RESUMO
A capillary zone electrophoresis (CZE) method was developed and validated to quantitate the monoclonal antibody denosumab (DmAb) and its charge variants in pharmaceutical products, demonstrating excellent precision, linearity and accuracy. Separations were obtained with migration times of 11.3 min for DmAb and the calibration curve was linear in the range of 0.95-20 mg/mL. The analytical comparability of seven batches of Prolia® showed mean differences of the estimated content/potencies of 1.87% lower, and 0.84 and 1.21% higher compared with the size-exclusion and reversed-phase liquid chromatography (SE-HPLC and RP-HPLC) methods and the osteoclast antiproliferative bioassay, respectively, with non-significant differences (P > 0.05). An RP-HPLC method with fluorescence detection (RP-HPLC-F), performed on a Kinetex® EVO C18 column (5 µm, 100 Å, 250 mm × 4.6 mm), was optimized to determine the levels of sialic acids of DmAb biomolecules, giving mean concentrations of 0.16 and 0.17 µg N-acetylneuraminic acid/mg DmAb for Prolia® and Xgeva® pharmaceutical products, respectively. The results demonstrated the capability of each one of the methods, and their use in combination constitutes a strategy to monitor instability, thereby assuring the quality and the batch-to-batch consistency of the biotechnology-derived medicine.
Assuntos
Denosumab , Ácidos Siálicos , Cromatografia Líquida de Alta Pressão , Eletroforese Capilar , Preparações FarmacêuticasRESUMO
Abstract Certolizumab pegol (CZP) is a Fab' fragment of the humanized antibody with anti-TNF-α activity that is indicated as therapy for Crohn's disease and rheumatoid arthritis. Using a BioSep-SEC-S3000 column (300 x 4.6 mm i.d., 5 µm particle size), a size exclusion liquid chromatography (SEC) method was developed. Mobile phase A consisted of 100 mM monobasic sodium phosphate and 200 mM sodium chloride (pH 7.0), while mobile phase B was ethanol (95:5, v/v), and the analysis was performed using a diode array detector (DAD) set to 214 nm and a flow rate of 0.5 ml min-1. In addition, a reversed-phase liquid chromatography (RP-LC) method based on gradient elution was developed on a Zorbax 300 SB C18 column (150 mm x 4.6 mm i.d., 3.5 µm particle size) kept at 80 °C. Mobile phase A was 0.1% (v/v) TFA in ultrapure water, and mobile phase B was a mixture of propanol, acetonitrile, ultrapure water and TFA (70 + 20 + 9.9 + 0.1, v/v) operated at a flow rate of 1.0 ml min-1, and DAD was applied at 214 nm. CZP elution was achieved with retention times of 5.6 min and 9.0 min for SEC and RP-LC, respectively.
Assuntos
Cromatografia Líquida/métodos , Estudo de Validação , Certolizumab Pegol/análise , Cromatografia de Fase Reversa/métodos , Anticorpos Monoclonais/classificaçãoRESUMO
Verbena litoralis is a plant popularly known as "gervãozinho-do-campo" in Portuguese. It is traditionally used for stomach, liver and gallbladder problems, and as an anti-inflammatory and anthelmintic. The goal of this study was to determine the chemical composition of the crude extract obtained from the aerial parts of V. litoralis by Ultra High Performance Liquid chromatography coupled with Mass Spectrometry in Tandem (UHPLC-MS/MS); assess the anti-inflammatory activity of ethyl acetate fraction and of crude extract; and verify liver, kidney and pancreas damage. In this study, the chemical composition of the extract was identified via UHPLC/MS/MS, assessing the anti-inflammatory activity of the crude extract and the acetate fraction in an induction model of the granulomatous tissue, as well as given liver, kidney and pancreas damage markers. Chlorogenic acid, luteolin, caffeic acid, apigenin, p-coumaric acid, vanillic acid, ferulic acid and quercetin were quantified in the extract. After the seven-day treatment, the granuloma of the animals treated with the plant extract and fraction presented values very close to the positive control (nimesulide). The V. litoralis crude extract and ethyl acetate fraction show anti-inflammatory activity similar to the nimesulide without evidence of liver, kidney and pancreas damage, which attributes the plant's pharmacological action to the flavonoids found.
