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TNF-related apoptosis-inducing ligand (TRAIL) is a death ligand that can induce apoptosis in cells expressing its cognate death receptors (DRs). Previously, we demonstrated the therapeutic potential of recombinant human TRAIL in experimental rheumatoid arthritis (RA) models. However, the mechanisms of how DR-mediated apoptosis elicits these actions is not known. Here, we show that systemically administering a potent, long-acting PEGylated TRAIL (TRAIL PEG ) is profoundly anti-rheumatic against two complementary experimental RA mouse models, collagen-induced arthritis (CIA) and collagen antibody-induced arthritis (CAIA), via targeting IL-17 secreting Th17 cells and regulatory T cells (Treg). Systemic administration of TRAIL PEG after disease onset ameliorated the severity of inflammatory arthritis including arthritis indices, paw thickness, cartilage damage and neutrophil infiltration in both CIA and CAIA models. Additionally, the levels of inflammatory molecules (p-p65, ICAM-1, Cox-2, MMP3, and iNOS), pro-inflammatory cytokines (TNF-α, IL-1β, IFN-γ, IL-6, IL-17) and accumulation of activated macrophages were significantly reduced after the TRAIL PEG treatment. Importantly, TRAIL PEG decreased the number of pro-inflammatory Th17 cells in inflamed arthritic joints through TRAIL-induced apoptosis while increasing anti-inflammatory Treg population in vivo. These results suggest that TRAIL PEG ameliorates autoimmunity by targeting the Th 17-Tregs axis, making it a promising candidate drug for the treatment of RA., (Copyright © 2017. Published by Elsevier B.V.)

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) has attracted great interest as a cancer therapy because it selectively induces death receptor (DR)-mediated apoptosis in cancer cells while sparing normal tissue. However, recombinant human TRAIL demonstrates limited therapeutic efficacy in clinical trials, possibly due to TRAIL-resistance of primary cancers and its inherent short half-life. Here we introduce drug delivery approaches to maximize in vivo potency of TRAIL in TRAIL-resistant tumor xenografts by (1) extending the half-life of the ligand with PEGylated TRAIL (TRAILPEG) and (2) concentrating a TRAIL sensitizer, selected from in vitro screening, in tumors via tumor-homing nanoparticles. Antitumor efficacy of TRAILPEG with tumor-homing sensitizer was evaluated in HCT116 and HT-29 colon xenografts. Western blot, real-time PCR, immunohistochemistry and cell viability assays were employed to investigate mechanisms of action and antitumor efficacy of the combination. We discovered that doxorubicin (DOX) sensitizes TRAIL-resistant HT-29 colon cancer cells to TRAIL by upregulating mRNA expression of DR5 by 60% in vitro. Intravenously administered free DOX does not effectively upregulate DR5 in tumor tissues nor demonstrate synergy with TRAILPEG in HT-29 xenografts, but rather introduces significant systemic toxicity. Alternatively, when DOX was encapsulated in hyaluronic acid-based nanoparticles (HAC/DOX) and intravenously administered with TRAILPEG, DR-mediated apoptosis was potentiated in HT-29 tumors by upregulating DR5 protein expression by 70% and initiating both extrinsic and intrinsic apoptotic pathways with reduced systemic toxicity compared to HAC/DOX or free DOX combined with TRAILPEG (80% vs. 40% survival rate; 75% vs. 34% tumor growth inhibition). This study demonstrates a unique approach to overcome TRAIL-based therapy drawbacks using sequential administration of a tumor-homing TRAIL sensitizer and long-acting TRAILPEG., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The purpose of this study was to demonstrate the biological potentials of PEGylated salmon calcitonin (PEG-sCT) derivatives administered intratracheally and their dependences on PEG Mw (1, 2, 5 kDa). Initially, three different PEG-sCT derivatives were site-specifically synthesized by attaching PEG to the Lys(18)-amine. In an attempt to examine the pulmonary feasibilities of these derivatives, the following evaluations were undertaken to determine their; (i) proteolytic resistances to pulmonary enzymes, (ii) bioactivities, and (iii) pulmonary pharmacokinetic and pharmacologic profiles. The results obtained showed that the pulmonary stabilities and pharmacokinetic properties of these derivatives were greatly improved by increasing PEG Mw. PEG-sCTs had 10.5-, 40.1-, and 1066.0-fold greater stabilities than that of sCT in rat lung homogenates. Moreover, all pharmacokinetic parameters (AUC(inf), C(max), t(1/2), and others) of these derivatives in endotracheally cannulated rats were significantly improved by PEGylation. Specifically, C(max) values increased on increasing PEG Mw, i.e., 78.1+/-21.1, 102.9+/-9.1, and 115.2+/-5.7 for 1, 2, 5 kDa, respectively, vs. 54.8+/-3.9 ng/mL for sCT. Their circulating t(1/2) values also increased to 53.9+/-6.0, 100.7+/-21.7, and 119.4+/-13.7 min, respectively, vs. 34.6+/-7.6 min for sCT. Despite having the best properties, Lys(18)-PEG(5k)-sCT was found to have significantly lower hypocalcemic efficacy than other PEG-sCTs, probably due to its reduced intrinsic bioactivity ( approximately 30% vs. sCT). Rather, Lys(18)-PEG(2k)-sCT showed the most promising pulmonary potential because of its well-preserved bioactivity (>80% of sCT). Taken together, our findings suggest that the site-specific substitution to peptides like sCT with a PEG of an appropriate size offers optimized therapeutic potential by dual advantages, i.e., (i) increased proteolytic stability and (ii) extended circulating half-life in terms of intrapulmonary delivery.

