v) CA, 0.5% polyvinylpyrrolidone (PVP) K30 as steric stabilizer, and 89.5% of distilled water, was characterized as follows: spherical or elliptical in shape, 200 nm in size, with low crystallinity. The in vitro dissolution of AA or MA from NSs was markedly faster compared to raw material, under sink condition. Penetration of AA, MA, and AS in the porcine skin was markedly elevated using the high-payload NS formula, providing 5-, 4-, and 4.5-fold higher accumulation in skin layer, compared to that of the marketed cream formula (CA 1%, Madeca cream). Moreover, topical application of high-payload NS was tolerable, showing neither erythema nor oedema in normal rats.Conclusion: The novel NS system is expected to be a virtuous approach for offering a better skin absorption of CA, without using an excess quantity of solubilizers.Keywords: Centella asiatica, asiatic acid, madecassic acid, asiaticoside, nanocrystal suspension, bead-milling, dissolution, skin absorption, skin irritation

Yoon Tae Goo,1,* Cheol-Ki Sa,1,* Ji Yeh Choi,2 Min Song Kim,1 Chang Hyun Kim,1 Hyeon Kyun Kim,1 Young Wook Choi1 1College of Pharmacy, Chung-Ang University, Seoul, Republic of Korea; 2Department of Psychology, York University, Toronto, Ontario, Canada*These authors contributed equally to this workCorrespondence: Young Wook ChoiCollege of Pharmacy, Chung-Ang University, 84 Heuksuk-Ro, Dongjak-Gu, Seoul, 06974, KoreaTel +82 2 820 5609Email ywchoi@cau.ac.krPurpose: To enhance the oral bioavailability of revaprazan (RVP), a novel solid, supersaturable micelle (SSuM) was developed.Methods: Surfactants and solid carriers were screened based on a solubility and a flowability test, respectively. Supersaturating agents, including Poloxamer 407 (P407), were screened. The SSuM was optimized using a Box-Behnken design with three independent variables, including Gelucire 44/14:Brij L4 (G44/BL4; X1) and the amounts of Florite PS-10 (FLO; X2) and Vivapur 105 (VP105; X3), and three response variables, ie, dissolution efficiency at 30 min (Y1), dissolution enhancing capacity (Y2), and Carr’s index (Y3). The solid state property was evaluated, and a dissolution test was conducted. RVP, Revanex®, solid micelle (P407-free from the composition of SSuM), and SSuM were orally administrated to rats (RVP 20 mg equivalent/kg) for in vivo pharmacokinetic study.Results: G44 and BL4 showed great solubility, with a critical micelle concentration range of 119.2– 333.0 μg/mL. P407 had an excellent supersaturating effect. FLO and VP105 were selected as solid carriers, with a critical solidifying ratio (g/mL) of 0.30 and 0.91, respectively. With optimized values of X1 (– 0.41), X2 (0.31), and X3 (– 0.78), RVP (200 mg)-containing SSuM consisting of G44 (253.8 mg), BL4 (106.2 mg), FLO (99.3 mg), VP105 (199.8 mg), and P407 (40 mg) was developed, resulting in Y1 (40.3%), Y2 (0.008), and Y3 (12.3%). RVP existed in an amorphous state in the optimized SSuM, and the SSuM formed a nanosized dispersion in the aqueous phase, with approximately 71.7% dissolution at 2 h. The optimized SSuM improved the relative bioavailability of RVP in rats by approximately 478%, 276%, and 161% compared to raw RVP, Revanex®, and solid micelle, respectively.Conclusion: The optimized SSuM has great potential for the development of solidified formulations of poorly water-soluble drugs with improved oral absorption.Keywords: revaprazan, supersaturation, solid micelle, Box-Behnken design, dissolution, oral bioavailability

Muhammad Naeem, Jiafu Cao, Moonjeong Choi, Woo Seong Kim, Hyung Ryong Moon, Bok Luel Lee, Min-Soo Kim, Yunjin Jung, Jin-Wook Yoo College of Pharmacy, Pusan National University, Busan, South Korea Abstract: Current colon-targeted drug-delivery approaches for colitis therapy often utilize single pH-triggered systems, which are less reliable due to the variation of gut pH in individuals and in disease conditions. Herein, we prepared budesonide-loaded dual-sensitive nanoparticles using enzyme-sensitive azo-polyurethane and pH-sensitive methacrylate copolymer for the treatment of colitis. The therapeutic potential of the enzyme/pH dual-sensitive nanoparticles was evaluated using a rat colitis model and compared to single pH-triggered nanoparticles. Clinical activity scores, colon/body weight ratios, myeloperoxidase activity, and proinflammatory cytokine levels were markedly decreased by dual-sensitive nanoparticles compared to single pH-triggered nanoparticles and budesonide solution. Moreover, dual-sensitive nanoparticles accumulated selectively in inflamed segments of the colon. In addition, dual-sensitive nanoparticle plasma concentrations were lower than single pH-triggered nanoparticles, and no noticeable in vitro or in vivo toxicity was observed. Our results demonstrate that enzyme/pH dual-sensitive nanoparticles are an effective and safe colon-targeted delivery system for colitis therapy. Keywords: azo-polyurethane, methacrylate copolymer, budesonide, colon-targeted nanoparticles, colitis

