Evaluation of an in vitro cell culture assay for the potency assessment of recombinant human erythropoietin.

Recombinant human erythropoietin is a sialoglycoprotein that stimulates erythropoiesis. To assess potency of human erythropoietin produced by recombinant technology, we investigated an in vitro TF-1 cell proliferation assay, which was applied in conjunction with a reversed-phase liquid chromatography method for the determination of the content of sialic acids. The results obtained, which were higher than 126.8ng/µg, were compared with those obtained with the in vivo normocythaemic mouse bioassay. The in vitro assay resulted in a non-significant lower mean difference of the estimated potencies (0.61% ± 0.026, p > 0.05). The use of this combination of methods represents an advance toward the establishment of alternative in vitro approaches, in the context of the Three Rs, for the potency assessment of biotechnology-derived medicines.

Assessment of recombinant human parathyroid hormone: correlation of LC methods with bioassays.

Reversed-phase liquid chromatography (RP-LC) and size exclusion liquid chromatography (SE-LC) methods were validated for the assessment of recombinant human parathyroid hormone (rhPTH 1-34). The gradient RP-LC method was carried out on a Zorbax 300 SB C(18) column (150 mm × 4.6 mm i.d.), maintained at 40 °C. The mobile phase A consisted of 0.1 M sodium sulphate buffer, pH 2.3, and the mobile phase B was acetonitrile. The SE-LC method was carried out on a BioSep-SEC-S 2000 column (300 mm × 7.8 mm i.d.), maintained at 25 °C. The mobile phase consisted of 0.1 M phosphoric acid buffer, pH 2.5, run isocratically at a flow rate of 0.7 mL min(-1). Chromatographic separation was obtained with retention times of 12.2 min, and 13.2 min, and was linear over the concentration range of 1-250 µg mL(-1) (r(2) = 0.9997) and 2-300 µg mL(-1) (r(2) = 0.9993), respectively, for RP-LC and SE-LC, with photodiode array (PDA) detection at 214 nm. Specificity was established in degradation studies, which also showed that there was no interference of the excipients. Equally, the accuracy was 100.49% and 100.22%, with bias lower than 1.12% and 0.81% respectively. Moreover, the in vitro cytotoxicity test of related proteins and higher molecular weight forms showed significant differences (p < 0.05). Chromatographic methods were applied for the content/potency assessment of rhPTH and related proteins in biopharmaceutical injectable dosage forms, and the results were correlated with those of in vitro and in vivo bioassays. It is concluded that the employment of the methods in conjunction allows a great improvement in monitoring stability, contributing to evaluate alternatives which improve the quality control and thereby assure the therapeutic efficacy of the biotechnology-derived medicine.

Uncaria tomentosa-Adjuvant Treatment for Breast Cancer: Clinical Trial.

Breast cancer is the most frequent neoplasm affecting women worldwide. Some of the recommended treatments involve chemotherapy whose toxic effects include leukopenia and neutropenia. This study assessed the effectiveness of Uncaria tomentosa (Ut) in reducing the adverse effects of chemotherapy through a randomized clinical trial. Patients with Invasive Ductal Carcinoma-Stage II, who underwent a treatment regimen known as FAC (Fluorouracil, Doxorubicin, Cyclophosphamide), were divided into two groups: the UtCa received chemotherapy plus 300 mg dry Ut extract per day and the Ca group that only received chemotherapy and served as the control experiment. Blood samples were collected before each one of the six chemotherapy cycles and blood counts, immunological parameters, antioxidant enzymes, and oxidative stress were analyzed. Uncaria tomentosa reduced the neutropenia caused by chemotherapy and was also able to restore cellular DNA damage. We concluded that Ut is an effective adjuvant treatment for breast cancer.

Validated stability-indicating RP-HPLC method for the determination of rimonabant in a pharmaceutical dosage form.

A simple RP-HPLC method was developed and validated for the determination of rimonabant in a pharmaceutical dosage form. The separation was performed on a C18 column (150 x 4.6 mm id, 5 microm) with acetonitrile-water (75 + 25, v/v) mobile phase. The detection was achieved with a diode array detector at 215 nm. The method was linear in the concentration range of 0.5-50 microg/mL (r = 1) with an LOQ of 0.24 microg/mL. The specificity and stability-indicating capability of the method were proved through forced degradation studies, and it was shown that there was no increase of the cytotoxicity. Rimonabant was exposed to hydrolytic, oxidative, and photolytic stress conditions, and the samples were analyzed by the proposed method. Under optimized conditions, rimonabant was successfully separated from its degradation products within 10 min, and the resolution was found to be greater than 2. The RSD values for intraday and interday precision were always less than 2%. Interday accuracy ranged from 98.1 to 101.7% (RSD = 1.0%). Moreover, method validation demonstrated acceptable results for sensitivity and robustness. The method was applied for the quantitative analysis of rimonabant in a tablet dosage form to demonstrate its use for improving the QC of pharmaceuticals containing this drug.

