Mono-lithocholated exendin-4-loaded glycol chitosan nanoparticles with prolonged antidiabetic effects.

Hydrophobically modified glycol chitosan (HGC) nanoparticles loaded with mono-lithocholic acid-conjugated exendin-4 at the Lys(27) residue (LAM1-Ex4) were prepared and characterized by particle size measurement, proteolytic stability, in vitro drug-release profile, and in vivo antidiabetic effects in a db/db diabetic mouse model. Compared with Ex-4-loaded HGC nanoparticles (Ex4/HGC NPs) prepared as a control, LAM1-Ex4-loaded HGC nanoparticles (LAM1-Ex4/HGC NPs) showed improved drug-loading efficiency, small particle size, enhanced resistance against proteolytic digestion, and an extended in vitro drug release profile. These findings may be attributable to the strong hydrophobic interaction between LAM1-Ex4 and the inner core of HGC. Furthermore, LAM1-Ex4/HGC NPs showed prolonged hypoglycemic efficacy in db/db mice, lasting 1 week after a single subcutaneous administration. The present study demonstrated that LAM1-Ex4/HGC NPs have considerable potential as a long-acting sustained-release antidiabetic system for type 2 diabetes.

Protein grafting of p53TAD onto a leucine zipper scaffold generates a potent HDM dual inhibitor.

Reactivation of the p53 pathway by a potential therapeutic antagonist, which inhibits HDM2 and HDMX, is an attractive strategy for drug development in oncology. Developing blockers towards conserved hydrophobic pockets of both HDMs has mainly focused on small synthetic compounds; however, this approach has proved challenging. Here we describe an approach to generate a potent HDM dual inhibitor, p53LZ2, by rational protein grafting of the p53 transactivation domain onto a homodimeric leucine zipper. p53LZ2 shows tight binding affinity to both HDMs compared with wild-type p53 in vitro. X-ray crystallographic, comparative modelling and small-angle X-ray scattering studies of p53LZ2-HDM complexes show butterfly-shaped structures. A cell-permeable TAT-p53LZ2 effectively inhibits the cancer cell growth in wild-type but not mutant p53 by arresting cell cycle and inducing apoptosis in vitro. Thus, p53LZ2, designed by rational grafting, shows a potential therapeutic approach against cancer.

Bioreducible hyaluronic acid conjugates as siRNA carrier for tumor targeting.

The successful clinical translation of siRNA-based therapeutics requires efficient carrier systems that can specifically deliver siRNA within the cytosol of the target cells. Although numerous polymeric nanocarriers forming ionic complexes with siRNA have been investigated for cancer therapy, their poor stability and lack of tumor targetability have impeded their in vivo applications. To surmount these limitations, we synthesized a novel type of biodegradable hyaluronic acid-graft-poly(dimethylaminoethyl methacrylate) (HPD) conjugate that can form complexes with siRNA and be chemically crosslinked via the formation of the disulfide bonds under facile conditions. The crosslinked siRNA-HPD (C-siRNA-HPD) complexes exhibited high stability in a 50% serum solution, as compared to the uncrosslinked siRNA-HPD (U-siRNA-HPD) complexes and free siRNA. Both the C-siRNA-HPD and U-siRNA-HPD complexes were efficiently taken up by the CD44-overexpressing melanoma cells (B16F10), but not by the normal fibroblast cells (NIH3T3). When the RFP-expressing B16F10 cells were treated with the complexes or free siRNA, the C-siRNA-HPD complexes showed the highest decrease in RFP expression. In vivo studies demonstrated the selective accumulation of C-siRNA-HPD complexes at the tumor site after their systemic administration into tumor-bearing mice, resulting in an efficient gene silencing effect. Overall, these results suggest that the HPD conjugate could be used as an efficient carrier for the tumor-targeted delivery of siRNA.

Effect of arginine-rich peptide length on the structure and binding strength of siRNA-peptide complexes.