Yu-Ri Kim,1,* Seung-Young Lee,3,* Eun Jeong Lee,1 Sung Ha Park,4 Nak-won Seong,3 Heung-Sik Seo,3 Sung-Sup Shin,3 Seon-Ju Kim,3 Eun-Ho Meang,3 Myeong-Kyu Park,3 Min-Seok Kim,3 Cheol-Su Kim,5 Soo-Ki Kim,5 Sang Wook Son,2 Young Rok Seo,6 Boo Hyon Kang,7 Beom Seok Han,8 Seong Soo A An,9 Beom-Jun Lee,10 Meyoung-Kon Kim1 1Department of Biochemistry and Molecular Biology, 2Department of Dermatology, Korea University Medical School and College, 3General Toxicology Team, Korea Testing and Research Institute, Seoul, Republic of Korea; 4Department of Biochemistry, University of Bath, Bath, UK; 5Department of Microbiology, Wonju College of Medicine, Yonsei University, Gangwon, 6Department of Life Science, Institute of Environmental Medicine for Green Chemistry, Dongguk University, Seoul, 7Nonclinical Research Institute, Chemon Inc, Gyeonggi, 8Toxicological Research Center, Hoseo University, Chungnam, 9Department of Bionanotechnology, Gachon University, Gyeonggi, 10College of Veterinary Medicine, Chungbuk National University, Chungbuk, Republic of Korea *These authors contributed equally to this work Abstract: This study was undertaken to investigate the potential toxicity and establish the no observed adverse effect level (NOAEL) and target organ(s) of negatively charged colloidal silica particles of different sizes, ie, SiO2EN,20(-) (20 nm) or SiO2EN,100(-) (100 nm), administered by gavage in Sprague-Dawley rats. After verification of the physicochemical properties of the SiO2 particles to be tested, a preliminary dose range-finding study and 90-day repeated dose study were conducted according to the Organisation for Economic Cooperation and Development test guideline. Based on the results of the 14-day dose range-finding study, a high dose was determined to be 2,000 mg/kg, and middle and low doses were set at 1,000 and 500 mg/kg, respectively. In the 90-day toxicity study, there were no animal deaths in relation to administration of SiO2 particles of either size. In addition, no treatment-related clinical changes or histopathological findings were observed in any of the experimental groups. Moreover, no difference in toxic effects from chronic exposure to SiO2EN,20(-) (20 nm) or SiO2EN,100(-) (100 nm) was observed. The results of this study indicate that the NOAEL for SiO2EN,20(-) and SiO2EN,100(-) would most likely be 2,000 mg/kg, and no target organ was identified in rats of either sex. Keywords: silica nanoparticles, particle size, 90-day oral dose toxicity, no observed adverse effect level