The purpose of this study was to develop and optimize a unique one-pot, two-step site-specific PEGylation method suitable for the high-yield production of mono-PEGylated (Lys(18)) salmon calcitonin (Lys(18)-PEG-sCT), which was previously demonstrated to have superior pharmaceutical properties to other conjugates. For the site-specific PEGylation, this study used the sCT derivative (FMOC(1,11)-sCT), which was FMOC protected at Cys(1)- and Lys(11)-amines among three PEGylation sites including Lys(18)-amine. This PEGylation process was achieved by the consecutive one-pot, two-step reaction: (i) the PEG conjugation to FMOC(1,11)-sCT; and (ii) the subsequent deprotection of FMOC group from the PEGylated FMOC(1,11)-sCT. The optimized reaction resulted in the high production yield of Lys(18)-PEG-sCT (about 86%), compared with that from conventional non-specific PEGylation (about 18%). The prepared Lys(18)-PEG-sCT conjugate showed improved biological stability without the loss in the in vitro and in vivo biological activity by PEGylation. Consequently, this site-specific PEGylation using an FMOC protection/deprotection strategy showed great usefulness in the production of the most promising Lys(18)-PEG-sCT conjugate with a high yield.

The purpose of this study was to investigate the potential of KSL, an antimicrobial decapeptide, which has been shown to inhibit the growth of oral bacterial strains associated with caries development and plaque formation, to act as an antiplaque agent in a chewing gum formulation. A reversed-phase high-performance liquid chromatography method was developed for KSL and found to be stability-indicating. KSL was stable in acetate buffer at pH 4 and artificial saliva. On the affinity of KSL to tooth-like materials, the KSL showed favorable interaction with hydroxyapatite discs pretreated with human saliva. A chewing gum formulation of KSL was prepared based on conventional procedures and the release of KSL from the gum was studied in vitro using the chewing apparatus and in vivo by a chew-out method. The gum formulations showed promising in vitro/in vivo release profiles, in which 70-80% KSL was released in a sustained manner over 20 min of chewing time. This study suggests that KSL in a gum formulation is suitable for the delivery in the oral cavity, thereby serving as a novel antiplaque agent.

The purpose of this research was to assess the acylation reactions of peptides, salmon calcitonin (sCT), human parathyroid hormone 1-34 (hPTH1-34) and leuprolide, in poly(lactic-co-glycolic acid) (PLGA) microspheres. Capillary electrophoresis (CE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) were used for determining and monitoring peptide acylation and quantitating acylation products in the degrading PLGA microspheres. In the degrading PLGA microspheres of sCT and hPTH1-34, the acylation products were observed and determined to be adducts with glycolic acid units from degradable PLGA polymer by MALDI-TOF MS. In the microsphere of leuprolide, however, the acylation product was not observed even after 28 days of incubation at the release medium, which represents the different stabilities among peptides according to the primary structure. As the leuprolide contains tyrosine and serine having hydroxyl group of nucleophilic amino acids, the acylation reaction of peptide is shown to be mainly due to the primary amino groups of N-terminus or lysine residue. The complementary use of CE and MALDI-TOF MS will be useful for searching the counter measures as well as determining the peptide acylation in the manufactured formulations on the market.