Estudo de biotransformação in vitro da ambrisentana e da rifampicina através de fungos filamentosos

A biotransformação de fármacos através de microrganismos é considerada uma tecnologia econômica e ecologicamente viável, sendo muito utilizada para modificar estruturas de compostos biologicamente ativos, estudar o metabolismo de novos fármacos e eliminar ou reduzir sua toxicidade. Estes estudos constituem uma alternativa promissora para elucidação dos metabólitos formados de diferentes classes farmacológicas. Este trabalho teve como objetivo empregar três diferentes fungos filamentosos: Cunninghamella elegans, Penicillium mineoluteum e Aspergillus niger, para estudar a biotransformação dos fármacos ambrisentana e rifampicina. Os fármacos foram escolhidos por apresentarem metabolismo via CYP450 e também por não haver relatos na literatura sobre estes estudos. Para o monitoramento da formação dos metabólitos foi necessário desenvolver metodologias analíticas com características adequadas para esta aplicação. O monitoramento da formação dos metabólitos foi empregado por cromatografia líquida de alta eficiência e para identificação destes foi empregado cromatografia líquida de ultra eficiência acoplada à espectrometria de massas sequencial (UHPLC-QTOF/MS). O Capítulo I apresenta a biotransformação da ambrisentana (AMB) através do fungo Cunnighamella elegans ATCC 9245. Primeiramente, foi realizada a otimização do estudo (meio de cultura, pH, concentração do fármaco e agitação) para identificar da melhor condição de biocatálise. A metodologia analítica para monitoramento dos metabólitos formados foi desenvolvida e validada. A biotransformação da AMB resultou na formação de um metabólito conhecido como glicosídeo da ambrisentana (C28H33N2O9), ainda não relatado na literatura, confirmado a capacidade do fungo em realizar fase II do metabolismo. O Capítulo II apresenta os resultados obtidos no estudo de biotransformação utilizando o mesmo fungo filamentoso para o fármaco rifampicina (RIF). A concentração de inóculo foi otimizada (1016 esporos), a fim de verificar o aumento na formação dos possíveis metabólitos. Os resultados relacionados à RIF mostram que ocorre o consumo do fármaco pela C. elegans, representado pela redução da concentração do mesmo, assim como formação de um metabólito já conhecido como produto de degradação, em maior concentração no meio biotransformado quando comparado com o controle negativo. Além disso, foi observada a presença de picos adicionais não observados nos controles. Posteriormente, as amostras foram analisadas em UHPLC-QTOF/MS para a elucidação estrutural dos sinais formados. Com isso, foi identificada a rifampicina quinona e um sinal de menor polaridade formado somente no meio de biotransformação (452 m/z), o qual foi proposto como uma mono-oxigenação da RIF. No Capítulo III, foi realizado o estudo de biotransformação da ambrisentana através dos fungos Aspergillus niger ATCC 9029 e Penicilium mineoluteum URM 6889, empregando meio de cultura Czapek. No meio reacional com o A.niger, ocorre formação de dois sinais mais polares que o fármaco, indicando a formação de possíveis metabólitos, que foram elucidados através da UHPLC-QTOF/MS. Foi possível propor dois metabólitos ainda não relatados na literatura de m/z 301,1339 e 313,1907, respectivamente. Entretanto, a biocatálise através do P.mineoluteum não apresentou formação de sinais adicionais quando comparado aos controles. No Capítulo IV, são apresentados os resultados obtidos da biotransformação da RIF através do fungo A.niger. O fungo foi capaz de reduzir drasticamente a concentração de RIF em meio reacional. Devido a isto, a amostra foi avaliada em UHPLC-QTOF/MS para verificar a possível formação de metabólitos. Foi observada a formação de dois compostos mais polares que a RIF com mesma massa e fragmentação (455.3115 m/z), caracterizando isômeros, além da RIF quinona. Posteriormente, foi investigada a atividade antimicrobiana da RIF após transformação através do método da microdiluição em poços preconizados por guia internacional, resultando em aumento da concentração inibitória mínima, consequentemente perda da atividade antimicrobiana. Os resultados obtidos neste estudo demonstram que biostranformações com fungos são uma importante alternativa para mimetizar a biotransformação em humanos e também para obtenção de metabólitos de fármacos. Os dois fungos propostos nesse estudo Cunninghamella elegans ATCC 9245 e Aspergillus niger ATCC 9029 apresentaram