Yu-Ri Kim,1,* Jong-Il Park,2,* Eun Jeong Lee,1 Sung Ha Park,3 Nak-won Seong,2 Jun-Ho Kim,2 Geon-Yong Kim,2 Eun-Ho Meang,2 Jeong-Sup Hong,2 Su-Hyon Kim,2 Sang-Bum Koh,2 Min-Seok Kim,2 Cheol-Su Kim,4 Soo-Ki Kim,4 Sang Wook Son,5 Young Rok Seo,6 Boo Hyon Kang,7 Beom Seok Han,8 Seong Soo A An,9 Hyo-In Yun,9 Meyoung-Kon Kim1 1Department of Biochemistry and Molecular Biology, Korea University Medical School and College, Seoul, Korea; 2General Toxicology Team, Korea Testing and Research Institute, Seoul, Korea; 3Department of Biochemistry, University of Bath, Bath, UK; 4Department of Microbiology, Wonju College of Medicine, Yonsei University, Wonju-si, Gangwon, Korea; 5Department of Life Science, Institute of Environmental Medicine for Green Chemistry, Dongguk University, Seoul, Korea; 6Nonclinical Research Institute, Chemon Inc., Yongin, Gyeonggi, Korea; 7Toxicological Research Center, Hoseo University, Ansan, Chungnam, Korea; 8Department of Bionanotechnology, Gachon University, Seongnam, Gyeonggi, Korea; 9College of Veterinary Medicine, Chungnam National University, Daejon, Korea *These authors contributed equally to this work Abstract: Nanoparticles (NPs) are used commercially in health and fitness fields, but information about the toxicity and mechanisms underlying the toxic effects of NPs is still very limited. The aim of this study is to investigate the toxic effect(s) of 100 nm negatively (ZnOAE100[-]) or positively (ZnOAE100[+]) charged zinc oxide (ZnO) NPs administered by gavage in Sprague Dawley rats, to establish a no observed adverse effect level, and to identify target organ(s). After verification of the primary particle size, morphology, hydrodynamic size, and zeta potential of each test article, we performed a 90-day study according to Organisation for Economic Co-operation and Development test guideline 408. For the 90-day study, the high dose was set at 500 mg/kg and the middle and low doses were set at 125 mg/kg and 31.25 mg/kg, respectively. Both ZnO NPs had significant changes in hematological and blood biochemical analysis, which could correlate with anemia-related parameters, in the 500 mg/kg groups of both sexes. Histopathological examination showed significant adverse effects (by both test articles) in the stomach, pancreas, eye, and prostate gland tissues, but the particle charge did not affect the tendency or the degree of the lesions. We speculate that this inflammatory damage might result from continuous irritation caused by both test articles. Therefore, the target organs for both ZnOAE100(-) and ZnOAE100(+) are considered to be the stomach, pancreas, eye, and prostate gland. Also, the no observed adverse effect level for both test articles was identified as 31.25 mg/kg for both sexes, because the adverse effects were observed at all doses greater than 125 mg/kg. Keywords: zinc oxide nanoparticles, surface charge, 90-day oral dose toxicity, no observed adverse effect level

Jeong-Sup Hong,1,2 Myeong-Kyu Park,1 Min-Seok Kim,1 Jeong-Hyeon Lim,1 Gil-Jong Park,1 Eun-Ho Maeng,1 Jae-Ho Shin,3 Meyoung-Kon Kim,4 Jayoung Jeong,5 Jin-A Park,2 Jong-Choon Kim,6 Ho-Chul Shin2 1Health Care Research Laboratory, Korea Testing and Research Institute, Gimpo, South Korea; 2College of Veterinary Medicine, Konkuk University, Seoul, South Korea; 3Department of Biomedical Laboratory Science, Eulji University, Seongnam-si, South Korea; 4Department of Biochemistry and Molecular Biology, Korea University Medical School and College, Seoul, South Korea; 5Toxicological Research Division, National Institute of Food and Drug Safety Evaluation, Chungcheongbuk-do, South Korea; 6College of Veterinary Medicine, Chonnam National University, Gwangju, South Korea Abstract: This study investigated the potential adverse effects of zinc oxide nanoparticles ([ZnOSM20(+) NPs] zinc oxide nanoparticles, positively charged, 20 nm) on pregnant dams and embryo–fetal development after maternal exposure over the period of gestational days 5–19 with Sprague-Dawley rats. ZnOSM20(+) NPs were administered to pregnant rats by gavage at 0, 100, 200, and 400 mg/kg/day. All dams were subjected to a cesarean section on gestational day 20, and all of the fetuses were examined for external, visceral, and skeletal alterations. Toxicity in the dams manifested as significantly decreased body weight after administration of 400 mg/kg/day NPs; reduced food consumption after administration of 200 and 400 mg/kg/day NPs; and decreased liver weight and increased adrenal glands weight after administration of 400 mg/kg/day NPs. However, no treatment-related difference in: number of corpora lutea; number of implantation sites; implantation rate (%); resorption; dead fetuses; litter size; fetal deaths and placental weights; and sex ratio were observed between the groups. On the other hand, significant decreases between treatment groups and controls were seen for fetal weights after administration of 400 mg/kg/day NPs. Morphological examinations of the fetuses demonstrated significant differences in incidences of abnormalities in the group administered 400mg/kg/day. Meanwhile, no significant difference was found in the Zn content of fetal tissue between the control and high-dose groups. These results showed that oral doses for the study with 15-days repeated of ZnOSM20(+) NPs were maternotoxic in the 200 mg/kg/day group, and embryotoxic in the 400 mg/kg/day group. Keywords: developmental toxicity, maternal toxicity, nanotoxicology, teratogenicity

