Design, synthesis, biological evaluation, and nitric-oxide release studies of a novel series of celecoxib prodrugs possessing a nitric-oxide donor moiety

A new group of hybrid nitric oxide-releasing anti-inflammatory drugs (NONO-coxibs), in which an O 2-acetoxymethyl-1-(N-ethyl-N-methylamino)diazen-1-ium-1,2-diolate NO-donor moiety is attached directly to the carboxylic acid group of 1-(4-aminosulfonylphenyl)-5-aryl-1H-pyrazol-3-carboxylic acids (6a-c), were synthesized. A low amount of NO was released from the diazen-1-ium-1,2-diolate compounds 6a-c upon incubation with phosphate buffer saline (PBS) at pH 7.4 (range: pH 7.97-8.51), whereas, the percentage of NO released was significantly higher (84.5%-85.05% of the theoretical maximal release of two molecules of NO/molecule of the parent hybrid ester prodrug) when the diazen-1-ium-1,2-diolate ester prodrugs were incubated in the presence of rat serum. These incubation studies demonstrated that both NO and the anti-inflammatory 1-(4-aminosulfonylphenyl)-5-(4-H, 4-F or 4-Me-phenyl)-1H-pyrazol-3-carboxylic acid (4a-c) would be released from the parent NONO-coxib upon in vivo cleavage by non-specific serum esterases. The parent compounds 4a-c displayed good anti-inflammatory effects (ID50=81.4-112.4 mg/kg p.o.) between those exhibited by the reference drugs, aspirin (ID50=114.3 mg/kg p.o.) and celecoxib (ID50=12.6 mg/kg p.o.). Hybrid ester anti-inflammatory/NO-donor prodrugs (NONO-coxibs) offer a potential drug-design concept directed toward the development of anti-inflammatory drugs that are lacking adverse ulcerogenic and/or cardiovascular effects.

Stability indicating method development and validation for simultaneous estimation of atorvastatin calcium and celecoxib in bulk and niosomal formulation by RP-HPLC

The present work describes development and validation of a specific, sensitive, precise and stability-indicating high-performance liquid chromatographic method of analysis of atorvastatin calcium and celecoxib, both as a bulk drug and in niosomal formulation. The analysis has been performed by using Cosmosil-C18 column (4.6 mm´250 mm, 5 m) at 25 °C using acetonitrile: ammonium acetate buffer pH 5.0: methanol (50:25:25 v/v/v) as mobile phase. The detection was carried out at 277nm with a flow rate of 1.0mL/min. The retention times of Atorvastatin calcium and Celecoxib were 6.195 and 3.989min, respectively. The method was validated according to ICH guidelines, for specificity, precision, linearity, accuracy and robustness. Atorvastatin calcium and Celecoxib were subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. The degradation was observed in oxidation and acid hydrolysis. The linearity for atorvastatin calcium and celecoxib were in the range of 100-500 µg/mL. The recovery study of atorvastatin and celecoxib were found to be in the range of 98.96 - 99.92% and 98.90-100%, respectively. The proposed method was validated and successfully applied to the estimation of Atorvastatin calcium and Celecoxib in combined in-house niosomal formulation.

O presente trabalho descreve o desenvolvimento e a validação de método de análise por cromatografia de alta eficiência específico, sensível, preciso e indicador de estabilidade de atorvastatina cálcica e celecoxibe, ambos como fármaco e como formulação niosômica. A análise foi realizada utilizando coluna Cosmosil-C18 (4,6 mm´250 mm, 5 m) a 25 °C, e acetonitrila: tampão acetato de amônio pH 5,0: metanol (50:25:25 v/v/v) como fase móvel. A detecção foi realizada a 277 nm, com fluxo de 1,0 mL/min. Os tempos de retenção de atorvastatina cálcica e de celecoxibe foram 6,195 e 3,989 min, respectivamente. O método foi validado de acordo com as regras da ICH para especificidade, precisão, exatidão e robustez. A atorvastatina cálcica e o celecoxibe foram submetidos a condições de estresse por hidrólise, oxidação, fotólise e degradação térmica. A degradação foi observada por oxidação e hidrólise ácida. Observou-se a linearidade da atorvastatina cálcica e do celecoxibe na faixa de 100-500 µg/mL. A recuperação da atorvastatina e do celecoxibe foi observada na faixa de 98,96-99,92% e 98,90-100%, respectivamente. O método proposto foi validado e aplicado com sucesso para a determinação de atorvastatina cálcica e celecoxibe em formulação niosômica caseira combinada.

