A new procedure for the synthesis of polyethylene glycol-protein adducts; effects on function, receptor recognition, and clearance of superoxide dismutase, lactoferrin, and alpha 2-macroglobulin

A new, simplified technique for the synthesis of polyethylene glycol (PEG) derivatives of proteins utilizing 1,1'-carbonyldiimidazole for PEG activation, is described. PEG derivatives of superoxide dismutase, alpha 2-macroglobulin, alpha 2-macroglobulin-trypsin, and lactoferrin were prepared and studied. Superoxide dismutase coupled to PEG preserved 95% of its original activity while its plasma half-life increased from 3.5 min to 9 or more hours depending on the PEG derivative studied. PEG-derivatized alpha 2-macroglobulin showed decreased protease binding activity but PEG derivatives of performed alpha 2-macroglobulin-trypsin demonstrated no loss of activity. The plasma clearance of PEG-alpha 2-macroglobulin-trypsin was prolonged significantly compared to native alpha 2-macroglobulin-trypsin, particularly when a high-molecular-weight PEG was coupled to the protease inhibitor complex. The plasma clearance half-life of lactoferrin was increased 5- to 20-fold by this modification. Trinitrobenzenesulfonic acid titration studies demonstrated that epsilon-amino groups of lysine residues are modified by the coupling of carbonyldiimidazole-activated PEG to proteins.     

Further studies on the site-specific protein modification by microbial transglutaminase

A guinea pig liver transglutaminase (G-TGase)-mediated procedure for the site-specific modification of chimeric proteins was recently reported. Here, an alternative method with advantages over the recent approach is described. This protocol utilizes a microbial transglutaminase (M-TGase) instead of the G-TGase as the catalyst. M-TGase, which has rather broad structural requirements as compared to the G-TGase, tends to catalyze an acyl transfer reaction between the gamma-carboxamide group of a intact protein-bound glutamine residue and various primary amines. To demonstrate the applicability of the M-TGase-catalyzed protein modification in a drug delivery system, we have utilized recombinant human interleukin 2 (rhIL-2) as the target protein and two synthetic alkylamine derivatives of poly(ethyleneglycol) (PEG12; MW 12 kDa) and galactose-terminated triantennary glycosides ((Gal)(3))) as the modifiers. For the M-TGase-catalyzed reaction with PEG12 and (Gal)(3), 1 mol of alkylamine was incorporated per mole of rhIL-2, respectively. Peptide mapping of (Gal)(3)-modified rhIL-2 ((Gal)(3)-rhIL-2) by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS) suggested that the Gln74 residue in rhIL-2 was site specifically modified with (Gal)(3). The PEG12-rhIL-2 and (Gal)(3)-rhIL-2 conjugates retained full bioactivity relative to the unmodified rhIL-2. In pharmacokinetic studies, PEG12-rhIL-2 was eliminated more slowly from the circulation than rhIL-2, whereas (Gal)(3)-rhIL-2 accumulated in the liver via hepatic asialoglycoprotein receptor binding. The results of this study expand the applicability of the TGase-catalyzed methodology for the preparation of protein conjugates for clinical use.     

Effect of divalent ions on protein precipitation with polyethylene glycol: mechanism of action and applications.

Polyethylene glycol (PEG) is extensively employed for protein purification by fractional precipitation. Efficiency of precipitation is highest when the solution pH is near the isoelectric point of the target protein. At pH values far from the isoelectric point of the target protein, proteins develop a net positive or negative charge and are not more resistant to precipitation. We have found that divalent cations (Ba2+, Sr2+, and Ca2+) or divalent anions (SO4(2-)) significantly change the pattern of PEG precipitation when the ion is chosen so as to counteract the expected net charge on the target protein. At moderate (5-50 mM) concentrations of Ba2+, negatively charged proteins can be precipitated from solution at pH values as high as 10 with efficiency unchanged from precipitation at pH values near their isoelectric point values. The mechanism of PEG precipitation of protein at these high pH values appears to be unchanged from the mechanism operative at the protein isoelectric point. Precipitation is rapid and the capacity for protein precipitation is high. There is no detectable coprecipitation of small molecules (AMP, ATP, and NADH) or soluble proteins (carbonic anhydrase) induced when large quantities of protein are precipitated by this method. The purification of bovine carbonic anhydrase from erythrocyte lysate is more efficient at pH 10 in the presence of Ba2+ than is conventional PEG precipitation carried out at the isoelectric point of carbonic anhydrase. Application of these observations should broaden the utility of protein purification by fractional precipitation with PEG.     