RESUMO
Botulinum neurotoxin type-A (BoNTA) is one of the seven different serotypes (A to G) produced by Clostridium botulinum. A stability-indicating size-exclusion chromatography (SEC) method was developed and validated, and the specificity was confirmed by forced degradation study, interference of the excipients, and peaks purity. The method was applied to assess the content and high-molecular-weight (HMW) forms of BoNTA in biopharmaceutical products, and the results were compared with those of the LD50 mouse bioassay, the T-47D cell culture assay, and the reversed-phase chromatography (RPC) method, giving mean values of 0.71% higher, 0.36% lower, and 0.87% higher, respectively. Aggregated forms showed significant effects on cytotoxicity, as well as a decrease in the bioactivity (p < 0.05). The employment of the proposed method in conjunction with the optimized analytical technologies for the analysis of the intact and altered forms of the biotechnology-derived medicines, in the correlation studies, enabled the demonstration of the capability of each one of the methods and allowed for great improvements, thereby assuring their safe and effective use.
Assuntos
Toxinas Botulínicas Tipo A/análise , Toxinas Botulínicas Tipo A/toxicidade , Animais , Bioensaio , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cromatografia em Gel , Cromatografia Líquida , Cromatografia de Fase Reversa , Feminino , Humanos , Dose Letal Mediana , Camundongos , Reprodutibilidade dos TestesRESUMO
Recombinant human interferon beta 1b (rhIFNß-1b) is clinically used to treat multiple sclerosis. A reversed-phase liquid chromatography (RP-LC) method was carried out on a Jupiter C4 column (250 mm × 4.6 mm i.d.). The mobile phase A consisted of 0.1% trifluoroacetic acid (TFA) in water, and the mobile phase B was acetonitrile with 0.1% TFA run at a flow rate of 1.0 mL/min. A size exclusion liquid chromatography (SE-LC) method was carried out on a BioSep-SEC-S 2000 column (300 mm × 7.8 mm i.d.). The mobile phase consisted of 1 mM monobasic potassium phosphate, 8 mM sodium phosphate dibasic and 200 mM sodium chloride buffer pH 7.4, run isocratically at a flow rate of 0.8 mL/min. Retention times were 31.87 and 17.78 min, and calibration curves were linear over the concentration range of 1-200 µg/mL (r2 = 0.9998) and 0.50-200 µg/mL (r2 = 0.9999), respectively, for RP-LC and SE-LC, with detection at 214 nm. Liquid chromatography (LC) methods were validated and employed in conjunction with the in vitro bioassay to assess the content/potency of rhIFNß-1b, contributing to improve the quality control and to ensure the efficacy of the biotherapeutic
Assuntos
Bioensaio/métodos , Humanos , Cromatografia de Fase Reversa/métodos , Interferon beta-1b/análise , Técnicas In Vitro , Biotecnologia/classificação , Estudo de ValidaçãoRESUMO
The monoclonal antibody Denosumab (DmAb) is clinically used to treat osteoporosis and bone loss. We developed a bioassay based on the ability of DmAb to inhibit the effect of human receptor activator of nuclear factor-κB ligand (RANKL) to stimulate the formation of osteoclasts derived from RAW 264.7 cells. This bioassay was applied in conjunction with size exclusion high-performance liquid chromatography (SE-HPLC) and reversed-phase high-performance liquid chromatography (RP-HPLC) methods, with diode array detection (DAD), validated for the quantitation of this biotechnology-derived medicine. The SE-HPLC(DAD) method was carried out on a TSKGel G2000SWXL column and the mobile phase consisted of potassium phosphate buffer with sodium chloride, pHâ¯7.4. The gradient RP-HPLC(DAD) method was carried out on a Vydac 214TP C4 column at 60⯰C. The mobile phases consisted of 0.1% v/v trifluoroacetic acid (TFA) in water and 0.1% v/v TFA in acetonitrile. Calibration curves were linear over the concentration ranges 6-200⯵gâ¯mL-1 and 6-300⯵gâ¯mL-1 for the SE-HPLC(DAD) and RP-HPLC(DAD) methods respectively. The bioassay results correlated with the LC methods results, indicating the capabilities of these methods to quantitate DmAb, which will contribute to ensure the batch-to-batch consistency and efficacy of this biotherapeutic.