Marcio Fronza

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.

Avaliação da atividade e caracterização de eritropoietina humana recombinante em produtos farmacêuticos

The identification of rhEPO in pharmaceutical products was carried out by polyacrylamide gel electrophoresis and detection with specific antibodies that revealed a typical single broad band similar to that obtained with the European Pharmacopoeia Biological Reference Preparation for erythropoietin. The isoelectric focusing with immunodetection revealed extensive heterogeneity with 5-6 isoforms, characteristic according to the manufacturer. The potency was assessed by the normocythaemic mouse assay with values within 67.6% and 119.4% calculated against the stated potency. The precision evaluated by the weight was within 200 and 389, for the combined assays. The toxicity, bacterial endotoxins, and pH gave variable results according to the batch. In conclusion, we emphasize the importance of the batch-to-batch tests and assays that would guarantee the quality and therapeutic efficacy of the pharmaceutical products.

Simultaneous Determination of Nimesulide and Valdecoxib by Micellar Electrokinetic Capillary Chromatography Method

Abstract A micellar electrokinetic capillary chromatography (MEKC) method for the analysis of nimesulide and valdecoxib, using celecoxib as internal standard, has been developed and validated. The MEKC method was carried out on a fused silica capillary (50 µm I.D., effective length 56 cm). The background electrolyte consisted of 35 mM borate buffer and 35 mM of anionic detergent SDS (pH 9.75)/acetonitrile (95:5, V/V). The capillary temperature was maintained at 35°C, the applied voltage was 30 kV, and the injection was performed using the pressure mode at 50 mbar for 5 s, with detection at 234 nm using a photodiode array detector. Method validation investigated parameters such as the linearity (r2=0.9999), range, precision, accuracy, robustness, and specificity, giving results within the acceptable range. The detection limit calculated for nimesulide and valdecoxib was 0.25 and 0.86 µg mL−1, respectively, and the quantitation limit evaluated experimentally was 2 µg mL−1 for both the compounds. The proposed method was successfully applied for the quality control analysis of pharmaceutical products and the results compared to the liquid chromatography method, showing non‐significant difference (P>0.05).

Validation of an LC‐Tandem MS/MS Method for the Determination of Etoricoxib in Human Plasma and Pharmaceutical Formulations

Abstract An analytical method based on liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) was developed and validated for the determination of etoricoxib in spiked human plasma and pharmaceutical dosage forms. Etoricoxib and piroxicam (internal standard) were extracted from the plasma by liquid–liquid extraction using tert‐butyl methyl ether as extraction solvent and separated on a C18 analytical column (50 mm×3.0 mm I.D.). The mobile phase consisted of acetonitrile:water (95:5)/0.1% acetic acid (90:10, v/v). Detection was carried out by positive electrospray ionization (ESI+) in multiple reaction monitoring (MRM) mode. The chromatographic separation was obtained within 2.0 min and was linear in the concentration range of 1–5000 ng/mL. The mean extraction recoveries of etoricoxib and piroxicam from plasma were 96.09 and 95.54%, respectively. Method validation investigated parameters such as the linearity, precision, accuracy, specificity, and stability, giving results within the acceptable range. Moreover, the proposed method was successfully applied for routine quality control analysis of pharmaceutical products and the results compared with those obtained by the RP‐HPLC method, showing significant correlation (P>0.05).

Development and Validation of an RP‐HPLC Method for the Dissolution Studies of Bisoprolol in Pharmaceutical Dosage Forms

Abstract An RP‐HPLC procedure was developed for the dissolution rate studies of bisoprolol in solid dosage formulations. The HPLC system was operated isocratically at controlled‐ambient temperature with reversed phase C18 column (150 mm × 4.6 mm i.d.; 5 µm particle size), using a mobile phase methanol∶phosphate buffer (pH 3.5; 0.01 M) (55∶45, v/v) at a flow rate of 1.0 mL min−1. Detection was achieved with a photodiode array (PDA) detector at 225 nm. Method validation investigated parameters such as the range, linearity (r 2 = 0.9999), precision, accuracy, and robustness. The dissolution test conditions and the medium were chosen as 0.1 M HCl at a stirring rate of 50 rpm, and the methodology was applied to bisoprolol fumarate tablets giving similar dissolution profiles compared by the difference and similarity factor (f 1, f 2), obtaining values lower than 7.22 and higher than 72.28, respectively.

Avaliação comparativa da atividade biológica de heparinas não-fracionadas em produtos farmacêuticos

Realizou-se a avaliacao biologica de potencia de heparinas convencionais contra o 5o Padrao Internacional de heparina suina pelos ensaios preconizados da inibicao da coagulacao do plasma ovino (ICPO), tempo de tromboplastina parcial ativada (TTPA) e anti-fator Xa. Correlacionaram-se os resultados, demonstrando que o ICPO fornece potencias em media 10,72%, significativamente superiores aos outros procedimentos. Analisou-se a influencia de diferentes lotes de plasma sobre o resultado do ensaio do ICPO observando-se variacao de ate 7,32%. Como alternativa, padronizou-se o ensaio do anti-fator IIa e efetuou-se a determinacao de potencia das amostras obtendo-se valores reprodutiveis, comparaveis ao metodo do anti-fator Xa, que demonstram a viabilidade da inclusao como ensaio harmonizado para o controle da qualidade. Os resultados de potencia, fornecidos lote a lote, em geral, cumprem as especificacoes farmacopeicas e demonstram a qualidade que garante a seguranca e eficacia clinica dos medicamentos.