PEG和DBBF修饰猪血红蛋白及其携氧性质

采用聚乙二醇 (PEG)修饰蛋白质可以增大蛋白质的分子量 ,改善其生物相容性和在生物体内的停留时间。而小分子交联修饰则可以稳定血红蛋白的高级结构 ,改善其对组织的递氧能力。比较了 4种方法活化的PEG衍生物对猪血红蛋白的修饰效率、修饰产物的携氧功能和稳定性等。PEG的分子量、轭合PEG的数量及变构效应物的存在与否都会影响修饰产物的性质 ;考察了双 3,5二溴水杨酸延胡索酸酯 (DBBF)修饰猪血红蛋白的反应条件以及修饰产物的物理特性和携氧能力 ,并进一步采用PEG和DBBF联合修饰猪血红蛋白。结果证明 ,联合修饰产物具有稳定的四聚体结构 ,分子量达 10 70 0 0 ,半饱和氧分压P50 在 3.33kPa左右 ,接近于生理条件下人体红细胞的P50 值。     

Transglutaminase-mediated dual and site-specific incorporation of poly(ethylene glycol) derivatives into a chimeric interleukin-2

To expand the applications of poly(ethylene glycol) (PEG)-protein conjugates for clinical use, we have developed a novel method for dual and site-specific incorporations of PEG derivatives into proteins using a substrate peptide (AQQIVM, named TG2) and transglutaminase (TGase). In our previous studies, TG2 was shown to be a special peptide with two adjacent Gln substrates for guinea pig liver transglutaminase (G-TGase). We have now constructed a chimeric protein (named rTG2-IL-2) of human interleukin-2 (IL-2), in which TG2 was fused to the N-terminus of IL-2. For the G-TGase-catalyzed reaction, rTG2-IL-2 was dually and site-specifically modified with alkylamine derivatives of PEG (PEG10, average M(r) 10 kDa) at both the Gln2 and Gln3 residues in the appended tag. To demonstrate the effectiveness of the G-TGase-catalyzed PEG-incorporation, we have compared the characteristics and the biological properties of PEG10-rTG2-IL-2 species with two PEG10 molecules attached to rTG2-IL-2 [(PEG10)(2)-rTG2-IL-2] with that of (PEG10)(2)-rhIL-2(R), in which PEG10 was randomly incorporated into rhIL-2 by a general procedure using a N-hydroxysuccinimidyl ester of PEG (PEG10-COOSu) (M(r) 10 kDa). (PEG10)(2)-rTG2-IL-2 was found to be superior in its in vitro bioactivities and equivalent in its pharmacokinetic profiles to (PEG10)(2)-rhIL-2(R). Unlike most previous methods, this approach can place dual PEG chains at designed sites on chimeric proteins without decreasing their bioactivities. Thus, TGase-catalyzed PEG-incorporation would improve the therapeutic utility of PEG-protein conjugates.     

The bundling of actin with polyethylene glycol 8000 in the presence and absence of gelsolin.

