Synergistic effect of copper and amino acid mixtures on the production of extracellular matrix proteins in skin fibroblasts.

The stimulation of extracellular matrix (ECM) protein production is an interesting target to maintain normal skin structure and delay skin aging. Copper has been shown to stimulate ECM protein synthesis by activating lysyl oxidase. Although copper increases elastin and collagen synthesis, the effect of copper and amino acid mixtures on gene expression and protein synthesis changes relating to the ECM have not been fully investigated. In this study, we showed that copper ions (Cu2+) and amino acid mixtures significantly increased the expression of genes and proteins related to the ECM in human dermal fibroblasts. The expression of genes involved in ECM production was evaluated through quantitative polymerase chain reaction in the presence of amino acid mixtures containing different Cu2+ concentrations. Cu2+ dose-dependently increased the gene expression of elastin and collagen I. In addition, a mixture of amino acids and Cu2+ increased the protein expression of elastin and collagen I. We further evaluated the effect of Cu2+ with or without amino acids. Although Cu2+ treatment increased the expression of genes encoding ECM proteins, the Cu2+ treatment without amino acids did not increase protein expression in the ECM. Our results demonstrated the synergistic effects of amino acids and a Cu2+ mixture on ECM protein synthesis in dermal fibroblasts.

Supersaturable self-microemulsifying drug delivery system enhances dissolution and bioavailability of telmisartan.

To enhance the dissolution and oral bioavailability of telmisartan (TMS), a poorly water-soluble anti-hypertensive drug, a supersaturable self-microemulsifying drug delivery system (SuSMEDDS) was developed. Amorphous alkalinized TMS (AAT) was formulated into a SMEDDS, composed of Capmul® MCM (oil), Cremophor® RH40 (surfactant), and tetraglycol (co-surfactant). Although the SMEDDS was rapidly dissolved (>80% within 5 min) in a limited condition (500 mL, pH 6.8), drug precipitation was observed over time, resulting in a decrease in dissolution levels. The precipitation was due to drug recrystallization, as determined by differential scanning calorimetry and powder X-ray diffraction analyses. Several polymers, including Soluplus® (SOL), were screened as precipitation inhibitors; ultimately, SuSMEDDS-SOL was prepared by admixing SOL and the SMEDDS at a 5:100 (w/w) ratio. SuSMEDDS-SOL was superior in terms of dissolution efficiency (>90% over 2 h) and dissolution-retaining time (no precipitation over 2 h). An in vivo pharmacokinetic study in rats revealed that the oral bioavailability of SuSMEDDS-SOL was 4.8-, 1.3-, and 1.2-fold greater than those of the TMS suspension, AAT solution, and SMEDDS, respectively. Therefore, SuSMEDDS-SOL is a promising candidate to enhance the dissolution and oral bioavailability of TMS.

Enhanced oral bioavailability of valsartan in rats using a supersaturable self-microemulsifying drug delivery system with P-glycoprotein inhibitors.

Valsartan (VST) is a poorly water-soluble drug and a P-glycoprotein (P-gp) substrate. To enhance the dissolution and oral absorption of VST, a novel supersaturable self-microemulsifying drug delivery system (Su-SMEDDS) was formulated. Based on the previously reported Su-SMEDDS composed of Capmul® MCM (oil), Tween® 20 (T20; surfactant), Transcutol® P (cosurfactant), and Poloxamer 407 (supersaturating agent), P-gp inhibitory surfactants including Tween® 80 (T80) and Cremophor® EL (CR) were newly introduced to replace T20. All Su-SMEDDS formulations had a droplet size of <200 nm and showed rapid (>90% within 5 min) and pH-independent dissolution characteristics. The effective permeability coefficient (Peff) in rat jejunum was obtained using an in situ single-pass intestinal perfusion study: Peff values of Su-SMEDDS-T20, Su-SMEDDS-T80, and Su-SMEDDS-CR were 2.3, 4.1, and 3.4 times greater, respectively, than that of the VST solution. After oral administration of various formulations to rats (equivalent dose of VST 10 mg/kg), plasma drug levels were measured by liquid chromatography-tandem mass spectrometry. The relative bioavailabilities of Su-SMEDDS-T20, Su-SMEDDS-T80, and Su-SMEDDS-CR were 262%, 470%, and 458%, respectively, compared with the VST suspension. Thus, we propose that the Su-SMEDDS-T80 formulation is a good candidate for improving the oral absorption of poorly water-soluble and P-gp substrate drugs such as VST.

Pharmacokinetics and four-week repeated-dose toxicity of hyaluronic acid and ketorolac combination following intra-articular administration in normal rats.

