Polyethylene Glycol Conjugation Research Articles

A PEGylation Technology of L-Asparaginase with Monomethoxy Polyethylene Glycol-Propionaldehyde

Polyethylene glycol (PEG) conjugation technology has been successfully applied to improve the performance of protein drugs. In this study, L-asparaginase was N-terminal site-specifically modified by alkylating PEG with monomethoxy polyethylene glycol-propionaldehyde (mPEG-ALD20000). The optimum reaction parameters were determined as pH 5.0, a molar ratio of mPEG-ALD2000 to L-asparaginase of 10:1, a reaction time of 16 h and temperature of 25 degrees C. PEG-L-asparaginase (PEG-L-ASNase) was isolated and purified with consecutive anion-exchange (XK, 16 x 20 cm, Q Sepharose FF) and gel-filtration (Tricorn, 10 x 600 cm, Sephacryl S-300 HR) chromatography, respectively. PEG-L-ASNase retained 43.5% of its activity and the N-terminal amino groups were modified to an extent of 3.67%.

Improved bioavailability of inhibitors of Trypanosoma cruzi trans-sialidase: PEGylation of lactose analogs with multiarm polyethyleneglycol

The trans-sialidase of Trypanosoma cruzi (TcTS) catalyzes the transfer of sialic acid from host glycoconjugates to terminal β-galactopyranosides in the mucins of the parasite. During infection, the enzyme is actively shed by the parasite to the bloodstream inducing hematological alterations. Lactitol prevents cell apoptosis caused by the TcTS, although it is rapidly eliminated from the circulatory system. Linear polyethyleneglycol (PEG) conjugates of lactose analogs were prepared but their clearance from blood was still quite fast. With the aim of improving their circulating half-lives in vivo, we now synthesized covalent conjugates of eight-arm PEG. The star-shape of these conjugates allows an increase in the molecular weight together with the loading of the active sugar. Two approaches were used for PEGylation of disaccharide derivatives containing β-D-Galp as the non-reducing unit. (1) Amide formation between benzyl β-D-galactopyranosyl-(1→6)-2-amino-2-deoxy-α-D-glucopyranoside and a succinimide-activated PEG. (2) Conjugation of lactobionolactone with amino end-functionalized PEG. Two 8-arm PEG derivatives (20 and 40 kDa) were used for each sugar. Substitution of all arms was proved by (1)H nuclear magnetic resonance (NMR) spectroscopy. The bioavailability of the conjugates in mice plasma was considerably improved with respect to the 5 kDa linear PEG conjugates retaining their inhibitory properties.

Abstract 378: Multifunctional bladder cancer targeting micelle nanocarriers with both imaging and therapeutic potentials

Abstract Background: Human and dog invasive bladder cancers share similar attributes, such as pathologic changes, response to chemotherapy, and extremely poor prognosis. Via combinatorial chemistry approach, we previously identified a novel bladder cancer-specific ligand, PLZ4, that can specifically bind to both human and dog bladder cancer cells in vitro and in vivo. We also developed a micelle nanocarrier drug delivery system with multifunctional loading capacity. Here, we assessed whether the targeting micelles impregnated with PLZ4 on the surface could promote targeting efficacy against both human and dog bladder cancers for diagnostic imaging and therapeutic purposes. Materials and Methods: Micelle-building monomers (i.e., telodendrimers) were synthesized through conjugation of polyethylene glycol with cholic acid cluster at one end and PLZ4 at the other end, which then self-assembled in aqueous solution to form micelles. Near-infrared dye DiD and chemotherapeutic drugs (paclitaxel or daunorubicin) were co-loaded for the experiments. Cellular uptake and distribution of nanomicelles were evaluated by fluorescence intensity and high resolution topography, while drug delivery efficacy was monitored by cytotoxicity assay. Using orthotopic/xenograft mouse model generated from human clinical bladder cancer specimen or dog bladder cancer line, in vivo and ex vivo fluorescence imaging as well as efficacy study were performed. Results: Compared to non-targeting micelles, targeting PLZ4-decorated-micelles loaded with an imaging agent DiD and chemotherapeutic drugs were more efficient in dye delivery, and caused a comparable to higher degree of cytotoxicity with free drugs in bladder cancer cell lines. According to topography, targeting micelles tended to localize at the membrane, perinuclear, and nucleus. In vivo and ex vivo orthotopic xenograft studies showed that both targeting (14.3X of free dye, p<0.01) and non-targeting micelles (9.6X, p<0.01) rapidly accumulated at the tumor sites, while targeting micelles exhibited significantly higher efficiency in homing property than non-targeting micelles (p<0.05). Considerably higher DiD signal was microscopically observed in targeting group than non-targeting group as well as free dye group, but all exhibited similar blood vessel density. Our preliminary efficacy study also supported a better drug delivery efficiency using targeting micelle formulation than non-targeting micelles/free drug in mouse xenografts formed from human clinical bladder tumors. Conclusions: Targeting micelles impregnated with PLZ4 can selectively and efficiently target both human and dog bladder cancer cells and can be potentially developed as imaging and therapeutic agents in both human and veterinary medicine. Preclinical studies of targeting micelles can be performed in dogs with spontaneous bladder cancer before proceeding to human patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 378. doi:1538-7445.AM2012-378

