Sung Rae Kim

RIPL peptide (IPLVVPLRRRRRRRRC)-conjugated liposomes for enhanced intracellular drug delivery to hepsin-expressing cancer cells

BACKGROUND To facilitate selective drug delivery to hepsin (Hpn)-expressing cancer cells, the RIPL peptide (IPLVVPLRRRRRRRRC; 16mer; 2.1 kDa) was synthesized as a novel cell penetrating/homing peptide (CPHP) and conjugated to a liposomal carrier. METHODS RIPL peptide-conjugated liposomes (RIPL-Lipo) were prepared by conjugating RIPL peptides to maleimide-derivatized liposomal vesicles via the thiol-maleimide reaction. Vesicle size and zeta potential were examined using a Zetasizer. Intracellular uptake specificity of the RIPL peptide, or RIPL-Lipo, was assessed by measuring mean fluorescence intensity (MFI) after treatment with a fluorescent marker in various cell lines: SK-OV-3, MCF-7, and LNCaP for Hpn(+); DU145, PC3, and HaCaT for Hpn(-). FITC-dextran was used as a model compound. Selective translocational behavior of RIPL-Lipo to LNCaP cells was visualized by fluorescence microscopy and confocal laser scanning microscopy. Cytotoxicities of the RIPL peptide and RIPL-Lipo were evaluated by WST-1 assay. RESULTS RIPL peptides exhibited significant Hpn-selectivity. RIPL-Lipo systems were of positively charged nanodispersion (165 nm in average; 6-24 mV depending on RIPL conjugation ratio). RIPL-Lipo with the conjugation of 2300 peptide molecules revealed the greatest MFI in all cell lines tested. Cellular uptake of RIPL-Lipo increased by 20- to 70-fold in Hpn(+) cells, and 5- to 7-fold in Hpn(-) cells, compared to the uptake of FITC-dextran. Cytosolic internalization of RIPL-Lipo was time-dependent: bound instantly; internalized within 30 min; distributed throughout the cytoplasm after 1 h. Cytotoxicities of RIPL peptide (up to 50 μM) and RIPL-Lipo (up to 10%) were minor (cell viability >90%) in LNCaP and HaCaT cells. CONCLUSION By employing a novel CPHP, the RIPL-Lipo system was successfully developed for Hpn-specific drug delivery.

Development of a solidified self-microemulsifying drug delivery system (S-SMEDDS) for atorvastatin calcium with improved dissolution and bioavailability

To improve the dissolution and oral bioavailability (BA) of atorvastatin calcium (ATV), we previously introduced an optimized self-microemulsifying drug delivery system (SMEDDS) using Capmul(®) MCM (oil), Tween(®) 20 (surfactant), and tetraglycol (cosurfactant). In this study, various solid carriers were employed to develop a solidified SMEDDS (S-SMEDDS): mannitol (M) and lactose (L) as water-soluble carriers, and Sylysia(®) 350 (S) and Aerosil(®) 200 (A) as water-insoluble carriers. Maximum solidifying capacities (SCmax) of water-insoluble carriers were significantly greater than those of water-soluble carriers were. The resultant powders were free flowing with an angle of repose <40° and Carrs index 5-20%, regardless of the solid carrier types. S-SMEDDS with mannitol (S(M)-SMEDDS) or lactose (S(L)-SMEDDS) had a smaller droplet size and greater dissolution than S-SMEDDS with Sylysia(®) 350 (S(S)-SMEDDS) or Aerosil(®) 200 (S(A)-SMEDDS). Following oral administration of various formulations to rats at a dose equivalent to 25mg/kg of ATV, plasma drug levels were measured by LC-MS/MS. The relative BAs (RBAs) of SMEDDS, S(M)-SMEDDS, and S(S)-SMEDDS were 345%, 216%, and 160%, respectively, compared to that of ATV suspension. Additionally, at a reduced dose of ATV equivalent to 5mg/kg, the RBAs of S(M)-SMEDDS and S(S)-SMEDDS compared to that of SMEDDS were 101% and 65%, respectively. These results suggest that S(M)-SEMDDS offers great potential for the development of solid dosage forms with improved oral absorption of drugs with poor water solubility.

Development and optimization of a self-microemulsifying drug delivery system for atorvastatin calcium by using D-optimal mixture design

In this study, we developed and optimized a self-microemulsifying drug delivery system (SMEDDS) formulation for improving the dissolution and oral absorption of atorvastatin calcium (ATV), a poorly water-soluble drug. Solubility and emulsification tests were performed to select a suitable combination of oil, surfactant, and cosurfactant. A d-optimal mixture design was used to optimize the concentration of components used in the SMEDDS formulation for achieving excellent physicochemical characteristics, such as small droplet size and high dissolution. The optimized ATV-loaded SMEDDS formulation containing 7.16% Capmul MCM (oil), 48.25% Tween 20 (surfactant), and 44.59% Tetraglycol (cosurfactant) significantly enhanced the dissolution rate of ATV in different types of medium, including simulated intestinal fluid, simulated gastric fluid, and distilled water, compared with ATV suspension. Good agreement was observed between predicted and experimental values for mean droplet size and percentage of the drug released in 15 minutes. Further, pharmacokinetic studies in rats showed that the optimized SMEDDS formulation considerably enhanced the oral absorption of ATV, with 3.4-fold and 4.3-fold increases in the area under the concentration-time curve and time taken to reach peak plasma concentration, respectively, when compared with the ATV suspension. Thus, we successfully developed an optimized ATV-loaded SMEDDS formulation by using the d-optimal mixture design, that could potentially be used for improving the oral absorption of poorly water-soluble drugs.