Hwa Jung Ryu,1,* Nak-won Seong,2,* Byoung Joon So,1 Heung-sik Seo,2 Jun-ho Kim,2 Jeong-Sup Hong,2 Myeong-kyu Park,2 Min-Seok Kim,2 Yu-Ri Kim,3 Kyu-Bong Cho,4 Mu yeb Seo,2 Meyoung-Kon Kim,3 Eun Ho Maeng,2 Sang Wook Son1 1Department of Dermatology, Korea University College of Medicine, Seoul, South Korea; 2Korea Testing and Research Institute, Gyunggi-Do, South Korea; 3Department of Biochemistry and Molecular Biology, Korea University College of Medicine, Seoul, South Korea; 4Department of Clinical Laboratory Science, Shinheung College, Uijeongbu, South Korea *These authors contributed equally to this work Abstract: Silica is a very common material that can be found in both crystalline and amorphous forms. Well-known toxicities of the lung can occur after exposure to the crystalline form of silica. However, the toxicities of the amorphous form of silica have not been thoroughly studied. The majority of in vivo studies of amorphous silica nanoparticles (NPs) were performed using an inhalation exposure method. Since silica NPs can be commonly administered through the skin, a study of dermal silica toxicity was necessary to determine any harmful effects from dermal exposures. The present study focused on the results of systemic toxicity after applying 20 nm colloidal silica NPs on rat skin for 90 days, in accordance with the Organization for Economic Cooperation and Development test guideline 411 with a good laboratory practice system. Unlike the inhalation route or gastrointestinal route, the contact of silica NPs through skin did not result in any toxicity or any change in internal organs up to a dose of 2,000 mg/kg in rats. Keywords: silica nanoparticles, toxicity, dermal route

Pankaj Ranjan Karn,1 Su-Eon Jin,2 Benjamin Joon Lee,1,2 Bo Kyung Sun,1,2 Min-Soo Kim,3 Jong-Hyuk Sung,1,2 Sung-Joo Hwang1,2 1Yonsei Institute of Pharmaceutical Sciences, 2College of Pharmacy, Yonsei University, Yeonsu-gu, Incheon, Republic of Korea; 3College of Pharmacy, Pusan National University, Geumjeong-gu, Busan, Republic of Korea Objectives: The objectives of this study were to prepare cyclosporin A (CsA)-containing proliposomes using the supercritical antisolvent (SAS) process and the conventional thin film method for the comparative study of proliposomal formulations and to evaluate the physicochemical properties of these proliposomes.Methods: CsA-containing proliposomes were prepared by the SAS process and the conventional film method, composed of natural and synthetic phospholipids. We investigated particle size, polydispersity index, and zeta potential of CsA-containing proliposomes. In addition, both production yield and entrapment efficiency of CsA in different proliposomes were analyzed. Physicochemical properties of CsA-containing proliposomes were also evaluated, using differential scanning calorimetry and X-ray diffraction. The morphology and size of ­CsA-containing proliposomes were confirmed, using scanning electron microscopy. We checked the in vitro release of CsA from CsA-containing proliposomes prepared by different preparation methods, comparing them with Restasis® as a positive control and the stability of SAS-mediated proliposomes was also studied.Results: CsA-containing proliposomes formed by the SAS process had a relatively smaller particle size, with a narrow size distribution and spherical particles compared with those of conventionally prepared proliposomes. The yield and entrapment efficiency of CsA in all proliposomes varied from 85% to 92% and from 86% to 89%, respectively. Differential scanning calorimetry and X-ray diffraction studies revealed that the anhydrous lactose powder used in this formulation retained its crystalline form and that CsA was present in an amorphous form. Proliposome powders were rapidly converted to liposomes on contact with water. The in vitro release study of proliposomal formulations demonstrated a similar pattern to Restasis®. The SAS-mediated CsA-containing proliposomes were stable on storage, with no significant changes in particle size, polydispersity index, and entrapment efficiency.Conclusion: These results show promising features of CsA-containing proliposomal formulations, using the SAS process for the large-scale industrial application. Keywords: proliposomes, cyclosporin A, supercritical antisolvent process, thin film method, Restasis®

