Pharmacological properties of granulocytic colony-stimulating factor pegylated using electron beam synthesis nanotechnologies.

Granulocytic CSF pegylated using electron-beam synthesis nanotechnology exhibits pronounced granulomonocytopoiesis-stimulating and SC-mobilizing activity. More potent stimulation of committed precursors against the background of less pronounced activation of polypotent hemopoietic cells is a peculiarity of hemostimulating action of pegylated using electron-beam synthesis nanotechnology granulocytic CSF in comparison with its non-modified analog. The mobilizing effect of pegylated using electron-beam synthesis nanotechnology granulocytic CSF on early progenitor elements surpasses that of non-conjugated cytokine.

Rational cytokine design for increased lifetime and enhanced potency using pH-activated "histidine switching".

We describe a method for the rational design of more effective therapeutic proteins using amino acid substitutions that reduce receptor binding affinity in intracellular endosomal compartments, thereby leading to increased recycling in the ligand-sorting process and consequently resulting in longer half-life in extracellular medium. We demonstrate this approach for granulocyte colony-stimulating factor by using computationally predicted histidine substitutions that switch protonation states between cell-surface and endosomal pH. Molecular modeling of binding electrostatics indicates two different single-histidine mutants that fulfill our design requirements; experimental assays demonstrate that each mutant indeed exhibits an order-of-magnitude increase in medium half-life along with enhanced potency due to increased endocytic recycling.

Non-covalent modification of granulocyte-colony stimulating factor (G-CSF) by coiled-coil technology.

We present here an approach to non-covalently combine an engineered model protein with a PEGylated peptide via coiled-coil binding. To this end a fusion protein of G-CSF and the peptide sequence (JunB) was created-one sequence of JunB was expressed at the N-terminal of GCSF. JunB is able to bind to the peptide sequence cFos, which was in turn covalently linked to a chain of poly(ethylene glycol) (PEG). The selected peptide sequences are leucine zipper motives from transcription factors and are known to bind to each other specifically by formation of a super secondary structure called coiled-coil. The binding between PEGylated peptides of various molecular weights and the modified protein was assessed by isothermal calorimetry (ITC), dynamic light scattering (DLS), circular dichroism (CD), and fluorescence anisotropy. Our findings show that the attachment of 2 and 5kDa PEG does not interfere with coiled-coil formation and thus binding of peptide to fusion protein. With this work we successfully demonstrate the non-covalent binding of a model moiety (PEG) to a protein through coiled-coil interaction.

Design Rationale and Development Approach for Pegfilgrastim as a Long-Acting Granulocyte Colony-Stimulating Factor.

Filgrastim, a recombinant methionyl human granulocyte colony-stimulating factor (G-CSF) (r-metHuG-CSF), is efficacious in stimulating neutrophil production and maturation to prevent febrile neutropenia (FN) in response to chemotherapy. Because of its relatively short circulating half-life, daily filgrastim injections are required to stimulate neutrophil recovery. In an effort to develop a long-acting form of filgrastim that was as safe and efficacious as filgrastim but had a longer in vivo residence time, a number of strategies were considered. Ultimately, fusion of filgrastim to polyethylene glycol (PEG) was selected. Following extensive analysis of conjugation chemistries as well as in vitro and in vivo characterization of a panel of PEGylated proteins, a construct containing a 20 kDa PEG moiety covalently conjugated to the N-terminus of filgrastim was chosen for advancement as pegfilgrastim. Pegfilgrastim is primarily cleared by neutrophils and neutrophil precursors (rather than the kidneys), meaning that clearance from the circulation is self-regulating and pegfilgrastim is eliminated only after neutrophils start to recover. Importantly, addition of PEG did not alter the mechanism of action and safety profile compared to filgrastim. Clinical evaluation revealed that a single 6 mg dose effectively reduces the duration of neutropenia and risk of FN in patients receiving chemotherapy. This work demonstrates the benefit of using PEGylation to generate pegfilgrastim, which allows for once-per-chemotherapy cycle administration while maintaining similar safety and efficacy profiles as those for multiple daily administration of filgrastim. Approaches that may provide advances for therapeutic agonists of G-CSF receptor are also discussed.