Cationic PLGA/Eudragit RL nanoparticles for increasing retention time in synovial cavity after intra-articular injection in knee joint.

Positively surface-charged poly(lactide-co-glycolide) (PLGA)/Eudragit RL nanoparticles (NPs) were designed to increase retention time and sustain release profile in joints after intra-articular injection, by forming micrometer-sized electrostatic aggregates with hyaluronic acid, an endogenous anionic polysaccharide found in high amounts in synovial fluid. The cationic NPs consisting of PLGA, Eudragit RL, and polyvinyl alcohol were fabricated by solvent evaporation technique. The NPs were 170.1 nm in size, with a zeta potential of 21.3 mV in phosphate-buffered saline. Hyperspectral imaging (CytoViva®) revealed the formation of the micrometer-sized filamentous aggregates upon admixing, due to electrostatic interaction between NPs and the polysaccharides. NPs loaded with a fluorescent probe (1,1′-dioctadecyl-3,3,3′,3′ tetramethylindotricarbocyanine iodide, DiR) displayed a significantly improved retention time in the knee joint, with over 50% preservation of the fluorescent signal 28 days after injection. When DiR solution was injected intra-articularly, the fluorescence levels rapidly decreased to 30% of the initial concentration within 3 days in mice. From these findings, we suggest that PLGA-based cationic NPs could be a promising tool for prolonged delivery of therapeutic agents in joints selectively.

Increased localized delivery of piroxicam by cationic nanoparticles after intra-articular injection.

Piroxicam (PRX), a potent nonsteroidal anti-inflammatory drug, is prescribed to relieve postoperative and/or chronic joint pain. However, its oral administration often results in serious gastrointestinal adverse effects including duodenal ulceration. Thus, a novel cationic nanoparticle (NP) was explored to minimize the systemic exposure and increase the retention time of PRX in the joint after intra-articular (IA) injection, by forming micrometer-sized electrostatic clusters with endogenous hyaluronic acid (HA) in the synovial cavity. PRX-loaded NPs consisting of poly(lactic-co-glycolic acid), Eudragit RL, and polyvinyl alcohol were constructed with the following characteristics: particle size of 220 nm, zeta potential of 11.5 mV in phosphate-buffered saline, and loading amount of 4.0% (w/w) of PRX. In optical and hyperspectral observations, the cationic NPs formed more than 50 μm-sized aggregates with HA, which was larger than the intercellular gaps between synoviocytes. In an in vivo pharmacokinetic study in rats, area under the plasma concentration–time curve (AUC0–24 h) and maximum plasma concentration (Cmax) of PRX after IA injection of the cationic NPs were <70% (P<0.05) and 60% (P<0.05), respectively, compared to those obtained from drug solution. Moreover, the drug concentration in joint tissue 24 h after dosing with the cationic NPs was 3.2-fold (P<0.05) and 1.8-fold (P<0.05) higher than that from drug solution and neutrally charged NPs, respectively. Therefore, we recommend the IA cationic NP therapy as an effective alternative to traditional oral therapy with PRX, as it increases drug retention selectively in the joint.

Docetaxel-loaded RIPL peptide (IPLVVPLRRRRRRRRC)-conjugated liposomes: Drug release, cytotoxicity, and antitumor efficacy

We previously synthesized the RIPL peptide (IPLVVPLRRRRRRRRC) to facilitate selective delivery into hepsin-expressing cancer cells and showed that RIPL peptide-conjugated liposomes (RIPL-L) enhanced the intracellular delivery of fluorescent probes in vitro. In this study, docetaxel-loaded RIPL-L (DTX-RIPL-L) were prepared and evaluated for in vitro drug release, cytotoxicity, and in vivo antitumor efficacy. DTX was successfully encapsulated by pre-loading, with an average encapsulation efficiency and drug loading capacity of 32.4% and 21.39±2.05 (μg/mg), respectively. A DTX release study using dialysis showed a biphasic release pattern, i.e., rapid release for 6h, followed by sustained release up to 72h. The first-order equation provided the best fit for drug release (r2=0.9349). In vitro cytotoxicity was dose-dependent, resulting in IC50 values of 36.10 (SK-OV-3) and 48.62ng/mL (MCF-7) for hepsin-positive, and 61.12 (DU145) and 53.04ng/mL (PC-3) for hepsin-negative cell lines. Live/dead cell imaging was carried out to visualize the proportion of viable and nonviable SK-OV-3 cells. Compared to DTX solution, DTX-RIPL-L significantly inhibited tumor growth and prolonged survival time in BALB/c nude mice with SK-OV-3 cell tumors. We suggest that DTX-RIPL-L is a good candidate for efficient drug targeting to hepsin-expressing cancer cells.