Actin filament and bundle formation occur in the cytosol under conditions of very high total macromolecular concentration. In this study we have utilized the inert molecule polyethylene glycol 8000 (PEG) as a means of simulating crowded conditions in vitro. Column-purified Ca-actin was polymerized in the absence and presence of gelsolin (to regulate mean filament lengths between 50 and 5000 mers) and PEG (2-8%) using various concentrations of KCl and/or 2 mM divalent cations. Bundling was characterized by the scattered light intensity and mean diffusion coefficients obtained from dynamic light scattering, as well as by fluorescence and phase-contrast microscopy. The minimum concentration of KCl required for bundling decreases both with increasing concentration of PEG at a fixed mean filament length, and with decreasing filament length at a fixed concentration of PEG. In the absence of divalent cation, bundling is reversible on dilution, as determined by intensity levels, diffusion coefficients, and microscopy. However, with either 2 mM Mg2+ or Ca2+ added, bundling is irreversible under conditions of higher PEG concentrations or longer filaments, indicating that osmotic pressure effects cannot fully explain actin bundling with PEG. Weaker divalent cation-binding sites on actin as well as disulfide bonds appear to be involved in the irreversible bundling.     

Molecular basis for the distinct divalent cation requirement in the uridylylation of the signal transduction proteins GlnJ and GlnB from Rhodospirillum rubrum

ABSTRACT: BACKGROUND: PII proteins have a fundamental role in the control of nitrogen metabolism in bacteria, through interactions with different PII targets, controlled by metabolite binding and post-translational modification, uridylylation in most organisms. In the photosynthetic bacterium Rhodospirillum rubrum, the PII proteins GlnB and GlnJ were shown, in spite of their high degree of similarity, to have different requirements for post-translational uridylylation, with respect to the divalent cations, Mg2+ and Mn2+. RESULTS: Given the importance of uridylylation in the functional interactions of PII proteins, we have hypothesized that the difference in the divalent cation requirement for the uridylylation is related to efficient binding of Mg/Mn-ATP to the PII proteins. We concluded that the amino acids at positions 42 and 85 in GlnJ and GlnB (in the vicinity of the ATP binding site) influence the divalent cation requirement for uridylylation catalyzed by GlnD. CONCLUSIONS: Efficient binding of Mg/Mn-ATP to the PII proteins is required for uridylylation by GlnD. Our results show that by simply exchanging two amino acid residues, we could modulate the divalent cation requirement in the uridylylation of GlnJ and GlnB. Considering that post-translational uridylylation of PII proteins modulates their signaling properties, a different requirement for divalent cations in the modification of GlnB and GlnJ adds an extra regulatory layer to the already intricate control of PII function.     

Polyethylene glycol conjugation enhances the nitrite reductase activity of native and cross-linked hemoglobin

Although stabilized hemoglobins have been evaluated as oxygen-carrying replacements for red cells in transfusions, in vivo evaluations have noted that these materials are associated with vasoactivity, a serious complication. Scavenging of endogenous nitric oxide by the deoxyheme sites of the stabilized proteins is one likely source of vasoactivity. Recent reports indicate that modification of cell-free hemoglobin derivatives with multiple chains of polyethylene glycol (PEG) suppresses vasoactivity. Gladwin and co-workers observed that the nitrite reductase activity of hemoglobin serves as a major endogenous source of nitric oxide. If PEG conjugation leads to enhanced nitrite reductase activity, this could compensate for scavenged endogenous nitric oxide. To test this possibility, the rates of conversion of nitrite ion to nitric oxide by altered hemoglobins with and without PEG were measured at 25 degrees C. Fumaryl (alpha99-alpha99) cross-linked hemoglobin reacts with nitrite with a bimolecular rate constant of 0.52 M (-1) s (-1), which is comparable to that associated with native hemoglobin (0.25 M (-1) s (-1)). Addition of PEG chains to the cross-linked hemoglobin at beta-Cys93 (alphaalpha-Hb-PEG5K 2) results in a material that produces nitric oxide much more rapidly ( k = 1.41 M (-1) s (-1)). R-State-stabilized hemoglobins with multiple PEG chains (Hb-PEG5K 2 and Hb-PEG5K 6) react 10 times faster with nitrite to produce nitric oxide than does native hemoglobin ( k = 2.5 and 2.4 M (-1) s (-1), respectively). These results, showing enhanced production of nitric oxide resulting from an increased proportion of the protein residing in the R-state, are consistent with the decrease in vasoactivity associated with PEG conjugation.     