Heparin decomplexation experiments, as well as all-atom (AA) and coarse-grained (CG) molecular dynamics (MD) simulations, were performed to determine the effect of the size of arginine(Arg)-rich peptides on the structure and binding strength of the siRNA-peptide complex. At a fixed peptide/siRNA mole ratio of 5:1 or 10:1, the siRNA complexes with peptides longer than nine Arg residues are more easily decomplexed by heparin than are those with nine Arg residues. At these mole ratios, peptides longer than nine Arg residues have cationic/anionic charge ratios in excess of unity, and produce more weakly bound complexes than nine Arg residue ones do. AA simulations of mixtures of peptides with a single siRNA show formation of an electrostatically induced complex, and the longer peptides produce a larger complex, but with no significant increase in the number of Arg residues bound to the siRNA. Larger-scale CG-MD simulations show that multiple siRNAs can be linked together by peptides into a large complex, as observed in the experiments. The peptides longer than nine residues, which at mole ratio 5:1 yield a peptide/siRNA charge ratio in excess of unity, include many noninteracting Arg residues, which repel each other electrostatically. This leads to a less dense complex than for 9-residue peptides, which can explain why these longer complexes are more easily decomplexed by heparin molecules, as observed in the experiments. The key role of the charge ratio is supported by simulations that show that, at a mole ratio of 2.5 peptides per siRNA, the longer 18-residue peptide has a charge ratio of roughly unity and also shows a tight complex, just as the 9-residue peptide does at a 5:1 mole ratio, where its charge ratio is also unity.

Robust PEGylated hyaluronic acid nanoparticles as the carrier of doxorubicin: mineralization and its effect on tumor targetability in vivo.

The in vivo stability and tumor targetability of self-assembled polymeric nanoparticles are crucial for effective drug delivery. In this study, to develop biostable nanoparticles with high tumor targetability, poly(ethylene glycol)-conjugated hyaluronic acid nanoparticles (PEG-HANPs) were mineralized through controlled deposition of inorganic calcium and phosphate ions on the nanoparticular shell via a sequential addition method. The resulting nanoparticles (M-PEG-HANPs) had a smaller size (153.7±4.5nm) than bare PEG-HANPs (265.1±9.5nm), implying that mineralization allows the formation of compact nanoparticles. Interestingly, when the mineralized nanoparticles were exposed to acidic buffer conditions (

Cell-penetrating peptide mimicking polymer-based combined delivery of paclitaxel and siRNA for enhanced tumor growth suppression.

Cancer chemotherapy is often limited, since more than one molecule is usually involved with the cancer pathogenesis. A combination of therapeutic drugs would be a promising approach to overcome the complexity of tumors. In this study, a conjugate (DA3) of deoxycholic acid and low molecular weight polyethylenimine (PEI 1.8 kDa), which has a property that mimics that of cell penetrating peptides (CPPs), was used for simultaneous delivery of an anticancer drug and siRNA. When complexed with siRNA, DA3 showed significantly higher target gene silencing efficiency than PEI 25 kDa. The gene silencing efficiency of DA3 was maintained even in the presence of endocytosis inhibitors, suggesting that the polymeric carrier can mediate an endocytosis-independent macromolecular transduction similar to CPPs. The capability of forming a micelle-like core-shell structure enables the conjugates to encapsulate and dissolve paclitaxel (PTX), a water-insoluble drug. The drug-loaded cationic micelles can then interact with siRNA to form stable complexes (PTX/DA3/siRNA). The PTX/DA3/siRNA showed significantly enhanced inhibition of cancer cell growth. When administered into tumor-bearing animals, the PTX/DA3/siRNA demonstrated significant suppression of tumor growth, providing potential usefulness in clinical settings.

Facial amphipathic deoxycholic acid-modified polyethyleneimine for efficient MMP-2 siRNA delivery in vascular smooth muscle cells.

Clinical applications of RNA interference-based therapeutics such as small interfering RNAs (siRNAs) have been limited mainly due to low intracellular delivery efficiency in vitro and in vivo. In this study, facially amphipathic deoxycholic acid (DA)-modified polyethyleneimine (PEI(1.8)) (DA-PEI(1.8)) was synthesized and used as a potent carrier system for siRNA targeted against matrix metalloproteinase-2 (MMP-2) to inhibit the migration of vascular smooth muscle cells (SMCs), which is the major pathomechanism in the development of atherosclerosis and restenosis after arterial injury. A representative facial amphipathic bile acid DA having a high membrane permeability was conjugated to the terminal amine groups of the low molecular weight PEI(1.8) via amide bonds. The DA-PEI(1.8) conjugates formed self-assembled nanoparticles with siRNA molecules in an aqueous phase and the DA-PEI(1.8)/siRNA polyplexes became stabilized and condensed as particle incubation time increased from 0 to 4h. Both cellular internalization and target gene silencing were enhanced as the DA-PEI(1.8)/siRNA polyplexes stabilized. When vascular SMCs were transfected with MMP-2 siRNA, the DA-PEI(1.8)/siRNA polyplex formulation led to a significant decrease in MMP-2 gene expression, resulting in the suppression of cell migration. These results suggest that the DA-PEI(1.8)/MMP-2 siRNA delivery system may be useful in anti-restenotic treatment for various vasculoproliferative disorders such as atherosclerosis, in-stent restenosis, and vein graft failure.

