Identification of the major positional isomer of pegylated interferon alpha-2b

Interferons display a wide range of antiviral, antiproliferative, and immunomodulatory activities on a variety of cell types and have been used to treat many diseases including hairy-cell leukemia and hepatitis B and C and have also been applied to other therapeutic areas. To improve the pharmacological properties of interferon (IFN) alpha-2b, a long-acting pegylated form (PEG-IFN) has been developed [PEG, monomethoxy poly(ethylene glycol) with average molecular mass of 12 000 Da]. PEG-IFN is a mixture of pegylated proteins with differing sites of PEG attachment. To identify the major positional isomer in the pegylated material [PEG-IFN(His-34)], NMR studies were conducted on a subtilisin-digested N-acetylated peptide of the major positional isomer [PEG-IFN(His-34)dig], synthetic peptide analogues containing His-34, as well as unmodified IFN and PEG-IFN(His-34). Our studies reveal a novel interferon-polymer attachment site as a histidine-linked interferon conjugate. We show that the major component of PEG-IFN is pegylated in the imidazole side chain of histidine-34. Chemical shift data suggest that pegylation occurs mainly at the N(delta)(1) position in the imidazole side chain of this residue. This positional isomer, PEG-IFN(His-34), comprises approximately 47% of the total pegylated species when PEG-IFN is synthesized under the current experimental conditions at pH 6.5 with an electrophilic derivative of PEG, succinimidyl carbonate PEG. The reversibility of the histidine modification was examined. The PEG-imidazole adduct in the intact protein, PEG-IFN(His-34), is labile but much more stable than in the peptide, PEG-IFN(His-34)dig. Apparently, the tertiary structure of the intact protein protects the His(34)-imidazole ring from depegylation.     

Rational design of a potent, long-lasting form of interferon: a 40 kDa branched polyethylene glycol-conjugated interferon alpha-2a for the treatment of hepatitis C

A potent, long-lasting form of interferon alpha-2a mono-pegylated with a 40 kilodalton branched poly(ethylene glycol) was designed, synthesized, and characterized. Mono-pegylated interferon alpha-2a was comprised of four major positional isomers involving Lys31, Lys121, Lys131, and Lys134 of interferon. The in vitro anti-viral activity of pegylated interferon alpha-2a was found to be only 7% of the original activity. In contrast, the in vivo antitumor activity was severalfold enhanced compared to interferon alpha-2a. Pegylated interferon alpha-2a showed no immunogenicity in mice. After subcutaneous injection of pegylated interferon alpha-2a, a 70-fold increase in serum half-life and a 50-fold increase in mean plasma residence time concomitant with sustained serum concentrations were observed relative to interferon alpha-2a. These preclinical results suggest a significantly enhanced human pharmacological profile for pegylated interferon alpha-2a. Results of Phase II/III hepatitis C clinical trials in humans confirmed the superior efficacy of pegylated interferon alpha-2a compared to unmodified interferon alpha-2a.     

Structural identification and biological activity of positional isomers of long-acting and mono-PEGylated recombinant human granulocyte colony-stimulating factor with trimeric-structured methoxy polyethylene glycol N-hydroxysuccinimidyl functional group

The individual positional isomers from the mono-PEGylated recombinant human granulocyte colony-stimulating factor (rhG-CSF) were successfully isolated with additional strong cation exchange chromatography using Source 15S. The three isolated individual positional isomers were found to be homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), analytical size exclusion high-performance liquid chromatography (SE-HPLC), and analytical cation exchange HPLC (CIE-HPLC) and were also characterized with respect to site of PEGylation by enzymatic digestion with endoproteinase Lys-C and N-terminal sequencing. In addition, in vitro biological activity was determined by cell proliferation assay. It was determined that the three isolated individual positional isomers were PEGylated at Lys35, Met(N-terminal), and Lys17 of the rhG-CSF molecule with a 23-kDa trimer-structured methoxy polyethylene glycol N-hydroxysuccinimidyl functional group (mPEG-NHS). All individual positional isomers (Lys35-PEGylated rhG-CSF, Met(N-terminal)-PEGylated rhG-CSF, and Lys17-PEGylated rhG-CSF) retained in vitro biological activity and were found to be 18.5%, 37.6%, and 7.1%, respectively, compared with the rhG-CSF molecule. The significantly different in vitro biological activities observed in the individual positional isomers could be presumably due to interference of receptor binding or active sites on the rhG-CSF molecule. In conclusion, the individual positional isomers isolated from the mono-PEGylated rhG-CSF were well characterized with respect to the site of PEGylation involving Lys35, Met(N-terminal), and Lys17. This characterization of the individual positional isomers would be critical to provide a basis for establishing consistency in the manufacturing process.     