Evaluation of the changes on hemostatic parameters induced by valdecoxib in male Wistar rats

3The effects of the cyclooxygenase (COX)-2 selective inhibitor, valdecoxib, on blood coagulation parameters were evaluated, along with aspirin in male Wistar rats. Groups of animals were administered a daily oral dose of 10 mg/kg rat of valdecoxib, 100 mg/kg rat of aspirin and the vehicle alone during 4 weeks. Blood samples were collected at the end of 1, 2, 3 and 4 weeks of administration period and the plasma concentrations of valdecoxib were determined by RP-HPLC giving mean values of 101.1, 113.5, 164.0 and 184.6 ng/mL, respectively. The same plasma samples were used for the analysis of hematological parameters and the results compared to the controls. Valdecoxib induced significant activated partial thromboplastin time reduction (18%) after 2 weeks and prothrombin time reduction (12.2%) after 3 weeks (P<0.05). There were no significant changes in the platelet count and fibrinogen levels. The antifactor Xa and anti-factor IIa activities showed slight reductions, but only significant for the anti-factor Xa on the 3rd week (6.7%). The results showed that valdecoxib at the dose tested with the plasmatic concentrations induced some changes in the hemostatic function of rats, which can be helpful to understand the side effects and the safe use of the drug. Rev. bras. hematol. hemoter. 2006;28(1):28-32.

Biological potency evaluation and characterization of rhG-CSF in pharmaceutical products

The identification of rhG-CSF was carried out in pharmaceutical preparations by non-reducing polyacrylamide gel electrophoresis and western blotting with specific antibodies, showing a single band in the 19 kDa region. The potency was assessed by the neutropenia mouse bioassay giving values between 88.4 and 122.4% of the stated potency. The precision index expressed by weight was between 141 and 432 for the independent assays. Batch-to-batch, the samples met the requirements for the safety test and bacterial endotoxins test. The biological and immunological results showed the quality of the products in clinical use and the specifications established contribute to assuring the safety and efficacy of biological medicines.

Induction of NAD (P)H: Quinone reductase 1 (QR1) and antioxidant activities in vitro of ‘Toranja Burarama’ (Citrus maxima [Burm.] Merr.): Chemoprevention of 'Toranja Burarama'

Toranja ‘Burarama’, Citrus maxima (Burm.) Merr. (Citrus grandis), is a new citrus discovered in the State of Espírito Santo, Brazil. As several varieties of citrus are known to possess antioxidant and cancer chemopreventive properties, the aim of the study was to evaluate in vitro if this Toranja possess these properties. The antioxidant activity, the potential to induce quinone reductase 1, and the influence on cell viability were measured. ESI(‐)FT‐ICR MS analysis was also performed and identified flavonoids, coumarins, and fatty acids in the extract. The ethyl acetate and methanolic extracts of the peels presented the highest antioxidant activity in vitro by DPPH (IC50 = 298.3 ± 2.6 μg/ml and 303.8 ± 0.4 μg/ml), ABTS assay (IC50 = 298.2 ± 6.4 μg/ml and 296.4 ± 2.5 μg/ml), and FRAP (IC50 = 234.6 ± 1.8 μg/ml and 398.1 ± 3.8 μg/ml). The ethyl acetate extract of the peel induced quinone reductase 1 activity in Hepa1c1c7 cells, indicating that C. maxima exhibited cancer chemopreventive properties.

Phytochemical profile of genotypes of Euterpe edulis Martius – Juçara palm fruits

Juçara fruit (Euterpe edulis) has received attention due to its similarities to Euterpe oleracea (Açaí). The aim of this study was to evaluate the cytotoxicity, bioactive compounds, antioxidant capacities and chemopreventive activities of the fruit pulps of six populations of E. edulis (J1-J6) and one population of E. espiritosantense from different ecological regions. ESI(-)-FT-ICR-MS was used to evaluate the pulp composition. The varieties J1 and J4 presented higher polyphenol contents, while J2 and J5 showed higher anthocyanin contents. ESI-FT-ICR MS identified cyanidin-3-rutinoside (J1, J2, J3, J4, J5, J7), protocatechuic acid, methylhydroxybenzoate hexoside and rutin (J1 to J7) and malvidin-glicoside (J2 to J5). The J2, J3, J4, J5 and J6 samples inhibited inducible nitric oxide synthase (iNOS). The chemoprevention biomarker quinone reductase was significantly induced by J6. Pulp from plants J3, J4, J6 and J7 significantly reduced the inflammatory cytokine TNF-α, and J6 was selected as having the most potential for cultivation and consumption.