Min-Soo Kim,1 In-hwan Baek21College of Pharmacy, Pusan National University, Geumjeong-gu, Busan, Republic of Korea; 2College of Pharmacy, Kyungsung University, Daeyeon-dong, Nam-gu, Busan, Republic of KoreaAbstract: The aim of this study was to fabricate valsartan composite nanoparticles by using the supercritical antisolvent (SAS) process, and to evaluate the correlation between in vitro dissolution and in vivo pharmacokinetic parameters for the poorly water-soluble drug valsartan. Spherical composite nanoparticles with a mean size smaller than 400 nm, which contained valsartan, were successfully fabricated by using the SAS process. X-ray diffraction and thermal analyses indicated that valsartan was present in an amorphous form within the composite nanoparticles. The in vitro dissolution and oral bioavailability of valsartan were dramatically enhanced by the composite nanoparticles. Valsartan–hydroxypropyl methylcellulose–poloxamer 407 nanoparticles exhibited faster drug release (up to 90% within 10 minutes under all dissolution conditions) and higher oral bioavailability than the raw material, with an approximately 7.2-fold higher maximum plasma concentration. In addition, there was a positive linear correlation between the pharmacokinetic parameters and the in vitro dissolution efficiency. Therefore, the preparation of composite nanoparticles with valsartan–hydroxypropyl methylcellulose and poloxamer 407 by using the SAS process could be an effective formulation strategy for the development of a new dosage form of valsartan with high oral bioavailability.Keywords: supersaturation, bioavailability, solid dispersion, dissolution, supercritical fluid

Min-Soo KimDepartment of Pharmaceutical Engineering, Inje University, Gimhae, Gyeongnam, Republic of KoreaAbstract: The objectives of this study were to develop a novel solid dutasteride formulation with improved physicochemical properties and oral bioavailability, and to examine the correlation between its in vitro dissolution and in vivo pharmacokinetic parameters. Hydroxypropyl-β-cyclodextrin (HP-β-CD) nanostructures with or without hydrophilic additives were manufactured using the supercritical antisolvent process. The dutasteride-loaded HP-β-CD nanoparticles formed aggregates with a mean particle size of less than 160 nm and a specific surface area greater than 100 m2/g. Increases in the supersaturation and dissolution rate for dutasteride were dependent on the type of additive; increases in maximum solubility and extended supersaturation were observed in dutasteride-loaded HP-β-CD nanostructures with hydroxypropylmethyl cellulose, whereas the dissolution rate was the highest for nanostructures containing d-α-tocopheryl polyethylene glycol 1000 succinate. In rats, the oral bioavailability of dutasteride increased with the supersaturation induced by the HP-β-CD nanostructures. In addition, compared with the in vitro drug release rate, the in vivo pharmacokinetic parameters were more closely correlated with in vitro parameters related to supersaturation (solubility). Further, the bioavailability of the dutasteride-loaded HP-β-CD nanostructures with hydroxypropylmethyl cellulose was similar to that of the commercially available soft gelatin capsule (Avodart®). In conclusion, preparation of dutasteride-loaded HP-β-CD nanostructures using the supercritical antisolvent process affords a viable alternative solid dosage form for dutasteride.Keywords: solubility, bioavailability, dutasteride, nanostructure, supercritical antisolvent

Wonkyung Cho,1,2 Min-Soo Kim,3 Jeong-Soo Kim,2 Junsung Park,1,2 Hee Jun Park,1,2 Kwang-Ho Cha,1,2 Jeong-Sook Park,2 Sung-Joo Hwang1,4 1Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon, Republic of Korea; 2College of Pharmacy, Chungnam National University, Daejeon, Republic of Korea; 3Department of Pharmaceutical Engineering, Inje University, Gimhae, Republic of Korea; 4College of Pharmacy, Yonsei University, Incheon, Republic of Korea Background: The aim of this study was to develop an optimized solid self-microemulsifying drug delivery system (SMEDDS) formulation for sirolimus to enhance its solubility, stability, and bioavailability. Methods: Excipients used for enhancing the solubility and stability of sirolimus were screened. A phase-separation test, visual observation for emulsifying efficiency, and droplet size analysis were performed. Ternary phase diagrams were constructed to optimize the liquid SMEDDS formulation. The selected liquid SMEDDS formulations were prepared into solid form. The dissolution profiles and pharmacokinetic profiles in rats were analyzed. Results: In the results of the oil and cosolvent screening studies, Capryol™ Propylene glycol monocaprylate (PGMC) and glycofurol exhibited the highest solubility of all oils and cosolvents, respectively. In the surfactant screening test, D-α-tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS) was determined to be the most effective stabilizer of sirolimus in pH 1.2 simulated gastric fluids. The optimal formulation determined by the construction of ternary phase diagrams was the T32 (Capryol™ PGMC:glycofurol:vitamin E TPGS = 30:30:40 weight ratio) formulation with a mean droplet size of 108.2 ± 11.4 nm. The solid SMEDDS formulations were prepared with Sucroester 15 and mannitol. The droplet size of the reconstituted solid SMEDDS showed no significant difference compared with the liquid SMEDDS. In the dissolution study, the release amounts of sirolimus from the SMEDDS formulation were significantly higher than the raw sirolimus powder. In addition, the solid SMEDDS formulation was in a more stable state than liquid SMEDDS in pH 1.2 simulated gastric fluids. The results of the pharmacokinetic study indicate that the SMEDDS formulation shows significantly greater bioavailability than the raw sirolimus powder or commercial product (Rapamune® oral solution). Conclusion: The results of this study suggest the potential use of a solid SMEDDS formulation for the delivery of poorly water-soluble drugs, such as sirolimus, through oral administration. Keywords: sirolimus, solubility, stability, bioavailability, self-emulsifying drug delivery systems, microemulsion

