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Exendin-4 (EX4), a glucagon-like peptide-1 receptor agonist, has been reported to attenuate myocardial ischemia and reperfusion injury and inflammatory or oxidative responses. The expression level of secretory group IIA phospholipase A2 (sPLA2-IIA) is elevated in inflammatory diseases. Lipopolysaccharide (LPS) upregulates the expression of sPLA2-IIA in human umbilical vein endothelial cells (HUVECs). Here, EX4 was examined for its effects on the expression and activity of sPLA2-IIA in HUVECs and mice. Pre-treatment of cells or mice with EX4 inhibited LPS-induced sPLA2-IIA expression and activity. Additionally, EX4 suppressed LPS-induced activation of cytosolic phospholipase A2 (cPLA2) and extracellular signal-regulated kinase (ERK) 1/2. Therefore, these results show that EX4 inhibited LPS-induced expression of sPLA2-IIA by suppressing cPLA2 and ERK 1/2., (Copyright © 2015 Elsevier Inc. All rights reserved.)

High mobility group box 1 (HMGB1) was recently shown to be an important extracellular mediator of severe vascular inflammatory disease, sepsis. Lysozyme (LYZ) has been shown to bind to bacterial lipopolysaccharide (LPS) and have a potential for playing a role in the therapy of inflammatory diseases. However, the effect of LYZ on HMGB1-induced septic response has not been investigated. Moreover, PEGylation effects on the antiseptic activity of LYZ are not known. Here, we show, for the first time, the anti-septic effects of PEGylated LYZ (PEG-LYZ) in HMGB1-mediated inflammatory responses in vitro and in vivo. Among four mono-PEGylated LYZs with different PEGylation sites (N-terminus, Lys(13), Lys(33), and Lys(97)), N-terminally PEGylated LYZ showed the highest activity. Subsequently, among three N-terminally PEGylated LYZs prepared with aldehyde-activated PEGs of 5, 10, and 20 kDa, 5 kDa-PEG-conjugated LYZ (P5-K(1)-LYZ) showed the highest antiseptic activity. The data showed that P5-K(1)-LYZ post-treatment effectively suppressed LPS-mediated release of HMGB1. P5-K(1)-LYZ also inhibited HMGB1-mediated hyperpermeability in human endothelial cells. Furthermore, P5-K(1)-LYZ reduced the cecal ligation and puncture (CLP)-induced release of HMGB1 and septic mortality. Collectively, these results suggest P5-K(1)-LYZ as a candidate therapeutic agent for the treatment of vascular inflammatory diseases via inhibition of the HMGB1 signaling pathway., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Although collagen pentapeptide (Lys-Thr-Thr-Lys-Ser, KTTKS) has received a great deal of attention owing to its collagen biosynthesis-stimulating effects, its enzymatic instability in the skin is an obstacle to effective topical application. PEGylation is a useful approach for improving the chemical and biological stability of peptides. However, the polydispersity of poly(ethylene glycol) (PEG) produces conjugates with different molecular sizes, which may create difficulties in chemical characterization and purity control, and in variability of biological properties. To overcome these difficulties, monodisperse PEG was site-specifically conjugated to the N-terminal amine of KTTKS to produce a single molecular conjugate, enabling more complete chemical characterization and more exact product specifications. PEG-KTTKS conjugates prepared using monodisperse PEG with two different molecular weights, monodisperse PEG220 and PEG572, were characterized by mass spectrometry. These monodisperse PEG-KTTKS conjugates showed no cytotoxicity (1-100 μM) and stimulated collagen biosynthesis in human skin fibroblasts. They also had high stability against proteolytic enzymes in rat skin. This study demonstrates the usefulness of monodisperse PEG for preparing chemically defined conjugates and suggests that monodisperse PEG-KTTKS would be a good candidate for use as a collagen biosynthesis-stimulating agent., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Exendin-4 (EX4) has been reported to attenuate myocardial ischemia and reperfusion (I/R) injury and inflammatory and oxidative responses. Nuclear DNA-binding protein high-mobility group box 1 (HMGB1) protein acts as a late mediator of severe vascular inflammatory conditions. However, the effect of EX4 on HMGB1-induced inflammatory response has not been studied. First, we accessed this question by monitoring the effects of posttreatment EX4 on lipopolysaccharide (LPS) and cecal ligation and puncture (CLP)-mediated release of HMGB1 and HMGB1-mediated regulation of proinflammatory responses in human umbilical vein endothelial cells (HUVECs) and septic mice. Posttreatment EX4 was found to suppress LPS-mediated release of HMGB1 and inhibited HMGB1-mediated hyperpermeability and leukocyte migration in septic mice. EX4 also induced downregulation of CLP-induced release of HMGB1, production of IL-6, and mortality. Collectively, these results suggest that EX4 may be regarded as a candidate therapeutic agent for treatment of vascular inflammatory diseases via inhibition of the HMGB1 signaling pathway.

Collagen pentapeptide (Lys-Thr-Thr-Lys-Ser, KTTKS) and its palmitoylated derivative (pal-KTTKS) have received a great deal of attention as cosmeceutical ingredients for their anti-wrinkle effects. The objective of this study was to evaluate stability and permeability of KTTKS and pal-KTTKS in hairless mouse skin. In this study, a liquid chromatography-tandem mass spectrometric method was developed for the quantification of pal-KTTKS, and used for stability and permeability studies. Stability studies were performed using skin extracts and homogenates. Both KTTKS and pal-KTTKS were rapidly degraded, but pal-KTTKS was more stable than KTTKS. When protease inhibitors were added, the stability of both compounds (KTTKS and pal-KTTKS) improved significantly. In the skin permeation study, neither KTTKS nor pal-KTTKS was detected in the receptor solution, which indicates that neither compound could permeate through the full-thickness hairless mouse skin in the experimental conditions of this study. While KTTKS was not detected in any of the skin layers (the stratum corneum, epidermis, and dermis), pal-KTTKS was observed in all skin layers: 4.2 ± 0.7 μg/cm(2) in the stratum corneum, 2.8 ± 0.5 μg/cm(2) in the epidermis, and 0.3 ± 0.1 μg/cm(2) in the dermis. In conclusion, this study indicated that pal-KTTKS had greater stability and permeability than that of un-modified KTTKS, and may be a useful anti-wrinkle and anti-aging cosmeceutical agent.