Abstract LB-356: Micelle-encapsulated thiostrepton: an effective nanomedicine for inhibiting tumor growth

Abstract The thiazole antiobiotic, thiostrepton, has been found to suppress the expression of FOXM1, the oncogenic forkhead transcription factor overexpressed in cancer cells. Thiostrepton has also been shown to induce cell death in cancer cells through a proteasome inhibitory mechanism. As thiostrepton is highly hydrophobic, encapsulation into nano-sized delivery vehicles not only aides its solubilization but also improves its targeting for tumors in vivo. This is achieved by encapsulating thiostrepton into the interior of an amphiphilic micelle, composed from an assembly of lipid-PEG (polyethylene glycol) conjugates. In this study, we examined the anti-tumor effects of micelle-thiostrepton in both subcutaneous xenograft models (HepG2 liver cancer, Capan-2 pancreatic cancer and MDA-MB-231 breast cancer) and endogenous liver tumor models (DEN/PB-induced adenocarcinomas). In the case of the xenograft tumor-bearing mice, biodistribution studies showed micelle-thiostrepton to accumulate into both the liver (30% of injected dose) and the xenograft tumors (60% of injected dose) from 4 hours post-administration. In the endogenous liver cancer models, micelle-thiostrepton accumulated into the liver only, allowing a high concentration of tumor-associated thiostrepton drug. Continuous administration of micelle-thiostrepton at doses of 40mg/kg, 3 times a week for 30 days retarded the growth rate of all subcutaneous tumors and reduced the occurrence of adenocarinomas in endogenous liver cancer models. Immunoblotting and immunohistochemical staining revealed that treatment with micelle-thiostrepton inhibited the expression of the oncogenic transcription factor FOXM1 in tumors, a reduction of which generally coincides with suppressed cell proliferation and increased cell death. Furthermore, it was found that the reduced tumor growth rate of thiostrepton-treated tumors was indeed related to increased apoptosis, as indicated by the elevated expression of cleaved caspase-3 in such tumors samples. Our data suggest that the thiazole antibiotic/proteasome inhibitor thiostrepton, when formulated into nanoparticles, may be highly suited as a nanomedicine for treating cancer, particularly that of liver cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-356. doi:1538-7445.AM2012-LB-356

Predictable and tunable half-life extension of therapeutic agents by controlled chemical release from macromolecular conjugates.

Conjugation to macromolecular carriers is a proven strategy for improving the pharmacokinetics of drugs, with many stable polyethylene glycol conjugates having reached the market. Stable conjugates suffer several limitations: loss of drug potency due to conjugation, confining the drug to the extracellular space, and the requirement for a circulating conjugate. Current research is directed toward overcoming such limitations through releasable conjugates in which the drug is covalently linked to the carrier through a cleavable linker. Satisfactory linkers that provide predictable cleavage rates tunable over a wide time range that are useful for both circulating and noncirculating conjugates are not yet available. We describe such conjugation linkers on the basis of a nonenzymatic β-elimination reaction with preprogrammed, highly tunable cleavage rates. A set of modular linkers is described that bears a succinimidyl carbonate group for attachment to an amine-containing drug or prodrug, an azido group for conjugation to the carrier, and a tunable modulator that controls the rate of β-eliminative cleavage. The linkers provide predictable, tunable release rates of ligands from macromolecular conjugates both in vitro and in vivo, with half-lives spanning from a range of hours to >1 y at physiological pH. A circulating PEG conjugate achieved a 56-fold half-life extension of the 39-aa peptide exenatide in rats, and a noncirculating s.c. hydrogel conjugate achieved a 150-fold extension. Using slow-cleaving linkers, the latter may provide a generic format for once-a-month dosage forms of potent drugs. The releasable linkers provide additional benefits that include lowering C(max) and pharmacokinetic coordination of drug combinations.