Soyoung Lee,1,* Mi-Sun Kim,1,* Dakeun Lee,2 Taeg Kyu Kwon,3 Dongwoo Khang,4 Hui-Suk Yun,5 Sang-Hyun Kim11CMRI, Laboratory of Immunotoxicology, Department of Pharmacology,School of Medicine, Kyungpook National University, Daegu, Republic of Korea; 2Department of Pathology, School of Medicine, Kyungpook National University, Daegu, Republic of Korea; 3Department of Immunology, School of Medicine, Keimyung University, Daegu, Republic of Korea; 4School of Nano and Advanced Materials Science and Engineering, Gyeongsang National University, Jinju, Republic of Korea; 5Engineering Ceramics Department, Powder and Ceramics Division, Korea Institute of Materials Science, Changwon, Republic of Korea*These authors contributed equally to this workBackground: Mesoporous silica (MPS) nanoparticles (NPs), which have a unique pore structure and extremely large surface area and pore volume, have received much attention because of their biomedical application potential. Using MPS NPs for biomedical devices requires the verification of their biocompatibility because the surface area of NPs is one of the most important determinants of toxicity, including the cellular uptake and immune response. We have previously reported that the cytotoxicity and inflammation potential of MPS NPs have been shown to be lower than those of general amorphous colloidal silica (Col) NPs in macrophages, but the low cytotoxicity does not guarantee high biocompatibility in vivo. In this study, we compared the in vivo immunotoxicity of MPS and Col NPs in the mouse model to define the effects of pore structural conditions of silica NPs.Materials and methods: Both MPS and Col NPs (2, 20, and 50 mg/kg/day) were intraperitoneally administered in female BALB/c mice for 4 weeks, and clinical toxicity, lymphocyte population, serum IgG/IgM levels, and histological changes were examined.Results: There was no overt sign of clinical toxicity in either MPS- or Col-treated mice. However, MPS NPs led to significant increases in liver and spleen weight and splenocyte proliferation. Mice treated with MPS NPs showed altered lymphocyte populations (CD3+, CD45+, CD4+, and CD8+) in the spleen, increased serum IgG and IgM levels, and histological changes. Despite slight changes in lymphocyte populations in the spleen, Col NPs did not alter other immunological factors.Conclusion: The results indicate that in vivo exposure to MPS NPs caused more damage to systemic immunity than that of Col NPs through the dysregulation of the spleen. The results for in vivo data are inconsistent with those for in vitro data, which show lower cytotoxicity for MPS NPs. These results suggest the importance of verifying biocompatibility both in vitro and in vivo during the design of new nanomaterials.Keywords: immunotoxicity, mesoporous silica nanoparticle, colloidal silica nanoparticle, spleen