Exendin-4 (EX4), a glucagon-like peptide-1 receptor agonist, has been reported to attenuate myocardial ischemia and reperfusion (I/R) injury, inflammatory and oxidative responses. Increasing evidence has demonstrated that beyond its role in activation of protein C, endothelial cell protein C receptor (EPCR) is involved in vascular inflammation. EPCR activity is markedly decreased by ectodomain cleavage and release as the soluble EPCR. EPCR can be shed from the cell surface, which is mediated by tumor necrosis factor-α converting enzyme (TACE). However, little is known about the effects of EX4 on EPCR shedding. Data from this study showed that EX4 induced potent inhibition of phorbol-12-myristate 13-acetate (PMA), tumor necrosis factor (TNF)-α, and interleukin (IL)-1β-induced EPCR shedding in human umbilical vein endothelial cells (HUVECs), and cecal ligation and puncture (CLP)-induced EPCR shedding in mice. EX4 also inhibited expression and activity of TACE induced by PMA in HUVECs. In addition, treatment with EX4 resulted in reduced PMA-stimulated phosphorylation of p38, extracellular regulated kinases (ERK) 1/2, and c-Jun N-terminal kinase (JNK). These results demonstrate the potential of EX4 as an anti-sEPCR shedding reagent against PMA and CLP-mediated EPCR shedding., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The objective of this study was to develop chromatographic methods for the determination of the modification degree and the characterization of poly(ethylene glycol)-modified polyamidoamine dendrimers (PEG-PAMAMs). The PEG-PAMAMs were prepared by reacting PAMAM generation 4 with monomethoxy PEG-nitrophenyl carbonate (mPEG-NPC). The modification degrees of PEG-PAMAMs were determined by quantifying 4-nitrophenol released from mPEG-NPC after PEGylation reaction using high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection. The PEG-PAMAMs, which have poor UV absorbances, were characterized by HPLC with charged aerosol detection. This study demonstrates that the combination of these two detectors is a powerful tool for the preparation and characterization of PEG-PAMAMs., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Caffeic acid phenethyl ester (CAPE) is a polyphenolic natural product that possesses numerous biological activities including anti-inflammatory effects. CAPE-mediated nuclear factor-erythroid 2 p45 (NF-E2)-related factor 2 (Nrf2) activation is likely responsible for some of its biological effects. CAPE was chemically modified to yield CAPE analogues that were subjected to experiments examining cellular Nrf2 activity. CAPE and the CAPE analogue with a catechol moiety, but not the other analogues, activated the Nrf2 pathway. In addition, only biotin-labeled CAPE analogues with the catechol moiety precipitated Kelch-like ECH associated protein 1 (Keap1) when incubated with cell lysates and streptavidin agarose beads. Sodium hypochlorite (NaOCl) oxidation of the catechol moiety in CAPE produced an oxidized, electrophilic form of CAPE (Oxi-CAPE) and greatly enhanced the ability of CAPE to activate Nrf2 and to bind to Keap1. Rectal administration of CAPE ameliorated 2,4,6-trinitrobenzene sulfonic acid-induced rat colitis and activated the Nrf2 pathway in the inflamed colon, and incubation of CAPE in the lumen of the inflamed distal colon generated Oxi-CAPE. However, these biological effects and chemical change of CAPE were not observed in the normal colon. Our data suggest that CAPE requires the catechol moiety for the oxidation-enhanced activation of the Nrf2 pathway and has potential as a pathologically targeted Nrf2-activating agent that is exclusively activated in pathological states with oxidative stress such as colonic inflammation., (© 2013 Elsevier Inc. All rights reserved.)

Antipsychotic drugs have been used to treat patients with schizophrenia and other psychotic disorders. Long-acting injectable antipsychotic drugs are useful for improving medication compliance with a better therapeutic option to treat patients who lack insight or adhere poorly to oral medication. Several long-acting injectable antipsychotic drugs are clinically available. Haloperidol decanoate and fluphenazine decanoate are first-generation depot drugs, but the use of these medicines has declined since the advent of second-generation depot agents, such as long-acting risperidone, paliperidone palmitate, and olanzapine pamoate. The second-generation depot drugs are better tolerated and have fewer adverse neurological side effects. Long-acting injectable risperidone, the first depot formulation of an atypical antipsychotic drug, was prepared by encapsulating risperidone into biodegradable microspheres. Paliperidone palmitate is an aqueous suspension of nanocrystal molecules, and olanzapine pamoate is a microcrystalline salt of olanzapine and pamoic acid suspended in aqueous solution. This review summarizes the characteristics and recent research of formulations of each long-acting injectable antipsychotic drug.

