An in vivo evaluation of amphiphilic, biodegradable peptide copolymers as siRNA delivery agents.

A series of amphiphilic, biodegradable polypeptide copolymers were prepared for the delivery of siRNA (short interfering ribonucleic acid). The molecular weight (or polymer chain length) of the linear polymer was controlled by reaction stoichiometry for the 11.5, 17.2, and 24.6 kDa polypeptides, and the highest molecular weight polypeptide was prepared using a sequential addition method to obtain a polypeptide having a molecular weight of 38.6 kDa. These polymers were used to prepare polymer conjugate systems designed to target and deliver an apolipoprotein B (ApoB) siRNA to hepatocyte cells and to help delineate the effect of polymer molecular weight or polymer chain length on siRNA delivery in vivo. A clear trend in increasing potency was found with increasing molecular weight of the polymers examined (at a constant polymer:siRNA (w/w) ratio), with minimal toxicity found. Furthermore, the biodegradability of these polymer conjugates was examined and demonstrates the potential of these systems as siRNA delivery vectors.

Development of a liver-targeted siRNA delivery platform with a broad therapeutic window utilizing biodegradable polypeptide-based polymer conjugates.

The greatest challenge standing in the way of effective in vivo siRNA delivery is creating a delivery vehicle that mediates a high degree of efficacy with a broad therapeutic window. Key structure-activity relationships of a poly(amide) polymer conjugate siRNA delivery platform were explored to discover the optimized polymer parameters that yield the highest activity of mRNA knockdown in the liver. At the same time, the poly(amide) backbone of the polymers allowed for the metabolism and clearance of the polymer from the body very quickly, which was established using radiolabeled polymers to demonstrate the time course of biodistribution and excretion from the body. The fast degradation and clearance of the polymers provided for very low toxicity at efficacious doses, and the therapeutic window of this poly(amide)-based siRNA delivery platform was shown to be much broader than a comparable polymer platform. The results of this work illustrate that the poly(amide) platform has a promising future in the development of a siRNA-based drug approved for human use.

Novel long-acting crystal formulation of human growth hormone.

PURPOSE: The aim of the study is to solve a significant challenge of extending the half-life of therapeutic proteins using crystalline biopharmaceuticals and without redesigning the molecules. METHODS: Crystals of recombinant human growth hormone were coated with a monomolecular layer of positively charged poly(arginine). The pharmacokinetics and pharmacodynamics of this poly(arginine)-coated human growth hormone crystalline formulation were determined in hypophysectomized rats and monkeys. RESULTS: Here we have demonstrated for the first time that crystals of human growth hormone coated with positively charged poly(arginine) allowed for in vivo pharmacokinetic release profiles of over several days in animal models. The efficacy of this crystalline formulation injected subcutaneously once a week was found to be equivalent to seven daily soluble injections in the standard weight gain assay using the hypophysectomized rat model and in measurement of serum insulin-like growth factor in monkeys. The nonviscous nature of the suspension facilitated easy administration through a fine, 30-gauge needle and should provide for improved patient convenience and compliance. CONCLUSIONS: The approach described here offers an exciting possibility of being broadly applicable to other therapeutic proteins.

Injectable controlled release formulations incorporating protein crystals.

Development of ready-to-inject in situ formable controlled release gel systems for proteins is extremely challenging due to poor stability of proteins in the organic solvents typically used to fabricate these systems and because of the need of initial drying of proteins. The focus of the present study was to develop and characterize injectable controlled release systems composed of crystals of amylase, a model protein, suspended in solutions of polymeric and non-polymeric matrix materials in organic solvents. In this study, alpha-amylase derived from Aspergillus oryzae was crystallized and crystals were suspended in a poly(DL-lactide-co-glycolide) (PLGA) solution in acetonitrile (PLGA/acetonitrile), or in sucrose acetate isobutyrate (SAIB) plasticized with ethanol (SAIB/ethanol) systems. The results indicate that the protein crystals could be incorporated in these in situ formable gels without the need for initial drying. The crystals withstand organic solvents and water/organic solvent interfaces, and provide high protein loading (>30%) in these systems. Moreover, changing the morphology of the amylase crystals successfully modulated amylase release profiles. Study of long-term stability at 4 degrees C revealed a greater stability of crystalline protein compared to amorphous amylase. The above-mentioned data suggest that protein crystals might offer greater feasibility in developing sustained release injectable in situ formable protein depot systems.

Crystalline monoclonal antibodies for subcutaneous delivery.

Therapeutic applications for mAbs have increased dramatically in recent years, but the large quantities required for clinical efficacy have limited the options that might be used for administration and thus have placed certain limitations on the use of these agents. We present an approach that allows for s.c. delivery of a small volume of a highly concentrated form of mAbs. Batch crystallization of three Ab-based therapeutics, rituximab, trastuzumab, and infliximab, provided products in high yield, with no detectable alteration to these proteins and with full retention of their biological activity in vitro. Administration s.c. of a crystalline preparation resulted in a remarkably long pharmacokinetic serum profile and a dose-dependent inhibition of tumor growth in nude mice bearing BT-474 xenografts (human breast cancer cells) in vivo. Overall, this approach of generating high-concentration, low-viscosity crystalline preparations of therapeutic Abs should lead to improved ease of administration and patient compliance, thus providing new opportunities for the biotechnology industry.

Recombinant human insulin IX. Investigation of factors, influencing the folding of fusion protein-S-sulfonates, biotechnological precursors of human insulin.

The peculiarities of molecular structures and the influence of reaction conditions on the folding efficiency of fusion proteins-biotechnological precursors of human insulin, expressed in Escherichia coli as inclusion bodies have been investigated. The fusion proteins contained proinsulin sequence with various leader peptides connected by an Arg residue to the insulin B-chain. The kind and the size of leader peptide do not have essential influence on folding efficiency. However, the efficiency of protein folding depends on the location of the (His)6 site, which is used for metal-chelating affinity chromatography. In our study the protein folding depends on the reaction medium composition (including additives), the presence of accompanied cell components, pH, temperature, concentrations of protein, and redox agents. A negative influence of nucleic acid and heavy metal ions on folding has been found. S-sulfonated fusion protein has proinsulin-like secondary structure (by CD-spectroscopy data) that is the key point for 95% efficient folding proceeding. Folded fusion proteins are transformed into insulin by enzymatic cleavage.

Methods for preparation of recombinant cytokine proteins V. mutant analogues of human interferon-gamma with higher stability and activity.

Mutant analogues of recombinant human immune interferon (IFN-gamma) with higher stability and biological activity were prepared. Depending on the analogue, protein structure modification might involve introduction of an intramonomer disulfide bond (through replacements of Glu7Cys and Ser69Cys), C-terminal shortening by 10 amino acid residues, as well as Gln133Leu substitution in truncated variant. Isolation, purification, and renaturation of the IFN-gamma analogues expressed in Escherichia coli as inclusion bodies were performed according to the scheme developed earlier for wild-type protein. The main idea of this scheme is to remove cellular impurities before recombinant protein renaturation. Folding kinetics of IFN-gamma was studied by reversed-phase HPLC. IFN-gamma and mutant proteins were characterized by their thermal stability and biological activity. Introduction of the intramolecular disulfide bond together with C-terminal shortening and replacement of C-terminal residue was shown to result in increasing the thermal stability by 19 degrees C and four times enhancement of biological activity compared with intact IFN-gamma molecule.