新一代PEG在修饰抗原和药物缓释中的应用

聚乙二醇及其衍生物是具有许多优良性质的高分子化合物,由于良好的生物相容性、无毒、无免疫原性,广泛用于生物医学领域。本文总结聚乙二醇的发展历史和新一代聚乙二醇的特点,阐述聚乙二醇化修饰的目的,特别是在抗原修饰、血型改造和细胞移植等方面的应用,重点对聚乙二醇在药物缓释方面的应用进行了系统的综述。     

A facile synthesis of azido-terminated heterobifunctional poly(ethylene glycol)s for "click" conjugation

New azido-terminated heterobifunctional poly(ethylene glycol) (PEG) derivatives having primary amine and carboxyl end groups, (Azide-PEG-NH 2 and Azide-PEG-COOH, respectively) were synthesized with high efficiency. An alpha-allyl-omega-hydroxyl PEG was prepared as the first step to Azide-PEG-X (X = NH 2 and COOH) through the ring-opening polymerization of ethylene oxide (EO) with allyl alcohol as an initiator, followed by two-step modification of the hydroxyl end to an azido group. To introduce primary amino or carboxyl functional groups, amination and carboxylation reactions of the allyl terminal ends was then conducted by a radical addition of thiol compounds. Molecular functionalities of both ends of the PEG derivatives thus prepared were characterized by (1)H, (13)C NMR, and MALDI-TOF MS spectra, validating that the reaction proceeded quantitatively. The terminal azido functionality is available to conjugate various ligands with an alkyne group through the 1,3-dipolar cycloaddition reaction condition ("click chemistry").     

Surface modifications based on the cyanobacterial siderophore anachelin: from structure to functional biomaterials design

This review describes the design, synthesis and evaluation of novel catechol based anchors for surface modification. The anachelin chromophore, the catecholate fragment of the siderophore anachelin from the cyanobacterium Anabaena cylindrica, allows for the immobilization of polyethylene glycol (PEG) on titania and glass surfaces thus rendering them protein resistant and antifouling. It is proposed that catecholate siderophores constitute a class of natural products useful for surface modification similar to dihydroxyphenylalanine and dopamine derived compounds found in mussel adhesive proteins. Second-generation dopamine derivatives featuring a quaternary ammonium group were found to be equally efficient in generating antifouling surfaces. The anachelin chromophore, merged via a PEG linker to the glycopeptide antibiotic vancomycin, allowed for the generation of antimicrobial surfaces through an operationally simple dip-and-rinse procedure. This approach offers an option for the prevention of nosocomial infections through antimicrobial implants, catheters and stents. Consequences for the mild generation of functional biomaterials are discussed and novel strategies for the immobilization of complex natural products, proteins and DNA on surfaces are presented.     

Prolonged circulating lives of single-chain Fv proteins conjugated with polyethylene glycol: a comparison of conjugation chemistries and compounds