Kwang-Ho Cha,1,3 Kyung-Jin Cho,3 Min-Soo Kim,4 Jeong-Soo Kim,3 Hee Jun Park,1,3 Junsung Park,1,3 Wonkyung Cho,1,3 Jeong-Sook Park,3 Sung-Joo Hwang1,21Yonsei Institute of Pharmaceutical Sciences, 2College of Pharmacy, Yonsei University, Incheon, Republic of Korea; 3College of Pharmacy, Chungnam National University, Daejeon, Republic of Korea; 4Department of Pharmaceutical Engineering, Inje University, Gimhae, Republic of KoreaBackground: The aim of this study was to enhance the bioavailability of fenofibrate, a poorly water-soluble drug, using a melt-adsorption method with supercritical CO2.Methods: Fenofibrate was loaded onto Neusilin® UFL2 at different weight ratios of fenofibrate to Neusilin UFL2 by melt-adsorption using supercritical CO2. For comparison, fenofibrate-loaded Neusilin UFL2 was prepared by solvent evaporation and hot melt-adsorption methods. The fenofibrate formulations prepared were characterized by differential scanning calorimetry, powder x-ray diffractometry, specific surface area, pore size distribution, scanning electron microscopy, and energy-dispersive x-ray spectrometry. In vitro dissolution and in vivo bioavailability were also investigated.Results: Fenofibrate was distributed into the pores of Neusilin UFL2 and showed reduced crystal formation following adsorption. Supercritical CO2 facilitated the introduction of fenofibrate into the pores of Neusilin UFL2. Compared with raw fenofibrate, fenofibrate from the prepared powders showed a significantly increased dissolution rate and better bioavailability. In particular, the area under the drug concentration-time curve and maximal serum concentration of the powders prepared using supercritical CO2 were 4.62-fold and 4.52-fold greater than the corresponding values for raw fenofibrate.Conclusion: The results of this study highlight the usefulness of the melt-adsorption method using supercritical CO2 for improving the bioavailability of fenofibrate.Keywords: fenofibrate, melt adsorption, supercritical CO2, bioavailability

Min-Soo Kim1, Jeong-Soo Kim1, Hee Jun Park1, Won Kyung Cho1,3, Kwang-Ho Cha1,3, Sung-Joo Hwang2,31College of Pharmacy, Chungnam National University, Daejeon, Republic of Korea, 2College of Pharmacy, 3Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon, Republic of KoreaBackground: The aim of this study was to improve the physicochemical properties and bioavailability of poorly water-soluble sirolimus via preparation of a solid dispersion of nanoparticles using a supercritical antisolvent (SAS) process.Methods: First, excipients for enhancing the stability and solubility of sirolimus were screened. Second, using the SAS process, solid dispersions of sirolimus-polyvinylpyrrolidone (PVP) K30 nanoparticles were prepared with or without surfactants such as sodium lauryl sulfate (SLS), tocopheryl propylene glycol succinate, Sucroester 15, Gelucire 50/13, and Myrj 52. A mean particle size of approximately 250 nm was obtained for PVP K30-sirolimus nanoparticles. Solid state characterization, kinetic solubility, powder dissolution, stability, and pharmacokinetics were analyzed in rats.Results: X-ray diffraction, differential scanning calorimetry, and high-pressure liquid chromatography indicated that sirolimus existed in an anhydrous amorphous form within a solid dispersion of nanoparticles and that no degradation occurred after SAS processing. The improved supersaturation and dissolution of sirolimus as a solid dispersion of nanoparticles appeared to be well correlated with enhanced bioavailability of oral sirolimus in rats. With oral administration of a solid dispersion of PVP K30-SLS-sirolimus nanoparticles, the peak concentration and AUC0→12h of sirolimus were increased by approximately 18.3-fold and 15.2-fold, respectively.Conclusion: The results of this study suggest that preparation of PVP K30-sirolimus-surfactant nanoparticles using the SAS process may be a promising approach for improving the bioavailability of sirolimus.Keywords: sirolimus, solubility, bioavailability, supercritical antisolvent, nanoparticles

Myung Joo Kang1, Jae Yoon Eum1, Mi Sook Jeong2, Sang Han Park1, Ki Young Moon1, Mean Hyung Kang1, Min Soo Kim1, Sun Eun Choi1, Min Won Lee1, Do Ik Lee1, Hyoweon Bang2, Chung Soo Lee2, Seong Soo Joo3, Kapsok Li2, Mi-Kyung Lee2, Seong Jun Seo2, Young Wook Choi11College of Pharmacy, ChungAng University, Heuksuk-dong, Dongjak-gu, Seoul, 2College of Medicine, Chung-Ang University, Heuksukdong, Dongjak-gu, Seoul, 3Division of Marine Molecular Biotechnology, Gangneung-Wonju National University, Gangneung, South KoreaBackground: The aim of the present study was to enhance a topical delivery of hirsutenone (HST), a naturally occuring immunomodulator, employing Tat peptide-admixed elastic liposomes (EL/T).Methods: HST-loaded EL, consisting of phosphatidylcholine and Tween 80 (85:15 w/w%), were prepared using thin film hydration method. By adding Tat peptide to EL (0.16 w/w%), EL/T were formulated. The in vitro skin permeation of HST was examined using a Franz diffusion cell mounted with depilated mouse skin. Lesions for atopic dermatitis (AD) were induced by a topical application of diphenylcyclopropenone to NC/Nga mice. Therapeutic improvements of AD were evaluated by clinical skin severity scores. Immunological analyses on inducible nitric oxide synthase and cyclooxygenase-2 levels in the skin and interleukin (IL)-4, IL-13, immunoglobulin E, and eosinophil levels in the blood were also performed.Results: EL systems were superior to conventional cream, revealing greater flux values in a permeation study. The addition of Tat peptide further increased the skin permeation of HST. In an efficacy study with AD-induced NC/Nga mice, an HST-containing EL/T formulation brought a significant improvement in both skin severity score and immune-related responses for the levels of nitric oxide synthase, cyclooxygenase-2, IL-4, IL-13, immunoglobulin E, and eosinophils.Conclusion: A novel EL/T formulation was successfully developed for topical delivery of HST to treat AD.Keywords: hirsutenone, elastic liposomes, atopic dermatitis, NC/Nga mice, Tat peptide