pH-Sensitive pentablock copolymer nanocapsules as nontoxic and efficient gene carriers.

A biodegradable amphiphilic pentablock copolymer PAE-PCL-PEG-PCL-PAE with a pH-sensitive unit was synthesized for use as a nontoxic, biodegradable carrier for gene delivery by forming nanocapsules entrapping nucleic acid drugs. The PAE block can interact with plasmid DNA to form polyelectrolyte complexes in an acidic environment. At physiological pH, the PAE blocks are deprotonated and form an insoluble skin, resulting in the formation of nanocapsules that encapsulate plasmid DNA. The surface charges of the nanocapsules became almost neutral at pH = 7.4, and their size ranged from 210 to 280 nm. The nanocapsule maintained most of its transfection efficiency even in the presence of serum. These nanocapsules are therefore potential carriers for systemic gene therapy.

Ionic complex systems based on hyaluronic acid and PEGylated TNF-related apoptosis-inducing ligand for treatment of rheumatoid arthritis.

The clinical applications of tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL), an emerging therapeutic protein for cancer and rheumatoid arthritis (RA), are limited by its instability and short biological half-life. In this study, efficient therapeutic modalities for RA treatment were developed in the form of nano-sized complexes (nanocomplexes) based on hyaluronic acid (HA) and polyethylene glycol (PEG)-derivatized TRAIL (PEG-TRAIL) formed by N-terminal specific PEGylation. The nanocomplexes were prepared by simply mixing the positively charged PEG-TRAIL and negatively charged HA, and showed negligible loss of bioactivity compared with the PEG-TRAIL. The in vivo biodistribution and diffusion kinetics of Cy5.5-labeled PEG-TRAIL in mice were observed using a near-infrared optical imaging system after subcutaneous injection of three different formulations: PEG-TRAIL in phosphate-buffered saline (PBS, pH 7.4), nanocomplex in PBS, or nanocomplex in 1% HA solution. The results suggested that PEG-TRAIL is released slowly in vivo from the nanocomplex in 1% HA. Experiments in a collagen-induced arthritis mouse model demonstrated that the magnitudes of therapeutic effects, as judged by clinical scores and histology, were significantly enhanced by the sustained delivery of PEG-TRAIL, with the order of nanocomplex in 1% HA>nanocomplex in PBS>PEG-TRAIL in PBS. In addition, sustained delivery of PEG-TRAIL from the nanocomplex in 1% HA resulted in significant reduction of serum inflammatory cytokines and collagen-specific antibodies that are responsible for the pathogenesis of RA. These results imply that HA/PEG-TRAIL nanocomplex formulations are promising therapeutic modalities for the treatment of RA.

Improved antitumor activity and tumor targeting of NH(2)-terminal-specific PEGylated tumor necrosis factor-related apoptosis-inducing ligand.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered an attractive anticancer agent due to its tumor cell-specific cytotoxicity. However, its low stability, solubility, unexpected side effects, and weak pharmacokinetic profiles restrict its successful clinical application. To develop efficient TRAIL-based anticancer biotherapeutics, a new version of trimeric TRAIL was constructed by incorporating trimer-forming zipper sequences (HZ-TRAIL), and then NH(2)-terminal-specific PEGylation was done to produce PEGylated TRAIL (PEG-HZ-TRAIL). The biological, physicochemical, and pharmaceutical characteristics of PEG-HZ-TRAIL were then investigated using various in vitro and in vivo experiments, including a cell-based cytotoxicity test, a solubility test, pharmacokinetic analysis, and antitumor efficacy evaluations. Although slight activity loss occurred after PEGylation, PEG-HZ-TRAIL showed excellent tumor cell-specific cytotoxic effects via apoptotic pathways with negligible normal cell toxicity. The stability and pharmacokinetic problems of HZ-TRAIL were successfully overcome by PEGylation. Furthermore, in vivo antitumor tests revealed that PEG-HZ-TRAIL treatment enhanced therapeutic potentials compared with HZ-TRAIL in tumor xenograft animal models, and these enhancements were attributed to its better pharmacokinetic properties and tumor-targeting performance. These findings show that PEG-HZ-TRAIL administration provides an effective antitumor treatment, which exhibits superior tumor targeting and better inhibits tumor growth, and suggest that PEG-HZ-TRAIL should be considered a potential candidate for antitumor biotherapy.