Development of Bone-Targeted Catalase Derivatives for Inhibition of Bone Metastasis of Tumor Cells in Mice

Removal of hydrogen peroxide by delivering catalase to the vicinity of metastasizing tumor cells is a promising approach for inhibiting tumor metastasis. To inhibit bone metastasis, catalase was conjugated with 3,5-di(ethylamino-2,2-bisphosphono)benzoic acid (Bip), a derivative of bone-seeking bisphosphonates, polyethylene glycol (PEG), or both. Bip-conjugated catalase derivatives, that is, catalase-Bip and PEG-catalase-Bip, exhibited a higher affinity for bone matrix as compared with their counterparts without Bip. The tissue distribution of (111) In-labeled catalase derivatives indicated that the accumulation of radioactivity in bones was increased by conjugation of either Bip or PEG with catalase. An experimental bone metastasis model was developed by injecting male C57BL/6 mice with murine melanoma B16-BL6/Luc cells, which stably express firefly luciferase into left ventricle. Repeated injections of catalase to tumor-bearing mice had no significant effect on the number of melanoma cells in tibiae and femurs, whereas injections of catalase-Bip, PEG-catalase, or PEG-catalase-Bip significantly reduced the number. These results indicate that targeted delivery of catalase to the bones can be achieved by conjugating the enzyme with either Bip or PEG, and this delivery is effective in inhibiting the bone metastasis of tumor cells.

Targetable Gold Nanorods for Epithelial Cancer Therapy Guided by Near‐IR Absorption Imaging

Well-designed nanoparticle-mediated, image-guided cancer therapy has attracted interest for increasing the efficacy of cancer treatment. A new class of smart theragnostic nanoprobes employing cetuximab (CET)-conjugated polyethylene glycol (PEG)ylated gold nanorods (CET-PGNRs) is presented; these nanoprobes target epithelial cancer cells using near-infrared light. The cetyltrimethylammonium bromide bilayer on GNRs is replaced with heterobifunctional PEG (COOH-PEG-SH) to serve as a biocompatible stabilizer and to increase specificity. The carboxylated GNRs are further functionalized with CET using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC-NHS) chemistry. To assess the potential of such GNRs, their optical properties, biocompatibility, colloidal stability, in vitro/in vivo binding affinities for cancer cells, absorption imaging, and photothermal therapy effects are investigated. CET-PGNRs exhibit excellent tumor targeting ability and strong potential for simultaneous absorption imaging and photothermal ablation of epithelial cancer cells.

Multifunctional targeting micelle nanocarriers with both imaging and therapeutic potential for bladder cancer

BackgroundWe previously developed a bladder cancer-specific ligand (PLZ4) that can specifically bind to both human and dog bladder cancer cells in vitro and in vivo. We have also developed a micelle nanocarrier drug-delivery system. Here, we assessed whether the targeting micelles decorated with PLZ4 on the surface could specifically target dog bladder cancer cells.Materials and methodsMicelle-building monomers (ie, telodendrimers) were synthesized through conjugation of polyethylene glycol with a cholic acid cluster at one end and PLZ4 at the other, which then self-assembled in an aqueous solution to form micelles. Dog bladder cancer cell lines were used for in vitro and in vivo drug delivery studies.ResultsCompared to nontargeting micelles, targeting PLZ4 micelles (23.2 ± 8.1 nm in diameter) loaded with the imaging agent DiD and the chemotherapeutic drug paclitaxel or daunorubicin were more efficient in targeted drug delivery and more effective in cell killing in vitro. PLZ4 facilitated the uptake of micelles together with the cargo load into the target cells. We also developed an orthotopic invasive dog bladder cancer xenograft model in mice. In vivo studies with this model showed the targeting micelles were more efficient in targeted drug delivery than the free dye (14.3×; P < 0.01) and nontargeting micelles (1.5×; P < 0.05).ConclusionTargeting micelles decorated with PLZ4 can selectively target dog bladder cancer cells and potentially be developed as imaging and therapeutic agents in a clinical setting. Preclinical studies of targeting micelles can be performed in dogs with spontaneous bladder cancer before proceeding with studies using human patients.

