Desenvolvimento de método analítico indicativo de estabilidade para o controle de qualidade químico e biofarmacêutico de linagliptina

Linagliptina (LGT) é um fármaco membro da classe de gliptinas que inibe a enzima dipeptidil - peptidase - 4 . Elas são usadas para reduzir os níveis de glicose no sangue em pacientes com Diabetes mellitus tipo 2. Devido a seu recente desenvolvimento e lançamento no mercado, a LGT não tem monografia em compêndio oficial, nem em registros nacionais ou internacionais disponíveis para determinações qualitativas e quantitativas indicativas de estabilidade do fármaco. O objetivo deste trabalho foi desenvolver um método cromatográfico para caracterizar LGT em forma de comprimido contendo 5 mg do fármaco, avaliar a cinética de degradação de LGT frente à condição alcalina (padrão e amostra), determinar a possível citotoxicidade dos produtos de degradação, tanto do fármaco quanto do padrão, em células mononucleares e sugerir os possíveis principais produtos de degradação, além de desenvolver método de dissolução para os comprimidos. Um método simples por cromatografia líquida de fase reversa indicativo de estabilidade foi otimizado e validado de acordo com o International Conference on Harmonization sob os critérios de aceitação como especificidade, linearidade, precisão, exatidão, robustez e adequabilidade do sistema. A coluna analítica C8 (150 mm x 4,6 mm x 5 μm) foi utilizada a 30 °C com uma mistura de água (trietilamina 1,0 %, ajustado para pH 4,5 com ácido ortofosfórico) e de acetonitrila (80:20, v/v), com vazão de 1,2 mL.min-1 e a detecção a 293 nm. Os excipientes não interferiram na determinação da LGT. Além disso, em estudos de degradação forçada, não foi observada interferência no pico do fármaco. A cinética de degradação em meio básico sob aquecimento a 60 °C foi determinada e tanto padrão e amostra seguem cinética de primeira ordem com constante (k) igual a 0,25 h-1 e 0,21 h-1, t ½ igual a 2,77 h e 3,31 h e coeficiente de determinação igual a 0,9153 e 0,9775 para o padrão e amostra, respectivamente. Foram avaliados os danos da membrana celular provocados pela LGT a 100 mg mL-1 e pelos produtos de degradação formados. Nas concentrações avaliadas e na degradação forçada até 50% e 100% dos comprimidos, não foi verificado nenhum dano celular tanto à luz UV-C quanto na condição alcalina. As estruturas moleculares foram sugeridas a partir de análises de espectro de massa para a identificação dos produtos de degradação formados. A LGT foi obtida após 6,3 min de análise e a resposta foi linear na faixa de 1-50 μg mL-1 (r = 0,9998 ), observou-se regressão linear significativa e desvio da linearidade não significativa por meio de análise de variância. O método foi preciso (DPR intradia e interdia < 1,10 %), exatidão (média de recuperação = 98,6%), e robusto. Limite de detecção e limite de quantificação foram de 0,14 e 0,45 mg.mL-1, respectivamente. Para análise biofarmacêutica foi desenvolvido método de dissolução utilizando meio citrato pH 3,5 com aparato cesta, velocidade de agitação de 75 rpm à temperatura de 37 ± 0,5 °C. De acordo com os resultados obtidos, o fármaco pode ser um candidato à bioisenção. As condições propostas foram aplicadas com sucesso para a análise quantitativa da LGT em comprimido, o que ajudará a melhorar o controle de qualidade de medicamentos e contribuir para estudos de estabilidade desta gliptina.Linagliptin (LGT) is drug a member of the class of gliptins that inhibits the enzyme dipeptidyl-peptidase-4. They are used to reduce glucose blood levels in patients with type 2 Diabetes mellitus. Due to its recent development and market launching, LGT has no official compendium monograph, national or international, or available registries for qualitative and quantitative determinations of this drug. The objective of this work was to develop a LC method to characterize LGT in tablet dosage form containing 5 mg, evaluating the kinetics of degradation of LGT (standard and sample), determining the possible cell membrane damage of drug degradation products in mononuclear cells and suggest the possible major degradation products. A simple and stability-indicating reversed-phase liquid chromatography method was optimized and validated according to International Conference on Harmonization acceptance criteria for specificity, linearity, precision, accuracy, robustness and system suitability. A C8 analytical column (150 mm x 4.6 mm x 5 μm) was operated at 30 °C with a mixture of triethylamine 1.0% and acetonitrile (80:20, v/v), adjusted to pH 4.5 with ortophosphoric acid, at a flow rate of 1.2 mL min-1 and detection at 293 nm. The excipients did not interfere in the determination of LGT. Furthermore, in forced degradation studies no interference in the drug peak was observed. The kinetics of degradation in basic medium under heating at 60 °C was determined and standard both standard and sample follows first order kinetic with constant (k) equal to 0,25 h-1 and 0,21 h-1, t½ equal to 2,77 h and 3,31 h and coefficient of determination equal to 0,9153 and 0,9775 for standard and sample, respectively. The cell membrane damage of LGT at 100 μg mL-1 and the formed products by its degradation were evaluated. At the appraised concentration and at forced degradation until 50% and 100% of the tablets, both UV-C light and alkaline conditions, no cell damage was verified. Structures were suggested for identifying the degradation products formed from mass spectral analyzes. The chromatography was obtained within 6.3 min and the response was linear in the range of 1–50 μg mL-1 (r= 0.9998) showed significant linear regression and non-significant linearity deviation by analysis of variance. The method was precise (intra-day relative standard deviation (RSD) and inter-day RSD values < 1.10%), accurate (mean recovery = 98.6%), and robust. Limit of detection and limit of quantitation were 0.14 and 0.45 μg mL-1, respectively. For biopharmaceutical analysis, a dissolution method was developed using citrate medium pH 3.5, basket apparatus, stirring rate of 75 rpm at temperature of 37 ± 0.5 °C. According to the results, the drug may be a biowaiver candidate. The proposed conditions were successfully applied for the quantitative analysis of LGT in tablet dosage form, which will help improving the drug quality control and contribute to stability studies of this gliptin

