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2.
Molecules ; 26(7)2021 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-33806101

RESUMO

Neutral [Ru(η6-arene)Cl2{Ph2P(CH2)3SPh-κP}] (arene = benzene, indane, 1,2,3,4-tetrahydronaphthalene: 2a, 2c and 2d) and cationic [Ru(η6-arene)Cl(Ph2P(CH2)3SPh-κP,κS)]X complexes (arene = mesitylene, 1,4-dihydronaphthalene; X = Cl: 3b, 3e; arene = benzene, mesitylene, indane, 1,2,3,4-tetrahydronaphthalene, and 1,4-dihydronaphthalene; X = PF6: 4a-4e) complexes were prepared and characterized by elemental analysis, IR, 1H, 13C and 31P NMR spectroscopy and also by single-crystal X-ray diffraction analyses. The stability of the complexes has been investigated in DMSO. Complexes have been assessed for their cytotoxic activity against 518A2, 8505C, A253, MCF-7 and SW480 cell lines. Generally, complexes exhibited activity in the lower micromolar range; moreover, they are found to be more active than cisplatin. For the most active ruthenium(II) complex, 4b, bearing mesitylene as ligand, the mechanism of action against 8505C cisplatin resistant cell line was determined. Complex 4b induced apoptosis accompanied by caspase activation.


Assuntos
Antineoplásicos , Complexos de Coordenação , Neoplasias/tratamento farmacológico , Rutênio , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Células MCF-7 , Neoplasias/metabolismo , Neoplasias/patologia , Rutênio/química , Rutênio/farmacologia
3.
J Med Chem ; 62(17): 7643-7655, 2019 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-31368705

RESUMO

Bruton's tyrosine kinase (BTK) inhibitors such as ibrutinib hold a prominent role in the treatment of B cell malignancies. However, further refinement is needed to this class of agents, particularly in terms of adverse events (potentially driven by kinase promiscuity), which preclude their evaluation in nononcology indications. Here, we report the discovery and preclinical characterization of evobrutinib, a potent, obligate covalent inhibitor with high kinase selectivity. Evobrutinib displayed sufficient preclinical pharmacokinetic and pharmacodynamic characteristics which allowed for in vivo evaluation in efficacy models. Moreover, the high selectivity of evobrutinib for BTK over epidermal growth factor receptor and other Tec family kinases suggested a low potential for off-target related adverse effects. Clinical investigation of evobrutinib is ongoing in several autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus.


Assuntos
Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Descoberta de Drogas , Doenças do Sistema Imunitário/tratamento farmacológico , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Administração Oral , Tirosina Quinase da Agamaglobulinemia/metabolismo , Relação Dose-Resposta a Droga , Humanos , Doenças do Sistema Imunitário/metabolismo , Estrutura Molecular , Piperidinas/administração & dosagem , Piperidinas/química , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Pirimidinas/administração & dosagem , Pirimidinas/química , Relação Estrutura-Atividade
4.
Biotechnol Bioeng ; 116(4): 816-830, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30552760

RESUMO

Glycosylation is a key critical quality attribute for monoclonal antibodies and other recombinant proteins because of its impact on effector mechanisms and half-life. In this study, a variety of compounds were evaluated for their ability to modulate glycosylation profiles of recombinant monoclonal antibodies produced in Chinese hamster ovary cells. Compounds were supplemented into the cell culture feed of fed-batch experiments performed with a CHO K1 and a CHO DG44 cell line expressing a recombinant immunoglobulin G1 (IgG1). Experiments were performed in spin tubes or the ambr®15 controlled bioreactor system, and the impact of the compounds at various concentrations was determined by monitoring the glycosylation profile of the IgG and cell culture parameters, such as viable cell density, viability, and titer. Results indicate that the highest impact on mannosylation was achieved through 15 µM kifunensine supplementation leading to an 85.8% increase in high-mannose containing species. Fucosylation was reduced by 76.1% through addition of 800 µM 2-F-peracetyl fucose. An increase of 40.9% in galactosylated species was achieved through the addition of 120 mM galactose in combination with 48 µM manganese and 24 µM uridine. Furthermore, 6.9% increased sialylation was detected through the addition of 30 µM dexamethasone in combination with the same manganese, uridine, and galactose mixture used to increase total galactosylation. Further compounds or combinations of additives were also efficient at achieving a smaller overall glycosylation modulation, required, for instance, during the development of biosimilars. To the best of our knowledge, no evaluation of the efficacy of such a variety of compounds in the same cell culture system has been described. The studied cell culture media additives are efficient modulators of glycosylation and are thus a valuable tool to produce recombinant glycoproteins.