The Tripeptide, GHK, Induces Programmed Cell Death in SH-SY5Y Neuroblastoma Cells

GHK-Cu is a tripeptide that is found in plasma and increases fibroblast proliferation and is widely used in wound healing creams. Studies show that GHK-Cu stimulates the level of p63, a member of the antitumor suppressor family which includes p53 and p73. Because of its effect on p63, we tested the hypothesis that GHK induces apoptosis in neuroblastoma cells, SH-SY5Y, and that modification of GHK by polyethylene glycol (PEG) conjugation, PEGylation, potentiates its effects. Initial studies on NIH-3T3 fibroblasts and U937 histiocytic lymphoma cells show that GHK-Cu and GHK-PEG stimulate fibroblast proliferation while attenuating U937 cell proliferation. SH-SY5Y cells were treated with GHK or GHK-PEG at 1 and 10 nM for 24 hours followed by cell proliferation, cell viability, cell cytotoxicity and apoptosis assays. Our results showed that 24 hour GHK treatment elevated apoptosis by 1.8 ± 0.17 and 3.3 ± 0.15 fold at 1 and 10 nM, respectively. GHK-PEG treatment, on the other hand, stimulated apoptosis by 3.2 ± 0.80 and 4.9 ± 0.9 fold at 1 and 10 nM, respectively. The treatments, while having no effects on cell cytotoxicity, reduced neuroblastoma cell viability and cell proliferation. Therefore, GHK induces apoptosis in neuroblastoma cells, an affect which was potentiated by PEGylation.

Randomized Comparison of IV PEG and IM E. Coli Asparaginase in Children and Adolescents with Acute Lymphoblastic Leukemia: Results of the DFCI ALL Consortium Protocol 05-01

Abstract 874E. coli L-asparaginase (E. coli ASP) is an important component of treatment for childhood ALL, but is associated with multiple toxicities, including allergy, pancreatitis, and thrombosis. It is typically given intramuscularly (IM). Because most pediatric ALL patients have indwelling venous catheters, intravenous (IV) administration of asparaginase would be a more convenient and less painful option than IM injection. PEG-asparaginase (PEG), the polyethylene glycol conjugate of E. coli ASP, has a longer circulating half-life and so may be given less frequently. We have previously demonstrated that a single dose of PEG 2500 IU/m2 given IV is tolerable in children with ALL, with potentially therapeutic serum enzyme activity (≥ 0.1 IU/mL) maintained for at least 18 days in most patients.[Blood 2010;115:1351-3] On DFCI ALL Consortium Protocol 05-01, all patients (pts) with newly diagnosed ALL aged 1–18 years (yrs) who achieved complete remission were eligible to participate in a randomized comparison of IM E. coli ASP and IV PEG during the 30-week (wk) multi-agent post-induction Consolidation phase. Beginning at week 7 of therapy, pts received either IM E. Coli ASP 25000 IU/m2 weekly × 30 wks or IV PEG 2500 IU/m2 every 2 wks × 30 wks. Serum samples were obtained every 6 wks just prior to an ASP dose and were assayed for ASP enzyme activity by a validated biochemical assay. Between 2005–2010, 463 pts were enrolled in the randomized comparison. Median age was 5 yrs (range 1.2–17.9 yrs). There was no significant difference in presenting characteristics between the two arms, except that more pts on the E. coli ASP arm presented with a mediastinal mass (9% vs 3%, p=0.04). Median follow-up was 2.8 years. Median nadir serum ASP activity (NSAA) at each assayed timepoint during the Consolidation phase was significantly higher with IV PEG than with IM E. coli ASP (Table 1). An NSAA of ≥ 0.1 IU/mL was achieved in ≥ 95% of IV PEG pts compared with < 50% of IM E. coli ASP pts (p<0.01 at each timepoint). There was no significant difference in ASP-related toxicities (allergy, pancreatitis, thrombosis) between the two types of ASP (Table 2). Older pts (≥ 10 yrs old) had a significantly higher overall rate (p<0.01) of pancreatitis (18% vs 7%) and thrombosis (18% vs 4%), but not of allergy (p=0.49) or infection (p=0.21), compared to younger pts. There was no significant difference in the rates of ASP-related toxicities when comparing IM E. coli ASP vs IV PEG separately within the two age groups (≥10 yrs and < 10 yrs). We conclude that every 2-week IV PEG is no more toxic than weekly IM E. coli ASP in children and adolescents with ALL, and is associated with higher serum ASP activity. Longer follow-up is necessary to determine whether there is any difference in event-free survival between the two treatment arms.Table 1:Nadir Serum ASP activity (NSAA) during 30-week Consolidation phaseIV PEGIM ECOLISample Time (wks)*NMedian IU/mL% pts with NSAA ≧ 0.10 IU/mLNMedian IU/mL% pts with NSAA ≧ 0.10 IU/mL5840.6795%920.09448%11700.7197%740.09447%17730.7697%860.09247%23600.70100%760.09446%29680.70100%630.09544%*Number of weeks after start of Consolidation phaseTable 2:Toxicities by ASP type during 30-week Consolidation phaseToxicityIV PEG # of pts (%)IM E. COLI # of pts (%)p-valueNumber of Patients232231Asparaginase Toxicity59 (25)58 (25)>0.99Allergy26 (11)20 (9)0.44Pancreatitis25 (11)21 (9)0.64 Mild/Moderate13 (6)13 (6) Severe12 (5)8 (3)Thrombosis14 (6)21 (9)0.22Infection (bacteremia, invasive fungal disease)35 (15)46 (20)0.18 Disclosures:Silverman:Enzon Pharmaceuticals: Honoraria. Supko:Enzon Pharmaceuticals: Research Funding. Sallan:Enzon Pharmaceuticals: Honoraria.