Avaliação do potencial antiproliferativo do Dendrímero de Poliglicerol associado ao celecoxibe em linhagens celulares de carcinoma epidermoide de cabeça e pescoço / Evaluation of antiproliferative potential of Polyglycerol Dendrimer conjugated to Celecoxib in Head and Neck Squamous Cell Carcinoma cell lines

Diversos mecanismos celulares estão associados à patogênese do Carcinoma Epidermoide de Cabeça e Pescoço (CECP). Algumas dessas alterações envolvem proteínas pertencentes à via de sinalização do Akt, e o fator de transcrição NF-kB, o qual têm importante papel na fisiologia normal e no câncer. A proteína COX-2, descrita em processos inflamatórios, também participa da carcinogênese e está associada com a via de sinalização do Akt e com o NF-kB. Dendrímeros são uma forma única de nanotecnologia, surgindo como nanotransportadores com a capacidade de penetrar na célula tumoral liberando drogas quimioterápicas em seu interior. Os benefícios desta tecnologia são o aumento da eficicácia do princípio ativo utilizado e a redução dos seus efeitos secundários tóxicos. O Celecoxibe, antiinflamatório não esteroidal, inibidor seletivo da COX-2, tem se mostrado um importante agente anticarcinogênico, no entanto seu mecanismo de ação no CECP não é totalmente compreendido. Neste trabalho, um Dendrímero de Poliglicerol associado ao Celecoxibe (PGLD-celecoxibe) foi sintetizado e caracterizado por técnicas de espectroscopia ¹H-RMN, ¹³C-RMN, Maldi-Tof, TLC e DSC. Além disso, o conjugado foi testado in vitro em três linhagens celulares de CECP. O PGLD-Celecoxibe foi sintetizado com sucesso e promoveu a redução da dose capaz de inibir a proliferação celular, reduzindo o IC 50 do Celecoxibe de forma significativa em todas as linhagens celulares, se aproximando da dose sérica alcançada por este medicamento, resultado corroborado pelo Ensaio de Migração Celular. O mecanismo de morte celular observado foi a apoptose, associada a diminuição significativa da expressão de COX-2 ou por uma via alternativa independente. Alguns dos grupos tratados apresentaram alteração na expressão das proteínas pAkt e NF-kB.

Several cellular mechanisms are associated with the pathogenesis of Head and Neck Squamous Cell Carcinoma (HNSCC). Some of these alterations involve proteins in the Akt signaling pathway and the transcription factor NF-kB, which plays an important role in normal physiology and in cancer. COX-2 protein, described in inflammatory processes, and also involved in the carcinogenesis is associated with the Akt signaling pathway and the NF-kB. Dendrimers are a unique form of nanotechnology, emerging as nanocarriers with the ability to penetrate the tumor cell releasing chemotherapeutic. This technology increases the active substance efficiency and reduces its toxic side effects. Celecoxib, a nonsteroidal anti-inflammatory, selective inhibitor of COX-2 has been shown to be an important anticancer agent, but its action mechanism in HNSCC is not fully understood. A polyglycerol dendrimer linked to celecoxib (PGLD-Celecoxibe) was synthesized and characterized by NMR spectroscopy ¹H-NMR, ¹³C-NMR,TLD, DSC and Maldi-Tof techniques. In addition, in vitro assays were performed in three HNSCC cell lines The PGLD-Celecoxibe was successfully synthesized and provided a decrease in the dose able to inhibit cell proliferation reducing the IC 50 index of Celecoxib significantly in all cell lines, approaching to the serum dose achieved for this product, result supported by Wound Healing Assay. The cell death mechanism observed was apoptosis, which can be associated with significant reduction of expression of COX-2 also may be occurring by a COX-2 independent pathway. Some of the treated groups showed alterations in pAkt and NF-kB proteins expression.