Site of pegylation and polyethylene glycol molecule size attenuate interferon-alpha antiviral and antiproliferative activities through the JAK/STAT signaling pathway

Therapeutic pegylated interferon-alphas (IFN-alpha) are mixtures of positional isomers that have been monopegylated at specific sites on the core IFN-alpha molecule. The pegylation results in lower in vitro specific activity associated with the core IFN-alpha molecule that is related to the site of pegylation and size of polyethylene glycol (PEG) attached. We prepared purified, homogeneous, positional pegylation isomers of IFN-alpha2b that were monopegylated using 5-30-kDa linear PEG molecules attached at 7 primary reactive amino acid residues: Cys(1), His(34), Lys(31), Lys(83), Lys(121), Lys(131), and Lys(134). The isomers were evaluated for STAT translocation and antiviral and antiproliferative activity. The site of pegylation strongly influenced activity relative to an IFN-alpha2b control. The highest residual activity was observed with the His(34) positional isomers, and the lowest was observed with the Cys(1) positional isomers. The Lys positional isomers demonstrated intermediate activity, with a general order of Lys(134) > Lys(83) approximately Lys(131) approximately Lys(121) > Lys(31). The progressive relationship between decreased activity and increased PEG size suggests that pegylation may interfere with interaction and binding of IFN-alpha to the IFNAR1-IFNAR2 heterodimeric receptor. The higher specific activity associated with the His(34) positional isomer suggests that this site may be favorable for pegylating IFN-alpha2b molecules.     

Isolation and structural analyses of positional isomers of 6(1),6(n)-di-O-(N-acetyl-beta-D-glucosaminyl)cyclomaltoheptaose (n=2, 3, and 4) and 6-O-[6-O-(N-acetyl-beta-D-glucosaminyl)-N-acetyl-beta-D-glucosaminyl]cyclomaltoheptaose.

6-O-[6-O-(N-acetyl-beta-D-glucosaminyl)-N-acetyl-beta-D-glucosaminyl]cyclomaltoheptaose (beta CD) and three positional isomers of 6(1),6(n)-di-O-(N-acetyl-beta-D-glucosaminyl)cyclomaltoheptaose (n=2, 3, and 4) in a mixture of products from beta CD and N-acetylglucosamine by the reversed reaction of beta-N-acetylhexosaminidase from jack bean were isolated and purified by HPLC. The structures of four isomers of di-N-acetylglucosaminyl-beta CDs were determined by FABMS and NMR spectroscopy. The degree of polymerization of the branched oligosaccharides produced by enzymatic degradation with bacterial saccharifying alpha-amylase (BSA) was established by LC-MS methods.     

干扰素及其聚乙二醇化修饰产物的概述

干扰素是一类由不同细胞产生的可溶性糖蛋白,并且具有抗病毒,抗肿瘤和免疫调节功能。目前,干扰素已被用于治疗多种疾病,包括毛细胞白血病、乙型肝炎、丙型肝炎以及其他治疗领域。然而,这种治疗的缺点是干扰素在血液中的短半衰期以及快速被清除。近年来,有资料已证明聚乙二醇（PEG）共轭结合生物分子比其相应的未修饰分子表现出优越的临床应用。然而,虽然蛋白共轭连接聚乙二醇能够延长它们在血液中的半衰期,但是通常会导致生物学和药理学活性的急剧降低甚至活性丧失。在此,对干扰素及其聚乙二醇修饰产物的药理学、药代动力学以及药效进行对比概述。     

Structural, kinetic, and thermodynamic analysis of the binding of the 40 kDa PEG-interferon-alpha2a and its individual positional isomers to the extracellular domain of the receptor IFNAR2