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacologia , Bioensaio , Cromatografia Líquida de Alta Pressão , Denosumab/química , Denosumab/farmacologia , Animais , Cromatografia de Fase Reversa/métodos , Camundongos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Reversed-phase and size-exclusion liquid chromatography methods were validated for the assessment of streptokinase. The reversed-phase method was carried out on a Jupiter C4 column (250 mm × 4.6 mm id) maintained at 25°C. The mobile phase consisted of 50 mM sodium sulfate solution pH 7.0 and methanol (90:10, v/v), run isocratically at a flow rate of 0.8 mL/min. The size-exclusion method was carried out on a Protein KW 802.5 column (300 mm × 8.0 mm id), at 25°C. The mobile phase consisted of 40 mM sodium acetate solution pH 7.0, run isocratically at a flow rate of 1.0 mL/min. Retention times were 19.3 min, and 14.1 min, and calibration curves were linear over the concentration range of 0.25-250 µg/mL (25.75-25 750 IU/mL) (r2 = 0.9997) and 5-80 µg/mL (515-8240 IU/mL) (r2 = 0.9996), respectively, for reversed-phase and size exclusion, with detection at 220 and 204 nm. Chromatographic methods were employed in conjunction with the in vitro bioassay for the content/potency assessment of Streptokinase, contributing to improve the quality control and ensure the efficacy of the biotherapeutic.
Assuntos
Bioensaio , Cromatografia Líquida , Ensaios Enzimáticos/métodos , Controle de Qualidade , Estreptoquinase/análise , Cromatografia em Gel , Reprodutibilidade dos Testes , Estreptoquinase/metabolismoRESUMO
A stability-indicating capillary zone electrophoresis (CZE) method was validated to assess the content/potency of the recombinant human parathyroid hormone (rhPTH 1-34), using ranitidine as internal standard (IS). A fused-silica capillary, (i.d. of 50µm; effective length of 40cm) was used at 25°C; the applied voltage was 20kV. The background electrolyte solution consisted of 50mmolL-1 sodium dihydrogen phosphate solution at pH 3.0. Injections were performed using a pressure mode at 50 mbar for 45s, with detection by photodiode array (PDA) detector set at 200nm. Separation was obtained with a migration time of 5.3min, and was linear over the concentration range of 0.25-250µgmL-1 (r2 =0.9992). Specificity and stability-indicating capability were established in degradation studies, which also showed that there was no interference of the excipients. The accuracy was 100.28% with bias lower than 0.85%. Analyses of the same batches showed mean differences of the estimated content/potencies of 0.61%, 1.31% higher and 0.86% lower as compared to the validated reversed-phase and size exclusion liquid chromatography methods, and to the UMR-106 cell culture bioassay, respectively, with non-significant differences (p>0.05). Degraded forms were also subjected to the in vitro cytotoxicity test. The results obtained showed the capabilities of each one of the methods, and constitute an alternative strategy to monitor stability, improve the quality control and ensure the batch-to-batch consistency of bulk and finished biotechnology-derived medicine.
Assuntos
Cromatografia em Gel/métodos , Cromatografia de Fase Reversa/métodos , Eletroforese Capilar/métodos , Hormônio Paratireóideo/metabolismo , Proteínas Recombinantes/metabolismo , Animais , Bioensaio/métodos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Células L , Camundongos , Hormônio Paratireóideo/genética , Hormônio Paratireóideo/farmacologia , Ranitidina/metabolismo , Ranitidina/normas , Ratos , Proteínas Recombinantes/farmacologia , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
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.