Biological potency evaluation and physicochemical characterization of unfractionated heparins

Unfractionated heparins are used clinically as anticoagulants. The biological potency of thirteen samples of raw material and pharmaceutical formulations were assessed utilizing the 5th International Standard of heparin using the sheep plasma coagulation inhibition assay, activated partial thromboplastin time, anti-factor Xa assay, and anti-factor IIa assay, resulting in mean potencies of 101.15 percent, 96.15 percent, 98.15 percent and 99.37 percent, respectively. The samples were also evaluated by the protamine neutralization test giving results within the range of 92 - 138 IU/mg. The anti-factor IIa assay was performed showing reproducibility and significant correlation with the pharmacopoeial assays, thus demonstrating it to be a feasible alternative to the sheep plasma coagulation inhibition assay. Moreover, an analysis by nuclear magnetic resonance and capillary electrophoresis showed some peaks attributable to oversulfated chondroitin sulfate. The results show that batch-to-batch variations and the quality of samples contributed to improvements in the quality control of pharmaceutical products and to assure the safe use and clinical efficacy of this biological medicine.

As heparinas não fracionadas são utilizadas clinicamente como anticoagulantes. A potência biológica de 13 amostras de matérias-primas e produtos farmacêuticos foram avaliadas em relação ao 5ª Padrão Internacional de heparina pelos ensaios da inibição da coagulação do plasma ovino, tempo de tromboplastina parcial ativada, anti-fator Xa e anti-fator IIa, que forneceram potências médias de 101,15 por cento, 96,15 por cento, 98,15 por cento e 99,37 por cento, respectivamente. As amostras foram também submetidas ao teste de neutralização pela protamina que apresentou resultados entre 92-138 UI/mg. Demonstrou-se reprodutibilidade e correlação significativa do ensaio do anti-fator IIa com os farmacopeicos, constituindo-se em alternativa ao ensaio da inibição da coagulação do plasma ovino. Além disso, as análises realizadas por ressonância magnética nuclear e eletroforese capilar mostraram picos correspondentesà condroitina supersulfatada. Os resultados mostraram variações lote-a-lote e a qualidade das amostras contribuindo para aprimorar o controle de qualidade dos produtos farmacêuticos e garantir a segurança e eficácia terapêutica desses produtos biológicos.

Validation of the normocythemic mice bioassay for the potency evaluation of recombinant human erythropoietin in pharmaceutical formulations.

The normocythemic mice bioassay was validated for the potency evaluation of the recombinant human erythropoietin (rhEPO) against the European Pharmacopoeia Biological Reference Preparation for erythropoietin. The bioassays were performed in 8-week-old female BALB/c mice, which received multiple daily injections of standard or sample solutions (3 + 3), for 4 days. The blood sampling was performed 24 h after the last injection and the reticulocytes were counted by automated flow cytometry. Method validation investigated parameters such as linearity, precision, accuracy, specificity, and robustness, giving results within the acceptable range. The dose-response curve was linear in the concentration range of 1-64 international units (IU)/mL, and the value of the determination coefficient (r2) was 0.9708. The bioassay was applied for the potency evaluation of rhEPO pharmaceutical products containing alfa or beta forms, expressed in different cell lines, giving biological potencies within 82.79 and 119.70% of the stated potency. The precision index calculated by the weight for the independent assays was >247. The results demonstrated the validity of the bioassay for the potency assessment of pharmaceutical formulations contributing to ensure the therapeutic efficacy of the biological medicine.

Evaluation of the changes in hemostatic parameters induced by etoricoxib in rat model.

The effects of etoricoxib on blood coagulation parameters were evaluated, along with acetylsalicylic acid, in male Wistar rats. Blood samples were collected at the end of the first, second, third and fourth weeks of the administration period and the plasma concentrations of etoricoxib determined by liquid chromatography-tandem mass spectrometry; the samples were also used for the analysis of the hematological parameters. There were no significant changes in the platelet count and fibrinogen levels, and a decrease by 1.9% of the prothrombin time was detected at the third week. The activated partial thromboplastin time assay showed a nonsignificant shortening. The antiactivated factor X and antiactivated factor II activities showed reductions lower than 2.8 and 1.3%, respectively. These findings and the comparison with acetylsalicylic acid can be helpful to support the benefit-to-risk profile, contributing to the safe therapeutic use.

Human granulocyte colony stimulating factor (hG-CSF): cloning, overexpression, purification and characterization.