Intra-articular (IA) injection of hyaluronic acid (HA) in combination with nonsteroidal anti-inflammatory drugs, such as ketorolac (KL), have been clinically investigated to provide more rapid and profound pain relief in patients with osteoarthritis. However, its safety, local tolerance, and potential for pharmacokinetic interaction have not been assessed. In this study, the pharmacokinetics and toxicity of a combination of HA and KL were evaluated in normal rats following four-week repeated-dose injection. Rats received HA or KL alone at 4 mg/kg or 16 mg/kg, respectively, or HA/KL combination at 4/4 mg/kg, 4/8 mg/kg, or 4/16 mg/kg on a weekly basis. The rats exhibited temporal, reversible changes in hematology, serum chemistry, and urinalysis caused primarily by KL treatment. No deleterious effects were observed on the joint following repeated IA HA/KL administration, which showed only minimal to mild levels of temporary inflammatory changes in synovial membrane. The plasma KL level following IA injection rose as fast as that of intra-muscular injection, with no alteration with the co-administered HA. In conclusion, repeated IA administration of HA/KL combination was tolerated well in normal rats, encouraging future studies of IA injection of HA/KL combination on osteoarthritis-induced animal models and even patients.

Improved skin delivery of voriconazole with a nanostructured lipid carrier-based hydrogel formulation.

In order to develop topical preparations of voriconazole (VRC) for the treatment of mycotic infections of the skin, a nanostructured lipid carrier-based hydrogel (NLC-gel) formulation was developed and its physical characteristics, in vitro skin permeation, and retention profiles were examined. A VRC-loaded NLC dispersion, consisting of Precirol ATO 5, Labrafil 1944 CS, and Tween 80, was prepared by high-pressure homogenization and embedded into Carbopol 940 hydrogel. The lipid nanoparticles in the hydrogel were approximately 210 nm in size, with a spherical shape and zeta potential of -30 mV. In a skin permeation study using a Franz diffusion cell mounted with depilated mouse skin, the NLC-gel was superior to conventional cream and microemulsion-based gel formulations, showing 2.8- and 1.7-fold greater flux values, respectively. In addition, the NLC-gel led to markedly greater accumulation of VRC in deeper skin layers as compared with the reference formulations. In conclusion, the novel topical formulation reported here represents an alternative treatment for skin infections such as candidiasis, with less potential for systemic adverse effects than oral therapy.

Tricaprylin-based drug crystalline suspension for intramuscular long-acting delivery of entecavir with alleviated local inflammation.

In order to ensure prolonged pharmacokinetic profile along with local tolerability at the injection site, tricaprylin-based drug crystalline suspension (TS) was designed and its local distribution, pharmacokinetics, and inflammatory response, were evaluated with conventional aqueous suspension (AS). As model drug particles, entecavir 3-palmitate (EV-P), an ester lipidic prodrug for entecavir (EV), was employed. The EV-P-loaded TS was prepared by ultra-sonication method. Prepared TS and conventional AS exhibited comparable morphology (rod or rectangular), median diameter (2.7 and 2.6 µm), crystallinity (melting point of 160-165°C), and in vitro dissolution profile. However, in vivo performances of drug microparticles were markedly different, depending on delivery vehicle. At AS-injected site, drug aggregates of up to 500 µm were formed upon intramuscular injection, and were surrounded with inflammatory cells and fibroblastic bands. In contrast, no distinct particle aggregation and adjacent granulation was observed at TS-injected site, with >4 weeks remaining of the oily vehicle in micro-computed tomographic observation. Surprisingly, TS exhibited markedly alleviated local inflammation compared to AS, endowing markedly lessened necrosis, fibrosis thickness, inflammatory area, and macrophage infiltration. The higher initial systemic exposure was observed with TS compared to AS, but TS provided prolonged delivery of EV for 3 weeks. Therefore, we suggest that the novel TS system can be a promising tool in designing parenteral long-acting delivery, with improved local tolerability.

Enhanced oral absorption of insulin: hydrophobic ion pairing and a self-microemulsifying drug delivery system using a D-optimal mixture design.

