Qualitative and quantitative reversed-phase high performance liquid chromatographic analysis of glycoprotein hormones in the presence of a large excess of human serum albumin.

The present work describes reversed-phase high performance liquid chromatographic methodologies (RP-HPLC) for the qualitative and quantitative analysis of the human glycoprotein hormones thyrotropin (hTSH), follitropin (hFSH), choriogonadotropin (hCG) and lutropin (hLH) in the presence of a large excess (up to 250:1) of human serum albumin (HSA). Chromatographic profiles with a good separation between the hormone and HSA were obtained by using a C4 column and specific gradient elution conditions for each hormone. Parameters such as resolution factor, tailing factor and relative retention time, were determined, and are useful for the evaluation of the quality of the separation obtained between the active pharmaceutical ingredient and the excipient present in the final formulation. The potential of each method for quantification of both HSA and the hormone was also demonstrated. Besides furnishing chromatographic quantifications that can substitute for in vivo bioassays and animal use, the chromatograms also provide a direct panorama of the quality and heterogeneity of the protein of interest.

Uncaria tomentosa for Reducing Side Effects Caused by Chemotherapy in CRC Patients: Clinical Trial.

To evaluate the effectiveness of Uncaria tomentosa in minimizing the side effects of chemotherapy and improving the antioxidant status of colorectal cancer (CRC) patients, a randomized clinical trial was conducted. Patients (43) undergoing adjuvant/palliative chemotherapy with 5-Fluorouracil/leucovorin + oxaliplatin (FOLFOX4) were split into two groups: the UT group received chemotherapy plus 300 mg of Uncaria tomentosa daily and the C group received only FOLFOX4 and served as a control. Blood samples were collected before each of the 6 cycles of chemotherapy, and hemograms, oxidative stress, enzymes antioxidants, immunologic parameters, and adverse events were analyzed. The use of 300 mg of Uncaria tomentosa daily during 6 cycles of FOLFOX4 did not change the analyzed parameters, and no toxic effects were observed.

A pilot study on potency determination of human follicle-stimulating hormone: a comparison between reversed-phase high-performance liquid chromatography method and the in vivo bioassay.

Reversed-phase high-performance liquid chromatography (RP-HPLC) was compared with the classical Steelman-Pohley bioassay (BA), based on animal use, for the determination of human follicle-stimulating hormone (hFSH) biological activity. A linear relationship (BA(IU)=0.9925 RP-HPLC(IU)-1.3165) with a highly significant correlation (r=0.9371; p<0.0001; n=24) was found for these two methods for six hFSH preparations of different origins. The mean difference between the bioactivity predicted from RP-HPLC data via this equation and the mean of the bioactivities obtained with the two methods for six other hFSH preparations was -1.4%, with a 95% confidence interval of -9.3 to +6.6%. The precision of these parameters was 1.63% and 2.82%, respectively. These results demonstrate that RP-HPLC is a viable physical-chemical alternative to the use of an in vivo bioassay for hFSH potency determination, applicable also to hFSH Standards containing large amounts of human serum albumin.

Analysis of human luteinizing hormone and human chorionic gonadotropin preparations of different origins by reversed-phase high-performance liquid chromatography.

Specific reversed-phase high-performance liquid chromatography conditions are reported for the analysis of recombinant and native human luteinizing hormone (hLH) and human chorionic gonadotropin (hCG) preparations. Heterodimeric hLH, hCG and their alpha- and beta-subunits migrated with significantly different retention times (t(R)) in the following order of increasing hydrophobicity: alpha-hCG

Biological evaluation of recombinant human erythropoietin in pharmaceutical products

The potencies of mammalian cell-derived recombinant human erythropoietin pharmaceutical preparations, from a total of five manufacturers, were assessed by in vivo bioassay using standardized protocols. Eight-week-old normocythemic mice received a single subcutaneous injection followed by blood sampling 96 h later or multiple daily injections with blood sampling 24 h after the last injection. Reticulocyte counting by microscopic examination was employed as the end-point using the brilliant cresyl blue or selective hemolysis methods, together with automated flow cytometry. Different injection schedules were investigated and dose-response curves for the European Pharmacopoeia Biological Reference Preparation of erythropoietin were compared. Manual and automated methods of reticulocyte counting were correlated with respect to assay validity and precision. Using 8 mice per treatment group, intra-assay precision determined for all of the assays in the study showed coefficients of variation of 12.1-28.4 percent for the brilliant cresyl blue method, 14.1-30.8 percent for the selective hemolysis method and 8.5-19.7 percent for the flow cytometry method. Applying the single injection protocol, a combination of at least two independent assays was required to achieve the precision potency and confidence limits indicated by the manufacturers, while the multiple daily injection protocol yielded the same acceptable results within a single assay. Although the latter protocol using flow cytometry for reticulocyte counting gave more precise and reproducible results (intra-assay coefficients of variation: 5.9-14.2 percent), the well-characterized manual methods provide equally valid alternatives for the quality control of recombinant human erythropoietin therapeutic products.

Comparative evaluation of the human whole blood and human peripheral blood monocyte tests for pyrogens.