Assuntos
Técnicas de Cultura de Células , Eritropoetina/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/farmacologiaRESUMO
A stability-indicating capillary zone electrophoresis (CZE) method was validated for the analysis of recombinant human interleukin-11(rhIL-11) using rupatadine fumarate, as internal standard (IS). A fused-silica capillary, (50 µm i.d.; effective length, 40 cm) was used at 25°C; the applied voltage was 20 kV. The background electrolyte solution consisted of 50 mmol L(-1) sodium dihydrogen phosphate solution at pH 3.0. Injections were performed using a pressure mode at 50 mbar for 45 s, with detection by photodiode array detector set at 196 nm. Specificity and stability-indicating capability were established in degradation studies, which also showed that there was no interference of the excipients. The method was linear over the concentration range of 1.0-300 µg mL(-1) (r(2)=0.9992) and the limit of detection (LOD) and limit of quantitation (LOQ) were 0.2 µg mL(-1) and 1.0 µg mL(-1), respectively. The accuracy was 100.4% with bias lower than 1.1%. Moreover, the in vitro cytotoxicity test of the degraded products showed significant differences (p<0.05). The method was applied for the content/potency assessment of rhIL-11 in biopharmaceutical formulations, and the results were correlated to those of a validated reversed-phase LC method (RP-LC) and an TF-1 cell culture assay, showing non-significant differences (p>0.05). In addition the CZE and RP-LC methods were applied for the analysis of rhIL-11 in human plasma. Therefore, the proposed alternative method can be applied to monitor stability, to assure the batch-to-batch consistency and quality of the bulk and finished biotechnology-derived medicine.
Assuntos
Cromatografia de Fase Reversa/métodos , Eletroforese Capilar/métodos , Interleucina-11/análise , Proteínas Recombinantes/análise , Animais , Bioensaio , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Estabilidade de Medicamentos , Humanos , Concentração Inibidora 50 , Interleucina-11/sangue , Interleucina-11/farmacologia , Estabilidade Proteica , Proteínas Recombinantes/farmacologia , Reprodutibilidade dos TestesRESUMO
A size-exclusion LC method was validated for the determination of interferon-a2a (rhlFN-alpha2a) in pharmaceutical formulations without interference from human serum albumin. Chromatographic separation was performed on a BioSep-SEC-S 2000 column (300 x 7.8 mm id). The mobile phase consisted of 0.001 M monobasic potassium phosphate, 0.008 M sodium phosphate dibasic; 0.2 M sodium chloride buffer, pH 7.4, run at a gradient flow rate and using photodiode array detection at 214 nm, was used. Chromatographic separation was achieved with a retention time of 17.2 min, and the analysis was linear over the concentration range of 1.98 to 198 microg/mL (r2 = 0.9996). The accuracy was 101.39%, with bias lower than 1.67%. The LOD and LOQ were 0.87 and 1.98 microg/mL, respectively. Moreover, method validation demonstrated acceptable results for precision and robustness. The method was applied to the assessment of rhlFN-alpha2a and related proteins in biopharmaceutical dosage forms, and the content/potencies were correlated to those given by a validated RP-LC method and an in vitro bioassay. It was concluded that use of the methods in conjunction allows a great improvement in monitoring stability and QC, thereby ensuring the therapeutic efficacy of the biotechnology-derived medicine.
Assuntos
Cromatografia Líquida/métodos , Interferon-alfa/química , Linhagem Celular Tumoral , Estabilidade de Medicamentos , Humanos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
No presente trabalho, avaliou-se a atividade anti-inflamatória do extrato hidroetanólico das folhas de Morus alba, através do modelo de indução de tecido granulomatoso e analisaram-se os efeitos toxicológicos sobre o fígado pela dosagem de AST e ALT e sobre o rim, pela dosagem de creatinina. O extrato hidroetanólico das folhas de M. alba foi administrado oralmente, três vezes ao dia, durante 6 dias. A nimesulida (5 mg/kg/dia) foi utilizada como controle positivo e o propilenoglicol 20% como controle negativo. Após o tratamento, foi avaliada a formação do granuloma e realizada a dosagem de AST, ALT e creatinina em todos os grupos. Os animais tratados com o extrato de M. alba apresentaram inibição do processo inflamatório de 20,24 ± 6,94%, enquanto que os tratados com controle positivo apresentaram 21,42 ± 6,52%. O extrato hidroetanólico de M. alba demonstrou atividade anti-inflamatória semelhante à nimesulida com ausência de indícios de hepatotoxicidade e nefrotoxicidade.
