Selective conjugation of poly(2-ethyl 2-oxazoline) to granulocyte colony stimulating factor.

Poly(2-ethyl 2-oxazoline) (PEOZ) is a water-soluble, stable and biocompatible polymer that was prepared in a linear form for the conjugation of protein biomolecules. Polymers of molecular weights ranging from 5 to 20 kDa, with an aldehyde or an amine functional terminal group, were synthesized with narrow polydispersities. To assess the suitability of the polymer for therapeutic application, granulocyte colony stimulating factor (G-CSF) was used as a model protein for PEOZ conjugation.

State of the art in PEGylation: the great versatility achieved after forty years of research.

In the recent years, protein PEGylation has become an established and highly refined technology by moving forward from initial simple random coupling approaches based on conjugation at the level of lysine ε-amino group. Nevertheless, amino PEGylation is still yielding important conjugates, currently in clinical practice, where the degree of homogeneity was improved by optimizing the reaction conditions and implementing the purification processes. However, the current research is mainly focused on methods of site-selective PEGylation that allow the obtainment of a single isomer, thus highly increasing the degree of homogeneity and the preservation of bioactivity.

Covalent conjugation of poly(ethylene glycol) to proteins and peptides: strategies and methods.

PEGylation, the covalent linking of PEG chains, has become the leading drug delivery approach for proteins. This technique initiated its first steps almost 40 years ago, and since then, a variety of methods and strategies for protein-polymer coupling have been devised. PEGylation can give a number of relevant advantages to the conjugated protein, such as an important in vivo half-life prolongation, a reduction or an abolishment of immunogenicity, and a reduction of aggregation.

Multivalent and flexible PEG-nitrilotriacetic acid derivatives for non-covalent protein pegylation.

Purpose: A new approach for non-covalent protein PEGylation is translated from immobilized metal ion affinity chromatography, and based on metal coordination bonds between a chelating agent linked to PEG, nitrilotriacetic acid (NTA), and the ring nitrogen of histidines in a protein.

Methods: PEG-NTA conjugates were synthesized differing in the number of NTA units and in the polymer structure. Three derivatives were investigated in association experiments with five model proteins.

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.

Polyoxazoline: chemistry, properties, and applications in drug delivery.

Polyoxazoline polymers with methyl (PMOZ), ethyl (PEOZ), and propyl (PPOZ) side chains were prepared by the living cationic polymerization method and purified by ion-exchange chromatography. The following properties of polyoxazoline (POZ) were measured: apparent hydrodynamic radius by aqueous size-exclusion chromatography, relative lipophilicity by reverse-phase chromatography, and viscosity by cone-plate viscometry. The PEOZ polymers of different molecular weights were first functionalized and then conjugated to model biomolecules such as bovine serum albumin, catalase, ribonuclease, uricase, and insulin.

Stabilization of a supplemental digestive enzyme by post-translational engineering using chemically-activated polyethylene glycol.

Many enzymes used as digestive aids exhibit, at best, moderate stability when incubated under gastrointestinal conditions. A supplemental β-galactosidase administered orally to treat lactose intolerance was conjugated to 40 kDa, branched polyethylene glycol (PEG). PEGylation increased the enzyme's relative activity at lower pH values (2.

PEGylation for improving the effectiveness of therapeutic biomolecules.

Proteins have gained an important role in clinical practice, and they are the treatment of choice or even the only therapy for several pathologies. Unfortunately, their great potential is often hampered by relevant shortcomings such as immunogenicity and fast body clearance. Polymer conjugation, especially with polyethylene glycol (PEG), has emerged as a suitable drug delivery solution to address these drawbacks.

Polymer-drug conjugates for combination anticancer therapy: investigating the mechanism of action.

We developed a family of polymer-drug conjugates carrying the combination of the anticancer agent epirubicin (EPI) and nitric oxide (NO). EPI-PEG-(NO)8, carrying the highest content of NO, displayed greater activity in Caco-2 cells while it decreased toxicity against endothelium cells and cardiomyocytes with respect to free EPI. FACS and confocal microscopy confirmed conjugates internalization.

PEG conjugates in clinical development or use as anticancer agents: an overview.

During the almost forty years of PEGylation, several antitumour agents, either proteins, peptides or low molecular weight drugs, have been considered for polymer conjugation but only few entered clinical phase studies. The results from the first clinical trials have shared and improved the knowledge on biodistribution, clearance, mechanism of action and stability of a polymer conjugate in vivo. This has helped to design conjugates with improved features.

Transglutaminase-mediated PEGylation of proteins: direct identification of the sites of protein modification by mass spectrometry using a novel monodisperse PEG.

Poly(ethylene glycol) (PEG) has been widely used to prolong the residence time of proteins in blood and to decrease their immunogenicity and antigenicity. A drawback of this polymer lies in its polydispersity that makes difficult the identification of the sites of protein modification. This is a mandatory requirement if a PEGylated protein should be approved as a drug.

Detection of sites of infection in mice using 99mTc-labeled PN(2)S-PEG conjugated to UBI and 99mTc-UBI: a comparative biodistribution study.

Unlabelled: The antimicrobial peptide ubiquicidin (UBI) directly labeled with technetium-99m ((99m)Tc) has recently been shown to be specifically taken up at sites of infection; however, its chemical structure is not well defined. To address this problem, the aim of the present study was to label UBI using poly(ethyleneglycol)-N-(N-(3-diphenylphosphinopropionyl)glycyl)-S-tritylcysteine ligand (PEG-PN(2)S) in order to compare its ability to detect infection sites with that of (99m)Tc-UBI.