capacidade de metabolizar os fármacos ambrisentana e rifampicina.Biotransformation of drugs by microorganisms is an economical and ecologically viable technology and is widely used to modify structures of some biologically active compounds, study the metabolism of molecules and eliminate or reduce their toxicity. These studies constitute a promising alternative for the elucidation of metabolites formed from different pharmacological classes. This work aims to employ three different filamentous fungi: Cunninghamella elegans, Penicillium mineoluteum e Aspergillus niger, to study the biotransformation of the drugs ambrisentan and rifampicin. These drugs were chosen because they have metabolism through CYP450 and also there are no reports in the literature about these studies. To monitor the formation of metabolites, it was necessary to develop and validate analytical methodologies with characteristics suitable for this application. High performance liquid chromatography was used to monitor the formation of the metabolites, and ultra-high performance liquid chromatography coupled to sequential mass spectrometry (UHPLC-QTOF/MS) was used to identify the metabolites. The first chapter presents the optimization of the study (culture medium, pH, drug concentration and agitation), the results obtained in the biotransformation of ambrisentan by the fungus Cunnighamella elegans ATCC 9245 in Czapek medium, the development and validation of the analytical methodology for monitoring the metabolites formed and the identification of their molecular structures. The biotransformation of AMB resulted in the formation of a metabolite known as ambrisentan glycoside (C28H33N2O9), not reported yet in the literature, confirming the ability of the fungus to perform phase II metabolism. Chapter II presents the results obtained in the biotransformation study using the same filamentous fungus for the drug rifampicin (RIF). The inoculum concentration was optimized (1016 spores) in order to verify the increase in the formation of the possible metabolites. The results related to RIF show that the consumption of the drug by C.elegans, represented by the reduction of the concentration of the drug, as well as the formation of a metabolite already known as degradation product, in a higher concentration in the biotransformed medium when compared to the negative control. In addition, the presence of additional peaks not observed in the controls was demonstrated. Subsequently the samples were analyzed in UHPLC-QTOF/MS for the structural elucidation of the formed signals. With this, rifampicin quinone was identified along to another compound of lower polarity formed only in the biotransformation medium (452 m/z), which was proposed as a mono-oxygenation of the RIF. In Chapter III, the biotransformation of ambrisentan was carried out through the fungus Aspergillus niger ATCC 9029 and Penicilium mineoluteum URM 6889, using Czapek culture medium. In the reaction medium with A.niger, formation of two more polar signals than the drug was observed, indicating the formation of possible metabolites, which were elucidated through UHPLC-QTOF/MS. It was possible to propose two metabolites not yet reported in the literature, with m/z 301.1339 and 313.1907, respectively. However, the biocatalysis through P.mineoluteum showed no additional signal formation when compared to the controls. In Chapter IV, the results obtained from the biotransformation of the RIF through the A.niger fungus are presented. The fungus was able to drastically reduce the concentration of RIF in reaction medium. Due to this, the sample was evaluated in UHPLC-QTOF/MS to verify the possible formation of metabolites. It was observed the formation of two more polar compounds than the RIF with the same mass and fragmentation (455.3115 m/z), characterizing isomers, besides RIF quinone. Subsequently, the antimicrobial activity of RIF was evaluated after transformation through the microdilution method in wells recommended by international guidelines, resulting in an increase in the minimum inhibitory concentration, consequently loss of antimicrobial activity. The results obtained in this study demonstrate that biotransformation with fungi are an important alternative to mimic the biotransformation in humans and also to obtain drug metabolites. The two fungi proposed in this study Cunninghamella elegans ATCC 9245 and Aspergillus niger ATCC 9029 were able to metabolize the drugs Ambrisentan and Rifampicin