Background: Colon-targeted oral nanoparticles (NPs) have emerged as an ideal, safe, and effective therapy for ulcerative colitis (UC) owing to their ability to selectively accumulate in inflamed colonic mucosa. Cyclosporine A (CSA), an immunosuppressive agent, has long been used as rescue therapy in severe steroid-refractory UC. In this study, we developed CSA-loaded dual-functional polymeric NPs composed of Eudragit ® FS30D as a pH-sensitive polymer for targeted delivery to the inflamed colon, and poly(lactic-co-glycolic acid) (PLGA) as a sustained-release polymer., Methods: CSA-loaded Eudragit FS30D nanoparticles (ENPs), PLGA nanoparticles (PNPs), and Eudragit FS30D/PLGA nanoparticles (E/PNPs) were prepared using the oil-in-water emulsion method. Scanning electron microscope images and zeta size data showed successful preparation of CSA-loaded NPs., Results: PNPs exhibited a burst drug release of >60% at pH 1.2 (stomach pH) in 0.5 h, which can lead to unwanted systemic absorption and side effects. ENPs effectively inhibited the burst drug release at pH 1.2 and 6.8 (proximal small intestine pH); however, nearly 100% of the CSA in ENPs was released rapidly at pH 7.4 (ileum-colon pH) owing to complete NP dissolution. In contrast to single-functional PNPs and ENPs, the dual-functional E/PNPs minimized burst drug release (only 18%) at pH 1.2 and 6.8, and generated a sustained release at pH 7.4 thereafter. Importantly, in distribution studies in the gastrointestinal tracts of mice, E/PNPs significantly improved CSA distribution to the colon compared with PNPs or ENPs. In a mouse model of colitis, E/PNP treatment improved weight loss and colon length, and decreased rectal bleeding, spleen weight, histological scoring, myeloperoxidase activity, macrophage infiltration, and expression of proinflammatory cytokines compared with PNPs or ENPs., Conclusion: Overall, this work confirms the benefits of CSA-loaded E/PNPs for efficiently delivering CSA to the colon, suggesting their potential for UC therapy., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

This study investigated the potential adverse effects of zinc oxide nanoparticles (ZnO(SM20[-]) NPs; negatively charged, 20 nm) on pregnant dams and embryo-fetal development after maternal exposure over the period of gestational days 5-19 with Sprague Dawley rats. ZnO(SM20(-)) NPs were administered to pregnant rats by gavage at 0 mg/kg/day, 100 mg/kg/day, 200 mg/kg/day, and 400 mg/kg/day. All dams were subjected to caesarean section on gestational day 20, and all the fetuses were examined for external, visceral, and skeletal alterations. Toxicity in the dams manifested as significantly decreased body weight at 400 mg/kg/day and decreased liver weight, and increased adrenal glands weight at 200 mg/kg/day and 400 mg/kg/day. However, no treatment-related difference in the number of corpora lutea, the number of implantation sites, the implantation rate (%), resorption, dead fetuses, litter size, fetal deaths, fetal and placental weights, and sex ratio were observed between the groups. Morphological examinations of the fetuses demonstrated no significant difference in the incidences of abnormalities between the groups. No significant difference was found in the Zn content of fetal tissue between the control and high-dose groups. These results showed that a 15-day repeated oral dose of ZnO(SM20(-)) was minimally maternotoxic at dose of 200 mg/kg/day and 400 mg/kg/day.