Modulation of the sustained delivery of myelopoietin (Leridistim) encapsulated in multivesicular liposomes (DepoFoam).

Myelopoietins (MPO) are novel chimeric growth factors containing IL-3 and G-CSF receptor agonists that enhance the biological properties of both cytokines. These cytokines, like many therapeutic proteins, clear rapidly from circulation and must be administered daily to provide efficacy. Therefore, a controlled and sustained delivery system comprised of a biocompatible and biodegradable matrix, would offer important therapeutic advantages in the clinic, such as significantly reducing dose frequency and providing efficacy without toxicity. We report here the encapsulation of Leridistim (a protein from the MPO family) in multivesicular liposomes (DepoFoam) for sustained delivery, and demonstrate that a single injection of DepoFoam-encapsulated Leridistim results in elevated neutrophil counts for 10 days, in contrast to only 2 days for un-encapsulated Leridistim. Moreover, varying the lipid content of the DepoFoam matrix modulated the duration of elevated neutrophils from 2-3 to 9-10 days. The encapsulated Leridistim was released in vivo from the multivesicular liposomes in a uniform manner, consistent with its pharmacodynamic duration. Finally, a reproducible pharmacodynamic effect was observed with several batches of a DepoLeridistim formulation, indicating consistency of the manufacturing process of the DepoFoam delivery system. The capability of altering the release rates by varying the lipid composition provides maximum flexibility for controlled delivery of cytokine therapeutics.

Design of long-acting formulation of protein drugs with a double-layer structure and its application to rhG-CSF.

In order to design a sustained-release formulation of protein drugs characterized by excellent long-acting properties without an initial burst, a new double-layer minipellet (DL-MP) in which the lateral side of a matrix-type sustained-release formulation 'minipellet' using collagen as a carrier was coated with collagen was designed, and its performance was evaluated. In a DL-MP using bovine serum albumin (BSA) as a model drug, the initial burst observed with a single-layer minipellet (SL-MP) was effectively inhibited in an in vitro release test, and the addition of additives such as chondroitin sulfate (CS) permitted control of release rate. This formulation of recombinant human granulocyte colony-stimulating factor (rhG-CSF) was then prepared, and its characteristics were determined in normal rats. It was found that blood rhG-CSF concentration was maintained for about 1 week after administration of a DL-MP with additional CS, with persistent increase in white cell count. The results of this study indicated that DL-MP was useful as a long-acting formulation of rhG-CSF characterized by excellent long acting properties without an initial burst.

Effect of divalent polyethylene glycol units, conjugated on human granulocyte colony-stimulating factor, on biological activities in vitro and in vivo.

New divalent (two-chain type) polyethylene glycol (PEG) conjugates of a derivative of human recombinant granulocyte colony-stimulating factor (rhG-CSF), ND28, were synthesized by a novel conjugation method using triazine ring and amino butyric acid, and separated into mono-, di- and tri-PEG2(two chains)-ND28 with high purity of more than 90%, to examine the effect of the number of PEG units on their biological properties. Three species of PEG2-ND28 conjugates showed reduced but clear in vitro bioactivity and receptor binding inhibitory activity, and an inverse correlation between the number of PEG units and the activity was seen. On the other hand, the in vivo granulopoietic effect of tri-PEG2-ND28 in mice was observed to be most potent and long-lasting for 6 days after only one administration, and was followed by di-PEG2-ND28 and mono-PEG2-ND28. The plasma concentration of tri-PEG2-ND28 was maintained at a high level for 3 days after administration, while that of PEG-unbound ND28 disappeared within 30 h. There was a positive correlation between the number of PEG units and both the granulopoietic effect and the plasma half-life. These results suggest that the number of PEG units attached to the rhG-CSF can increase their stability during circulation in the plasma of mice, in turn resulting in a long-lasting granulopoietic effect in vivo.

Construction of protein analogues by site-specific condensation of unprotected fragments.