The objective of this study was to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine the stability of collagen pentapeptide (KTTKS), which is a subfragment of collagen and has been proved to promote the extracellular release of collagen in skin fibroblast, in rat skin. The chromatographic condition was optimized on an Acclaim C-18 column (2.1 mm × 150 mm, 3 μm) under isocratic elution using a mobile phase consisting of deionized water and acetonitrile (87:13, v/v) mixture containing 5mM pentafluoropropionic acid as an ion-pairing reagent. The quantitation of KTTKS was performed on a triple quadrupole mass spectrometer in multiple reaction monitoring mode. The calibration curve showed good linearity in the concentration range of 0.05-10.0 μg/mL (r(2)>0.999). The intra- and inter-day precisions were 0.8-6.5% and 2.4-5.8%, respectively, and the intra- and inter-day accuracies were 96.3-102.7% and 92.8-98.5%, respectively. The developed LC-MS/MS method was successfully applied to investigate the degradation rate and sites of KTTKS in rat skin homogenate. KTTKS was found to be very susceptible to the peptide bond cleavage by aminopeptidases present in the skin., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The purpose of this study was to develop a novel method to inhibit the formation of acylated peptide impurities in poly(D,L-lactide-co-glycolide) (PLGA) formulations by reversely blocking the amino groups of octreotide with maleic anhydride (MA). Two mono-MA conjugates with different modification sites (N terminus and Lys residue) and di-MA conjugate of octreotide were prepared and isolated by reversed-phase high-performance liquid chromatography (RP-HPLC). The polymer interaction of peptides and the formation of acylated peptides were monitored by RP-HPLC. The stability of MA-octreotide conjugates in PLGA films was studied in 0.1 M phosphate buffer (pH 7.4) at 37°C. The conjugation of MA to octreotide substantially inhibited the interaction of peptide with PLGA polymer and the subsequent formation of acylated peptide impurities. The MA-octreotides were successfully converted to intact octreotide as pH drops by PLGA hydrolysis. In PLGA films, MA-octreotide also showed complete inhibition of peptide acylation. In conclusion, MA conjugation provides a viable approach for stabilizing peptides in PLGA delivery systems.

As the use of herbal medicines increases, the public health consequences of drug-herb interactions are becoming more significant. Herbal medicines share the same drug metabolizing enzymes and drug transporters, including cytochrome P450 enzymes (CYPs), glucuronosyltransferases (UGTs), and P-glycoprotein, with several clinically important drugs. Interactions of several commonly used herbal medicines, such as Ginko biloba, milk thistle, and St. John's wort, with therapeutic drugs including warfarin, midazolam, alprazolam, indinavir, saquinavir, digoxin, nifedipine, cyclosporine, tacrolimus, irinotecan, and imatinib in humans have been reported. Many of these drugs have very narrow therapeutic indices. As the herb-drug interactions can significantly alter pharmacokinetic and pharmacodynamic properties of administered drugs, the drugs interacting with herbal medicines should be identified by appropriate in vitro and in vivo methods. A good understanding of the mechanisms of herb-drug interactions is also essential for assessing and minimizing clinical risks. In vitro methods are useful for providing mechanistic information and evaluating multiple components in herbal medicines. This review describes major factors affecting the metabolism of herbal medicines, mechanisms of herb-drug interactions mediated by CYPs and UGTs, and several in vitro methods to assess the herb-drug interactions. Finally, drug interactions of Ginkgo biloba and St. John's wort, as representative herbal medicines, are described.

Objective: The purpose of this study was to compare cognitive flexibility abilities, stress, and anxiety between starters and non-starter athletes., Methods: A total of 30 male professional-soccer and 40 professional-baseball athletes were recruited. Wisconsin Card Sorting Test (WCST) and Trail Making Test A & B (TMT A & B) were administered to assess cognitive flexibility during competition. The Korean version of the STAI form Y (STAI-KY) and Visual analogue scale for anxiety and stress were used to assess the anxiety and stress., Results: The starter group had better cognitive function (fewer perseverative errors and rapid TMTB times) (Z=3.32, p<0.01; Z=2.20, p=0.03, respectively) and lower stress and anxiety (F=4.34, p=0.01; F=6.61, p<0.01, respectively) during competition than the non-starter group., Conclusion: The better cognitive performances were negatively correlated with stress and anxiety. Current results suggested that cognitive flexibility would enhance human performance by modulation of the anxiety and stress during competition.

Styraxlignolide A is a pharmacologically active ingredient isolated from Styrax japonica Sieb. et Zucc. A rapid, selective, and sensitive liquid chromatographic method with electrospray ionization tandem mass spectrometry was developed for use in the quantification of styraxlignolide A in rat plasma. Styraxlignolide A was extracted from rat plasma using ethyl acetate at neutral pH. The analytes were separated on an Atlantis dC18 column using a mixture of methanol and ammonium formate (10 mM, pH 3.0) (70:30, v/v) and detected by tandem mass spectrometry in multiple reaction monitoring mode. The standard curve was linear (r(2) =0.9978) over the concentration range of 100-10000 ng/mL. The lower limit of quantification was 100 ng/mL using 50 μL of plasma sample. The coefficient of variation and relative error for intra- and inter-assays at four QC levels were 1.6-8.3% and from -12.0 to -1.7%, respectively. The present method was applied successfully to the pharmacokinetic study of styraxlignolide A after intravenous administration of styraxlignolide A at a dose of 10 mg/kg in male Sprague-Dawley rats., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Novel thiazolidinedione analogues as 15-hydroxyprostaglandin dehydrogenase (15-PGDH) inhibitors were synthesized. Compounds 2, 3, and 4 exhibited IC(50) of 25, 8, and 19 nM, respectively. They also significantly increased levels of PGE(2) in A549 cells. To assess the influence of 15-PGDH inhibitor on cochlear blood flow (CBF), 2 was applied intravenously to guinea pigs. It increased their CBFs. Scratch wounds were also analyzed in confluent monolayers of HaCaT cells. Cells exposed to 4 showed significantly improved wound healing with respect to a control.

The purpose of this study was to evaluate the microchip CGE (MCGE) for the analysis of PEG-modified granulocyte-colony stimulating factor (PEG-G-CSF) prepared with PEG-aldehydes. The unmodified and PEG-modified G-CSFs were analyzed by Protein 80 and 230 Labchips on the Agilent 2100 Bioanalyzer. The MCGE allowed size-based separation and quantitation of PEG-G-CSF. The Protein 80 Labchip was useful for PEG-5K-G-CSF, while the Protein 230 Labchip was more suitable for PEG-20K-G-CSF. The MCGE was also used to monitor a search for optimal PEG-modification (PEGylation) conditions to produce mono-PEG-G-CSF. This study demonstrates the usefulness of MCGE for monitoring and optimizing the PEGylation of G-CSF with the advantages of speed, minimal sample consumption, and automatic quantitation.

