[Recombinant human erythropoietin: method of in vivo evaluation using normocythemic mice]. / Eritropoyetina recombinante humana: Método de valoración in vivo con ratones normocitémicos.

PURPOSE: The implementation of an in vivo assay to determine the biological activity of human recombinant erythropoietin (Hu-r EPO) is essential. The purpose of this study was to perform and optimize the conditions of an easy in vivo bioassay suitable for routine testing of quality control of Hu-r EPO preparations. MATERIAL AND METHODS: Normocythemic 8 weeks female mice treated with different Hu-r EPO doses were employed. The reticulocyte response was measured by flow cytometry and by visual count in a Neubauer cell count chamber, after selective red blood cell haemolysis. A unique subcutaneous injection with blood extraction 96 hours later was the schedule employed. The reticulocyte count measured by both methods was plotted against the log dose of Hu-r EPO. RESULTS: The dose-response curve obtained was linear between 5 and 160 UI/mouse and the doses chosen for future assays were 10, 30 and 90 UI/mouse. The use of at least 6 animals per dose and not less than 3 assays to obtain reliable limits according to international regulations is convenient. Thirty assays were performed in four different samples and were analyzed by parallel lines (3 + 3) relating the response with the log dose. The coefficient of correlation between both methods was 0.989, so they are equivalent. CONCLUSIONS: This method is suitable because fewer animals and bioassays are necessary to obtain fiducial limits according to international requirements. It is in agreement with the tendency to reduce the number of animals used for bioassay because ethical and economic reasons.

Preparation of norfloxacin spherical agglomerates using the ammonia diffusion system.

Agglomerated crystals of norfloxacin were prepared by a spherical crystallization technique using the ammonia diffusion system (ADS). This technique makes it possible to agglomerate amphoteric drugs like norfloxacin, which cannot be agglomerated by conventional procedures. When an ammonia-water solution of norfloxacin is poured into an acetone dichloromethane mixture under agitation, a small amount of ammonia is liberated in the system. The ammonia-water solution plays a role both as a good solvent for norfloxacin and as a bridging liquid, allowing the crystals' collection to take place in one step. It has been proven that the agglomeration mechanism follows three steps: first acetone enters into the droplets of ammonia-water (this emulsion is formed because of the system characteristics); dissolved norfloxacin is consequently precipitated while the droplets collect the crystals; simultaneously, a part of the ammonia contained in the agglomerates diffuses to the outer organic solvent phase, thereby forming the norfloxacin spherical agglomerates. The correct selection of solvents has enabled us to obtain a suitable stable crystalline shape.