Assuntos
Meios de Cultura/metabolismo , Imunoglobulina G/metabolismo , Animais , Reatores Biológicos , Biotecnologia/métodos , Células CHO , Técnicas de Cultura de Células/métodos , Cricetinae , Cricetulus , Meios de Cultura/química , Glicosilação , Humanos , Imunoglobulina G/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
6.
Sci Rep ; 6: 39291, 2016 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-27982100

RESUMO

Spontaneous isopeptide bond formation, a stabilizing posttranslational modification that can be found in gram-positive bacterial cell surface proteins, has previously been used to develop a peptide-peptide ligation technology that enables the polymerization of tagged-proteins catalyzed by SpyLigase. Here we adapted this technology to establish a novel modular antibody labeling approach which is based on isopeptide bond formation between two recognition peptides, SpyTag and KTag. Our labeling strategy allows the attachment of a reporting cargo of interest to an antibody scaffold by fusing it chemically to KTag, available via semi-automated solid-phase peptide synthesis (SPPS), while equipping the antibody with SpyTag. This strategy was successfully used to engineer site-specific antibody-drug conjugates (ADCs) that exhibit cytotoxicities in the subnanomolar range. Our approach may lead to a new class of antibody conjugates based on peptide-tags that have minimal effects on protein structure and function, thus expanding the toolbox of site-specific antibody conjugation.


Assuntos
Anticorpos/metabolismo , Imunoconjugados/metabolismo , Peptídeos/metabolismo , Preparações Farmacêuticas/metabolismo , Engenharia Química , Tecnologia Farmacêutica
7.
Eur Arch Otorhinolaryngol ; 273(5): 1079-93, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-25613297

RESUMO

The UEMS Otorhinolaryngology-Head and Neck Surgery section is a dedicated body formed to promote the standardisation and harmonisation of European Otorhinolaryngology (ORL). The European Examination Board of Otorhinolaryngology and Head and Neck Surgery was created to establish a supranational final exam and accreditation for ORL Surgeons. It is open to candidates both from the European Union and outside the EU. The exam is composed of a written examination to assess mainly the theoretical knowledge of Otorhinolaryngological diseases. The second part, a viva voce examination, is designed to test the clinical application of knowledge based on case scenarios and clinical conditions presented to the candidates. The inaugural examination written component took place in Mannheim/Germany in 2009 and the inaugural Viva Voce examination in Vienna/Austria in 2010. Up to and including the year 2013, 858 participants have attempted one of the two exam components. Of the 858 participants, 305 were successful in both examinations and obtained the accreditation of the European Diploma (European Board Certification). The historical origins, development of the examination, its formal arrangements and the format of the examination are presented in this article.


Assuntos
Certificação , Avaliação Educacional , Otolaringologia , Europa (Continente) , União Europeia , Humanos
8.
J Biotechnol ; 218: 53-63, 2016 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-26654938

