High-yield production of biologically active mono-PEGylated salmon calcitonin by site-specific PEGylation

Youn, Y.S.,Na, D.H.,Lee, K.C. Elsevier Science Publishers 2007 Journal of controlled release Vol.117 No.3

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<SUP>18</SUP>) salmon calcitonin (Lys<SUP>18</SUP>-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<SUP>1,11</SUP>-sCT), which was FMOC protected at Cys<SUP>1</SUP>- and Lys<SUP>11</SUP>-amines among three PEGylation sites including Lys<SUP>18</SUP>-amine. This PEGylation process was achieved by the consecutive one-pot, two-step reaction: (i) the PEG conjugation to FMOC<SUP>1,11</SUP>-sCT; and (ii) the subsequent deprotection of FMOC group from the PEGylated FMOC<SUP>1,11</SUP>-sCT. The optimized reaction resulted in the high production yield of Lys<SUP>18</SUP>-PEG-sCT (about 86%), compared with that from conventional non-specific PEGylation (about 18%). The prepared Lys<SUP>18</SUP>-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<SUP>18</SUP>-PEG-sCT conjugate with a high yield.

Oxidative Stabilization of Conjugated Linoleic Acid by One-Pot PEGylation

문현석,조종수,서지혜,Ding-Ding Guo,이홍구,Prati Bajracharya,최윤재,정근수,박종래 한국고분자학회 2011 Macromolecular Research Vol.19 No.8

This study describes a novel and one-step procedure for the PEGylation of conjugated linoleic acid (CLA)in the melting state without solvents or catalysts for oxidative stabilization of CLA. The double bonds of CLA in PEGylated CLA (PCLA) were clearly detected by 1H NMR (nuclear magnetic resonance) spectroscopy, indicating the successful PEGylation of CLA without oxidation during PEGylation, even at high temperatures. The level of headspace oxygen depletion by PCLA was lower than CLA, indicating that the oxidation of CLA was protected by PEGylation. This easy and simple one-pot PEGylation of CLA is an effective way of preparing fatty acids with increased stability to oxidation, which would be quite useful for nutritional and pharmaceutical studies.

In vitro refolding of PEGylated lipase

Kim, M.Y.,Kwon, J.S.,Kim, H.J.,Lee, E.K. Elsevier Science Publishers 2007 Journal of biotechnology Vol.131 No.2

Covalent modification of proteins with polyethylene glycol (PEG) has become a well established drug enhancement strategy in the biopharmaceutical industry. The general benefits of PEGylation, such as prolonged serum half-lives or reduced in vivo immunogenicity, are well known. To date, the PEGylation process has been performed with purified proteins, which often requires additional multi-step purification steps to harvest the desired PEGylate. However, it would be beneficial for bioprocessing if 'renaturation,' i.e. in vitro refolding and 'modification,' and PEGylation can be integrated, especially for inclusion body proteins. We investigated the feasibility of protein PEGylation under denaturing conditions and of protein refolding with the attached PEG molecule. Using lipase as a model protein, PEGylation occurred in 8M urea and covalently attached PEG did not appear to hinder subsequent refolding.

Improved intestinal delivery of salmon calcitonin by Lys¹⁸-amine specific PEGylation: Stability, permeability, pharmacokinetic behavior and in vivo hypocalcemic efficacy

Youn, Yu Seok,Jung, Ju Young,Oh, Seung Hyun,Yoo, Sun Dong,Lee, Kang Choon Elsevier 2006 Journal of controlled release Vol.114 No.3