The purpose of this study was to evaluate the effect of PEGylation on the stabilization of peptide in poly(D,L-lactide-co-glycolide) (PLGA) microspheres for sustained release delivery. As model peptide, growth hormone-releasing peptide-6 (GHRP-6) was conjugated with succinimidyl propionate monomethoxy poly(ethylene glycol) (PEG) with an average molecular weight of 2000 Da. The mono-PEG-GHRP-6 was separated by ion-exchange chromatography, and its molecular mass was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The microspheres encapsulating native GHRP-6 or mono-PEG-GHRP-6 were prepared using the single oil-in-water emulsion solvent evaporation method. During incubation in a 0.1 M phosphate buffer (pH 7.4) for one month at 37 degrees C, native GHRP-6 microspheres were identified to form several acylated peptides by reversed-phase HPLC and MALDI-TOF MS, whereas the mono-PEG-GHRP-6 microspheres was not affected from peptide acylation by PLGA. This study demonstrates that PEGylation can stabilize peptide against the acylation reaction occurred in PLGA microspheres.

A microchip electrophoretic method was applied to monitor and characterize the covalent attachment of poly(ethylene glycol) (PEGylation) of two proteins, alpha-lactalbumin and bovine serum albumin, using several poly(ethylene glycol) (PEG) derivatives with molecular weights from 1 to 20 kDa. This method effectively separated multi-PEGylated proteins in a size-based manner and allowed monitoring of the PEGylation pattern with the advantages of high speed, minimal sample consumption, and high reproducibility. Microchip electrophoresis would be a very useful tool for protein PEGylation studies such as reaction monitoring, purity checks, and characterization of PEGylated protein products., (Copyright 2010 Elsevier Inc. All rights reserved.)

Ecklonia stolonifera is a rich source of phlorotannins, which are responsible for the potent pharmacological effects associated with this seaweed. The purpose of this study was to develop a reversed-phase high-performance liquid chromatography method for the simultaneous determination of three major phlorotannins, eckol, dieckol, and phlorofucofuroeckol-A, in the extracts of Ecklonia stolonifera. The optimal chromatographic conditions were achieved on a Thermo Hypersil Gold C-18 column (250 x 4.6 mm i.d., 5 microm) using linear gradient elution of acetonitrile and water containing 0.1% formic acid at UV 254 nm. The separated phlorotannins were identified by liquid chromatography-mass spectrometry. The high-performance liquid chromatography method showed good linearity (r2 > 0.998), precision (1.4-9.5%), and accuracy (93.9-108.7%). The limits of detection ranged from 0.06 to 0.30 microg/mL and the lower limits of quantitation ranged from 0.2 to 1.0 microg/mL. Among phlorotannins, dieckol was the most abundant in both ethanol and ethyl acetate extracts of Ecklonia stolonifera.

We evaluated the utility of capillary electrophoretic methods for analyzing poly(ethylene glycol) (PEG)-modified granulocyte-colony stimulating factor (G-CSF), a long-acting form of GCSF for the treatment of cancer therapy-induced neutropenia. Low- and high-molecularweight PEG-G-CSF conjugates prepared with aldehyde-activated PEG-5K and PEG-20K were separated by high-performance size-exclusion chromatography (HP-SEC), capillary zone electrophoresis (CZE), and sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE). HPSEC showed low resolution for separating mono- and di-PEG-G-CSFs. SDS-CGE had higher resolution, but required a long analysis and had low peak efficiency. CZE could successfully separate both PEG-5K- and PEG-20K-conjugated G-CSFs with a running time of 20 min and high peak efficiency. In conclusion, CZE was better than SDS-CGE for separating PEG-G-CSF conjugates and will be useful for PEGylation studies, such as reaction monitoring for optimization of the PEGylation reaction, and purity and stability tests of PEG-G-CSF.

The purpose of this study was to develop a reversed-phase high-performance liquid chromatographic method (RP-HPLC) for separating each positional isomer from low- to high-molecular-weight mono-PEGylated octreotides prepared by polyethylene glycol (PEG) derivatives with various molecular weights (2, 5, or 20kDa). In the gradient elution using acetonitrile and 10mM phosphate buffer at pH 7.0 on a Phenomenex Gemini C-18 column (250mmx4.6mm id, 5microm), each positional isomer of the mono-PEGylated octreotides was completely resolved with good resolution (PEG-2K: 7.6, PEG-5K: 6.6, and PEG-20K: 3.1). The optimal RP-HPLC condition also resolved the degradation products of mono-PEG-octreotide isomers in thermal stability studies at 55 degrees C and enzymatic stability studies with trypsin. In conclusion, the developed RP-HPLC method will be valuable for studying the effect of PEGylation site and the attached PEG size on the physicochemical and pharmacological properties of PEGylated octreotides.

The purpose of this study was to develop reversed-phase HPLC method for the simultaneous determination of the flavonoids from the leaves of Nelumbo nucifera, which have been known to exhibit antioxidant, anti-HIV, antihyperlipidemic and antiobesity effects. HPLC separation was achieved on C(18) column using gradient elution with mobile phase consisting of acetonitrile-water containing 0.1% formic acid. The separated peaks were identified by electrospray ionization mass spectrometry. The HPLC method was validated and applied for the simultaneous determination of the bioactive flavonoids from the leaves of Nelumbo nucifera. In the methanol extract, six flavonoids, quercetin, rutin, quercetin 3-O-beta-D-galactopyranoside (Qc-3-Gal), quercetin 3-O-beta-D-glucopyranoside (Qc-3-Glc), quercetin 3-O-beta-D-glucuronide (Qc-3-Gln) and quercetin 3-O-alpha-arabinopyranosyl-(1-->2)-beta-galactopyranoside (Qc-3-AraGal), were identified. Among them, Qc-3-Glc and Qc-3-Gln were found to be major component in the methanol extract of Nelumbo nucifera leaves.