Type-I Interferons exert antiviral and antiproliferative activities through the binding to a common cell surface receptor comprising two subunits, IFNAR1 and IFNAR2. Human recombinant Interferon-alpha(2a) (IFNalpha(2a)) is a potent drug (Roferon-A) used to treat various cancers and viral diseases including Hepatitis B/C infections. To significantly improve the pharmacological properties of the drug, a pegylated form of IFNalpha(2a) was developed (PEGASYS). This 40 kDa PEG-conjugated IFNalpha(2a) ((40)PEG-IFNalpha(2a)) is obtained by the covalent binding of one 40 kDa branched PEG-polymer to a lysine side-chain of IFNalpha(2a). Here, we report the detailed structural, kinetic, and thermodynamic analysis of the binding to the extracellular domain of the receptor IFNAR2 of (40)PEG-IFNalpha(2a) and its isolated positional isomers modified at K31, K134, K131, K121, K164, and K70, respectively, in comparison with unmodified IFNalpha(2a). Our binding studies, using the surface plasmon resonance technique, show that the pegylation does not abolish the binding to the receptor, but significantly reduces the affinity mainly due to a change of the association rate. The results are supported by modeling and simulation of the binding, using Self-Avoiding-Walk calculations for the polymer conformations. A correlation between the structural parameters and the kinetic and thermodynamic parameters of the binding of the positional isomers could be established. For the Isomer-K31 and -K164, the PEG-polymer attachment point is located in proximity to the binding interface, and the isomers display affinity in the range 150-520 nM in an enthalpy-driven binding process. In contrast for the Isomer-K134, -K131, -K121, and -K70, the PEG-polymer is attached remotely from the binding interface, and the isomers exhibit a higher affinity (32-76 nM) in an entropy-driven binding process. This study constitutes an essential collection of knowledge on which the interaction of (40)PEG-IFNalpha(2a) and its positional isomers with its cellular receptors can be better understood.     

Occurrence of positional isomers of octadecenoic and hexadecenoic acids in human depot fat

Positional isomers of hexadecenoic aud octadecenoic acids of human adipose tissue have been separated by gas-liquid chromatography and their amounts determined by oxidative cleavage (MnO(4) and IO(4)). The following isomeric octadecenoic acids were present: 7-octadecenoic acid (0.4%), 8- (1.9%), 9- (73.0%), 10- (2.5%), 11- (19.0%) and 12- (3.2%). The hexadecenoic acids have also been shown to be a mixture of positional isomers, in which the cis-9-isomer predominates. 10-Hexadecenoic and 12-octadecenoic acids could conceivably be precursors of linoleic acid. The following branched fatty acids have also been determined in human depot fat: 13-methyltetradecanoic, 12-methyltetradecanoic, 14-methylpentadecanoic, 14-methylhexadecanoic, and 16-methylheptadecanoic acid. They were present in percentages of 0.02-0.6% and their identification rests solely on comparison of their gas-liquid chromatographic retention times with those of synthetic compounds.     

2-Propanol in the mobile phase reduces the time of analysis of CLA isomers by silver ion-HPLC

Individual isomers of octadecadienoic acid (C18:2) with conjugated double bonds (conjugated linoleic acids; CLA) exert different biological activities. Their distribution in food and tissues differs. Therefore, the separation of the various positional and geometric isomers is important. The time of analysis using silver ion-high performance liquid chromatography can extend up to 90 min. The aim of this study was to reduce this time. The time of analysis reduced from ca. 90 min onto 45 to 35 min, respectively, by the addition of 0.05% or 0.1% (v/v) 2-propanol to the mobile phase [acetonitrile (0.1%; v/v) and diethyl ether (0.5%; v/v) in n-hexane]. There was no effect on resolution of the 17 individual CLA isomers of the CLA mixture. Regarding the lowest coefficient of variation and an adequate baseline separation the use of 0.05% 2-propanol in the mobile phase is recommended, without any disadvantages and adverse effects on the service life of columns. In conclusion, adding 0.05% or 0.1% 2-propanol to the mobile phase shortens the time of analysis of CLA isomers, saves solvents and reduces costs.     