Avaliação da permeação/retenção cutânea in vitro de formulações semissólidas comerciais contendo aciclovir e estudo de sua relação com a concentração antiviral efetiva

Avaliar a permeação/retenção cutânea de formulações semissólidas em pele animal pode ser útil para complementar a equivalência farmacêutica destes produtos. O aciclovir (ACV) é um fármaco derivado da guanosina, com alta especificidade para o vírus herpes, disponível no mercado nacional sob forma de creme dermatológico de vários fabricantes. O objetivo do trabalho foi a avaliação da permeação/retenção cutânea in vitro de formulações comerciais contendo ACV (referência, genérico e similar – dois lotes de cada), com determinação da concentração do fármaco no tecido cutâneo, nas duas principais camadas, para correlacionar com a concentração antiviral efetiva. Os estudos foram conduzidos utilizando células de Franz e pele suína. Primeiramente, foi validado o método analítico para a quantificação do fármaco nas formulações e, principalmente, nas camadas da pele suína. As análises foram conduzidas em cromatógrafo a líquido de alta eficiência, com coluna C8, detecção em 254 nm e fase móvel constituída de uma mistura de água:metanol (95:5, v/v). A metodologia analítica demonstrou ter adequada sensibilidade (LQ 0,38 μg/mL), especificidade e recuperação do fármaco a partir das matrizes biológicas na faixa de 85 – 102%. Quanto ao estudo de retenção cutânea in vitro, o produto genérico, do primeiro lote analisado forneceu quantidade de fármaco na pele (1,56 ± 0,65 μg de ACV/mg de tecido) que apresentou diferença estatisticamente significativa em relação ao referência e similar (0,85 ± 0,40 e 0,88 ± 0,43 μg/mg, respectivamente), enquanto que no segundo lote todos os produtos apresentaram similaridade de penetração (p>0,05) (referência - 0,74 ± 0,45; genérico - 1,25 ± 0,90 e similar - 1,23 ± 0,85 μg/mg). Para compreender se estes resultados e sua variabilidade poderiam trazer comprometimento ao efeito terapêutico, foi realizada uma simulação empregando um modelo de efeito inibitório para replicação viral. Com esta avaliação pode-se verificar que os produtos analisados ofereceriam efeito antiviral desejado no tecido cutâneo, mesmo em contato com cepas de Herpes simples de IC50 elevado.Evaluate the permeation/retention of semisolid formulations in animal skin could be useful to supplement the pharmaceutical equivalence of these products. Acyclovir (ACV) is a guanosine derivative drug with high specificity for the herpes vírus. The objective was to evaluate the skin in vitro permeation/retention of commercial formulations containing ACV (reference, generic and similar - two batches each) with determination of drug concentration in the skin tissue in different layers to correlate with effective antiviral concentration. Studies were conducted using Franz cells and porcine skin. Was validated analytical method for quantification of the drug in the formulations and layers of pig skin. Tests were conducted in high performance liquid chromatograph, eqquiped with C8 column, UV detection at 254 nm and the mobile phase consisted of a mixture of water: methanol (95:5, v/v). The analytical methodology has demonstrated good sensitivity (LOQ 0.38 mg/mL), and specific, with drug recovery from biological matrices in the range of 85-102%. As for the study of skin retention in vitro, the generic product (1.56 ± 0.65 ACV/mg of tissue), the first batch analysis showed a significant difference from the reference and similar (0.85 ± 0.40 and 0.88 ± 0.43 μg/mg, respectively), while in the second batch all products showed similar penetration (reference - 0.74 ± 0.45; generic - 1.25 ± 0.90 and similar - 1.23 ± 0.85 μg/mg). To understand whether this difference could bring change in therapeutic effect, a simulation was performed using an inhibitory effect model for viral replication. With the simulation study, it may be suggested that the formulations analyzed would provide wanted antiviral effect even in contact with Herpes simplex strains of high IC50

In vitro evaluation of cutaneous penetration of acyclovir from semisolid commercial formulations and relation with its effective antiviral concentration

The evaluation of drug permeation/penetration of semisolid formulations into animal skin can be useful to supplement the pharmaceutical equivalence. This paper describes the in vitro assessment of acyclovir (ACV) into porcine skin from commercial formulations with etermination of drug concentration in different layers of cutaneous tissue to correlate with effective antiviral concentration in order to improve the equivalence decision. Studies were conducted using Franz cells and porcine skin. Selected pharmaceutical creams containing ACV had identical (reference and generic) and different (similar) excipients. A software program was employed for the simulation of antiviral effectiveness in the skin. Regarding ACV skin penetration, the first batch of the generic product showed a significant difference from reference and similar products, while in the second batch all products demonstrated equivalent drug penetration in the skin. Simulation studies suggest that formulations analysed exhibit a pharmacological effect even when in contact with Herpes simplex strains of high IC50 (inhibitory concentration required to reduce viral replication by 50%). According to results, it can be assumed that the in vitro cutaneous permeation/penetration study does not supply sensitivity information regarding small alterations of ACV semisolid formulations due to the variability inherent to the method, although it can be relevant to pharmaceutical equivalence studies in the development of semisolid products

Ceftaroline fosamil: development a rapid HPLC method indicating stability and bioassay for determination in pharmaceutical formulation, stability and cytotoxicity studies