A sensitive LC-MS method was developed and validated for the determination of niflumic acid (NFA), the active metabolite of the talniflumate formulation, in human plasma. The analyses were performed on C(18) column using acetonitrile-ammonium acetate buffer (pH 5.7, 40:60) as a mobile phase with quadrupole MS detection of NFA at m/z 281 in a negative ion-monitoring mode. Calibration curve was linear in the concentration range of 1-1000ng/mL in human plasma. The higher sensitivity of LC-MS allowed low concentrations of NFA to be determined at initial drug absorption and terminal elimination phases following oral administration of talniflumate tablet.

The preventive and therapeutic potency against oxidative stress and diabetic complications of Nelumbo nucifera were evaluated via the 1,1-diphenyl-2-picrylhydrazyl (DPPH), Trolox equivalent antioxidant capacity (TEAC), and total reactive oxygen species (ROS) assays, as well as the rat lens aldose reductase (RLAR) and advanced glycation endproducts (AGE) assays. The leaf extract of N. nucifera exerted potent antioxidant effects as well as marked inhibitory effects for RLAR and AGE formation, corresponding to high values for total phenolic content (TPC) and total flavonoid content (TFC). Among several solvent fractions, the EtOAc and n-BuOH fractions, having prominent TPC and TFC values, showed significant antioxidant effects in the DPPH and TEAC assays. Moreover, the EtOAc fraction exhibited superior inhibitory effects in the total ROS, RLAR, and AGE assays, with IC(50) values of 9.4, 2.4, and 28.2microg/ml, respectively. Also, the HPLC profiles of the active EtOAc fraction indicated that quercetin 3-O-beta-d-glucopyranoside (Qc-3-Glc) and Qc 3-O-beta-d-glucuronopyranoside (Qc-3-Gln) were two of its major components, as well as Qc 3-O-beta-d-galactopyranoside (Qc-3-Gal) as a minor compound. Therefore, the results suggest that two key antioxidant flavonoids, Qc-3-Glc and Qc-3-Gln, may play important roles in the antioxidant and RLAR inhibitory effects of N. nucifera leaves. Also, the leaves, and the flavonoids contained within them, would clearly have potential uses in the development of therapeutic or preventive agents for diabetic complications and oxidative stress-related diseases.

The purpose of this study was to develop a fast reversed-phase high-performance liquid chromatography (HPLC) method for monitoring the octreotide PEGylation reaction in order to find optimal conditions for the production of the desired mono-PEGylated octreotide. The fast HPLC method could separate the positional isomers of two mono-PEGylated octreotides, di-PEGylated octreotide, and unmodified octreotide within 4.5 min. The PEGylation pattern was monitored at various pH conditions and molar ratios of reactants to allow optimization of the PEGylation reaction conditions for the production of N-terminally mono-PEGylated octreotide.

A gastroretentive drug delivery system of DA-6034, a new synthetic flavonoid derivative, for the treatment of gastritis was developed by using effervescent floating matrix system (EFMS). The therapeutic limitations of DA-6034 caused by its low solubility in acidic conditions were overcome by using the EFMS, which was designed to cause tablets to float in gastric fluid and release the drug continuously. The release of DA-6034 from tablets in acidic media was significantly improved by using EFMS, which is attributed to the effect of the solubilizers and the alkalizing agent such as sodium bicarbonate used as gas generating agent. DA-6034 EFMS tablets showed enhanced gastroprotective effects in gastric ulcer-induced beagle dogs, indicating the therapeutic potential of EFMS tablets for the treatment of gastritis.

This study was designed to demonstrate the utility of capillary electrophoresis (CE) for separating high-molecular-weight poly(ethylene glycol) (PEG)-conjugated proteins. As a CE method, sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) was applied to analyze interferon alpha (IFN) modified with branched and trimer-structured PEG molecules. Five mono-PEG-IFN conjugates prepared with two branched PEGs (MW 20 and 40 kDa) and three trimer-structured PEGs (MW 23.5, 43.5, and 47 kDa) were purified by cation-exchange chromatography and their masses were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The SDS-CGE method showed high separation capacity by differentiating PEG-IFN conjugates with small differences in molecular size, such as PEG(40K)-, PEG(43.5K)-, and PEG(47K)-IFNs, and it was useful for checking the purity of each mono-PEG-IFN. This study shows that SDS-CGE can well be utilized in the development and quality control of PEGylated proteins prepared with various types of PEG.

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 investigate in vitro release property of mono-PEGylated growth hormone-releasing peptide-6 (GHRP-6) microspheres. The microspheres encapsulating native GHRP-6 or mono-PEG-GHRP-6 were prepared using the single oil-in-water emulsion solvent evaporation method. In vitro release study was performed in 0.1M phosphate buffer, pH 7.4, containing 0.02% Tween 80 and sodium azide at 37 or 55 degrees C. The mono-PEG-GHRP-6 microspheres showed a lower initial burst compared with native GHRP-6 microspheres and zero-order release profile for a 1-month period. The release period was dependent on the PEG size attached to the GHRP-6 with more rapid drug release being observed with the smaller PEG size. This study suggests that PEGylated peptide has good potential as a source for a sustained release microsphere delivery system.