Platelet sparing effect of COX II inhibition used with pegylated interferon alfa-2a for the treatment of chronic hepatitis C: a short term pilot study

The role of a cyclooxygenase (COX) II inhibitor in reducing microvascular inflammation and the platelet count associated with interferon (IFN) plus ribavirin therapy of chronic hepatitis C (HCV) was assessed. Three plasma mediators (biomarkers) associated with platelet activation, inflammation and fibrosis were measured. Eighteen IFN na?ve patients were studied. Nine were treated with pegylated IFN alfa-2a (PEG-IFN alpha-2a) plus ribavirin and rofecoxib; nine were treated with PEG-IFN alpha-2a plus ribavirin. A complete blood count, liver panel and HCV-RNA were assayed weekly. Human soluble P-selectin (hs-P-selectin), human interleukin-8 (IL-8), human interleukin-13 (IL-13) and human thrombopoietin (TPO) were assayed at 4 week intervals. The COX II inhibitor reduced the platelet reduction experienced with PEG-IFN alpha-2a treatment of HCV despite a reduction in the plasma TPO level. Hs-P-selectin was increased in both groups. In contrast, human IL-8 levels declined to undetectable levels in virologic responders. Similarly, human IL-13 levels declined with therapy (P < 0.001). These data suggest that: (1) a COX II inhibition is associated with an increase in the platelet count despite a reduction in the TPO level; (2) human IL-8 and human IL-13 but not hs-P-selectin levels decline in those who experience an early virologic response.     

Immunochemical mapping of alpha-2 interferon

A panel of five monoclonal antibodies, designated U1-U5, produced by murine hybridoma clones has been raised to recombinant interferon (IFN) alpha-2, and one monoclonal antibody, designated U6, has been raised to a mixture of cyanogen bromide fragments of IFN alpha-2. These antibodies have been characterized with respect to (1) neutralization of IFN antiviral and antiproliferative activities, (2) binding to four cloned IFN alpha subtypes (alpha-1, alpha-2, alpha-4, and alpha-7) that are naturally occurring and to two novel products of recombinant DNA technology (delta-4 alpha-1 and delta-4 alpha-2/alpha-1 hybrid), and (3) binding to three cyanogen bromide fragments of IFN alpha-2. Four of the six monoclonal antibodies inhibited IFN antiviral activity. In conjunction with the previously reported monoclonal antibodies III/21 [Arnheiter, H., Thomas, R. M., Leist, T., Fountoulakis, M., & Gutte, B. (1981) Nature (London) 294, 278-280] and NK-2 [Secher, D. S., & Burke, D. C. (1980) Nature (London) 285, 446-450], eight unique epitopes have been described. Analysis of cross-reactivity patterns with IFN alpha fragments and subtypes indicated that monoclonal antibodies U1 and NK-2, which neutralized both antiviral and antiproliferative activities, and U2, which was nonneutralizing in these assays, were directed to distinct epitopes located in a polypeptide consisting of the amino-terminal 15 amino acid residues linked to residues 60-110 by a disulfide bond. The epitope recognized by U1 was determined to reside, at least in part, between residues 5 and 15. Competitive binding studies indicated that neutralizing monoclonal antibody U3, which did not bind to any of the cyanogen bromide fragments, was directed to an epitope partially overlapping that of NK-2. Epitopes to which neutralizing monoclonal antibodies U3, U4, and U5 and nonneutralizing antibody U6 were directed were readily distinguished by cross-reactivity with IFN alpha subtypes. The nonneutralizing monoclonal antibody U6 was determined to be directed to an epitope between residues 22 and 58. The fact that delta-4 alpha-1 and the delta-4 alpha-2/alpha-1 hybrid were active in an antiviral assay indicated a lack of direct functional significance for the first four amino-terminal amino acid residues and the Cys1-Cys98 disulfide bond. However, reduction with 2-mercaptoethanol of IFN alpha-2 altered the integrity of four of the eight epitopes. These data support a critical role for disulfide linkages in maintaining the native conformation of IFN alpha-2 and provide a potential basis for predicting the location of functionally important domains.     

Fatty acids,Part 22: Infrared and Raman studies of all positional isomers of furan-containing C18 fatty esters

The infrared and Raman spectra of all 2,5-disubstituted C₁₈ furanoid fatty esters were studied. Where the furan system was located at the end of the alkyl chain or conjugated to the carbomethoxy group, such positional isomers could be readily identified through their unique absorption features in the infrared and Raman spectra. Other positional isomers of the series gave less differentiating spectral absorption characteristics.     