The extreme sensitivity to periodate of 1-amino, 2-hydroxy compounds permits the selective conversion of N-terminal serine and threonine to an aldehydic group. We have used this reaction to construct analogues of human granulocyte colony stimulating factor (G-CSF) by allowing such oxidized peptides to react with others that have had a hydrazide derivative attached to the C-terminus by reversed proteolysis. Two recombinant analogues of G-CSF were used as starting materials. Both had only a single lysine residue (at position 62 and 75, respectively) followed immediately by a serine. Digestion of each analogue by the lysine-specific protease from Achromobacter lyticus gave two fragments, one of which could be N-terminally oxidized and the other converted to the C-terminal hydrazide derivative by reversed proteolysis using the same enzyme. After preliminary studies with model peptides, we first reacted the corresponding peptide pairs together and then, in order to eliminate the 64-74 disulfide loop, fragment 1-62 from the first analogue with fragment 76-174 from the second. Reactions are efficient (up to 80% product based on the oxidized fragment) and take place under very mild conditions. The hydrazone bond can easily be stabilized by reduction with NaBH3CN. This method represents a new, reasonably general route for the construction of large protein chimeras of precisely controlled structure.

Chemo-enzymic backbone engineering of proteins. Site-specific incorporation of synthetic peptides that mimic the 64-74 disulfide loop of granulocyte colony-stimulating factor.

We present the concept of chemo-enzymic backbone engineering of proteins. Recombinant DNA techniques are used to produce appropriate proteins that are enzymically fragmented to give the starting materials. These fragments are modified specifically at their chain termini either enzymically (coupling of a hydrazide to the C terminus) or chemically (periodate oxidation of N-terminal serine to a glyoxylyl function). The modified fragments, which need no side protection whatever, are mixed together and religate themselves spontaneously under mild conditions. The hydrazone bond thus formed can be reduced if desired, which stabilizes the linkage and enhances the flexibility of the local conformation. In this way biologically or chemically derived structures can be incorporated into the protein, and the choice of the chemical ones is free of all of the constraints of the genetic code. We believe that this combined approach gives access to constructions that could not be derived by either recombinant or chemical methods alone. We illustrate the particularity of this concept by the engineered modifications of the 64-74 disulfide loop region of human granulocyte colony-stimulating factor. Analogs constructed include one which, in spite of having a nonpeptide link in its backbone, has full biological activity.

Fourier transform infared spectroscopy investigation of protein conformation in spray-dried protein/trehalose powders.

Spray drying is a way to generate protein solids (powders), which is also true for lyophilization. Sugars are used to protect proteins from conformational changes and chemical degradations arising from drying processes and storage conditions such as the humidity. The influence of trehalose and humidity on the conformation and hydration of spray-dried recombinant human granolucyte colony stimulating factor (rhG-CSF) and recombinant consensus interferon-alpha (rConIFN) was investigated using Fourier transform IR spectroscopy. The spectral analysis of spray-dried powders in the amide I region demonstrated that trehalose stabilized the alpha-helical conformation of both rhG-CSF and rConIFN proteins. Exposure of the pure protein powders to 33% relative humidity (RH) resulted in the formation of beta sheets and loss of turns but no change in alpha-helical structure. Trehalose reduced the magnitude of the changes in beta sheets and turns. Exposure of the pure protein powders to 75% RH resulted in the loss of alpha-helical conformation with a corresponding increase in beta structures (beta sheets and turns). Trehalose did not protect proteins from the loss of alpha-helical structures, but it reduced the formation of antiparallel beta sheets. Hydrogen-deuterium exchange (H-D exchange) was used to further characterize these hydration-induced conformational changes. At 33% RH the percent exchange of the protein decreased with increasing trehalose content, indicating a greater protection of the protein from H-D exchange by a higher concentration of trehalose. Such protection correlates with decreased conformational changes of the protein by trehalose at this humidity. At 75% RH the degree of H-D exchange of the protein was insensitive to the powder composition in all powders. Surprisingly, the H-D exchange of trehalose was low at about 20-25%, which was nearly independent of the protein/trehalose ratio and humidity, indicating that the exchangeable protons on trehalose molecules are highly protected in protein-containing powders. The observed protein hydration is related to the effect of trehalose on the conformational changes of the protein under humidity.