The present study reports the development and validation of a microbiological assay. To assess this methodology, the method was developed and validated for the quantification of CEF by high performance liquid chromatography (HPLC). The validation of the microbial assay by diffusion method in 3x3 cylinder agar presented showed satisfactory results as to specificity, linearity in the range of 2.0 - 8.0 μg.mL-1, precision (109.42 %), accuracy (102.3 %), and robustness. The development and validation of the method by HPLC were evaluated according to specificity, linearity, precision, accuracy and robustness. A high performance liquid chromatography from Shimadzu with Agilent® C18 column, mobile phase (water with triethylamine 1.0 % pH 5.0: acetonitrile 87:13 v/v was used in the chromatographic method. The validated microbiological and chromatographic methods were compared statistically and there was no significant difference between them when compared by Student's t-test. In the preliminary stability study, it was found stable in acid hydrolysis (0.1M) and UVA light in the period evaluated, and unstable against thermal degradation (40 and 60 °C), oxidative with hydrogen peroxide, basic in NaOH (0.1 M and 0.01M) and UVC light. Samples exposed to UVC light and thermal degradation at 60°C showed degradation kinetics following zero order and second order, respectively. The cytotoxicity assay showed no difference between the normal condition and the sample submitted to forced degradation, suggesting that the possible degradation products formed did not change the result. The methods developed did not present a significant difference, therefore, they are interchangeable, and so can be used for routine quality control analysis

STUDY OF FLAVONOIDS PRESENT IN POMELO (Citrus Maxima) BY DSC, UV-VIS, IR, 1H AND 13C NMR AND MS

Flavonoids are among the most important plant metabolites. Due to their potential benefits, there is a considerable interest in this natural product. In genus Citrus, some plants have not yet been much exploited in Brazil, as in the case of grapefruit (Citrus maxima), whose main flavonoids are naringin and their aglycone naringenin. The physico-chemical characteristics are important pre-requisites of reference chemical in future studies. In this context, the objective of this study was to determine the characterization of naringin and naringenin by melting point, DSC, UV-VIS, 1H and 13C NMR, IR and MS. Results revealed that, naringin and naringenin after characterization, can be used as a chemical of reference in future studies and contribute to seeking possible technological applications

Study of flavonoids presente in Pomelo (Citrus máxima) by DSC, UV-VIS, IR, 1H and 13C NMR and MS

Flavonoids are among the most important plant metabolites. Due to their potential benefits, there is a considerable interest in this natural product. In genus Citrus, some plants have not yet been much exploited in Brazil, as in the case of grapefruit (Citrus maxima), whose main flavonoids are naringin and their aglycone naringenin. The physico-chemical characteristics are important pre-requisites of reference chemical in future studies. In this context, the objective of this study was to determine the characterization of naringin and naringenin by melting point, DSC, UV-VIS, 1H and 13C NMR, IR and MS. Results revealed that, naringin and naringenin after characterization, can be used in future studies and contribute to seeking possible technological applications

Polymeric nanoparticles loaded naringin and naringenin: effect of solvent, characterization, photodegradation and stability studies

Naringin (NAR) and naringenin (NGE) are flavonoids with important effects, such as antioxidant, nephroprotective and anti-inflammatory action. However, factors such as poor solubility and oral bioavailability, gastrointestinal instability and extensive first pass metabolism lead to limited deliverability. As far as we know, there are no papers describing the use of combination of NAR and NGE in nanoparticles. This paper describes the development and characterization of new nanoparticles containing NAR and NGE (NAR-NGE-NPs) which were prepared by nanoprecipitation using ethanol or mixture of solvents. Size distribution of NAR-NGE-NPs demonstrated a narrow distribution (121 nm), low polydispersity (< 0.1), and encapsulation efficiencies were greater than 80%. Infrared spectroscopy analyses confirmed the structure of NAR-NGE-NPs and in transmission electron microscopy, NAR-NGE-NPs presented a spherical and regular shape. A degradation study by UV-C, NAR-NGE-NPs improved photostability and conferred protection against NAR and NGE degradation. Minimal inhibitory concentrations of NAR and NGE were evaluated, however samples did not show antimicrobial activity. In this investigation, new NAR-NGE-NPs were successfully developed by a nanoprecipitation technique, using Endragit®L100 as polymer and ethanol as solvent.