<P><B>Abstract</B></P><P>Peptides like salmon calcitonin (sCT) are subjected to aggressive proteolytic attack by various intestinal enzymes, and fractions that enter the systemic circulation via the intestinal route are rapidly inactivated by tissue accumulation and glomerular filtration. Here, we describe the beneficial effects of the Lys<SUP>18</SUP>-amine specific PEGylation of sCT on the intestinal delivery of sCT. Two key properties were enhanced by the PEGylation process: (i) the resistance of sCT to intestinal enzymes and (ii) the systemic clearance of sCT that had entered the circulation. Initially, we evaluated the cAMP-secreting activities of PEG<SUB>2K</SUB>-sCT isomers substituted at Cys<SUP>1</SUP>-, Lys<SUP>11</SUP>- or Lys<SUP>18</SUP>-amine position in T47D cells, and found that sCT PEGylated at Lys<SUP>18</SUP>-amine (Lys<SUP>18</SUP>-PEG<SUB>2K</SUB>-sCT) had the highest bioactivity. We then investigated the stability of Lys<SUP>18</SUP>-PEG<SUB>2K</SUB>-sCT in the presence of intestinal enzymes, its abilities to traverse the intestinal membrane, its pharmacokinetic behavior and in vivo hypocalcemic efficacy. Results show that Lys<SUP>18</SUP>-PEG<SUB>2K</SUB>-sCT has significantly increased resistance to pancreatic peptidases and brush-border peptidases. Despite the molecular size increase caused by PEGylation, Lys<SUP>18</SUP>-PEG<SUB>2K</SUB>-sCT was found to have an intestinal permeability similar to that of unmodified sCT (<I>p</I>>0.59) over an apical concentration range 12.5–100?μM in a Caco-2 cell monolayer transport system. In particular, tissue distribution results showed that <SUP>125</SUP>I-labeled Lys<SUP>18</SUP>-PEG<SUB>2K</SUB>-sCT markedly resists liver accumulation and glomerular filtration; levels were reduced by 75% and 50% vs. sCT. Finally, the hypocalcemic efficacy of intestinally administered Lys<SUP>18</SUP>-PEG<SUB>2K</SUB>-sCT, measured as total serum calcium in a rat model, was 5.8 and 3.0 times that of sCT at 100 and 200?IU/kg (<I>p</I><0.025). Our findings suggest that this site-specific conjugation of peptides with PEG of proper size enhances pharmacokinetic properties by increasing their abilities to resist both proteolysis and systemic clearance without significantly reducing their membrane permeabilities or bioactivities. We believe that this concept, namely, dual effects by PEGylation, has great potential value because it presents a practical means of enhancing the efficacies of the peroral/intestinal pharmacologic route.</P>

A study of PEGylated-oligopeptide derivatives: synthesis and biological activity

김미진,김보미,양두리,정성훈,채규윤 한국공업화학회 2015 한국공업화학회 연구논문 초록집 Vol.2015 No.1

In this study, some known, basic oligopeptide derivatives were synthesized and then conjugated with gallic acid (GA) through the peptide bindings between the amine (-NH₂) and carboxylic acid (-COOH) moieties to improve their anti-wrinkle effect. The solubility and bioactivity of these novel conjugated oligopeptides were investigated for the development of new drugs and functional cosmetic materials. For the synthesis of the designed conjugated oligopeptides, first, the known, basic tripeptide (Gly-His-Lys : GHK), pentapeptide (Lys-Thr-Thr-Lys-Ser : KTTKS), and hexapeptide (Glu-Glu-Met-Gln-Arg-Arg : EEMQRR) were linked to the resin by the solid phase peptide synthesis (SPPS). The linking yields were depended on the chains of the amino acid sequences in the peptides. The longer chain of the amino acid sequence gave the lower yield. Second, PEGylated 3,4,5-triacetoxy benzoic acid (GA-PEG) was incorporated with the basic solid peptides by using the DIC and HOBt coupling reagents at 30℃. The Kaiser test of the free amino acid groups was used to monitor the reaction. Finally, the trifluoroacetic acid (TFA) cleavages from the solid supports gave the desired oligopeptides in the moderate yields such as 38-45% after 10-14 h for PEGylated 3,4,5-triacetoxy benzoic tripeptide (GA-PEG-GHK), PEGylated 3,4,5-triacetoxy benzoic pentapeptide (GA-PEG-KTTKS) and PEGylated 3,4,5- triacetoxy benzoic hexapeptide (GA-PEG-EEMQRR). The solubility of the conjugated oligopeptides was examined. In almost cases, the synthesized oligopeptides were soluble in water, methanol, and dichloromethane and were very soluble in DMSO. However, hexapeptide derivatives GA-PEG-EEMQRR was only dissolved in CHCl₃. The biological activity of the conjugated oligopeptides was investigated. We tried anti-oxidation activity, anti-inflammatory effect via nitric oxide inhibition, anti-wrinkle effect experiment and get good results. It can be inferred from our experimental results that GA-PEG-GHK and GA-PEGKTTKS may be recognized as the new potential drugs and functional cosmetic materials for industrial applications.