CHARACTERIZATION OF LINAGLIPTIN USING ANALYTICAL TECHNIQUES

Linagliptin (LGT) is a member of the class of gliptins that inhibit the enzyme dipeptidyl-peptidase-4. They are used to reduce glucose blood levels in patients with type 2 Diabetes mellitus. Due to its recent development and launching on the market, LGT has no official compendium monograph, national or international, or available registries for the qualitative determination of this drug. The objective of this work was to characterize LGT by using thermal techniques, nuclear magnetic resonance, mass and infrared spectrometry, liquid chromatography and ultraviolet spectrophotometry to be used as a chemical reference substance. The range and melting point obtained are in accordance with that described in the literature. The main groups of LGT molecule were observed in infrared spectroscopy and the molecular ion m/z 473.25 ratio was found in mass spectroscopy analysis. In UV spectroscopy, the maximum wavelength absorption of the substance in different solvents can be observed. The chromatographic methods provide selectivity for LGT and can be used to analyze it qualitatively. The proposed conditions have been successfully applied for identification and qualitative analysis of LGT as a chemical reference substance, contributing to studies of this gliptin, and to the quality control of medicines that contain it.Linagliptin (LGT) is a member of the class of gliptins that inhibit the enzyme dipeptidyl-peptidase-4. They are used to reduce glucose blood levels in patients with type 2 Diabetes mellitus. Due to its recent development and launching on the market, LGT has no official compendium monograph, national or international, or available registries for the qualitative determination of this drug. The objective of this work was to characterize LGT by using thermal techniques, nuclear magnetic resonance, mass and infrared spectrometry, liquid chromatography and ultraviolet spectrophotometry to be used as a chemical reference substance. The range and melting point obtained are in accordance with that described in the literature. The main groups of LGT molecule were observed in infrared spectroscopy and the molecular ion m/z 473.25 ratio was found in mass spectroscopy analysis. In UV spectroscopy, the maximum wavelength absorption of the substance in different solvents can be observed. The chromatographic methods provide selectivity for LGT and can be used to analyze it qualitatively. The proposed conditions have been successfully applied for identification and qualitative analysis of LGT as a chemical reference substance, contributing to studies of this gliptin, and to the quality control of medicines that contain it

Evaluation of linagliptin dissolution from tablets using HPLC and UV methods

Linagliptin (LGT) is used to reduce glucose blood levels in patients with type 2 Diabetes mellitus. The proposed conditions for a biowaiver guide can be applied due to high solubility of linagliptin in aqueous media. The aim of the present study was to develop and validate a dissolution test for 5 mg linagliptin coated tablets. After diverse dissolution procedure evaluation, the selected method was achieved using USP apparatus 1 (basket) at 75 rpm and 900 mL of citrate buffer pH 3.5 as dissolution medium. The conditions proposed by biowaiver guide were also applied to this drug, using USP apparatus 2 (paddle) and 900 mL of 0.1 M HCl, pH 4.5 acetate buffer and pH 6.8 phosphate buffer as dissolution medium. HPLC and UV spectrophotometry were used to LGT quantitation and validated for this purpose. The chromatographic and spectrophotometric methods in the dissolution context proved to be linear in 0.5 – 20.0 μg.mL-1 range, precise with RSD values less than 1.0 % and 2.0%, respectively, and accurate (mean recovery 100.29 % and 100.59%). The parameters such as specificity, linearity, accuracy, precision and robustness were according to international guidelines for both methods HPLC and UV. Dissolved linagliptin results were compared using the Student’s t-test and the data found were not significant different (P=0.068). In most dissolution conditions evaluated, LGT presented more than 85% drug dissolved from the tablets in fifteen minutes. The proposed methods can be applied for quality control of this gliptin. According to the results, linagliptin may be a biowaiver candidate