In this study, the anti-inflammatory activity of a hydroethanolic extract of leaves of Morus alba (white mulberry) was assessed in a model of granulomatous tissue induction and the toxicological effects on the liver were analyzed by assaying AST and ALT, and those on the kidney by determining creatinine. The hydroethanolic leaf extract was administered orally three times a day six days. Nimesulide (5 mg /kg/day) was used as a positive control and 20% propylene glycol as a negative control. After the 6-day treatment, granuloma formation was assessed and the levels of AST, ALT and creatinine assayed in all groups. Animals treated with the extract showed inflammation inhibition of 20.24 ± 6.94%, while those treated with positive control showed 21.42 ± 6.52%. The hydroethanolic extract of M. alba thus exhibited anti-inflammatory activity similar to nimesulide, with an absence of hepatotoxicity or nephrotoxicity.
Assuntos
Animais , Masculino , Ratos , Rim , Fígado , Morus/toxicidade , Ratos WistarRESUMO
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.
Assuntos
Cromatografia em Gel/métodos , Cromatografia de Fase Reversa/métodos , Hormônio Paratireóideo/análise , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Galinhas , Humanos , Limite de Detecção , Masculino , Hormônio Paratireóideo/farmacologia , Proteínas Recombinantes/análise , Proteínas Recombinantes/farmacologiaRESUMO
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.
RESUMO
A stability-indicating capillary zone electrophoresis (CZE) method was validated for the analysis of granulocyte-macrophage colony-stimulating factor (rhGM-CSF) using leuprorelin acetate (LA), as internal standard (IS). A fused-silica capillary (75 µm i.d.; effective length, 72 cm) was used at 25 °C; the applied voltage was 12 kV. The background electrolyte solution consisted of 50mM di-sodium hydrogen phosphate solution at pH 8.8. Injections were performed using a pressure mode at 50 mbar for 9s, with detection by photodiode array detector set at 200 nm. Specificity and stability-indicating capability were established in degradation studies, which also showed that there was no interference of the excipients. The method was linear over the concentration range of 2.5-200 µg mL(-1) (r(2)=0.9995) and the limit of detection (LOD) and limit of quantitation (LOQ) were 0.79 µg mL(-1) and 2.5 µg mL(-1), respectively. The accuracy was 99.14% with bias lower than 1.40%. The method was applied to the quantitative analysis of biopharmaceutical formulations, and the results were correlated to those of a validated reversed-phase LC method (RP-LC), and an in vitro bioassay, showing non-significant differences (p>0.05).
Assuntos
Eletroforese Capilar/métodos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/análise , Proteínas Recombinantes/análise , Bioensaio , Calibragem , Linhagem Celular , Cromatografia de Fase Reversa , Humanos , Concentração de Íons de Hidrogênio , Leuprolida/análise , Limite de Detecção , Fosfatos/química , Dióxido de Silício/químicaRESUMO
O presente trabalho avaliou a atividade antimicrobiana in vitro do extrato hidroetanólico e frações hexânica, clorofórmica, acetato de etila e butanólica das folhas de Morus alba L. A concentração inibitória mínima (CIM) foi determinada frente à Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Aspergillus fumigatus e Prothoteca zophii. As frações que apresentaram melhores respostas para a atividade antimicrobiana foram acetato de etila e clorofórmica com CIM de 256 ?g/mL. Não foi possível detectar atividade antimicrobiana para Aspergillus fumigatus em nenhuma das concentrações testadas. A citotoxidade do extrato hidroetanólico foi avaliada através de culturas de células de ovário de hamster chinês (CHO) e células do tecido conectivo de camundongo (NCTC) clone 929, determinando o índice de citotoxidade (IC50). O IC50 foi de 0,34 mg/mL para as células CHO e 3,24 mg/mL para as células NCTC 929. De modo geral, as frações acetato de etila e clorofórmica das folhas de M. alba L. apresentaram moderada atividade antimicrobiana e o extrato bruto demonstrou ação citotóxica in vitro frente as células CHO e NCTC 929.