Methods: The PN(2)S-PEG-UBI conjugate was prepared and labeled with (99m)Tc, and its radiochemical purity was subsequently assessed.

A new PEG-beta-alanine active derivative for releasable protein conjugation.

A new PEGylating agent, PEG-betaAla-NHCO-OSu, has been studied for protein amino conjugation using human growth hormone (hGH) and granulocyte colony stimulating factor (G-CSF) as model therapeutic proteins. This new activated PEG possesses a convenient property for protein modification when compared to other activated carboxylate PEGs, namely, lower reactivity. When this polymer reacts with a protein, its features lead to fewer PEG-protein conjugate isomers because it preferentially binds the most nucleophilic and exposed amines.

PEGylation: Posttranslational bioengineering of protein biotherapeutics.

Polymer conjugation, especially by poly(ethylene glycol), has become a leading technology for the delivery of proteins. Nowadays, biotech drugs represent an increasing share of the new approved drugs, but their use is often prevented by drawbacks and safety concern. In particular, short in vivo half-life and immunogenicity are significant problems faced by the researchers dealing with the development of protein and peptide drugs.

The impact of PEGylation on biological therapies.

The term PEGylation describes the modification of biological molecules by covalent conjugation with polyethylene glycol (PEG), a non-toxic, non-immunogenic polymer, and is used as a strategy to overcome disadvantages associated with some biopharmaceuticals. PEGylation changes the physical and chemical properties of the biomedical molecule, such as its conformation, electrostatic binding, and hydrophobicity, and results in an improvement in the pharmacokinetic behavior of the drug. In general, PEGylation improves drug solubility and decreases immunogenicity.

Antitumoral activity of PEG-gemcitabine prodrugs targeted by folic acid.

Gemcitabine, 2',2'-difluoro-2'-deoxycytidine (dFdC), is an antitumor agent effective in the treatment of several solid tumors but its use is hampered by short plasma half-life, rapid metabolism and low selectivity towards tumor tissue. To overcome these limits, bioconjugates of gemcitabine were studied using poly(ethylene glycol) as polymeric carrier. Two types of conjugates were prepared, non-targeted and folic acid targeted conjugates.

Synthesis and characterization of poly(2-ethyl 2-oxazoline)-conjugates with proteins and drugs: suitable alternatives to PEG-conjugates?

Poly(2-ethyl-2-oxazoline) (POZ) was synthesized by living cationic ring-opening polymerization under microwave irradiation yielding polymers with low polydispersity indices (PDI, 1.15). The polymerization was quenched with sodium carbonate yielding a hydroxyl end-group with a high degree of functionality.

Site-specific pegylation of G-CSF by reversible denaturation.

A new strategy has been developed for extending the possibility of poly(ethylene glycol) (PEG) modification to accessible thiol groups of biologically active proteins. In particular, thiol-reactive PEGs have been coupled to the cysteine 17 of granulocyte colony stimulating factor (G-CSF), which is known to be partially buried in a hydrophobic protein pocket. The PEG linking was accomplished by partial protein denaturation with 3 M guanidine.

Site-specific modification and PEGylation of pharmaceutical proteins mediated by transglutaminase.

Transglutaminase (TGase, E.C. 2.

Anti-cancer PEG-enzymes: 30 years old, but still a current approach.

PEGylation (i.e. the covalent link of PEG strands) is a well known technique used to improve pharmaceutical properties of bioactive proteins and peptides.

Cardiac safety and antitumoral activity of a new nitric oxide derivative of pegylated epirubicin in mice.

The use of epirubicin is limited by the risk of a dilatory congestive heart failure that develops as a consequence of induction of a mitochondrial-dependent cardiomyocyte apoptosis. In a previous in-vitro study, we have provided evidence that a new formulation of pegylated epirubicin- bearing moieties that release nitric oxide, named BP-747, exerted a potent antitumoral activity against a colon cancer cell line, which was completely devoid of cytotoxic activity against cardiomyocytes. The aim of this study was to investigate the antitumoral and cardiotoxic profile of BP-747 in Caco-2 and SKOV-2 tumor-bearing mice.

Histaminase PEGylation: preparation and characterization of a new bioconjugate for therapeutic application.

Copper amine oxidase catalyses the oxidative deamination of primary amino groups of several biogenic amines, one of which is histamine, the principal chemical mediator of the first phase of allergic reactions. Looking forward to a possible future therapeutic application of this enzyme in the field of histamine-mediated afflictions, we developed a simple method for the purification of a histaminase from grass pea shoots, a source particularly enriched with the enzyme. Furthermore, in order to improve its therapeutic potential, in particular to reduce the high impurity due to its heterologous source, we conjugated the protein with poly(ethylene glycol) and tested the molecular, immunogenic and pharmacokinetic properties of the native and modified forms.

Novel monodisperse PEG-dendrons as new tools for targeted drug delivery: synthesis, characterization and cellular uptake.

Dendrimers, dendrons, and hyperbranched polymers are gaining popularity as novel drugs, imaging agents, and drug delivery systems. They present advantages of well-defined molecular weight, multivalent surfaces, and high drug carrying capacity. Moreover, it is emerging that such architectures can display unique endocytic properties.

PEGylation, successful approach to drug delivery.

PEGylation defines the modification of a protein, peptide or non-peptide molecule by the linking of one or more polyethylene glycol (PEG) chains. This polymer is non-toxic, non-immunogenic, non-antigenic, highly soluble in water and FDA approved. The PEG-drug conjugates have several advantages: a prolonged residence in body, a decreased degradation by metabolic enzymes and a reduction or elimination of protein immunogenicity.