Purpose: To investigate the stability of KSL, an antimicrobial decapeptide, and its analogues, in human saliva and simulated gastric fluid for delivery in the oral cavity., Materials and Methods: The degradation products of KSL in human saliva and simulated gastric fluid were separated by reversed-phase HPLC and their structures were identified by electrospray ionization-mass spectrometry. Analogues of KSL were synthesized by solid-phase synthesis procedure. Their enzymatic stabilities and antimicrobial activities were studied., Results: KSL was degraded by the peptide bond cleavages at Lys(6)-Val(7) in the human saliva and Phe(5)-Lys(6) in simulated gastric fluids. Three analogues of KSL were synthesized; the Lys(6) residue was either methylated (KSL-M), or replaced with Trp (KSL-W), or the d-form of Lys (KSL-D). The KSL analogues were much more stable than the native KSL, with the rank order of stability being KSL-D > KSL-W > KSL-M > KSL in human saliva. However, in simulated gastric fluid, while KSL-D was still stable, KSL-W was significantly degraded. In addition, KSL-D significantly lost the antimicrobial activity, whereas KSL-W completely preserved the activity against several oral bacteria. In a chewing gum formulation, KSL-W showed a more sustained release profile as compared with the native KSL., Conclusion: This study suggests that KSL-W could be used as an antiplaque agent in a chewing gum formulation.

The purpose of this study was to investigate the formation of acylated impurity resulting from a chemical reaction between the growth hormone-releasing peptide-6 (GHRP-6) and poly(lactide-co-glycolide) (PLGA) and the effect of peptide acylation on the in vivo biological activity of GHRP-6. The peptide acylation pattern of GHRP-6 by hydrophilic PLGA polymers with different molecular weights was characterized by reversed-phase high-performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Higher levels of acylated GHRP-6 were produced with the higher molecular weight PLGA, which might be due to the slower degradation rate of the polymer. The evaluation of the biological activity in rats showed that the acylated GHRP-6 had a much lower activity than the intact GHRP-6. This finding suggests that the acylation reaction would decrease the effectiveness of the GHRP-6 formulation such as PLGA microspheres. Therefore, a strategy for stabilizing the GHRP-6 will be necessary for the development of a successful formulation of PLGA microspheres.

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.

Ricin A-chain, which exhibits excellent cytotoxicity to tumor cells, has been widely used as an immunotoxin source. However, it has the fatal shortcoming of poor pharmacokinetics due to the tremendous liver uptake via carbohydrate-mediated recognition. Modification of proteins with polyethylene glycol, PEGylation, has the advantages of shielding the specific sites and prolonging the biological half-life. In this study, the carbohydrate-specific PEGylation of ricin A-chain was considered to be a novel approach to overcome this limitation. The carbohydrate group of ricin A-chain was oxidized by sodium m-periodate and further specifically conjugated with hydrazide-derivatized PEG. For a comparative study, the PEGylated ricin A-chain at amino groups was prepared using the hydroxysuccinimide ester-derivatized PEG. The carbohydrate-specifically PEGylated ricin A-chain showed a markedly lower liver uptake and systemic clearance compared with the amine-directly PEGylated ricin A-chain as well as the unmodified ricin A-chain. Furthermore, carbohydrate-specifically PEGylated ricin A-chain showed a significantly higher in vitro ribosome-inactivating activity than the amine-directly PEGylated ricin A-chain. These findings clearly demonstrate that the carbohydrate-specificity as well as PEGylation plays an important role in improving the in vivo pharmacokinetic properties and in vitro bioactivity. Therefore, these results suggest that the therapeutic use of immunotoxins constructed using this carbohydrate-specifically PEGylated ricin A-chain has potential as a cancer therapy.

Since acylated peptide impurities were isolated from octreotide microspheres following incubation in an in vivo environment, the present investigation was undertaken to determine the dosage form dynamics responsible for facilitating acylation. In particular, microsphere batches made with poly(L-lactide) (PLA) and poly(lactide-co-glycolide) (PLGA) 85:15 were studied for in vitro drug release, mass balance relationships, mass loss behavior, hydration uptake, and solid-state stability. Furthermore, native octreotide was incubated in a varying pH stability model (heat treated lactic acid solutions 42.5%, w/w) to determine the effects of acidity on impurity formation. From a review of the experimental results, the appearance of octreotide impurities or related substances occurred with the onset of polymeric mass loss. In fact, the significant formation of acylated peptide did not appear until >90% mass loss, which was observed at 14 days. It was surmised that because of water uptake, the hydrolytic cleavage of the polymeric backbone created an acidic microenvironment to facilitate the covalent coupling of peptide with polymer. The lactic acid solution stability model corroborated with greater evidence of acylation at pH 2.25 where the presence lactoyl (+72 m/z) derivatives of octreotide were confirmed by MALDI-TOF mass spectrometry.

Purpose: : To determine the optimal polyethylene glycol (PEG)-conjugate of octreotide by evaluating the effects of PEGylation chemistry on the biological activity and pharmacokinetic properties., Methods: : Octreotide was chemically modified by reaction with succinimidyl propionate monomethoxy PEG (SPA-mPEG, molecular weight 2000) or succinimidyl butyraldehyde-mPEG (ALD-mPEG, molecular weight 2000 and 5000). The structural conformation of PEG-octreotides was evaluated by circular dichroism (CD), the biological activity was assessed by measuring the decrease of serum insulin-like growth factor-I levels in rats, and a pharmacokinetic study was performed after subcutaneous administration in rats. The stability against acylation was investigated with poly(D,L -lactide-co-glycolide) (PLGA)., Results: : ALD-mPEG was site-specific in PEGylating octreotide at the N-terminus. The mono-PEG-octreotides prepared with ALD-mPEG (mono-ALDPEG-octreotide), which alkyl bond preserves the amine's positive charge, showed complete preservation of biological activity, whereas the PEG-octreotides prepared with SPA-mPEG showed lower activity. In the CD analysis, the spectra of the mono-ALDPEG-octreotides were nearly superimposable with that of native octreotide. The mono-ALDPEG-5K-octreotide showed significantly improved pharmacokinetic properties compared with mono-ALDPEG-2K-octreotide as well as native octreotide. Both mono-ALDPEG-2K- and mono-ALDPEG-5K-octreotides were stable against acylation by degrading PLGA., Conclusions: : The mono-PEGylation of octreotide at N-terminus with ALD-mPEG produced a conjugate that is biologically and structurally active and stable against acylation by PLGA, and therefore it may serve as a candidate for somatostatin microsphere formulations.