Pharmacokinetic and pharmacodynamic evaluation of site-specific PEGylated glucagon-like peptide-1 analogs as flexible postprandial-glucose controllers

Chae, Su Young,Chun, Young Goo,Lee, Seulki,Jin, Cheng-Hao,Lee, Eun Seong,Lee, Kang Choon,Youn, Yu Seok Wiley Subscription Services, Inc., A Wiley Company 2009 journal of pharmaceutical sciences Vol.98 No.4

<P>The rapid elimination of glucagon-like peptide-1 (GLP-1) is the main impediment to its anti-diabetic utility. Here, we tried to improve its poor pharmacokinetic/pharmacodynamic profiles using PEGylation. The site-specific (Lys<SUP>34</SUP>) PEGylated GLP-1s were synthesized with PEGs of 2, 5, and 10 kDa, respectively. Oral glucose tolerance tests using db/db mice showed that these three PEGylated GLP-1s (5 nmol/kg) specifically stabilized plasma glucose levels when intraperitoneally (i.p.) administered at 30, 30–120, or 120–360 min preoral glucose treatment, respectively (total hypoglycemic degree: 60.5 ± 5.0%, ∼67.2 ± 2.3%, and ∼59.4 ± 4.3%, respectively). Particularly, Lys<SUP>34</SUP>-PEG<SUB>10K</SUB>-GLP-1 showed an stable hypoglycemic efficacy when administered up to 360 min preglucose. The different anti-diabetic effects of PEGylated GLP-1s were attributed to their augmented pharmacokinetics and metabolic resistance. These analogs had higher metabolic stabilities in rat plasma, liver and kidney homogenates, and extended pharmacokinetic profiles with the greater circulating half-lives (26.6, 64.5, and 105.5 min for Lys<SUP>34</SUP>-PEG<SUB>2,5,10 K</SUB>-GLP-1s, respectively, vs. 8.5 min for GLP-1, at elimination phases after i.p. injections) in ICR mice. Our findings suggest that GLP-1 substituted with a PEG of an appropriate Mw at Lys<SUP>34</SUP> could be used as a promising type 2 anti-diabetic agent to timely control postprandial glucose levels. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:1556–1567, 2009</P>

Oligopeptide의 PEGylation과 반응 생성물의 생물학적 연구

김보미 ( Kim Bo Mi ),송미선 ( Mi Seon Song ),강경희 ( Kyung Hee Kang ),이덕희 ( Duckhee Lee ),채규윤 ( Kyu Yun Chai ) 한국키틴키토산학회 2014 한국키틴키토산학회지 Vol.19 No.1

PEGylated peptide (GHK and GPHyp), the collagen peptides with hydrophilic mPEG to improve stability, soubility, and degree of absorbance in physiology condition, were successfully synthesized in solid phase by coupling reactions through three steps. Their applications as cosmetic materials to improve the skin absorbablity will be evaluated. Acrylonitrile using mPEG ishydrolysis to yield the stylized oxide, which was separated by using Sephadex ion exchange chromatography. The good result from bioactive experiments of the effects on cytotoyicity, collagen activation and antioxidant were obtained.

PEGylation and HAylation via catechol: α-Amine-specific reaction at N-terminus of peptides and proteins

Song, I.T.,Lee, M.,Lee, H.,Han, J.,Jang, J.H.,Lee, M.S.,Koh, G.Y.,Lee, H. Elsevier BV 2016 ACTA BIOMATERIALIA Vol.43 No.-