Hemodynamic responses to a hemoglobin bis‐tetramer and its polyethylene glycol conjugate

The design of hemoglobin-based oxygen carriers (HBOCs) poses a significant challenge as clinical trials of many materials have reported adverse side effects that may come from the scavenging of the vasodilator nitric oxide (NO). A compensating reaction, reduction of endogenous nitrite by hemoglobin (Hb) and its derivatives, generates NO. Polyethylene glycol (PEG) conjugation of Hb enhances the rate of the reaction. Hemoglobin bis-tetramers (BT) and their PEGylated derivative (BT-PEG) bind oxygen with a degree of cooperativity and also have significantly enhanced nitrite reductase activity compared to the native protein. Circulatory evaluation will test if the properties of BT and BT-PEG are reflected in their effects in vivo. BT and BT-PEG were evaluated as infusions into healthy wild-type (WT) and diabetic (db/db) mouse models. The effects were compared to infusions of murine Hb. The materials were found not to cause significant increases in systemic blood pressure in either WT mice or db/db mice. The latter are highly sensitive to NO scavenging. Further hemodynamic measurements in WT mice indicate that while a slight increase in systemic vascular resistance (SVR) was observed after infusion of BT, the extent is not significant. No change in SVR from baseline was observed after infusion of BT-PEG. The enlarged Hb derivatives do not evoke unfavorable circulatory responses that have been noted to result from infusion of Hb derivatives. These results suggest that a compromise between the P(50) , n(50) , and nitrite reductase activity of a Hb derivative can serve as the basis for producing HBOCs that can be tested for vasoactivity.

Genomic instability induced by mutant succinate dehydrogenase subunit D (SDHD) is mediated by O2-• and H2O2

SDHD mutations are associated with human cancers but the mechanisms that may contribute to transformation are unknown. The hypothesis that mutations in SDHD increase levels of superoxide leading to genomic instability was tested using site-directed mutagenesis to generate a truncated SDHD cDNA that was expressed in Chinese hamster fibroblasts. Stable expression of mutant SDHD resulted in 2-fold increases in steady-state levels of superoxide that were accompanied by a significantly increased mutation rate as well as a 70-fold increase in mutation frequency at the hprt locus. Overexpression of MnSOD or treatment with polyethylene glycol conjugated (PEG)-catalase suppressed mutation frequency in SDHD mutant cells by 50% (P<0.05). Simultaneous treatment with PEG-catalase and PEG-SOD suppressed mutation frequency in SDHD mutant cells by 90% (P<0.0005). Finally, 95% depletion of glutathione using l-buthionine-[S,R]-sulfoximine (BSO) in SDHD mutant cells caused a 4-fold increase in mutation frequency (P<0.05). These results demonstrate that mutations in SDHD cause increased steady-state levels of superoxide which significantly contributed to increases in mutation rates and frequency mediated by superoxide and hydrogen peroxide. These results support the hypothesis that mutations in SDHD may contribute to carcinogenesis by increasing genomic instability mediated by increased steady-state levels of reactive oxygen species.