BACKGROUND: Biopharmaceutical drugs are mainly recombinant proteins produced by biotechnological tools. The patents of many biopharmaceuticals have expired, and biosimilars are thus currently being developed. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that acts on cells of the neutrophil lineage causing proliferation and differentiation of committed precursor cells and activation of mature neutrophils. Recombinant hG-CSF has been produced in genetically engineered Escherichia coli (Filgrastim) and successfully used to treat cancer patients suffering from chemotherapy-induced neutropenia. Filgrastim is a 175 amino acid protein, containing an extra N-terminal methionine, which is needed for expression in E. coli. Here we describe a simple and low-cost process that is amenable to scaling-up for the production and purification of homogeneous and active recombinant hG-CSF expressed in E. coli cells. RESULTS: Here we describe cloning of the human granulocyte colony-stimulating factor coding DNA sequence, protein expression in E. coli BL21(DE3) host cells in the absence of isopropyl-beta-D-thiogalactopyranoside (IPTG) induction, efficient isolation and solubilization of inclusion bodies by a multi-step washing procedure, and a purification protocol using a single cationic exchange column. Characterization of homogeneous rhG-CSF by size exclusion and reverse phase chromatography showed similar yields to the standard. The immunoassay and N-terminal sequencing confirmed the identity of rhG-CSF. The biological activity assay, in vivo, showed an equivalent biological effect (109.4%) to the standard reference rhG-CSF. The homogeneous rhG-CSF protein yield was 3.2 mg of bioactive protein per liter of cell culture. CONCLUSION: The recombinant protein expression in the absence of IPTG induction is advantageous since cost is reduced, and the protein purification protocol using a single chromatographic step should reduce cost even further for large scale production. The physicochemical, immunological and biological analyses showed that this protocol can be useful to develop therapeutic bioproducts. In summary, the combination of different experimental strategies presented here allowed an efficient and cost-effective protocol for rhG-CSF production. These data may be of interest to biopharmaceutical companies interested in developing biosimilars and healthcare community.

Development and validation of a stability-indicating micellar electrokinetic chromatography method for the determination of ezetimibe in pharmaceutical formulations.

A micellar electrokinetic chromatography (MEKC) method was validated for the analysis of ezetimibe. The method was carried out on a fused-silica capillary (50 microm i.d.; effective length, 40 cm). The background electrolyte consisted of a 25 mM borate buffer and 25 mM anionic detergent SDS (pH 9.75)/methanol (90:10, v/v). The capillary temperature was maintained at 35 degrees C, the applied voltage was 30 kV; the injection was performed using a pressure mode at 50 mbar for 5 s, with detection at 232 nm. The method was linear in the range of 2-150 microg/mL (R2=0.9999). The specificity and the stability-indicating capability were proven through degradation studies, which also showed that there was no interference of the excipients. The limits of quantitation and detection were 2 and 0.41 microg/mL, respectively. The method was applied for the analysis of ezetimibe pharmaceutical formulations, and the results were compared to those of the liquid-chromatography method.

Evaluation of the changes on hemostatic parameters induced by valdecoxib in male Wistar rats / Avaliação dos efeitos do valdecoxibe sobre os parâmetros hemostáticos de ratos Wistar

The 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 percent) after 2 weeks and prothrombin time reduction (12.2 percent) after 3 weeks (P<0.05). There were no significant changes in the platelet count and fibrinogen levels. The anti-factor Xa and anti-factor IIa activities showed slight reductions, but only significant for the anti-factor Xa on the 3rd week (6.7 percent). 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.

Avaliaram-se os efeitos do valdecoxibe, um inibidor seletivo da cicloxigenase-2 (COX-2), sobre os parâ­metros sangüíneos da coagulação em ratos Wistar utilizando-se paralelamente a aspirina. Os animais foram divididos em grupos e submetidos à administração oral diária de 10 mg/kg animal para o valdecoxib, 100 mg/kg animal para a aspirina e veículo para o grupo controle, durante quatro semanas. A coleta do sangue foi efetuada após uma, duas, três e quatro semanas, e as concentrações plasmáticas do valdecoxibe determinadas por cromatografia líquida, obtendo-se valores médios de 101,1 - 113,5 - 164 e 184,6 ng/ml, respectivamente. As amostras de plasma foram também usadas para as análises hematológicas e os resultados comparados aos dos controles. O valde­coxibe apresentou redução significativa no TTPA (18 por cento) após duas semanas, e redução do TP (12,2 por cento) após três semanas (P<0.05). Os efeitos observados na contagem de pla­quetas e nos níveis plasmáticos de fibrinogênio não foram significativos. As atividades anti-fator Xa e anti-fator IIa apresentaram redução, porém os resultados foram significativos somente para o anti-fator Xa na terceira semana (6,7 por cento). Os resultados experimentais com as concentrações plasmáticas alcan­çadas demonstram que o valdecoxibe pode induzir alterações nos parâ­metros hematológicos dos ratos. Esses dados poderão contribuir para a melhor compreensão dos efeitos colaterais e uso do fármaco com segurança.

