Design, modeling, and expression of erythropoietin cysteine analogs in Pichia pastoris: improvement of mean residence times and in vivo activities through cysteine-specific PEGylation.

In this study, the low-cost production of recombinant human erythropoietin cysteine analogs (Cys-rhEPOs) from Pichia pastoris and the potential to increase their serum residency and in vivo activity through cysteine-specific PEGylation were investigated. Three-dimensional structures of several Cys-rhEPOs were generated using homology modeling, and three stable Cys-rhEPOs were selected on the basis of model stability in molecular dynamics simulation and surface accessibility of the inserted cysteine. cDNAs encoding Cys-rhEPOs were constructed by site-directed mutagenesis and expressed as secreted proteins in flask cultures of P.

Evaluation of bioactivity and pharmacokinetic characteristics of PEGylated P.pastoris-expressed erythropoietin.

High costs of production and relatively short serum half-life of mammalian cell-derived recombinant human erythropoietin (rHuEpo) necessitate finding and developing superior hosts/technologies for more efficient production of longer-acting erythropoietic agents. With these aims, we provide the first report on reductive alkylation of low-cost P.pastoris-expressed rHuEpo (PPEpo) with PEG-aldehyde.

High Expression of Methylotrophic Yeast-Derived Recombinant Human Erythropoietin in a pH-Controlled Batch System.

To accomplish the worldwide demand for recombinant human erythropoietin (rHuEpo) as a therapeutic, application of cost-efficient expression system of methylotrophic yeast Pichia pastoris (P. pastoris) rather than mammalian cells is indispensable. Herein, a report on high levels secreted-expression of Pichia-derived rHuEpo by batch fermentation in a pH stabilized format is presented.

The utility of rat jejunal permeability for biopharmaceutics classification system.

Purpose: The biopharmaceutical classification system has been developed to provide a scientific approach for classifying drug compounds based on their dose/solubility ratio and human intestinal permeability. Therefore in this study a new classification is presented, which is based on a correlation between rat and human intestinal permeability values.

Methods: In situ technique in rat jejunum was used to determine the effective intestinal permeability of tested drugs.

Effect of dimethyl-beta-cyclodextrin concentrations on the pulmonary delivery of recombinant human growth hormone dry powder in rats.

The aim of this article is to prepare and characterize inhalable dry powders of recombinant human growth hormone (rhGH), and assess their efficacy for systemic delivery of the protein in rats. The powders were prepared by spray drying using dimethyl-beta-cyclodextrin (DMbetaCD) at different molar ratios in the initial feeds. Size exclusive chromatography was performed in order to determine protecting effect of DMbetaCD on the rhGH aggregation during spray drying.

Characterization and aerodynamic evaluation of spray dried recombinant human growth hormone using protein stabilizing agents.

The effect of the protein stabilizers on the stability and aerosol performance of spray dried recombinant human growth hormone (SD rhGH) was investigated. rhGH solution was spray dried alone, with polysorbate 20 (at three concentrations of 0.05%, 0.

Predicting human intestinal permeability using single-pass intestinal perfusion in rat.

Purpose: The aim of the study was the prediction of human intestinal permeability and fraction absorbed of oral dose using single-pass intestinal perfusion technique (SPIP) in rats.

Methods: Permeability coefficients in anaesthetized rats were determined for 14 compounds. Drug solution in phosphate buffered saline (PBS) was perfused through a ingle-pass intestinal perfusion (SPIP) with flow rate of 0.

Intranasal bioavailability of insulin from carbopol-based gel spray in rabbits.

The purpose of this study was to investigate the nasal absorption of insulin from a carbopol-based nasal gel spray in rabbits. An insulin nasal gel was prepared by dispersing carbopol in distilled water, followed by the addition of insulin solution, then neutralization and viscosity adjustment. The nasal absorption of insulin from the gel, in conscious rabbits, was evaluated in comparison with absorption from an insulin solution.

A rapid and sensitive method for simultaneous determination of insulin and A21-desamido insulin by high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatography (RP-HPLC) method with UV-detection at room temperature has been developed for the analysis of insulin and its main degradation product, A21-desamido insulin. Octadecylsilica was used as stationary phase and a mixture of water and acetonitrile containing tetrametylammonium hydroxide as eluent. The method produced linear response over the concentration range of 10-100 microg/ml, with an average accuracy of 97.

Carrier erythrocytes: an overview.

Application of erythrocytes, the most abundant cells of the human body with desirable physiologic and morphologic characteristics, in drug delivery has been exploited extensively. These cellular carriers, having remarkable biocompatibility, biodegradability, and life-span in circulation, can be loaded by a wide spectrum of compounds of therapeutic value using different chemically, as well as physically, based methods. Most of the characteristics of the erythrocytes, including shape, membrane fragility, deformability, and hematologic indices undergo some degree of irreversible changes during the loading procedure.