Artificial Extracellular Matrix Proteins Containing Phenylalanine Analogues Biosynthesized in Bacteria Using T7 Expression System and the PEGylation

In vivo incorporation of phenylalanine (Phe) analogues into an artificial extracellular matrix protein (aECM-CS5-ELF) was accomplished using a bacterial expression host that harbors the mutant phenylalanyl-tRNA synthetase (PheRS) with an enlarged binding pocket. Although the Ala294Gly/Thr251Gly mutant PheRS (PheRS**) under the control of T5 promoter allows incorporation of some Phe analogues into a protein, the T5 system is not suitable for material science studies because the amount of materials produced is not sufficient due to the moderate strength of the T5 promoter. This limitation can be overcome by using a pair of T7 promoter and T7 RNA polymerase instead. In the T7 expression system, it is difficult, however, to achieve a high incorporation level of Phe analogues, due to competition of Phe analogues for incorporation with the residual Phe that is required for synthesis of active T7 RNA polymerase. In this study, we prepared the PheRS** under T7 promoter and optimized culture condition to improve both the incorporation level of recombinant aECM protein and the incorporation level of Phe analogues. Incorporation and expression levels tend to increase in the case of p-azidophenylalanine, p-iodophenylalanine, and p-acetylphenylalanine. We evaluated the lower critical transition temperature, which is dependent on the incorporation ratio and the turbidity decreased when the incorporation level increased. Circular dichromism measurement indicated that this tendency is based on conformational change from random coil to β-turn structure. We demonstrated that polyethylene glycol (PEG) can be conjugated at reaction site of Phe analogues incorporated. We also demonstrated that the increased hydrophilicity of elastin-like sequences in the aECM-CS5-ELF made by PEG conjugation could suppress nonspecific adhesion of human umbilical vein endothelial cells (HUVEC).

Surface modification with polyethylene glycol–corn trypsin inhibitor conjugate to inhibit the contact factor pathway on blood-contacting surfaces

Blood contacting surfaces bind plasma proteins and trigger coagulation by activating factor XII (FXII). The objective of this work was to develop blood contacting surfaces having the dual properties of protein resistance and inhibition of coagulation. Gold was used as a model substrate because it is amenable to facile modification using gold–thiol chemistry and to detailed surface characterization. The gold was modified with both polyethylene glycol (PEG) and corn trypsin inhibitor (CTI), a potent and specific inhibitor of activated FXII (FXIIa). Two methods of surface modification were developed; sequential and direct. In the sequential method PEG was first chemisorbed on gold; CTI was then attached to the PEG. In the direct method a conjugate of PEG and CTI was first prepared; the conjugate was then immobilized on gold. The surfaces were characterized by water contact angle and XPS. Biointeractions with the modified surfaces were assessed by measuring fibrinogen adsorption from buffer and plasma and by immunoblot analysis of eluted proteins after plasma exposure. Inhibition of FXIIa, autoactivation of FXII, and clotting times of plasma in contact with the surfaces were also measured. Both the sequential and direct surfaces showed reduced protein adsorption, increased FXIIa inhibition and longer clotting times compared with controls. Although the CTI density was lower on surfaces prepared using the sequential method, surfaces so prepared exhibited greater CTI activity than those generated by the direct method. It is concluded that the activity of immobilized PEG–CTI depends on the method of attachment and that immobilized CTI may be useful in rendering biomaterials more blood compatible.