Purpose: To investigate the mechanism by which polyethylene glycol (PEG) conjugation (PEGylation) prevents the acylation of octreotide by poly(d,l-lactide-co-glycolide) (PLGA)., Methods: Octreotide was chemically modified by reaction with succinimidyl propionate-monomethoxy PEG. Each PEGylated octreotide species with different PEG number and modified position was separated by reversed-phase high-performance liquid chromatography (RP-HPLC) and characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with endoproteinase Lys-C digestion. Acylation of octreotide and PEGylated octreotides was observed with hydrophobic and hydrophilic PLGA., Results: Two mono- and one di-PEGylated octreotides were separated by RP-HPLC. MALDI-TOF MS of the PEGylated products after Lys-C digestion at different pH revealed that the two mono-PEGylated octreotides were modified at the N-terminus and Lys(5) residue, respectively. The interaction of octreotide with PLGA involved an initial adsorption followed by acylation and the subsequent release of octreotide and acylated octreotide. The initial adsorption of octreotide was dependent on the acidity of PLGA. PEGylation of octreotide significantly inhibited the initial adsorption and acylation by PLGA. In particular, the acylation could be completely prevented by mono-PEGylation at the N-terminus of octreotide., Conclusions: This study shows that the N-terminus of octreotide is the preferred PEGylation site to prevent acylation in degrading PLGA microspheres. The mono-N-terminally PEGylated octreotide may possibly serve as a new source for somatostatin microsphere formulation.

Glucagon-like peptide-1-(7-36) (GLP-1) is a hormone derived from the proglucagon molecule, which is considered a highly desirable antidiabetic agent mainly due to its unique glucose-dependent stimulation of insulin secretion profiles. However, the development of a GLP-1-based pharmaceutical agent has a severe limitation due to its very short half-life in plasma, being primarily degraded by dipeptidyl peptidase IV (DPP-IV) enzyme. To overcome this limitation, in this article we propose a novel and potent DPP-IV-resistant form of a poly(ethylene glycol)-conjugated GLP-1 preparation and its pharmacokinetic evaluation in rats. Two series of mono-PEGylated GLP-1, (i) N-terminally modified PEG(2k)-N(ter)-GLP-1 and (ii) isomers of Lys(26), Lys(34) modified PEG(2k)-Lys-GLP-1, were prepared by using mPEG-aldehyde and mPEG-succinimidyl propionate, respectively. To determine the optimized condition for PEGylation, the reactions were monitored at different pH buffer and time intervals by RP-HPLC and MALDI-TOF-MS. The in vitro insulinotropic effect of PEG(2k)-Lys-GLP-1 showed comparable biological activity with native GLP-1 (P = 0.11) in stimulating insulin secretion in isolated rat pancreatic islet and was significantly more potent than the PEG(2k)-N(ter)-GLP-1 (P < 0.05) that showed a marked reduced potency. Furthermore, PEG(2k)-Lys-GLP-1 was clearly resistant to purified DPP-IV in buffer with 50-fold increased half-life compared to unmodified GLP-1. When PEG(2k)-Lys-GLP-1 was administered intravenously and subcutaneously into rats, PEGylation improved the half-life, which resulted in substantial improvement of the mean plasma residence time as a 16-fold increase for iv and a 3.2-fold increase for sc. These preliminary results suggest a site specifically mono-PEGylated GLP-1 greatly improved the pharmacological profiles; thus, we anticipated that it could serve as potential candidate as an antidiabetic agent for the treatment of non-insulin-dependent diabetes patients.

A one-step chromatographic method capable of separating all isomers of polyethylene glycol (PEG)-growth hormone-releasing factor (GRF) (1-29) conjugates was developed. The unmodified GRF (1-29) and seven different isomers of PEG-GRF (1-29) conjugates were separated by using a simple reversed-phase HPLC method depending on the differences of hydrophobicity due to the number and site of PEG attachment. The PEGylation sites of all isomers of PEG-GRF (1-29) conjugates were identified by determining the molecular masses of the Lys-C digested fragments with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This study is a first report for the separation of all PEG-conjugate isomers and would be useful for further studies to find the promising conjugate by evaluating biological activity and stability of each isomer.

A capillary electrophoretic method (CE) for characterizing PEGylated human parathyroid hormone 1-34 (PTH) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is described. CE was used to optimize the PEGylation of PTH through control of the reaction pH and the molar ratio of reactants with the advantages of minimal sample consumption and high separation capacity. The mono-PEGylated PTH (mono-PEG-PTH) was isolated and then digested with endoproteinase Lys-C. Resistance to Lys-C digestion on the PEGylation sites in the mono-PEG-PTH resulted in patterns of CE electropherograms different from that of the native PTH, and the PEGylation sites were assigned accordingly. The extent of positional isomers present in the mono-PEG-PTH was also determined by quantifying PEGylated fragments in the same CE electropherogram. In conclusion, the CE analysis of the Lys-C-digested sample allowed for simultaneous analysis of the PEGylation site and the extent of positional isomers in the mono-PEG-PTH. The results were confirmed by MALDI-TOF MS. This method will be applicable for characterizing PEGylation of other therapeutic peptides.

A quantitative determination of peptides incorporated into poly(d,l-lactide-co-glycolide) microspheres by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was accomplished in a single step without pretreatment for extracting the peptide from the microsphere. The conventional extraction methods often underestimate the actual amount of peptide because of incomplete extraction from the microspheres or loss during the procedures. In this study, the microspheres dissolved in acetonitrile containing 0.1% trifluoroacetic acid were mixed with matrix solution containing the internal standard, and the peptide content was directly determined by MALDI-TOF MS. The drug content values determined by MALDI-TOF MS in both the leuprolide- and salmon calcitonin-incorporated microspheres were closer to the theoretical contents than those determined by the conventional extraction method. This method using MALDI-TOF MS could be a good alternative to time-consuming and less-accurate conventional methods.