The utility of single-chain Fv proteins as therapeutic agents would be substantially broadened if the circulating lives of these minimal antigen-binding polypeptides were both prolonged and adjustable. Poly(ethylene glycol) (PEG) bioconjugate derivatives of the model single-chain Fv, CC49/218 sFv, were constructed using six different linker chemistries that selectively conjugate either primary amines or carboxylic acid groups. Activated PEG polymers with molecular weights of 2000, 5000, 10 000, 12 000, and 20 000 were included in the sFv bioconjugate evaluation. Additionally, the influence of PEG conjugate geometry in branched PEG strands (U-PEG) and the effect of multimeric PEG-sFv bioconjugates on circulating life and affinity were examined. Although random and extensive PEG polymer conjugations have been achievable in highly active derivatives of the prototypical PEG-enzymes, PEGylation of CC49/218 sFv required stringent adjustment of reaction conditions in order to preserve antigen-binding affinity as measured in either mucin-specific or whole cell immunoassays. Purified bioconjugates with PEG:sFv ratios of 1:1 through 2:1 were identified as promising candidates which exhibit sFv affinity (K(d)) values within 2-fold of the unmodified sFv protein. Interestingly, PEG conjugation to carboxylic acid moieties, using a PEG-hydrazide chemistry, achieved significant activity retention in bioconjugates at a higher PEG:sFv ratio (5:1) than with any of the amine-reactive activated PEG polymers. Prolonged circulating life in mice was demonstrated for each of the PEG conjugates. An increase in PEG polymer length was found to be more effective for serum half-life extension than a corresponding increase in total PEG mass. For example, CC49/218 sFv conjugated to either one strand of PEG-20000, or four strands of PEG-5000, displayed about 20- or 14-fold increased serum half-life, respectively, relative to the unmodified sFv. The demonstrated suitability of established random conjugation chemistries for PEGylation of sFv proteins, in conjunction with innovative site-specific conjugation methods, indicates that production of a panoply of sFv proteins with both engineered affinity and tailored circulating life may now be achievable.     

Chemically modified porcine hemoglobins and their biological properties

Hemoglobin cross-linked with small molecular modifiers turns out to be more stable. Modifications of proteins with polyethylene glycol (PEG) have been proven to enlarge the molecular size of proteins, to prolong their retention time in the circulation as well as blunt immune reactions. In the present study, the optimal conditions for porcine hemoglobin (pHb) modification with bis (3, 5-dibromosalicyl) fumarate (DBBF) and PEG were evaluated. The derivative of DBBF cross-linked pHb (DBBF-pHb) showed improved oxygen affinity and the ability to resist the dissociation of the alpha2beta2 tetramer compared with the natural protein. DBBF-pHb was then bound to the activated PEG. The results indicated that the pHb modified with DBBF and PEG had more stable tetrameric conformation with a molecular weight of 107000. Their oxygen half-saturation pressure (P50) is around 3.33 kPa, which approximates the physiological P50 of human red blood cells. Both routine and reinforced immunizing methods were adopted to study the immunogenicity of modified products and the results showed that the products had very low immunogenicity evaluated by enzyme-linked immunoadsordent assay (ELISA). Somewhat beneficial effects were shown in the treatment of hemorrhagic shock where modified hemoglobin solutions were used as resuscitation fluids in the hemorrhagic shock Sprague-Dawley (SD) rats model.     

Enhanced activity of Candida rugosa lipase modified by polyethylene glycol derivatives

The derivatives of polyethylene glycol (PEG) were prepared by reacting PEG with propylene oxide to enhance its hydrophobicity and introduce a branched structure. The PEG derivatives were activated with cyanuric chloride and used to modify the lipase fromCandida rugosa. The maximum specific activity of lipase modified with the PEG derivatives was about 2-fold of that modified with PEG for the esterification of oleic acid and lauryl alcohol in hexane.     

UV resonance Raman study of beta93-modified hemoglobin A: chemical modifier-specific effects and added influences of attached poly(ethylene glycol) chains.