A double antigen bridging immunogenicity ELISA for the detection of antibodies to polyethylene glycol polymers

Introduction Polyethylene glycol (PEG) polymers attached to biotherapeutic molecules enhance in vivo delivery and stability of these large molecular weight drugs. However, these polymers may by themselves be immunogenic and elicit antibodies that can reduce the efficacy of the drug and contribute to potential patient morbidity. A double antigen bridging ELISA immunogenicity assay for the detection of anti-drug antibodies (ADAs) specific to PEG polymers of various sizes has been developed. Methods Hapten-labeled conjugate of 40 kDa PEG polymer was synthesized and used in a double antigen bridging ELISA. The hapten-labeled PEG is incubated with the patient sample, then this mixture is added to a 96-well microplate precoated with 40 kDa PEG, allowing PEG-specific ADA to form a bridge complex with the PEG conjugate and the PEG coated on the microplate. After incubation, the reaction mixture is removed and replaced by horseradish peroxidase (HRP)-labeled anti-hapten antibody. After sufficient incubation, the plate is washed and substrate reagent is added. Enzyme color development, directly proportional to ADA, is stopped after 20 min with 2N sulfuric acid and the absorbance in each well is measured at 450/630 nm. Dose response, drug tolerance, matrix effects, reproducibility, specificity/free drug depletion experiments and screening cut-point determination of 350 naïve normal human sera were performed. Results Using an anti-PEG mouse monoclonal IgM as a positive control, a reproducible dose response curve was demonstrated for the PEG Immunogenicity ELISA. Pre-existing PEG-specific antibodies which were proven to be highly specific to the PEG polymer structure were found in 15 human serum samples in a total population of 350 naïve donors. The assay exhibited no significant matrix effects and was shown to be highly reproducible. Discussion A double antigen bridging immunogenicity assay for the detection of antibodies to PEG in the typical polymer size ranges used in biotherapeutics has been successfully developed in ELISA format. The antibodies detected in positive samples displayed a diverse spectrum of specificities for different PEG polymer lengths and linking functional groups. The discovery of 15 confirmed positive samples among 350 naïve patient samples calls into focus the need for testing PEG-specific immunogenicity of PEGylated biotherapeutics.

RAFT-synthesized graft copolymers that enhance pH-dependent membrane destabilization and protein circulation times

Here we describe a new graft copolymer architecture of poly(propylacrylic acid) (polyPAA) that displays potent pH-dependent, membrane-destabilizing activity and in addition is shown to enhance protein blood circulation kinetics. PolyPAA containing a single telechelic alkyne functionality was prepared via reversible addition–fragmentation chain transfer (RAFT) polymerization with an alkyne–functional chain transfer agent (CTA) and coupled to RAFT polymerized poly(azidopropyl methacrylate) (polyAPMA) through azide-alkyne [3 + 2] Huisgen cycloaddition. The graft copolymers become membrane destabilizing at endosomal pH values and are active at significantly lower concentrations than the linear polyPAA. A biotin terminated polyPAA graft copolymer was prepared by grafting PAA onto polyAPMA polymerized with a biotin functional RAFT CTA. The blood circulation time and biodistribution of tritium labeled avidin conjugated to the polyPAA graft copolymer was characterized along with a clinically utilized 40 kDa branched polyethylene glycol (PEG) also possessing biotin functionalization. The linear and graft polyPAA increase the area under the curve (AUC) over avidin alone by 9 and 12 times, respectively. Furthermore, polyPAA graft copolymer conjugates accumulated in tumor tissue significantly more than the linear polyPAA and the branched PEG conjugates. The collective data presented in this report indicate that the polyPAA graft copolymers exhibit robust pH-dependent membrane-destabilizing activity, low cytotoxicity, significantly enhanced blood circulation time, and increased tumor accumulation.

A new method to increase selectivity of transglutaminase mediated PEGylation of salmon calcitonin and human growth hormone

Modification of therapeutic proteins and peptides by polyethylene glycol (PEG) conjugation is a well-known approach to improve the pharmacological properties of drugs. Several chemical procedures of PEG coupling are already in use but an alternative method based on microbial transglutaminase (mTGase) was recently devised. The enzyme catalyzes the link of mPEG-NH 2 to glutamines (Gln) of a substrate protein. In this case the advantage resides in the fact that usually only few Gln(s) in a protein are substrate of mTGase. In order to further restrict the selectivity of the enzyme, we investigated a new approach leading to the formation of a single conjugate isomer as well as for those proteins containing two or more Gln(s) as mTGase substrates. It was found that the addition of co-solvents in the reaction mixture influenced both the secondary structure of the targeted protein and the mTGase activity. The enzymatic PEGylation under these conditions yielded only mono- and selectively modified conjugates. The method was investigated with salmon calcitonin (sCT) and human growth hormone (hGH). In the case of sCT we also demonstrated the importance of site-selective conjugation for the preservation of in vivo activity.

