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

<p>Abstract</p> <p>Background</p> <p>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 <it>Escherichia coli </it>(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 <it>E. coli</it>. 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 <it>E. coli </it>cells.</p> <p>Results</p> <p>Here we describe cloning of the human granulocyte colony-stimulating factor coding DNA sequence, protein expression in <it>E. coli </it>BL21(DE3) host cells in the absence of isopropyl-β-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, <it>in vivo</it>, 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.</p> <p>Conclusion</p> <p>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.</p

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

Variedades crioulas de milho para a realidade da agricultura familiar no semiárido sergipano.

bitstream/item/153480/1/cot-190.pd

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

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 (B-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. (C) 2001 Elsevier B.V. B.V. All rights reserved

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-mu m 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

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%) 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 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%). 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 parâ&shy;metros 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 valde&shy;coxibe apresentou redução significativa no TTPA (18%) após duas semanas, e redução do TP (12,2%) após três semanas (P<0.05). Os efeitos observados na contagem de pla&shy;quetas 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%). Os resultados experimentais com as concentrações plasmáticas alcan&shy;çadas demonstram que o valdecoxibe pode induzir alterações nos parâ&shy;metros hematológicos dos ratos. Esses dados poderão contribuir para a melhor compreensão dos efeitos colaterais e uso do fármaco com segurança