ABSTRACT
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 full length cDNA of rHuEpo was inserted into pPICZ alpha A vector under control of AOX[1] promoter, downstream of the secretion-alpha-factor and electroporated into P. pastoris strain X33. The highest expression transformant was selected by screening among the colonies surviving high concentration of Zeocin [1.0 mg/ml], followed by comparative small scale expression analysis by ELISA. Stabilization of pH around 6.0 by adding phosphoric acid into the culture media during induction period, improved the yield of expression to 150 mg/l of the media. Single-step Nickel-affinity chromatography was employed for purification of rHuEpo-6xHis to 80% purity. Analyses by SDSPAGE, Western blot and N-terminal protein sequencing confirmed the authenticity of the 33 kDa expressed rHuEpo with a native N-terminal indicating the proper cleavage of secretion-signal. Results of this study, further confirmed the possibility of employing methylotrophic yeast for scaled up production aims of rHuEpo as a cost-efficient expression system when provided evidence for higher expression yields through application of pH-controlled systems
Subject(s)
Yeasts , Hydrogen-Ion Concentration , Pichia , FermentationABSTRACT
Poly-[alkylcyanoacrylate] ester particles have been used for the preparation of different formulations ranging from ophthalmic delivery systems to cancer chemotherapy carriers in clinic. The aim of this study was to prepare and characterize poly-[methyl ethyl cyanoacrylate] [PMECA] particles containing 5-aminosalicylic acid [5-ASA]. PMECA particles were prepared using the inverse emulsification polymerization technique. The effects of various formulation parameters such as dispersed phase volume, polymer to drug weight ratio, and surfactant concentration on loading efficiency and size of the prepared particles were investigated. The amount of drug was determined by UV spectrophotometery at 331 nm. Morphological characteristics of particles and particle size analysis were studied by Scanning Electron Microscopy [SEM] and Laser Light Scattering techniques, respectively. The results showed that there was no linear relationship between the dispersed phase volume [DPV] and loading efficiency of 5-ASA in the range of 20-50%. Increasing polymer/drug weight ratio in the range of 1-15, enhanced the loading efficiency from 11.4 to 78.2% in a linear mode [r=0.987]. Increasing the surfactant concentration in the range of 1-3% did not increase the loading efficiency of the prepared particles, and increasing the concentration to 5% even decreased the loading efficiency of particles. Laser light scattering and SEM evaluations showed that in all prepared formulations, particles were in a mixture of micro and nanospheres and micro and nanocapsules and 50 percent of particles had mean sizes in the range of 6.4-10.1 micrometer. Although our results showed significant differences in the mean particle size between some of the prepared formulations, this variation was not practically important. It was concluded that by using proper conditions, it is possible to use PMECA as a polymeric matrix for loading of 5-ASA and the other hydrophilic drugs
Subject(s)
/chemical synthesis , Drug Delivery SystemsABSTRACT
The use of secondary metrics has become special interest in bioequivalency studies. The applicability of partial area method, truncated AUC and Cmax/AUC has been argued by many authors. This study aims to evaluate the possible superiority of these metrics to primary metrics [i.e. AUCinf, Cmax and Tmax]. The suitability of truncated AUC for assessment of absorption extent as well as Cmax/AUC and partial AUC for the evaluation of absorption rate in bioequivalency determination was investigated following administration of same product as test and reference to 7 healthy volunteers. Among the pharmacokinetic parameters obtained, Cmax/AUCinf was a better indicator or absorption rate and the AUCinf was more sensitive than truncated AUC in evaluation of absorption extent
Subject(s)
Humans , Therapeutic EquivalencyABSTRACT
The bioequivalence of two preparations of enalapril maleate [20 mg tablets] manufactured in Iran has been exploited in reference to a standard preparation [Xanef 20 tablets, MSD, Germany] in 14 healthy volunteers. Following oral dosing of a single tablet of each of test and standard products, as a randomized crossover design with 10-day washout intervals, the blood samples were collected in predetermined time points and using a synthetic substrate, Hippuryl-Histidy-Leucine [HHL], the release of hippuric acid from the substrate was determined as Angiotensin-Converting-Enzyme [ACE] activity of serum fractions. The percent of ACE inhibition in each sample was calculated and plotted against time, from which three pharmacodynamic parameters, i.e. Emax, tmax and AUC0-24 were derived. The results of statistical comparison of these parameters showed that both of the test preparations are bioequivalent with reference standard preparation