The lipophilicity of a peptide drug can be considerably increased by hydrophobic ion pairing with amphiphilic counterions for successful incorporation into lipid-based formulations. Herein, to enhance the oral absorption of insulin (INS), a self-microemulsifying drug delivery system (SMEDDS) formulation was developed. Prior to optimization, INS was complexed with sodium n-octadecyl sulfate (SOS) to increase the loading into the SMEDDS. The INS-SOS complex was characterized via scanning electron microscopy, Fourier transform infrared spectroscopy, differential scanning calorimetry, and its dissociation behavior. The SMEDDS was optimized using a D-optimal mixture design with three independent variables including Capmul MCM (X1, 9.31%), Labrasol (X2, 49.77%), and Tetraglycol (X3, 40.92%) and three response variables including droplet size (Y1, 115.2 nm), INS stability (Y2, 46.75%), and INS leakage (Y3, 17.67%). The desirability function was 0.766, indicating excellent agreement between the predicted and experimental values. The stability of INS-SOS against gastrointestinal enzymes was noticeably improved in the SMEDDS, and the majority of INS remained in oil droplets during release. Following oral administration in diabetic rats, the optimized SMEDDS resulted in pharmacological availabilities of 3.23% (50 IU/kg) and 2.13% (100 IU/kg). Thus, the optimized SMEDDS is a good candidate for the practical development of oral delivery of peptide drugs such as INS.

Optimization of solid self-dispersing micelle for enhancing dissolution and oral bioavailability of valsartan using Box-Behnken design.

A novel solid self-dispersing micelle (S-SDM) was developed to enhance the oral bioavailability of valsartan (VST) and to reduce the total mass of solidified supersaturable self-microemulsifying drug delivery system (S-SuSMEDDS), composed of Capmul MCM, Tween 80 (T80), Gelucire 44/14 (G44), Poloxamer 407, Florite PS-10 (FLO), and low-substituted hydroxypropyl cellulose B1 (HPC). Excluding oil component from S-SuSMEDDS, S-SDM was optimized using a Box-Behnken design with three independent variables: X1 (T80/G44, 0.63), X2 (FLO/HPC, 0.41), and X3 (solid carrier, 177.6 mg); and three response factors: Y1 (droplet size, 191.9 nm), Y2 (dissolution efficiency at 15 min, 55.0%), and Y3 (angle of repose, 32.4°). The desirability function was 0.636, showing an excellent agreement between the predicted and experimental values. With approximately 75% weight of S-SuSMEDDS, no distinct crystallinity of VST was observed in S-SDM, resulting in critical micelle concentration value of 32 µg/mL. Optimized S-SDM showed an approximate 4-fold improved dissolution (pH 1.2, 500 mL) compared with raw VST. Following oral administration in rats, optimized S-SDM improved relative bioavailability by approximately 235%, 216%, and 127% versus raw VST, Diovan® (commercial reference), and S-SuSMEDDS, respectively. Thus, optimized S-SDM could be a selectable candidate for developing water-insoluble drugs in reduced quantity.

Intravesical delivery of rapamycin via folate-modified liposomes dispersed in thermo-reversible hydrogel.

PURPOSE: To develop an intravesical instillation system for the treatment of bladder cancer, rapamycin (Rap) was encapsulated into liposomes and then homogeneously dispersed throughout a poloxamer 407 (P407)-based hydrogel. METHODS: Rap-loaded conventional liposomes (R-CL) and folate-modified liposomes (R-FL) were prepared using a film hydration method and pre-loading technique, and characterized by particle size, drug entrapment efficiency, and drug loading. The cellular uptake behavior in folate receptor-expressing bladder cancer cells was observed by flow cytometry and confocal laser scanning microscopy using a fluorescent probe. In vitro cytotoxic effects were evaluated using MTT assay, colony forming assay, and Western blot. For in vivo intravesical instillation, Rap-loaded liposomes were dispersed in P407-gel, generating R-CL/P407 and R-FL/P407. Gel-forming capacities and drug release were evaluated. Using the MBT2/Luc orthotopic bladder cancer mouse model, in vivo antitumor efficacy was evaluated according to regions of interest (ROI) measurement. RESULTS: R-CL and R-FL were successfully prepared, at approximately <160 nm, 42% entrapment efficiency, and 57 µg/mg drug loading. FL cellular uptake was enhanced over 2-fold than that of CL; folate receptor-mediated endocytosis was confirmed using a competitive assay with folic acid pretreatment. In vitro cytotoxic effects increased dose-dependently. Rap-loaded liposomes inhibited mTOR signaling and induced autophagy in urothelial carcinoma cells. With gelation time of <30 seconds and gel duration of >12 hrs, both R-CL/P407 and R-FL/P407 preparations transformed into gel immediately after instillation into the mouse bladder. Drug release from the liposomal gel was erosion controlled. In orthotopic bladder cancer mouse model, statistically significant differences in ROI values were found between R-CL/P407 and R-FL/P407 groups at day 11 (P=0.0273) and day 14 (P=0.0088), indicating the highest tumor growth inhibition by R-FL/P407. CONCLUSION: Intravesical instillation of R-FL/P407 might represent a good candidate for bladder cancer treatment, owing to its enhanced retention and FR-targeting.

Improved Dissolution and Oral Bioavailability of Valsartan Using a Solidified Supersaturable Self-Microemulsifying Drug Delivery System Containing Gelucire® 44/14.