Preparation and characterization of 6I,6n-di-O-(L-fucopyranosyl)-beta-cyclodextrin (n=II-IV) and investigation of their functions

Three positional isomers of 6(I),6(n)-di-O-(beta-L-fucopyranosyl)-cyclomaltoheptaose [6(I),6(n)-di-O-(beta-L-Fuc)-beta-cyclodextrin, -betaCD, n=II-IV] were chemically synthesized using the corresponding authentic compounds, 6(I),6(n)-di-O-(tert-butyldimethylsilyl)-betaCD (n=II-IV), as the fucosyl acceptors, and 2,3,4-tri-O-acetyl-L-fucopyranosyl trichloroacetimidate as the fucosyl donor. Their structures were analyzed by HPLC, MS, and NMR spectroscopy. The hemolytic activities of L-Fuc-betaCDs were lower than that of betaCD, while the solubilities of these branched CDs in water were much higher than that of betaCD. The molecular interaction between these compounds and the fucose-binding lectin Aleuria aurantia lectin (AAL) was investigated using an optical biosensor based on a surface plasmon resonance (SPR) technique. The order of binding affinity, as a function of the fucose-binding position, was 6(I),6(IV)->6(I),6(III)->6(I),6(II)-di-O-(beta-L-Fuc)-betaCD>6-O-(beta-L-Fuc)-betaCD.     

Poly(2-methacryloyloxyethyl phosphorylcholine) for protein conjugation

The water-soluble, biocompatible polymer poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) was evaluated for protein conjugation. PMPC is a zwitterionic polymer that is able to form a more compact conformation in aqueous solution than poly(ethylene glycol) (PEG). While a terminally functionalized N-hydroxysuccinimide derivative of PMPC was not efficient for conjugation to an amine moiety on interferon-alpha2a (IFN), we found that a bis-thiol specific derivative of PMPC could be conjugated after reduction of the disulfide bonds in IFN. Utilizing PMPC that displayed a similar hydrodynamic volume to 20 kDa PEG, we evaluated the in vitro antiviral and antiproliferative activity and pharmacokinetics of a PMPC-IFN conjugate. As a hygroscopic zwitterionic polymer, PMPC is able to form a compact conformation in aqueous solution, which was found to be more compact than PEG. This suggests that PMPC protein conjugates may display different plasma elimination characteristics than PEG protein conjugates. PMPC-IFN displayed marked resistance to antibody binding in Western blot analysis with a polyclonal anti-IFN antibody while displaying comparable in vitro antiviral and antiproliferative activity to PEG-IFN. During an in vivo pharmacokinetic study, the absorption t(1/2) for PMPC-IFN was considerably extended compared to the native IFN and 20 kDa PEG analogue. This is also consistent with the SDS-PAGE result where an apparent reduction in mobility through a hydrated medium was observed. The elimination t(1/2) was also vastly extended over the native IFN and twice the value of 20 kDa PEG-IFN. This suggests that tissue migration of PMPC-IFN occurs more slowly than the 20 kDa PEG-IFN despite their similarity in hydrodynamic volume, leading to an an improved depot effect, which could explain the longer elimination t(1/2). In this study, we demonstrate a potential use of PMPCylation as a novel tool for enhancing the pharmacokinetic profile of therapeutic proteins in ways that complement PEGylation.     

Construction,Purification, and Characterization of a Homodimeric Granulocyte Colony-Stimulating Factor

Granulocyte colony-stimulating factor (G-CSF) has found widespread clinical application, and modified forms with improved biopharmaceutical properties have been marketed as well. PEGylation, the covalent modification of G-CSF with polyethylene glycol (PEG), has a beneficial effect on drug properties, but there are concerns connected to the immunogenicity of PEGylated compounds and bioaccumulation of the synthetic polymer. To overcome challenges connected with chemical modifications, we developed fusion proteins composed of two G-CSF molecules connected via different peptide linkers. Three different homodimeric G-CSF proteins were purified, and their in vitro and in vivo activities were determined. A G-CSF dimer, GCSF-Lα, was constructed using an alpha-helix-forming peptide linker, and it demonstrated an extended half-life in serum with a stronger neutrophil response as compared to the monomeric G-CSF protein. The GCSF-Lα protein, therefore, might be selected for further studies as a potential drug candidate.     