Enhanced topical delivery of tacrolimus by a carbomer hydrogel formulation with Transcutol P

Abstract Tacrolimus (TAC), a non-steroidal anti-inflammatory and immunosuppressive agent, is used for the treatment of atopic dermatitis (AD) and skin immune diseases. TAC-loaded topical hydrogel formulations composed of carbomer, carnosine, transcutol P (diethylene glycol monoethyl ether) and humectant were prepared. For comparison, TAC-loaded topical cream-type formulations were also prepared and commercially available TAC ointment was used as a reference. A drug release study in vitro revealed that the total amount of TAC released from hydrogels over 24 h was approximately 30 times greater than that for the reference formulation. Compared to the reference ointment and creams, carbomer gel formulations showed higher skin permeation and retention of TAC (significantly different at p < 0.05), especially those with more than 10% of transcutol P. Therefore, carbomer gel formulations with sufficient levels of transcutol P are good candidates for skin delivery of TAC and have potential as therapeutic agents for the treatment of AD or immune skin disorders.

Formulation of controlled-release pelubiprofen tablet using Kollidon(®) SR.

To develop a matrix-type, controlled-release tablet formulation of pelubiprofen (PLB), a recently developed non-steroidal anti-inflammatory drug, polymeric excipients including hypromellose, hydroxypropylcellulose, Eudragit(®) RS PO, and Kollidon(®) SR were screened. A formulation containing 12.4% w/w Kollidon(®) SR (K2 tablet) was found to be the most promising and stable for 6 months in an accelerated stability test. PLB release from K2 tablet was limited at pH 1.2, but gradually increased at pH 6.8 with a surface-erosion, resulting in the best fit to Hixson-Crowell equation. Comparative human PK studies were performed using a randomized, 2-way crossover design. LC-MS/MS assay revealed that the plasma level of PLB-transOH, an active metabolite, was significantly higher than that of PLB. After multiple dosing of immediate-release tablet (R) and K2 tablet (T), the T/R ratios of AUC were 1.02 and 1.04 for PLB and PLB-transOH, respectively. Level A in vitro-in vivo correlation was established for the K2 tablet-administered group. PK profile of PLB-transOH was not influenced by food intake, while that of PLB was altered. We suggest that K2 tablet could be administered twice a day without being affected by food intake, thereby enhancing patient compliance.

Development of a chitosan based double layer-coated tablet as a platform for colon-specific drug delivery.

A double layer-coated colon-specific drug delivery system (DL-CDDS) was developed, which consisted of chitosan (CTN) based polymeric subcoating of the core tablet containing citric acid for microclimate acidification, followed by an enteric coating. The polymeric composition ratio of Eudragit E100 and ethyl cellulose and amount of subcoating were optimized using a two-level factorial design method. Drug-release characteristics in terms of dissolution efficiency and controlled-release duration were evaluated in various dissolution media, such as simulated colonic fluid in the presence or absence of CTNase. Microflora activation and a stepwise mechanism for drug release were postulated. Consequently, the optimized DL-CDDS showed drug release in a controlled manner by inhibiting drug release in the stomach and intestine, but releasing the drug gradually in the colon (approximately 40% at 10 hours and 92% at 24 hours in CTNase-supplemented simulated colonic fluid), indicating its feasibility as a novel platform for CDD.

Solid formulation of a supersaturable self-microemulsifying drug delivery system for valsartan with improved dissolution and bioavailability

In order to improve the dissolution and oral bioavailability of valsartan (VST), and reduce the required volume for treatment, we previously formulated a supersaturable self-microemulsifying drug delivery system (SuSMEDDS) composed of VST (80 mg), Capmul® MCM (13.2 mg), Tween® 80 (59.2 mg), Transcutol® P (59.2 mg), and Poloxamer 407 (13.2 mg). In the present study, by using Florite® PS-10 (119.1 mg) and Vivapur® 105 (105.6 mg) as solid carriers, VST-loaded solidified SuSMEDDS (S-SuSMEDDS) granules were successfully developed, which possessed good flow properties and rapid drug dissolution. By introducing croscarmellose sodium (31 mg) as a superdisintegrant, S-SuSMEDDS tablets were also successfully formulated, which showed fast disintegration and high dissolution efficiency. Preparation of granules and tablets was successfully optimized using D-optimal mixture design and 3-level factorial design, respectively, resulting in percentage prediction errors of <10%. In pharmacokinetic studies in rats, the relative bioavailability of the optimized granules was 107% and 222% of values obtained for SuSMEDDS and Diovan® powder, respectively. Therefore, we conclude that novel S-SuSMEDDS formulations offer great potential for developing solid dosage forms of a liquefied formulation such as SuSMEDDS, while improving oral absorption of drugs with poor water solubility.