To improve the dissolution and oral bioavailability of valsartan (VST), we previously formulated a supersaturable self-microemulsifying drug delivery system (SuSMED) composed of Capmul® MCM (oil), Tween® 80 (surfactant), Transcutol® P (cosurfactant), and Poloxamer 407 (precipitation inhibitor) but encountered a stability problem (Transcutol® P-induced weight loss in storage) after solidification. In the present study, replacing Transcutol® P with Gelucire® 44/14 resulted in a novel SuSMED formulation, wherein the total amount of surfactant/cosurfactant was less than that of the previous formulation. Solidified SuSMED (S-SuSMED) granules were prepared by blending VST-containing SuSMED with selective solid carriers, L-HPC and Florite® PS-10, wherein VST existed in an amorphous state. S-SuSMED tablets fabricated by direct compression with additional excipients were sufficiently stable in terms of drug content and impurity changes after 6 months of storage at accelerated conditions (40 ± 2 °C and 75 ± 5% relative humidity). Consequently, enhanced dissolution was obtained (pH 1.2, 2 h): 6-fold for S-SuSMED granules against raw VST; 2.3-fold for S-SuSMED tablets against Diovan® (reference tablet). S-SuSMED tablets increased oral bioavailability in rats (10 mg/kg VST dose): approximately 177⁻198% versus raw VST and Diovan®. Therefore, VST-loaded S-SuSMED formulations might be good candidates for practical development in the pharmaceutical industry.

Enhanced oral bioavailability of valsartan using a polymer-based supersaturable self-microemulsifying drug delivery system.

A novel, supersaturable self-microemulsifying drug delivery system (S-SMEDDS) was successfully formulated to enhance the dissolution and oral absorption of valsartan (VST), a poorly water-soluble drug, while reducing the total quantity for administration. Poloxamer 407 is a selectable, supersaturating agent for VST-containing SMEDDS composed of 10% Capmul® MCM, 45% Tween® 20, and 45% Transcutol® P. The amounts of SMEDDS and Poloxamer 407 were chosen as formulation variables for a 3-level factorial design. Further optimization was established by weighting different levels of importance on response variables for dissolution and total quantity, resulting in an optimal S-SMEDDS in large quantity (S-SMEDDS_LQ; 352 mg in total) and S-SMEDDS in reduced quantity (S-SMEDDS_RQ; 144.6 mg in total). Good agreement was observed between predicted and experimental values for response variables. Consequently, compared with VST powder or suspension and SMEDDS, both S-SMEDDS_LQ and S-SMEDDS_RQ showed excellent in vitro dissolution and in vivo oral bioavailability in rats. The magnitude of dissolution and absorption-enhancing capacities using quantity-based comparisons was in the order S-SMEDDS_RQ > S-SMEDDS_LQ > SMEDDS > VST powder or suspension. Thus, we concluded that, in terms of developing an effective SMEDDS preparation with minimal total quantity, S-SMEDDS_RQ is a promising candidate.

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.

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.

In situ intestinal permeability and in vivo oral bioavailability of celecoxib in supersaturating self-emulsifying drug delivery system.

In order to characterize the in situ intestinal permeability and in vivo oral bioavailability of celecoxib (CXB), a poorly water-soluble cyclooxygenase (COX)-2 inhibitor, various formulations including the self-emulsifying drug delivery system (SEDDS) and supersaturating SEDDS (S-SEDDS) were compared. The S-SEDDS formulation was obtained by adding Soluplus as a precipitation inhibitor to SEDDS, composed of Capryol 90 as oil, Tween 20 as surfactant, and Tetraglycol as cosurfactant (1:4.5:4.5 in volume ratio). An in situ single pass intestinal perfusion study in rats was performed with CXB-dissolved solutions at a concentration of 40 µg/mL. The effective permeability (Peff) of CXB in the control solution (2.5 v/v% Tween 20-containing PBS) was 6.39 × 10(-5) cm/s. The Peff value was significantly increased (P < 0.05) by the lipid-based formulation, yielding 1.5- and 2.9-fold increases for the SEDDS and S-SEDDS solutions, respectively, compared to the control solution. After oral administration of various formulations to rats at the equivalent dose of 100 mg/kg of CXB, the plasma drug level was measured by LC-MS/MS. The relative bioavailabilities of SEDDS and S-SEDDS were 263 and 355 %, respectively, compared to the CXB suspension as a reference. In particular, S-SEDDS revealed the highest Cmax and the smallest Tmax, indicating rapid and enhanced absorption with this formulation. This study illustrates the potential use of the S-SEDDS formulation in the oral delivery of poorly water-soluble compounds.