Two different in vitro tests for pyrogens, using human peripheral blood monocytes (PBMNC) and diluted whole blood (WBC), respectively, were applied to different classes of parenteral medicinal products. Many of these products did not have a specified endotoxin limit concentration that was established as the maximum valid dilution to comply with the test. The results of the in vitro tests for pyrogens were compared with the results from the Limulus amoebocyte lysate (LAL) and rabbit pyrogen tests. The Second International Standard for endotoxin was used to calibrate all of the assays and the International Standard for IL-6 was used to calibrate the IL-6 ELISA which provided the readout for the in vitro tests for pyrogens. Preparatory tests were conducted to ensure that the "criteria for validity and precision of the standard curve" were satisfied and that the drugs being tested did not interfere in the tests. The PBMNC/IL-6 test had a detection limit of 0.06 EU/ml and spike recoveries were 62-165%. The whole blood/IL-6 test also had a detection limit of 0.06 EU/ml and spike recoveries were 58-132%. The application to the detection of non-endotoxin pyrogens needs to be evaluated in more detail, but the two in vitro tests for pyrogens showed good agreement overall, both with each other and with the LAL test and the rabbit pyrogen test for the detection of endotoxins.

Biological evaluation of recombinant human erythropoietin in pharmaceutical products.

The potencies of mammalian cell-derived recombinant human erythropoietin pharmaceutical preparations, from a total of five manufacturers, were assessed by in vivo bioassay using standardized protocols. Eight-week-old normocythemic mice received a single subcutaneous injection followed by blood sampling 96 h later or multiple daily injections with blood sampling 24 h after the last injection. Reticulocyte counting by microscopic examination was employed as the end-point using the brilliant cresyl blue or selective hemolysis methods, together with automated flow cytometry. Different injection schedules were investigated and dose-response curves for the European Pharmacopoeia Biological Reference Preparation of erythropoietin were compared. Manual and automated methods of reticulocyte counting were correlated with respect to assay validity and precision. Using 8 mice per treatment group, intra-assay precision determined for all of the assays in the study showed coefficients of variation of 12.1-28.4% for the brilliant cresyl blue method, 14.1-30.8% for the selective hemolysis method and 8.5-19.7% for the flow cytometry method. Applying the single injection protocol, a combination of at least two independent assays was required to achieve the precision potency and confidence limits indicated by the manufacturers, while the multiple daily injection protocol yielded the same acceptable results within a single assay. Although the latter protocol using flow cytometry for reticulocyte counting gave more precise and reproducible results (intra-assay coefficients of variation: 5.9-14.2%), the well-characterized manual methods provide equally valid alternatives for the quality control of recombinant human erythropoietin therapeutic products.

Inclusion complex of piroxicam with beta-cyclodextrin and incorporation in cationic microemulsion. In vitro drug release and in vivo topical anti-inflammatory effect.

Topical formulations of piroxicam were evaluated by determination of their in vitro release and in vivo anti-inflammatory effect. The in vitro release assay demonstrated that the microemulsion (ME) systems provided a reservoir effect for piroxicam release. However, the incorporation of the ME into carboxyvinilic gel provoked a greater reduction in the release of piroxicam than the ME system alone. Anti-inflammatory activity was carried out by the cotton pellet granuloma inhibition bioassay. Topical anti-inflammatory effect of the piroxicam inclusion complex/ME contained in carboxyvinilic gel showed significant inhibition of the inflammation process (36.9%, P<0.05). Subcutaneous administration of the drug formulations showed a significant effect on the inhibition of inflammation, 68.8 and 70.5%, P<0.05, when the piroxicam was incorporated in ME and in the combined system beta-cyclodextrin (beta-CD)/ME, respectively, relative to the buffered piroxicam (42.2%). These results demonstrated that the ME induced prolonged effects, providing inhibition of the inflammation for 9 days after a single dose administration.

Piroxicam encapsulated in liposomes: characterization and in vivo evaluation of topical anti-inflammatory effect.

Liposomes of soya phosphatidylcholine, cholesterol, and stearylamine (molar ratio 6/3/1) and 0.1% alpha-tocopherol were prepared by the extrusion of multilamellar vesicles through 0.2-micron polycarbonate membrane. Liposomes were characterized by electron transmission microscopy, and the mean structure diameter was 278 nm. The encapsulation efficiency obtained was 12.73%. The topical anti-inflammatory effect was evaluated in vivo by the cotton pellet granuloma method. We analyzed free piroxicam at 4 mg/kg, piroxicam encapsulated in liposomes added to 1.5% hydroxyethylcellulose (HEC) gel at 1.6 mg/kg, and piroxicam encapsulated in liposomes added to HEC gel at 4 mg/kg; the inhibition of inflammation obtained was 21.1%, 32.8%, and 47.4%, respectively. These results showed that the encapsulation of piroxicam produced an increase of topical anti-inflammatory effect, suggesting that the inhibition of inflammation can be obtained with lower drug concentrations.

Determinacao da atividade anticoagulante da heparina / Determination of anticoagulant activity of heparin

Foi realizada a dosagem biologica de produtos comerciais de heparina e de amostras do Primeiro Padrao Nacional Argentino de Heparina da mucosa intestinal suina. Os resultados foram comparados com o Quarto Padrao Internacional. Foi empregado o metodo de Reinert e Winterstein, modificado preconizado pelas Farmacopeias Americana e Brasileira. Foram discutidas as metodologias descritas na literatura e indicada a importancia destas para a avaliacao da atividade anticoagulante das heparinas nao-fracionadas e das de baixo peso molecular.

[Determination of anticoagulant activity of heparin]. / Determinação da atividade anticoagulante da heparina.

Heparin samples from four manufacturers and the First Argentinian Standard Heparin obtained from porcine mucosa were assayed by the Reinert and Winterstein method, modified as suggested by USP and Brazilian Pharmacopeias. Other methods described in the literature and their importance for the evaluation of anticoagulant activity of unfractionated heparins with low molecular weight are discussed.