This study evaluated the in vitro antimicrobial activity of hydroethanolic extract and hexane, chloroform, ethyl acetate and butanol fractions from Morus alba L. leaves. The minimum inhibitory concentration (MIC) was determined using Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Aspergillus fumigatus and Prothoteca zophii. The fractions that better responded the antimicrobial activity were ethyl acetate and chloroform with CIM of 256 ?g/mL. It was unable to detect antimicrobial activity for Aspergillus fumigatus in the tested concentrations. The cytotoxicity of the hydroethanolic extract was evaluated by cell cultures of chinese hamster ovary (CHO) and connective tissue of mouse clone 929 (NCTC), determining the level of cytotoxicity (IC50). The IC50 obtained was 0.34 mg/mL for CHO and 3.24 mg/ml for NCTC 929 cells. In general, ethyl acetate and chloroform fractions from M. alba L. leaves showed moderate antimicrobial activity and its extract presented in vitro cytotoxicity against CHO and NCTC 929 cells.
Assuntos
Anti-Infecciosos , Moraceae , MorusRESUMO
A stability-indicating reversed-phase liquid chromatography (RP-LC) method was validated for the assessment of recombinant human interleukin-11 (rhIL-11), based on the ICH guidelines. The method was carried out on a Jupiter C(4) column (250 mm × 4.6 mm i.d.), maintained at 25°C. The mobile phase A consisted of 0.1% trifluoroacetic acid (TFA) and the mobile phase B was acetonitrile with 0.1% TFA, run at a flow rate of 1 mL/min, and using a photodiode array (PDA) detection at 214 nm. Separation was obtained with a retention time of 27.6 min, and was linear over the concentration range of 1-200 µg/mL (r(2) = 0.9995). Specificity was established in degradation studies, which also showed that there was no interference of the excipients. The accuracy was 100.22% with bias lower than 1.25%. Moreover, the in vitro cytotoxicity test of the degraded products showed non-significant differences (p > 0.05). The method was applied to the assessment of rhIL-11 and related proteins in biopharmaceutical dosage forms, and the results were correlated to those of a bioassay.
Assuntos
Bioensaio , Cromatografia de Fase Reversa/métodos , Interleucina-11/isolamento & purificação , Interleucina-11/farmacologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Humanos , Interleucina-11/análise , Interleucina-11/toxicidade , Limite de Detecção , Controle de Qualidade , Proteínas Recombinantes/análise , Proteínas Recombinantes/toxicidadeRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: The Asháninkas, indigenous people of Peru, use cat's claw (Uncaria tomentosa) to restore health. Uncaria tomentosa has antioxidant activity and works as an agent to repair DNA damage. It causes different effects on cell proliferation depending on the cell type involved; specifically, it can stimulate the proliferation of myeloid progenitors and cause apoptosis of neoplastic cells. Neutropenia is the most common collateral effect of chemotherapy. For patients undergoing cancer treatment, the administration of a drug that stimulates the proliferation of healthy hematopoietic tissue cells is very desirable. It is important to assess the acute effects of Uncaria tomentosa on granulocyte-macrophage colony-forming cells (CFU-GM) and in the recovery of neutrophils after chemotherapy-induced neutropenia, by establishing the correlation with filgrastim (rhG-CSF) treatment to evaluate its possible use in clinical oncology. MATERIALS AND METHODS: The in vivo assay was performed in ifosfamide-treated mice receiving oral doses of 5 and 15 mg of Uncaria tomentosa and intraperitoneal doses of 3 and 9 µg of filgrastim, respectively, for four days. Colony-forming cell (CFC) assays were performed with human hematopoietic stem/precursor cells (hHSPCs) obtained from umbilical cord blood (UCB). RESULTS: Bioassays showed that treatment with Uncaria tomentosa significantly increased the neutrophil count, and a potency of 85.2% was calculated in relation to filgrastim at the corresponding doses tested. An in vitro CFC assay showed an increase in CFU-GM size and mixed colonies (CFU-GEMM) size at the final concentrations of 100 and 200 µg extract/mL. CONCLUSIONS: At the tested doses, Uncaria tomentosa had a positive effect on myeloid progenitor number and is promising for use with chemotherapy to minimize the adverse effects of this treatment. These results support the belief of the Asháninkas, who have classified Uncaria tomentosa as a 'powerful plant'.