Long acting hyaluronate--exendin 4 conjugate for the treatment of type 2 diabetes.

Despite clinical exploitation of exendin 4 for the treatment of type 2 diabetes, the significantly short half-life requiring twice a day injection has limited the wide applications. In this work, a protocol for the synthesis of long acting hyaluronate (HA) - exendin 4 conjugate was successfully developed using Michael addition chemistry between vinyl sulfone modified HA (HA-VS) and thiolated exendin 4. The exendin 4 content could be controlled in the range of 5-30 molecules per single HA chain with a bioconjugation efficiency higher than 90%. The conjugation of exendin 4 with HA resulted in about 20 times improved in vitro serum stability maintaining the hypoglycemic and gluco-regulatory bioactivities of exendin 4. HA - exendin 4 conjugates showed excellent glucose-lowering capabilities in type 2 db/db mice demonstrating protracted hypoglycemic effect up to 3 days after a single subcutaneous injection. Furthermore, insulin immunohistochemical analysis of islets in db/db mice confirmed the improved insulinotropic activity of HA - exendin 4 conjugates. The HA - exendin 4 conjugates will be investigated further as a twice a week injection dosage form for clinical applications.

The fatty acid conjugated exendin-4 analogs for type 2 antidiabetic therapeutics.

Improved glucagon-like peptide-1 (GLP-1) receptor activation is considered one of the most effective targets for antidiabetic therapy. For this purpose, we modified the GLP-1 analog of exendin-4 using two fatty acids (FA) either lauric acid (LUA, C12) or palmitic acid (PAA, C16) at its two lysine residues, to produce; Lys(12)-FA-Exendin-4 (FA-M2), Lys(27)-FA-Exendin-4 (FA-M1), or Lys(12,27)-diBA-Exendin-4 (FA-Di). The structural, biological, and pharmaceutical characteristics of these exendin-4 analogs were then investigated. Biological activity tests demonstrated that LUA-M1 had well-preserved in vivo antidiabetic activity and in vitro insulinotropic activity with minimum GLP-1 receptor binding affinity loss as compared with exendin-4. Furthermore, pharmacokinetic studies in rats revealed that s.c. administration of LUA-M1 significantly enhanced pharmacokinetic parameters, such as, elimination half-life, mean residence time, and AUC values as compared with exendin-4. The protracted antidiabetic effects of LUA-M1 were also confirmed by prolonged normoglycemia observed in type 2 diabetic mice (20nmol/mouse single injection of exendin-4 or LUA-M1 induced normoglycemia for 6 or 24h, respectively). These findings suggest that FA conjugated exendin-4s should be considered potential candidates for the treatment of diabetes.

Effect of tumor necrosis factor-related apoptosis-inducing ligand on the reduction of joint inflammation in experimental rheumatoid arthritis.

This study focused on the potential therapeutic effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on collagen-induced arthritis (CIA) and on the elucidation of the mechanisms involved. DBA/1J mice with established CIA were treated with various amount of recombinant soluble human TRAIL. The effects of TRAIL on the development and severity of CIA in this DBA/1J mouse model were assessed clinically and histologically, and a detailed investigation was conducted on proinflammatory cytokine and anticollagen-specific antibody levels. Cellular immunity was evaluated by investigating the proliferative responses and cytokine release profiles of splenocytes after TRAIL treatment. TRAIL treatment significantly reduced the severity and incidence of CIA, joint swelling, erythema, and edema. Histologic evaluations revealed that inflammatory cell infiltration, cartilage destruction, and bone erosion were significantly reduced in joints of TRAIL-treated mice with dose-dependent manner. TRAIL treatment also strongly decreased and/or normalized the productions of proinflammatory cytokines and of anti-collagen-specific antibodies in the sera of CIA mice. Furthermore, in vitro studies with primary splenocytes showed the cytotoxic effect of TRAIL on activated lymphocytes, with reduction of inflammatory cytokine release. These findings show that TRAIL administration is an effective anti-inflammatory treatment that prevents the development and progression of CIA in DBA/1J mice, and they suggest that TRAIL might be considered a potential treatment for human RA.

