Modulation of protein aggregation by polyethylene glycol conjugation: GCSF as a case study.

Polyethylene glycol (PEG) conjugation to proteins has emerged as an important technology to produce drug molecules with sustained duration in the body. However, the implications of PEG conjugation to protein aggregation have not been well understood. In this study, conducted under physiological pH and temperature, N-terminal attachment of a 20 kDa PEG moiety to GCSF had the ability to (1) prevent protein precipitation by rendering the aggregates soluble, and (2) slow the rate of aggregation relative to GCSF.

Design of PEGylated soluble tumor necrosis factor receptor type I (PEG sTNF-RI) for chronic inflammatory diseases.

A recombinant C-terminal truncated form of the human soluble tumor necrosis factor receptor type I (sTNF-RI) was produced in E. coli. This soluble receptor contains the first 2.

Mono-N-terminal poly(ethylene glycol)-protein conjugates.

A site-directed method of joining proteins to poly(ethylene glycol) is presented which allows for the preparation of essentially homogeneous PEG-protein derivatives with a single PEG chain conjugated to the amine terminus of the protein. This selectivity is achieved by conducting the reductive alkylation of proteins with PEG-aldehydes at lower pH. Working examples demonstrating the application of this method to improve the delivery characteristics and therapeutic value of several proteins are provided.

Development of a novel, nonimmunogenic, soluble human TNF receptor type I (sTNFR-I) construct in the baboon.

Pharmacokinetics and immunogenicity of six different recombinant human soluble p55 tumor necrosis factor (TNF) receptor I (sTNFR-I) constructs were evaluated in juvenile baboons. The constructs included either an sTNFR-I IgG1 immunoadhesin (p55 sTNFR-I Fc) or five different sTNFR-I constructs covalently linked to polyethylene glycol. The constructs were administered intravenously three times, and pharmacokinetics and immunogenicity were examined over 63 days.

Pegylated brain-derived neurotrophic factor shows improved distribution into the spinal cord and stimulates locomotor activity and morphological changes after injury.

The neurotrophin brain-derived neurotrophic factor (BDNF) shows promise for the treatment of central nervous system (CNS) trauma and disease. Effective delivery methods are required, however, for BDNF to be useful as a therapeutic agent. To this end, we examined the penetration of intrathecally infused N-terminal pegylated BDNF (peg-BDNF) compared to similar infusion of native BDNF after spinal cord injury (SCI).

A new form of Filgrastim with sustained duration in vivo and enhanced ability to mobilize PBPC in both mice and humans.

Granulocyte colony-stimulating factor (G-CSF) has proven effective in the prophylaxis of chemotherapy-induced neutropenia and as a mobilizer of peripheral blood progenitor cells. The longevity of G-CSF action is limited by its removal from the body by two mechanisms. The first is thought to be mediated via receptors (receptor mediated clearance [RMC]) predominantly on neutrophils, the second process is likely the result of renal clearance.

Characterization and stability of N-terminally PEGylated rhG-CSF.

Purpose: The liquid stability of rhG-CSF was investigated after polyethylene glycol (PEG) with an average molecular weight of 6000 daltons was covalently attached to the N-terminal methionine residue.

Methods: The conjugation methods chosen for modifying the N-terminal residue were alkylation and acylation. The N-terminally PEGylated rhG-CSF conjugates were purified by cation exchange chromatography.

Systemic hematologic effects of PEG-rHuMGDF-induced megakaryocyte hyperplasia in mice.

PEG-rHuMGDF injected daily in normal mice causes a rapid dose-dependent increase in megakaryocytes and platelets. At the same time that platelet numbers are increased, the mean platelet volume (MPV) and platelet distribution width (PDW) can be either decreased, normal, or increased depending on the dose and time after administration. Thus, PEG-rHuMGDF at a low dose causes decreases in MPV and PDW, MGDF at an intermediate dose causes an initial increase followed by a decrease in MPV and PDW, and PEG-rHuMGDF at higher doses causes an increase in MPV and PDW followed by a gradual normalization of these platelet indices.

Pegylated megakaryocyte growth and development factor abrogates the lethal thrombocytopenia associated with carboplatin and irradiation in mice.

Megakaryocyte growth and development factor (MGDF) is a potent inducer of megakaryopoiesis in vitro and thrombopoiesis in vivo. The effects of MGDF appear to be lineage-selective, making this cytokine an ideal candidate for use in alleviating clinically relevant thrombocytopenias. This report describes a murine model of life-threatening thrombocytopenia that results from the combination treatment of carboplatin and sublethal irradiation.

Prolonged circulation of recombinant human granulocyte-colony stimulating factor by covalent linkage to albumin through a heterobifunctional polyethylene glycol.

Purpose: Recombinant human granulocyte-colony stimulating factor (rhG-CSF) was covalently conjugated to both rat and human serum albumin (RSA and HSA respectively) to increases the circulating half life (t1/2) of rhG-CSF.

Methods: Conjugates of RSA (MW 67,000) and HSA (MW 66,000) were prepared by linking the two proteins through a heterobifunctional maleimido-carboxyl polyethylene glycol (PEG) and were tested in the rat. The conjugates were injected intravenously (IV) at the equivalent dose of 50 micrograms/kg of rhG-CSF, and white blood cell (WBC) counts and plasma concentrations of drug were determined.

The pulmonary absorption of aerosolized and intratracheally instilled rhG-CSF and monoPEGylated rhG-CSF.

Purpose: The objective of this study was to highlight differences in the pulmonary absorption of a monoPEGylated rhG-CSF and rhG-CSF after intratracheal instillation and aerosol delivery.

Methods: Male Sprague Dawley rats (250 g) were anesthetized and intratracheally instilled (IT) with protein solution or were endotracheally intubated and administered aerosol for 20 min via a Harvard small animal ventilator. A DeVilbiss "Aerosonic" nebulizer containing 5 ml of protein solution at approximately 3 mg/ml was used to generate aerosol.

Mode of action of 5'-linked cholesteryl phosphorothioate oligodeoxynucleotides in inhibiting syncytia formation and infection by HIV-1 and HIV-2 in vitro.

A phosphorothioate homocytidine 10-mer containing a cholesteryl moiety covalently linked to the 5'-end (Chol-SdC10) inhibited syncytium formation in susceptible T cells induced by HIV-1 and HIV-2. The syncytium inhibition effect was minimal with unmodified cytidine homopolymer of the same net charge. Chol-SdC10 was shown to protect CEM cells against infection by cell-free HIV-1 particles without any apparent toxicity to the growth of CD4+ T cells.

Synthesis and properties of modified oligonucleotides.

Phosphorothioate oligonucleotide analogs conjugated to cholesteryl by a neutral, 6 atom linker are more effective inhibitors of HIV-1 in cell culture than the corresponding analogs conjugated via a phosphorothioate group. The antiviral activity correlates with the hydrophobic character of the oligonucleotide. Some new synthetic methodology is also discussed.

[High molecular weight L-asparaginase derivatives based on a water-soluble carboxymethyl cellulose: biological properties].

L-asparaginase, covalently bound with water-soluble CM-cellulose, exhibited the elevated antileukemic activity in mice with inoculated lymphoid leukemia L5178y as compared with the native enzyme. The antileukemic activity of the immobilized enzyme was shown to depend on the content of the polymer bound with the enzyme; the polymer amount may be altered during the enzyme modification. The prolonged effect of immobilized L-asparaginase was observed in rabbit circulation.