Comparative evaluation of pyrogens tests in pharmaceutical products

Realizou-se a comparação de metodologia para avaliação de pirogênios em produtos farmacêuticos. Otimizou-se o teste da hipertermia em coelhos elaborando a curva dose-resposta com o 2º Padrão Internacional de endotoxinas bacterianas, com base na qual determinou-se a concentração de 13,81 UE/mL por kg de peso corporal, necessária para produzir aumento de temperatura de 0,5ºC. Observou-se que o limite de 0,5ºC forneceu resultados comparáveis com as doses pirogênicas para o homem. Padronizou-se o teste do lisado de amebócitos do Limulus (LAL) com determinação do ponto final cromogênico e por geleificação, que foram utilizados para a avaliação de produtos farmacêuticos obtendo-se resultados concordantes. Avaliaram-se as respostas de reagentes LAL reativos e não-reativos a b-glicanos, observando diferenças que poderiam reprovar amostras com base em resultados falso-positivos. Executou-se o teste de interferências, validou-se o procedimento e estabeleceu-se a máxima diluição válida para produtos farmacêuticos sem especificações farmacopéicas. Os resultados enfatizam a importância e as limitações dos ensaios preconizados para avaliação da pureza e controle da qualidade de produtos farmacêuticos parenterais, contribuindo para aprimorar as metodologias existentes no contexto da redução e substituição dos modelos animais.

Biological potency evaluation and characterization of rhG-CSF in pharmaceutical products / Avaliação biológica da potência e caracterização de rhG-CSF em produtos farmacêuticos

Realizou-se a identificação do fator estimulador da colônia de granulócitos humanos recombinante em produtos farmacêuticos por eletroforese em gel de poliacrilamida não redutora e imunodetecção com anticorpos específicos, que apresentaram banda única na região de, aproximadamente, 19 kDa. A avaliação de potência baseada na contagem do número de neutrófilos em camundongos com neutropenia forneceu valores entre 88,4 ù 122,4 % em relação à potência declarada. A precisão expressa pela ponderação, calculada nos ensaios independentes, forneceu valores entre 141 e 432. As amostras lote-a-lote cumpriram os requisitos dos testes de toxicidade e endotoxinas bacterianas. Os resultados dos ensaios biológicos e imunológicos demonstram a qualidade dos produtos farmacêuticos em uso clínico, e as especificações sugeridas contribuem para assegurar a inocuidade e eficácia terapêutica dos produtos biológicos.

Avaliação comparativa da atividade biológica de heparinas não-fracionadas em produtos farmacêuticos / Comparison of the biological activities of unfractionated heparins in pharmaceutical products

Realizou-se a avaliação biológica de potência de heparinas convencionais contra o 5º Padrão Internacional de heparina suína pelos ensaios preconizados da inibição da coagulação do plasma ovino (ICPO), tempo de tromboplastina parcial ativada (TTPA) e anti-fator Xa. Correlacionaram-se os resultados, demonstrando que o ICPO fornece potências em média 10,72 por cento, significativamente superiores aos outros procedimentos. Analisou-se a influência de diferentes lotes de plasma sobre o resultado do ensaio do ICPO observando-se variação de até 7,32 por cento. Como alternativa, padronizou-se o ensaio do anti-fator IIa e efetuou-se a determinação de potência das amostras obtendo-se valores reprodutíveis, comparáveis ao método do anti-fator Xa, que demonstram a viabilidade da inclusão como ensaio harmonizado para o controle da qualidade. Os resultados de potência, fornecidos lote a lote, em geral, cumprem as especificações farmacopéicas e demonstram a qualidade que garante a segurança e eficácia clínica dos medicamentos

Avaliaçäo da atividade e caracterizaçäo de eritropoietina humana recombinante em produtos farmacêuticos / Activity evaluation and characterization of recombinant human erythropoietin in pharmaceutical products

Realizou-se a identificaçäo de eritropoietina humana recombinante em produtos farmacêuticos comerciais por eletroforese em gel de poliacrilamida e detecçäo com anticorpos específicos, demonstrando-se típica banda larga, semelhante ao padräo de rhEPO da Farmacopéia Européia. Igualmente por focalizaçäo isoelétrica e imunodetecçäo, observaram-se 5-6 isoformas características de acordo com o laboratório produtor. A avaliaçäo de potência efetuada através de ensaio biológico em camundongos normocitêmicos forneceu valores entre 67,6 por cento e 119,4 por cento em relaçäo à declarada. A precisäo dos ensaios combinados, calculada pela ponderaçäo, variou de 200 a 389. Os testes de endotoxinas bacterianas, toxicidade e pH apresentaram valores variáveis conforme o lote. Concluiu-se destacando a importância dos testes e ensaios de controle para assegurar a qualidade lote a lote e garantir a eficácia terapêutica dos produtos farmacêuticos