The reactive sulfhydryl group on Cys beta93 in human adult hemoglobin (HbA) has been the focus of many studies because of its importance both as a site for synthetic manipulation and as a possible binding site for nitric oxide (NO) in vivo. Despite the interest in this site and the known functional alterations associated with manipulation of this site, there is still considerable uncertainty as to the conformational basis for these effects. UV resonance Raman (UVRR) spectroscopy is used in this study to evaluate the conformational consequences of chemically modifying the Cys beta93 sulfhydryl group of both the deoxy and CO-saturated derivatives of HbA using different maleimide and mixed disulfide reagents. Included among the maleimide reagents are NEM (n-ethylmaleimide) and several poly(ethylene glycol) (PEG)-linked maleimides. The PEG-based reagents include both different sizes of PEG chains (PEG2000, -5000, and -20000) and different linkers between the PEG and the maleimide. Thus, the effect on the conformation of both linker chemistry and PEG size is evaluated. The spectroscopic results reveal minimal perturbation of the global structure of deoxyHbA for the mixed disulfide modification. In contrast, maleimide-based modifications of HbA perturb the deoxy T state of HbA by "loosening" the contacts associated with the switch region of the T state alpha(1)beta(2) interface but do not modify the hinge region of this interface. When the NEM-modified HbA is also subjected to enzymatic treatment to remove the C-terminal Arg alpha141 (yielding NESdes-ArgHb), the resulting deoxy derivative exhibits the spectroscopic features associated with a deoxy R state species. All of the CO-saturated derivatives exhibit spectra that are characteristic of the fully liganded R structure. The deoxy and CO derivatives of HbA that have been decorated on the surface with large PEG chains linked to the maleimide-modified sulfhydryl through a short linker group all show a general intensity enhancement of the tyrosine and tryptophan bands in the UVRR spectrum. It is proposed that this effect arises from the osmotic impact of a large, close PEG molecule enveloping the surface of the protein.     

Improving the sensitivity of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry by using polyethylene glycol modified polyurethane MALDI target

Previous studies in our group have shown that the analyte signal in a matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) experiment is strongly influenced by the binding interactions between the target surface and the analyte. Specifically, the analyte signal increases with decreases in surface binding affinity, which has been attributed to more unbound analyte being available for incorporation within the MALDI matrix. In this work, polyethylene glycol (PEG) was chemically grafted onto a polyurethane (PU) film to produce a MALDI target having reduced surface-protein binding affinity, and the effect of this modification on protein MALDI ion signals was investigated. The proteins myoglobin, lysozyme, and albumin were used to evaluate the PEG PU modified target as compared with a PU target and a commercial stainless steel target. It is shown that there are enhancements in the protein MALDI ion signals on the PEG PU modified target and that the limit of detection for these proteins is decreased by a factor of 2 to 6 in comparison with the unmodified PU and the commercial stainless steel targets.     

Structure‐Based Site‐Specific PEGylation of Fibroblast Growth Factor 2 Facilitates Rational Selection of Conjugate Sites

Polyethylene glycol modification (PEGylation) can enhance the pharmacokinetic properties of therapeutic proteins by the attachment of polyethylene glycol (PEG) to the surface of a protein to shield the protein surface from proteolytic degradation and limit aggregation. However, current PEGylation strategies often reduce biological activity, potentially as a result of steric hindrance of PEG. Overall, there are no structure‐based guidelines for selection of conjugate sites that retain optimal biological activity with improved pharmacokinetic properties. In this study, site‐specific PEGylation based on the FGF2‐FGFR1‐heparin complex structure is performed. The effects of the conjugate sites on protein function are investigated by measuring the receptor/heparin binding affinities of the modified proteins and performing assays to measure cell‐based bio‐activity and in vivo stability. Comprehensive analysis of these data demonstrates that PEGylation of FGF2 that avoids the binding sites for fibroblast growth factor receptor 1 (FGFR1) and heparin provides optimal pharmacokinetic enhancement with minimal losses to biological activity. Animal experiments demonstrate that PEGylated FGF2 exhibits greater efficacy in protecting against traumatic brain injury‐induced brain damage and neurological functions than the non‐modified FGF2. This rational structure‐based PEGylation strategy for protein modification is expected to have a major impact in the area of protein‐based therapeutics.     

A liquid chromatographic method for determination of the modification degree of proteins: PEGylated arginase as an example

A liquid chromatographic method was developed to determine the modification degree of PEGylated proteins. This method effectively separated free polyethylene glycol (PEG) from other species in conjugation mixtures on a C4 reversed-phase column using water-acetonitrile gradient elution. Then the concentrations of free PEG were determined according to the integrated area under the curve of its evaporative light scattering detector (ELSD) signal, which was normalized by the PEG standard with similar molecular weights. The actual numbers of PEG attached to proteins, not those of lysines modified, were calculated. This method was performed with PEGylated arginase mixtures as an example and showed clear advantages over 2,4,6-trinitrobenzenesulfonic acid (TNBS) assays.     