A new direction for anticoagulants: Inhibiting fibrin assembly with PEGylated fibrin knob mimics

Current anticoagulants target coagulation factors upstream from fibrin assembly and polymerization (i.e., formation of fibrin clot). While effective, this approach requires constant patient monitoring since pharmacokinetics and pharmacodynamics vary from patient to patient. To address these limitations, we developed an alternative anticoagulant that effectively inhibits fibrin polymerization. Specifically, we investigated PEGylated fibrin knob "A" peptides, evaluating the effect of both polyethylene glycol (PEG) chain length (0, 2, 5, and 10-30 kDa) and knob peptide sequence (GPRPAAC, GPRPFPAC, and GPRPPERC) on inhibiting fibrin polymerization (i.e., clot formation). Thrombin-initiated clotting assays with purified fibrinogen were performed to compare clot formation with each peptide-PEG conjugate. Results indicated a biphasic effect of PEG chain length, whereby, active-PEG conjugates demonstrated increasingly enhanced inhibition of fibrin polymerization from 0 to 5 kDa PEG. However, the anticoagulant activity diminished to control levels for PEG chains above 5 kDa. Ultimately, we observed a 10-fold enhancement of anticoagulant activity with active peptides PEGylated with 5 kDa PEG compared to non-PEGylated knob peptides. The sequence of the active peptide significantly influenced the anticoagulant properties only at the highest 1:100 molar ratio where GPRPFPAC-5 kDa PEG and GPRPPERC-5 kDa PEG demonstrated significantly lower percent clottable protein than GPRPAAC-5 kDa PEG. Moreover, human plasma treated with the active 5 kDa PEG conjugate exhibited delayed prothrombin time to within the therapeutic range specified for oral anticoagulants. Collectively, this study demonstrated the utility of PEGylated fibrin knob peptides as potential anticoagulant therapeutics. Biotechnol. Bioeng. 2011;108: 2424-2433. © 2011 Wiley Periodicals, Inc.

Protein Chip Array Profiling of Molecular Variants of Hirudins Using Surface Enhanced Laser Desorption Ionization (SELDI) Technique

Various molecular variants of natural hirudin are developed for pharmaceutical purposes. Most of these are molecular variants, their conjugates with PEG Hirudin and fragment conjugates. Desirudin and Refludin represent recombinant hirudin variants with minor structural differences. PEG Hirudin represents a polyethylene glycol conjugate. Angiomax represents a terminal decapeptide of hirudin which is complex with a tripeptide and linker polypeptide. Although these products are homogeneous, their immunogenic responses reportedly vary. The purpose of this investigation was to investigate the molecular profile of each of these agents utilizing the SELDI technique employing a gold chip. Desirudin showed a single peak at 6.97kda, where is Refludin showed a single peak at 6.98kda. PEG Hirudin showed a heterogeneous peak at 17.8kda Angiomax showed a major peak at 2.18kda and a minor peak at 2.39kda. All of these products were free of other minor components below 1kda. These results show that ProteinChip array can be used to determine the molecular weight profile of each of these proteins and to check the presence of other impurities such as peptide fragment and other contaminants.

A New Method for In Vivo Visualization of Vessel Remodeling Using a Near‐Infrared Dye

Vascular obstructive events can be partially compensated for by remodeling processes that increase vessel diameter and collateral tortuosity. However, methods for visualizing remodeling events in vivo and with temporal comparisons from the same animal remain elusive. Using a novel infrared conjugated polyethylene glycol dye, we investigated the possibility of intravital vascular imaging of the mouse ear before and after ligation of the primary feeder artery. For comparison, we used two different mouse models known to have impaired vascular remodeling after ligation (i.e., aged and PAI-1(-/-) mice). The results obtained with the infrared dye were confirmed using immunofluorescence labeling of the ear microvasculature with confocal microscopy. After ligation, increases in vessel diameter (between 10% and 60%) and tortuosity (approximately 15%) were observed in C57Bl/6 mice using both the infrared dye and the immunofluorescence technique. However, aged C57Bl/6 and PAI-1(-/-) mice did not show vascular remodeling following ligation. Vascular remodeling can be visualized and accurately quantified using a new infrared dye in vivo. This analysis technique could be generally employed for quantitative investigations of changes in vascular remodeling.