Biochemical, pharmaceutical and therapeutic properties of long-acting lithocholic acid derivatized exendin-4 analogs.

Alterations in the physicochemical characteristics of peptide drugs can transform their biological and pharmaceutical features. In the present study, we explored the potentials of lithocholic acid (LCA)-modified exendin-4 derivatives as novel long-acting GLP-1 receptor agonists. Exendin-4 was modified with lithocholic acid at two lysine residues to produce three derivatives that were obtained by reverse-phase HPLC separation, namely, Lys(12)-LCA-exendin-4 (LCA-M2), Lys(27)-LCA-exendin-4 (LCA-M1), and Lys(12,27)-LCA-exendin-4 (LCA-Di)). The biological, pharmacological, and physicochemical characteristics of these three exendin-4 analogues were then investigated. Although slight reductions in the GLP-1 receptor binding capacity and insulinotropic activity of exendin-4 were observed after derivatization, the mono-LCA substitutions, especially LCA-M1, well-preserved antidiabetic activity in type 2 diabetic mice when administered subcutaneously or intraperitoneally. Furthermore, the pharmacokinetic characteristics were dramatically enhanced, that is, absorption was delayed and elimination half-life was increased (1.6+/-0.4 and 9.7+/-1.4h by exendin-4 and LCA-M1, respectively). The enhanced long-acting characteristics of the derivative was found to be due to albumin binding and nanoparticle formation, and these were verified by the restoration of normoglycemia in type 2 diabetic mice after single injection (>24h, >10 nmol/kg, s.c.) and daily injections (15 nmol/kg/day) maintained normoglycemia for the 4-week administration period. Furthermore, antidiabetic potentials, such as, glucose clearance kinetics and percentage areas occupied by pancreatic beta-cells were also enhanced by long-term LCA-M1 administration. The present study demonstrates that the derivatization of exendin-4 with LCA offers a possible means of producing a long-acting GLP-1 receptor agonist.

Preparation and structural, biochemical, and pharmaceutical characterizations of bile acid-modified long-acting exendin-4 derivatives.

To develop an effective long-acting antidiabetic, the GLP-1 analogue of exendin-4 was modified with three different bile acids (BAs; cholic, deoxycholic, or lithocholic acid), at its two lysine residues. The biological, pharmaceutical, and physicochemical characteristics of these exendin-4 analogues were carefully investigated. Biological activity tests demonstrated that the monobile acid substitutions of exendin-4 showed well preserved receptor binding efficacy without noticeable insulinotropic or antidiabetic activity loss. However, physicochemical and pharmacokinetic studies revealed that the albumin-binding properties and in vivo elimination half-lives of BAM1-Ex4s (Lys(27)-BA-Ex4s) were significantly enhanced by increasing the hydrophobicities of the conjugated BAs. Furthermore, the protracted antidiabetic effects of the BAM1-Ex4s were also verified by the prolonged restoration of normoglycemia in type 2 diabetic mice. Accordingly, the present study suggests that the derivatization of exendin-4 with BAs offers a means of producing long-acting GLP-1 receptor agonists for type 2 diabetic therapy.

A new orally available glucagon-like peptide-1 receptor agonist, biotinylated exendin-4, displays improved hypoglycemic effects in db/db mice.