RESUMO

Industrial fed-batch cultivation of mammalian cells is used for the production of therapeutic proteins such as monoclonal antibodies. Besides medium ensuring initial growth, feeding is necessary to improve growth, viability and antibody production. Established processes include a slight acidic main feed and a separate alkaline feed containing l-tyrosine and l-cysteine. Since l-cysteine is not stable at neutral pH, a new derivative, S-sulfocysteine, was tested in neutral pH feeds. In small scale fed-batch processes, the S-sulfocysteine process yielded a comparable maximum viable cell density, prolonged viability and increased titer compared to the two feed system. Bioreactor experiments confirmed the increase in specific productivity. In depth characterization of the monoclonal antibody indicated no change in the glycosylation, or charge variant pattern whereas peptide mapping experiments were not able to detect any integration of the modified amino acid in the sequence of the monoclonal antibody. Finally, the mechanism of action of S-sulfocysteine was investigated, and results pointed out the anti-oxidative potential of the molecule, mediated through an increase in superoxide dismutase enzyme levels and in the total intracellular glutathione pool. Finally, we propose that the increase in specific productivity obtained in the S-sulfocysteine process results from the anti-oxidative properties of the molecule.


Assuntos
Antioxidantes/farmacologia , Técnicas de Cultura Celular por Lotes/métodos , Meios de Cultura , Cisteína/análogos & derivados , Aminoácidos/metabolismo , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/efeitos dos fármacos , Anticorpos Monoclonais/metabolismo , Antioxidantes/metabolismo , Reatores Biológicos , Células CHO , Cricetinae , Cricetulus , Cisteína/metabolismo , Cisteína/farmacologia , Glutationa/metabolismo , Glicosilação/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Mapeamento de Peptídeos , Tirosina/metabolismo
9.
Glob Health Action ; 8: 29227, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26498745

RESUMO

BACKGROUND: Collaborations for global surgery face many challenges to achieve fair and safe patient care and to build sustainable capacity. The 2004 terrorist attack on a school in Beslan in North Ossetia in the Russian North Caucasus left many victims with complex otologic barotrauma. In response, we implemented a global surgery partnership between the Vladikavkaz Children's Hospital, international surgical teams, the North Ossetian Health Ministry, and civil society organizations. This study's aim was to describe the implementation and 5-year results of capacity building for complex surgery in a postconflict, mid-income setting. DESIGN: We conducted an observational study at the Children's Hospital in Vladikavkaz in the autonomous Republic of North Ossetia-Alania, part of the Russian Federation. We assessed the outcomes of 15 initial patients who received otologic surgeries for complex barotrauma resulting from the Beslan terrorism attack and for other indications, and report the incidence of intra- and postoperative complications. RESULTS: Patients were treated for trauma related to terrorism (53%) and for indications not related to violence (47%). None of the patients developed peri- or postoperative complications. Three patients (two victims of terrorism) who underwent repair of tympanic perforations presented with re-perforations. Four junior and senior surgeons were trained on-site and in Germany to perform and teach similar procedures autonomously. CONCLUSIONS: In mid-income, postconflict settings, complex surgery can be safely implemented and achieve patient outcomes comparable to global standards. Capacity building can build on existing resources, such as operation room management, nursing, and anesthesia services. In postconflict environments, substantial surgical burden is not directly attributable to conflict-related injury and disease, but to health systems weakened by conflicts. Extending training and safe surgical care to include specialized interventions such as microsurgery are integral components to strengthen local capacity and ownership. Our experience identified strategies for fair patient selection and might provide a model for potentially sustainable surgical system building in postconflict environments.


Assuntos
Barotrauma/cirurgia , Fortalecimento Institucional/métodos , Orelha Média/cirurgia , Cooperação Internacional , Desenvolvimento de Programas/métodos , Procedimentos Cirúrgicos Operatórios/educação , Adolescente , Adulto , Barotrauma/complicações , Criança , Pré-Escolar , Orelha Média/lesões , Explosões , Feminino , Alemanha , Saúde Global , Acesso aos Serviços de Saúde , Hospitais Pediátricos , Humanos , Lactente , Intercâmbio Educacional Internacional , Masculino , Federação Russa
10.
J Biol Inorg Chem ; 20(3): 575-83, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25827592