Assuntos
Unha-de-Gato , Proliferação de Células/efeitos dos fármacos , Células Progenitoras Mieloides/efeitos dos fármacos , Neutropenia/tratamento farmacológico , Neutrófilos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Administração Oral , Animais , Unha-de-Gato/química , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Sangue Fetal/citologia , Filgrastim , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Células Progenitoras de Granulócitos e Macrófagos/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Ifosfamida , Injeções Intraperitoneais , Masculino , Medicina Tradicional , Camundongos , Camundongos Endogâmicos BALB C , Células Progenitoras Mieloides/metabolismo , Células Progenitoras Mieloides/patologia , Neutropenia/induzido quimicamente , Neutropenia/metabolismo , Neutropenia/patologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/administração & dosagem , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Plantas Medicinais , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/administração & dosagemRESUMO
The granulocyte-macrophage colony stimulating factor (GM-CSF) is a cytokine that regulates the proliferation and differentiation of hematopoietic cells and activates granulocytes and macrophages. A reversed-phase liquid chromatography (RP-LC) method was validated for the assessing of the stability of non-glycosylated recombinant rhGM-CSF (Molgramostim) in biopharmaceutical formulations. The RP-LC method was carried out on a Jupiter C(4) column (250 mm × 4.6 mm i.d.), maintained at 45 °C. The mobile phase A consisted of 0.1% TFA and the mobile phase B was acetonitrile with 0.1% TFA in acetonitrile, run at a flow rate of 1 mL/min, and using photodiode array (PDA) detection at 214 nm. Chromatographic separation was obtained with a retention time of 29.2 min, and was linear over the concentration range of 2-300 µg/mL (r(2) = 0.9992). Specificity was established in degradation studies. Moreover, the in vitro cytotoxicity test of the degraded products showed significant differences (p < 0.05). The method was applied to the assessment of rhGM-CSF and related proteins in biopharmaceutical dosage forms, and the results were correlated to those of a bioassay. It is concluded that the employment of RP-LC in conjunction with current methods allows a great improvement in monitoring stability, quality control and thereby assures the therapeutic efficacy.
Assuntos
Estabilidade de Medicamentos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Preparações Farmacêuticas , Cromatografia Líquida , Limite de Detecção , Reprodutibilidade dos TestesRESUMO
A stability-indicating capillary zone electrophoresis (CZE) method was validated for the analysis of entecavir in pharmaceutical formulations, using nimesulide as an internal standard. A fused-silica capillary (50 µm i.d.; effective length, 40 cm) was used while being maintained at 25°C; the applied voltage was 25 kV. A background electrolyte solution consisted of a 20 mM sodium tetraborate solution at pH 10. Injections were performed using a pressure mode at 50 mbar for 5 s, with detection at 216 nm. The specificity and stability-indicating capability were proven through forced degradation studies, evaluating also the in vitro cytotoxicity test of the degraded products. The method was linear over the concentration range of 1-200 µg mL(-1) (r(2) = 0.9999), and was applied for the analysis of entecavir in tablet dosage forms. The results were correlated to those of validated conventional and fast LC methods, showing non-significant differences (p > 0.05).