An orally active glucagon-like peptide-1 (GLP-1) formulation would have great advantages over conventional injectable therapies for the treatment of diabetic patients. Because GLP-1 absorption in the intestine is restricted by its natural physiological characteristics, biotinylated exendin-4 analogues might useful as orally active GLP-1 receptor agonists. Three different biotinylated exendin-4 analogues, Lys(27)-Biotin-Exendin-4 (MB1-Ex-4), Lys(12)-Biotin-Exendin-4 (MB2-Ex-4), and Lys(12, 27)-Biotin-Exendin-4 (DB-Ex-4) were prepared, and their biological activities and enzymatic stabilities were studied in vitro. The hypoglycemic effects and pharmacokinetics of these analogues after oral administration were evaluated in db/db mice and Sprague-Dawley rats, respectively. These biotinylated exendin-4 analogues preserved GLP-1 receptor binding affinity and stimulated insulin secretion in RIN-m5F murine insulinoma cells and in isolated rat islets, respectively, and were as potent as exendin-4. In particular, DB-Ex-4 showed 9.0-fold better stability against rat intestinal fluid than exendin-4. When 0.1, 1, and 10 microg/mouse of DB-Ex-4 were orally administered, mean total hypoglycemic degrees (HGD) were increased by 36.8+/-1.2, 46.9+/-1.8, and 54.3+/-4.5%, respectively, whereas 1 microg/mouse of native exendin-4 showed an increase of 8.8+/-7.3%. This study demonstrates that biotinylated exendin-4 analogues are absorbed in the intestine and that they have biological efficacies of exendin-4. Furthermore, it indicates that biotinylated exendin-4 analogues could be used as potential oral antidiabetic agent for the treatment of type 2 diabetes.

Conjugated chitosan as a novel platform for oral delivery of paclitaxel.

A new platform for oral delivery of paclitaxel (PTX) was developed through chemical conjugation of PTX to a low molecular weight chitosan (LMWC). The LMWC-PTX conjugate contained approximately 12 wt % PTX and showed greatly enhanced water solubility (>1 mg/mL) as compared to native PTX. The conjugate showed comparable IC 50 values to that of the parent PTX against human cancer cell lines. The pharmacokinetic data revealed approximately 42% of bioavailability after oral administration of 5 mg PTX/kg of the conjugate. When the conjugate (10 mg/kg based on PTX content) was administered orally to mice bearing xenograft or allograft tumors, the conjugate-treated group showed significant inhibition of tumor growth, which was comparable to that seen with PTX of the clinically available injected form, formulated in cremophor EL/ethanol (iv) but with much lower toxicity. Tracking I (125)-labeled conjugate showed that LMWC-PTX was likely to be absorbed mainly from the ileum and reach the blood as the intact conjugate.

Stability and bioactivity of nanocomplex of TNF-related apoptosis-inducing ligand.

The tumor necrosis factor-related apoptosis inducing ligand (TRAIL) has gained much attention due to its potent therapeutic effect for cancer and rheumatoid arthritis. In this study, we attempted to develop the injectable formulations which can stabilize TRAIL and thus show prolonged blood circulation in vivo. The positively charged TRAIL was mixed with hyaluronic acid (HA), resulting in the formation of nanocomplexes. The zeta-potentials of nanocomplexes and their mean diameters were significantly dependent on the feed ratio of HA to TRAIL. The increase in the feed ratio of HA reduced the particle size and decreased the value of the zeta-potential. The bioactivity of TRAIL in the complexes was comparable to that of native TRAIL, indicating that the complex formation did not affect the activity of TRAIL. Furthermore, the stability of TRAIL in the complexes was retained for 6 days, during which the bioactivity of native TRAIL disappeared. When native TRAIL was subcutaneously injected into the rats, its plasma concentration was not detectable after 12h. In contrast, for HA/TRAIL nanocomplexes in 1% HA solution, substantial amount of TRAIL was circulated in blood for up to 5 days. These results imply that HA-based formulations of TRAIL hold the potential as the therapeutics.

Improved intrapulmonary delivery of site-specific PEGylated salmon calcitonin: optimization by PEG size selection.

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.

Biological and physicochemical evaluation of the conformational stability of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

Tumor necrosis factor (TNF)-related, apoptosis-inducing ligand (Apo2L/TRAIL) has a unique homotrimeric structure, and its conformational stability is essential for its apoptotic activity. The conformational stability of a modified version of TRAIL(114-281) with two additional domains of histidine tag and isoleucine zipper [His-ILZ-TRAIL(114-281)] was evaluated in various pH environments according to three different biological or physicochemical considerations: cytotoxicity, antibody-binding affinity, and tertiary structure. The biological properties of His-ILZ-TRAIL(114-281) were the most stably maintained at pH 6.0. The physicochemical analyses (circular dichroism and fluorescence spectroscopy) demonstrate that its bioactivity loss by pH challenge was originated from its structural collapse as a homotrimer.