Systematic investigation of polyamidoamine dendrimers surface-modified with poly(ethylene glycol) for drug delivery applications: synthesis, characterization, and evaluation of cytotoxicity

Surface modification of amine-terminated polyamidoamine (PAMAM) dendrimers by poly(ethylene glycol) (PEG) groups generally enhances water-solubility and biocompatibility for drug delivery applications. In order to provide guidelines for designing appropriate dendritic scaffolds, a series of G3 PAMAM-PEG dendrimer conjugates was synthesized by varying the number of PEG attachments and chain length (shorter PEG 550 and PEG 750 and longer PEG 2000). Each conjugate was purified by size exclusion chromatography (SEC) and the molecular weight (MW) was determined by (1)H NMR integration and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). NOESY experiments performed in D 2O on selected structures suggested no penetration of PEG chains to the central PAMAM domain, regardless of chain length and degree of substitution. CHO cell cultures exposed to PAMAM-PEG derivatives (< or =1 microM) showed a relatively high cell viability. Generally, increasing the degree of PEG substitution reduced cytotoxicity. Moreover, compared to G3 PAMAM dendrimers that were N-acetylated to varying degrees, a lower degree of surface substitution with PEG was needed for a similar cell viability. Interestingly, when longer PEG 2000 was fully incorporated on the surface, cell viability was reduced at higher concentrations (32 muM), suggesting increased toxicity potentially by forming intermolecular aggregates. A similar observation was made for anionic carboxylate G5.5 PAMAM dendrimer at the same dendrimer concentration. Our findings suggest that a lower degree of peripheral substitution with shorter PEG chains may suffice for these PAMAM-PEG conjugates to serve as efficient universal scaffolds for drug delivery, particularly valuable in relation to targeting or other ligand-receptor interactions.     

Enhanced circulating half-life and antitumor activity of a site-specific pegylated interferon-alpha protein therapeutic

Recombinant interferon alpha-2 (IFN-alpha2) has proven useful for treating a variety of human cancers and viral diseases. IFN-alpha2 has a short circulating half-life in vivo, which necessitates daily or thrice weekly administration to patients. It is possible to extend the circulating half-life of IFN-alpha2 by random modification of lysine residues in the protein with polyethylene glycol (PEG); however, such preparations have heterogeneous structures and low specific activities, and may not provide optimal therapeutic benefits to patients. A long-acting, site-specific, monoPEGylated IFN-alpha2 protein has now been created by targeted attachment of a 20 kDa or a 40 kDa maleimide-PEG to a cysteine analogue of IFN-alpha2, M111C. In vitro bioactivities of the purified 20 kDa and 40 kDa PEG-M111C proteins were within 2- to 3-fold of those of wild type IFN-alpha2 and 7- to 10-fold better than that of a 40 kDa PEG IFN-alpha2 protein created using nontargeted, amine-PEGylation methodology. The 20 kDa and 40 kDa PEG-M111C proteins demonstrated 26- to 38-fold longer half-lives, respectively, than IFN-alpha2 following subcutaneous administration to rats. The 20 kDa PEG M111C protein inhibited growth of human NIH:OVCAR-3 cells transplanted into nude mice by >90%, as measured by tumor size, tumor weight, and number of animals with detectable tumors at necropsy, and was significantly more effective than a comparable dose of IFN-alpha2. These data extend our previous findings that bioactivity of IFN-alpha2 can be largely preserved by targeted attachment of PEG moieties to nonessential sites in the protein and provide evidence that site-specific PEGylated IFN-alpha2 proteins possess enhanced tumoricidal properties in vivo.