Structural and biophysical characterization of the 40 kDa PEG-interferon-alpha2a and its individual positional isomers

The human recombinant Interferon-alpha(2a) (IFNalpha(2a)) is a potent drug (Roferon-A) to treat various types of cancer and viral diseases including Hepatitis B/C infections. To improve the pharmacological properties of the drug, a new pegylated form of IFNalpha(2a) was developed (PEGASYS). This 40 kDa PEG-conjugated IFNalpha(2a) ((40)PEG-IFNalpha(2a)) is obtained by the covalent binding of one 40 kDa branched PEG-polymer to a lysine side chain of IFNalpha(2a). (40)PEG-IFNalpha(2a) is a mixture of mainly six monopegylated positional isomers modified at K31, K134, K131, K121, K164, and K70, respectively. Here we report the detailed structural and biophysical characterization of (40)PEG-IFNalpha(2a) and its positional isomers, in comparison with IFNalpha(2a), using NMR spectroscopy, analytical ultracentrifugation, circular dichroism, fluorescence spectroscopy, and differential scanning calorimetry. Our results show that the three-dimensional structure of IFNalpha(2a) is not modified by the presence of the polymer in all positional isomers constituting (40)PEG-IFNalpha(2a). Regardless of where the PEG-polymer is attached, it adopts a very mobile and flexible random coil conformation, producing a shield for the protein without a permanent coverage of the protein surface. Hydrodynamic data indicate that the protein-attached PEG has a slightly more compact random-coil structure than the free PEG-polymer. Our results also provide evidence of significant structural and physicochemical advantages conferred by the pegylation: increase of the effective hydrodynamic volume and modification of the molecular shape, higher temperature stability, and reduced tendency for aggregation. These results are of tremendous pharmacological interest and benefit as was clinically shown with PEGASYS. This study constitutes a new standard for the characterization of pegylated proteins and enables an important step toward the understanding on a molecular level of the binding of (40)PEG-IFNalpha(2a) and its positional isomers to its cellular receptors.     

Enhancement of ribonucleotide reductase activity at low enzyme concentrations by polyethylene glycol

The estimation of ribonucleotide reductase in cell extracts has been problematical on account of abnormally low activities at low enzyme concentrations, presumably due to subunit dissociation. This problem can be alleviated by assaying the enzyme in the presence of polyethylene glycol. The presence of 15% polyethylene glycol during the assay greatly stimulated ribonucleotide reductase activity at low enzyme concentrations and allowed measurement of enzyme activity in as little as 10(5) mouse L929 cells, a 30-fold enhancement of assay sensitivity. Enzyme activity measured in the presence of 15% polyethylene glycol was proportional to enzyme concentration, thus making possible the accurate measurement of very low levels of ribonucleotide reductase.     

Synthesis of (+),(-)-neamine and their positional isomers as potential antibiotics

The syntheses of (+)-neamine 1, (-)-neamine ent-1 and their positional isomers 2, 3, ent-2 and ent-3 are reported as potential new scaffolds for novel aminoglycoside antibiotics. These isomers exhibit similar inhibitory activities, as shown using an in vitro translation assay. A simple model is proposed to explain this lack of stereospecific binding to the ribosomal RNA.     

Slow absorption of conjugated linoleic acid in rat intestines, and similar absorption rates of 9c,11t-conjugated linoleic acid and 10t,12c-conjugated linoleic acid

We have previously shown that the 9c,11t-conjugated linoleic acid (CLA) concentration was always significantly higher than the 10t,12c-CLA concentration following the administration of these compounds to mice and rats, and considered that structural differences between the conjugated double bonds in these isomers affected absorption in the small intestine. This study investigates the absorption of CLA in the rat intestine by a lipid absorption assay of lymph from the thoracic duct. In Study 1, we used safflower oil and a triacylglycerol form of CLA (CLA-TG), while in Study 2, we used 9c,11t-CLA and 10t,12c-CLA. The cumulative recovery of CLA was lower than that of linoleic acid until two hours after sample administration. There was no difference in the extent of lymphatic recovery of 9c,11t-CLA and 10t,12c-CLA after the administration of CLA-TG, 9c,11t-CLA, and 10t,12c-CLA to the rats, suggesting that geometrical and positional isomerism of the conjugated double bonds did not influence the absorption.     

The Structure of a Novel Lignan,Phrymarolin-II from Phryma leptostachya L.

The ginsenosides in Panax ginseng have vast structural and pharmacological efficacies. We covalently conjugated polyethylene glycol on the surface of CK (PEG-CK) through an acid-labile ester-linkage that showed increased solubility of CK. HPLC analysis showed that the release of CK was enhanced at acidic pH 5, whereas it was dramatically decreased at physiological pH 7.4. This might enhance the efficacy of CK.