The development of chemoselective, site-specific chemistries for proteins/peptides is essential for biochemistry, pharmaceutical chemistry, and other fields. In this work, we found that catechol, which has been extensively utilized as an adhesive molecule for material-independent surface chemistry and as a crosslinker in hydrogel preparation, specifically reacts with N-terminal α-amines, avoiding the ε-amine group in lysine. A conjugate of methoxy-poly(ethylene glycol)-catechol called mPEG-cat chemoselectively reacts with N-terminal amine groups at neutral pH resulting in site-specific PEGylation. To demonstrate the versatility of this catechol chemoselective reaction, we used four proteins (lysozyme, basic-fibroblast growth factor (bFGF), granulocyte-colony stimulating factor (G-CSF), insulin, and erythropoietin (EPO)) as well as two peptides (hinge-3 and laminin-derived peptide (LDP)). All the tested macromolecules showed N-terminal site-specific modifications. Furthermore, we prepared another catechol grafted conjugate called hyaluronic acid-catechol (HA-cat) to demonstrate that this catechol-involved chemoselective chemistry is not specific for PEG conjugates. This new catechol chemoselective chemistry could be a new platform for the functionalization of proteins and peptides for a variety of purposes. Statement of Significance: Considering the fact that biological activities of proteins or peptides depend largely on their 3-dimensional conformation, the orientation-controllable reaction is very important for preserving the intrinsic functionality of them. In addition to PEG, many other bio-polymers such as oligonucleotides, antibodies, and oligosaccharides have been conjugated with proteins or peptides for various biomedical applications. Although several chemoselective conjugation chemistries have been reported, conjugation efficiencies are different depending on types of proteins or polymers, and thus there've been strong needs for the development of alternative strategy of chemoselective conjugation that can be applied for a variety of therapeutic proteins towards high biological activities. We are certain this new catechol chemoselective chemistry could be a new platform for the functionalization of proteins and peptides for various purposes.

Development of paclitaxel-loaded liposomal nanocarrier stabilized by triglyceride incorporation

Hong, Soon-Seok,Choi, Ju Yeon,Kim, Jong Oh,Lee, Mi-Kyung,Kim, So Hee,Lim, Soo-Jeong DOVE MEDICAL PRESS 2016 INTERNATIONAL JOURNAL OF NANOMEDICINE Vol.11 No.-

<P>Studies have highlighted the challenge of developing injectable liposomes as a paclitaxel (PTX) carrier, a challenge attributable to the limitations in liposomal stability caused by PTX loading. Poor stability of PTX-loaded liposomes is caused by PTX-triggered aggregation or fusion of liposomal membranes and is exacerbated in the presence of PEGylated lipid. In the present study, the effect of triglyceride incorporation on the stability of PTX-loaded/PEGylated liposomes was explored. Incorporation of a medium chain triglyceride Captex 300 into saturated phosphatidylcholine (PC)-based liposomes (1,2-dimyristoyl-<I>sn</I>-glycero-3-phosphocholine [DMPC]:cholesterol [CHOL]:N-(Carbonyl-methoxypolyethyleneglycol 2000)-1, 2-distearoyl-<I>sn</I>-glycero-3-phospho-ethanolamine [PE-PEG]), produced a fine, homogeneous, and membrane-filterable PTX-loaded liposomes fulfilling the requirement of an injectable lipid formulation. Triglyceride incorporation also greatly inhibited the time-dependent leakage of PTX from saturated PC-based liposomes, which appears to be mediated by the inhibition of liposome fusion. In contrast, triglyceride incorporation induced the destabilization and PTX leakage of unsaturated PC-based liposomes, indicating the opposite effect of triglyceride depending on the fluidity status of PC constituting the liposomal membrane. PTX release profile and the in vitro and in vivo anticancer efficacy of triglyceride-incorporated DMPC:CHOL:PE-PEG liposomes were similar to Taxol<SUP>®</SUP> while the toxicity of liposomal PTX was significantly lower than that of Taxol. Taken together, triglyceride incorporation provided an injectable PTX formulation by functioning as a formulation stabilizer of PEGylated/saturated PC-based liposomes.</P>

Bioinspired Adhesive Coating on PET Film for Antifouling Surface Modification

허성범,김지흥,전영실,김승일,김수현 한국고분자학회 2014 Macromolecular Research Vol.22 No.2

One of the most common approaches to preparing antifouling surfaces is the immobilization of poly(ethyleneglycol), which is known as PEGylation. In addition, the catechol functional group of dopamine (3,4-dihydroxyphenethylamine)has the capacity to create a strong coordinate bond with inorganic and organic surfaces in an aqueousenvironment. We synthesized novel polyaspartamide derivatives containing poly(ethylene glycol) and catechol pendantgroups, and the same polymers in a pH-controlled aqueous solution were successfully employed to modify apoly(ethylene terephthalate) (PET) surface using a simple immersion method. Contact angle measurements, X-rayphotoelectron spectroscopy, attenuated total reflectance-infrared spectroscopy, and atomic force microscopy were usedto verify the surface coating on the PET substrate. Also, the cytotoxicity and antifouling properties were evaluatedby protein adsorption and platelet adhesion tests of the coated materials. This biocompatible polymer has potentialas an adhesive coating and for the surface modification of various biomaterials.