Capillary electrophoresis (CE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) have been employed as highly efficient methods to characterize ricin, its subunits, and the chemically deglycosylated forms. As a CE method, sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) was used because of its merit over the conventional slab gel techniques. SDS-CGE showed higher resolution capability over other analytical tools in the analysis of the ricin mixture as well as in each of its purified forms. The high resolution was considered to be a result of the presence of carbohydrates on ricin subunits, and this property was useful for identifying the native ricin or its A chain from their chemically deglycosylated forms. However, this method exhibited an overestimation of the molecular mass due to the carbohydrate moieties on ricin subunits, and the inaccuracies were observed to be dependent on the carbohydrate content of the subunits. The exact molecular masses were measured by MALDI-TOF MS, and the results were almost consistent with the expected values. This study clearly illustrates the usefulness and necessity of complementary use of two powerful analytical techniques to characterize ricin and its subunits in a various research fields such as poisoning and immunotoxin research.

The purpose of this study was to evaluate the stabilization of salmon calcitonin (sCT) by PEGylation in nasal mucosa. Degradation of native sCT in the homogenates of rat nasal mucosa was investigated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The initial cleavage of sCT was due to tryptic-like endopeptidase activity, and the subsequent degradation followed the sequential pattern of aminopeptidase activity. To prepare PEGylated sCT resistant to the proteolytic degradation, the lysine residues susceptible to tryptic activity were selectively PEGylated by controlling reaction pH. The PEGylated sCT showed strong resistance against enzymatic degradation in rat nasal mucosa, with 56-fold prolonged half-life compared with that of native sCT. In the MALDI-TOF MS spectrum, the PEGylated sCT did not show any degradation peak for incubation of 120 min in the homogenates of rat nasal mucosa. The improved stability may be responsible for enhancing nasal absorption of PEGylated sCT., (Copyright 2004 Wiley-Liss, Inc. and the American Pharmacists Association)

Sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) using a hydrophilic replaceable polymer network matrix was applied to characterize the polyethylene glycol(PEG)ylated interferon alpha (PEG-IFN). The SDS-CGE method resulted in a clearer resolution in both the PEG-IFN species and the native IFN species. The distribution profile of PEGylation determined by SDS-CGE was consistent with that obtained by SDS-polyacrylamide gel electrophoresis (PAGE) with Coomassie blue or barium iodide staining. The result was also compared using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry. SDS-CGE was also useful for monitoring the PEGylation reaction to optimize the reaction conditions, such as reaction molar ratio. This study shows the potential of SDS-CGE as a new method for characterizing the PEGylated proteins with advantages of speed, minimal sample consumption and high resolution.

The purpose of this study was to prepare poly(ethylene glycol) (PEG)ylated octreotide and investigate the stability against acylation by polyester polymers such as poly(lactic acid) and poly(lactic-co-glycolic acid). Octreotide was modified by reaction with monomethoxy PEG-propionaldehyde (molecular weight 5,000) in the presence of sodium cyanoborohydride. The mono-PEGylated fraction was isolated by reversed-phase high-performance liquid chromatography (HPLC) and characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Circular dichroism demonstrated no significant secondary structural differences between mono-PEGylated octreotide (mono-PEG-octreotide) and intact octreotide. As a test system for the stability study against acylation reaction, lactic acid (LA) solutions with various concentrations and pH values were prepared with water dilution and subsequent accelerated equilibration at 90 degrees C for 24 hours. Native octreotide was found to be acylated in all the diluted LA solutions with different concentrations (42.5%, 21.3%, and 8.5%, wt/wt) and pH values (2.25, 1.47, and 1.85, respectively). The remaining amounts of intact octreotide continuously decreased to 50% through 30 days of incubation at 37 degrees C. MALDI-TOF MS identified the octreotide to be acylated by LA units. However, acylation reaction of mono-PEG-octreotide in LA solutions was negligible, and the remaining amounts of intact one through 30 days of incubation in LA solutions were also comparable to the initial concentration. These data suggest that mono-PEG-octreotide may prevent the acylation reaction in degrading PLA microspheres and possibly serve as a new source for somatostatin microsphere formulation.

To evaluate the hypocalcemic effect of polyethylene gtycol-conjugated salmon calcitonins (PEG-sCT) in rats, mono-PEGylated sCTs (mono-PEG-sCTs) and unmodified sCT were administered via the intranasal route and serum calcium levels were measured by colorimetric assay using o-cresolphthalein. Mono-PEG-sCTs were prepared with different sizes of succinimidyl succinate monomethoxy PEG molecules (PEG2K), PEG5K, PEG12K) and characterized by HPLC and MALDI-TOF mass spectrometry. Nasal instillation of mono-PEG2K-sCT at a dose of 2 IU/kg resulted in sustained reduction in serum calcium levels over 8 hr, with a maximum reduction (% maxd) of 13% after 6 hr of application. Whereas unmodified sCT showed a transient decrease in serum calcium levels with the maximum reduction (5%) observed after 30 min of administration. The overall reductions in serum calcium levels expressed as the net change in AUC relative to control in 8 hr were 11.9 +/- 0.2, 4.6 +/- 0.7, and 2.6 +/- 0.7% for mono-PEG2K-, mono-PEG5K-, and mono-PEG12K-sCT, respectively, compared to 3.2 +/- 0.6% for unmodified sCT. The relative bioavailability of nasally administered 2 IU/kg of mono-PEG2K-sCT was approximately 4-fold higher than nasally administrated unmodified sCT, and the absolute bioavailability was approximately 91% of intravenously injected sCT in 8 hr. It can be concluded that the intranasal absorption of mono-PEG-sCTs was inversely related to the molecular weights of the PEG attached. Of the PEGylated sCTs examined, mono-PEG2K-sCT showed the most pronounced hypocalcemic effect. Therefore the intranasal application would probably be an alternative route of administration for mono-PEG-sCTs in achieving sustained calcium-lowering effects.

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.