BIOTRANSFORMATION OF METRONIDAZOLE BY CUNNINGHAMELLA ELEGANS ATCC 9245

Drug biotransformation studies appear as an alternative to pharmacological studies of metabolites, development of new drug candidates with reduced investment as well as the most efficient production of chemical structures involves and drug quality control studies. A wide range of reactions in biotransformations process is catalyzed by microorganisms. Fungi can be considered as a promising source of new biotransformation reactions. The aim of this study was to evaluate the capacity of metronidazole biotransformation through the filamentous fungus Cunninghamella elegans ATCC 9245. The monitoring of metabolite formation was performed by high-performance liquid chromatography (HPLC) coupled to ultraviolet (UV) spectrophotometry. The results of the biotransformation of metronidazole showed drug consumption in culture and the formation of four new chromatographic peaks of chemical structures not elucidated. The method showed it became linear over 10-70 μg/mL (r = 0.999953). Accuracy, precision and stability studies agree with international guidelines.  Results are consistent in accordance with the principles of green chemistry as the experimental conditions had a low environmental impact, and few solvents use. 

Avaliação da permeação/retenção cutânea in vitro de formulações semissólidas comerciais contendo aciclovir e estudo de sua relação com a concentração antiviral efetiva

Avaliar a permeação/retenção cutânea de formulações semissólidas em pele animal pode ser útil para complementar a equivalência farmacêutica destes produtos. O aciclovir (ACV) é um fármaco derivado da guanosina, com alta especificidade para o vírus herpes, disponível no mercado nacional sob forma de creme dermatológico de vários fabricantes. O objetivo do trabalho foi a avaliação da permeação/retenção cutânea in vitro de formulações comerciais contendo ACV (referência, genérico e similar – dois lotes de cada), com determinação da concentração do fármaco no tecido cutâneo, nas duas principais camadas, para correlacionar com a concentração antiviral efetiva. Os estudos foram conduzidos utilizando células de Franz e pele suína. Primeiramente, foi validado o método analítico para a quantificação do fármaco nas formulações e, principalmente, nas camadas da pele suína. As análises foram conduzidas em cromatógrafo a líquido de alta eficiência, com coluna C8, detecção em 254 nm e fase móvel constituída de uma mistura de água:metanol (95:5, v/v). A metodologia analítica demonstrou ter adequada sensibilidade (LQ 0,38 μg/mL), especificidade e recuperação do fármaco a partir das matrizes biológicas na faixa de 85 – 102%. Quanto ao estudo de retenção cutânea in vitro, o produto genérico, do primeiro lote analisado forneceu quantidade de fármaco na pele (1,56 ± 0,65 μg de ACV/mg de tecido) que apresentou diferença estatisticamente significativa em relação ao referência e similar (0,85 ± 0,40 e 0,88 ± 0,43 μg/mg, respectivamente), enquanto que no segundo lote todos os produtos apresentaram similaridade de penetração (p>0,05) (referência - 0,74 ± 0,45; genérico - 1,25 ± 0,90 e similar - 1,23 ± 0,85 μg/mg). Para compreender se estes resultados e sua variabilidade poderiam trazer comprometimento ao efeito terapêutico, foi realizada uma simulação empregando um modelo de efeito inibitório para replicação viral. Com esta avaliação pode-se verificar que os produtos analisados ofereceriam efeito antiviral desejado no tecido cutâneo, mesmo em contato com cepas de Herpes simples de IC50 elevado.Evaluate the permeation/retention of semisolid formulations in animal skin could be useful to supplement the pharmaceutical equivalence of these products. Acyclovir (ACV) is a guanosine derivative drug with high specificity for the herpes vírus. The objective was to evaluate the skin in vitro permeation/retention of commercial formulations containing ACV (reference, generic and similar - two batches each) with determination of drug concentration in the skin tissue in different layers to correlate with effective antiviral concentration. Studies were conducted using Franz cells and porcine skin. Was validated analytical method for quantification of the drug in the formulations and layers of pig skin. Tests were conducted in high performance liquid chromatograph, eqquiped with C8 column, UV detection at 254 nm and the mobile phase consisted of a mixture of water: methanol (95:5, v/v). The analytical methodology has demonstrated good sensitivity (LOQ 0.38 mg/mL), and specific, with drug recovery from biological matrices in the range of 85-102%. As for the study of skin retention in vitro, the generic product (1.56 ± 0.65 ACV/mg of tissue), the first batch analysis showed a significant difference from the reference and similar (0.85 ± 0.40 and 0.88 ± 0.43 μg/mg, respectively), while in the second batch all products showed similar penetration (reference - 0.74 ± 0.45; generic - 1.25 ± 0.90 and similar - 1.23 ± 0.85 μg/mg). To understand whether this difference could bring change in therapeutic effect, a simulation was performed using an inhibitory effect model for viral replication. With the simulation study, it may be suggested that the formulations analyzed would provide wanted antiviral effect even in contact with Herpes simplex strains of high IC50