RESUMO

T cell differentiation into distinct T helper (Th) subpopulations is crucial in governing acquired immune responses as well as some inflammatory and autoimmune disorders. This study investigated potential of the novel neutral binuclear ruthenium(II) complexes 1-8 with general formula [{RuCl2(η(6)-p-cym)}2µ-(N(∩)N)] (N(∩)N = bis(nicotinate)- and bis(iso-nicotinate)-polyethylene glycol esters; (3-py)COO(CH2CH2O) n CO(3-py) and (4-py)COO(CH2CH2O) n CO(4-py); n = 1-4), as well as [RuCl2(η(6)-p-cym)(nic)] (R1, nic = nicotinate) and [RuCl2(η(6)-p-cym)(inic)] (R2, inic = isonicotinate) as an immunomodulatory agents capable to direct Th cell differentiation. From all investigated complexes, [{RuCl2(η(6)-p-cym)}2µ-{(3-py)COO(CH2CH2O)4CO(3-py)}] (4) was selected for further study because it did not affect splenocyte viability (in concentration up to 50 µM), but significantly reduced secretion of representative Th1 cytokine, IFN-γ induced by T cell mitogen. Besides IFN-γ, 4 inhibited dose dependently expression and production of representative Th17 cytokine, IL-17, in these cells. Otherwise, the production of anti-inflammatory cytokines IL-4 and IL-10 was upregulated. Also, 4 significantly increased CD4(+)CD25(+)FoxP3(+) Treg cell frequency in the activated splenocytes. Moreover, ConA-induced expression of Th1 transcription factors, T-bet and STAT1, as well as of Th17-related protein STAT3 was attenuated upon exposure to 4, while the expression of Th2-related transcription factor GATA3 remained stable. In conclusion, ruthenium(II) complex 4 modulates immune system cell functions in vitro by inhibiting T cell differentiation towards pathogenic Th1/Th17 phenotype and inducing a regulatory phenotype characterized by IL-10 and IL-4 production, which may provide novel therapeutic opportunities for immune-inflammatory and/or autoimmune disorders.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Complexos de Coordenação/farmacologia , Ésteres/farmacologia , Mitógenos/farmacologia , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação/química , Cimenos , Ésteres/química , Immunoblotting , Camundongos , Modelos Moleculares , Monoterpenos/química , Polietilenoglicóis/química , Reação em Cadeia da Polimerase , Rutênio/química , Linfócitos T Auxiliares-Indutores/citologia
12.
Anal Biochem ; 395(2): 178-88, 2009 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-19699707

RESUMO

A common technique for analysis of protein glycosylation is HPLC coupled to mass spectrometry (LC-MS). However, analysis is challenging due to a low abundance of glycopeptides in complex protein digests, microheterogeneity at the glycosylation site, ion suppression effects, and competition for ionization by coeluting peptides. Specific sample preparation is necessary for a comprehensive and site-specific glycosylation analysis by MS. In this study we qualitatively compared hydrophilic interaction chromatography (HILIC) and hydrazine chemistry for the enrichment of all N-linked glycopeptides and titanium dioxide for capturing sialylated glycopeptides from a complex peptide mixture. Bare silica, microcrystalline cellulose, amino-, amide- (TSKgel Amide-80), and sulfobetaine-(ZIC-HILIC) bonded phases were evaluated for HILIC enrichment. The experiments revealed that ZIC-HILIC and TSKgel Amide-80 are very specific for capturing glycopeptides under optimized conditions. Quantitative analysis of N-glycosidase F-released and 2-aminobenzamide-labeled glycans of a ZIC-HILIC-enriched monoclonal antibody demonstrated that glycopeptides could be enriched without bias for particular glycan structures and without significant losses. Sialylated glycopeptides could be efficiently enriched by titanium dioxide and in addition to HILIC both methods enable a comprehensive analysis of protein glycosylation by MS. Enrichment of N-linked glycopeptides by hydrazine chemistry resulted in lower peptide recovery using a more complex enrichment scheme.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Glicopeptídeos/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Glicopeptídeos/isolamento & purificação , Glicosilação , Hidrazinas/química , Neuraminidase/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Titânio/química , ortoaminobenzoatos/química
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