Incidence of severe critical events in paediatric anaesthesia (APRICOT): a prospective multicentre observational study in 261 hospitals in Europe

Background Little is known about the incidence of severe critical events in children undergoing general anaesthesia in Europe. We aimed to identify the incidence, nature, and outcome of severe critical events in children undergoing anaesthesia, and the associated potential risk factors. Methods The APRICOT study was a prospective observational multicentre cohort study of children from birth to 15 years of age undergoing elective or urgent anaesthesia for diagnostic or surgical procedures. Children were eligible for inclusion during a 2-week period determined prospectively by each centre. There were 261 participating centres across 33 European countries. The primary endpoint was the occurence of perioperative severe critical events requiring immediate intervention. A severe critical event was defined as the occurrence of respiratory, cardiac, allergic, or neurological complications requiring immediate intervention and that led (or could have led) to major disability or death. This study is registered with ClinicalTrials.gov, number NCT01878760. Findings Between April 1, 2014, and Jan 31, 2015, 31â127 anaesthetic procedures in 30â874 children with a mean age of 6·35 years (SD 4·50) were included. The incidence of perioperative severe critical events was 5·2% (95% CI 5·0â5·5) with an incidence of respiratory critical events of 3·1% (2·9â3·3). Cardiovascular instability occurred in 1·9% (1·7â2·1), with an immediate poor outcome in 5·4% (3·7â7·5) of these cases. The all-cause 30-day in-hospital mortality rate was 10 in 10â000. This was independent of type of anaesthesia. Age (relative risk 0·88, 95% CI 0·86â0·90; p<0·0001), medical history, and physical condition (1·60, 1·40â1·82; p<0·0001) were the major risk factors for a serious critical event. Multivariate analysis revealed evidence for the beneficial effect of years of experience of the most senior anaesthesia team member (0·99, 0·981â0·997; p<0·0048 for respiratory critical events, and 0·98, 0·97â0·99; p=0·0039 for cardiovascular critical events), rather than the type of health institution or providers. Interpretation This study highlights a relatively high rate of severe critical events during the anaesthesia management of children for surgical or diagnostic procedures in Europe, and a large variability in the practice of paediatric anaesthesia. These findings are substantial enough to warrant attention from national, regional, and specialist societies to target education of anaesthesiologists and their teams and implement strategies for quality improvement in paediatric anaesthesia. Funding European Society of Anaesthesiology

Incidence of severe critical events in paediatric anaesthesia (APRICOT): a prospective multicentre observational study in 261 hospitals in Europe

Background Little is known about the incidence of severe critical events in children undergoing general anaesthesia in Europe. We aimed to identify the incidence, nature, and outcome of severe critical events in children undergoing anaesthesia, and the associated potential risk factors. Methods The APRICOT study was a prospective observational multicentre cohort study of children from birth to 15 years of age undergoing elective or urgent anaesthesia for diagnostic or surgical procedures. Children were eligible for inclusion during a 2-week period determined prospectively by each centre. There were 261 participating centres across 33 European countries. The primary endpoint was the occurence of perioperative severe critical events requiring immediate intervention. A severe critical event was defined as the occurrence of respiratory, cardiac, allergic, or neurological complications requiring immediate intervention and that led (or could have led) to major disability or death. This study is registered with ClinicalTrials.gov, number NCT01878760. Findings Between April 1, 2014, and Jan 31, 2015, 31 127 anaesthetic procedures in 30 874 children with a mean age of 6.35 years (SD 4.50) were included. The incidence of perioperative severe critical events was 5.2% (95% CI 5.0-5.5) with an incidence of respiratory critical events of 3.1% (2.9-3.3). Cardiovascular instability occurred in 1.9% (1.7-2.1), with an immediate poor outcome in 5.4% (3.7-7.5) of these cases. The all-cause 30-day in-hospital mortality rate was 10 in 10 000. This was independent of type of anaesthesia. Age (relative risk 0.88, 95% CI 0.86-0.90; p<0.0001), medical history, and physical condition (1.60, 1.40-1.82; p<0.0001) were the major risk factors for a serious critical event. Multivariate analysis revealed evidence for the beneficial effect of years of experience of the most senior anaesthesia team member (0.99, 0.981-0.997; p<0.0048 for respiratory critical events, and 0.98, 0.97-0.99; p=0.0039 for cardiovascular critical events), rather than the type of health institution or providers. Interpretation This study highlights a relatively high rate of severe critical events during the anaesthesia management of children for surgical or diagnostic procedures in Europe, and a large variability in the practice of paediatric anaesthesia. These findings are substantial enough to warrant attention from national, regional, and specialist societies to target education of anaesthesiologists and their teams and implement strategies for quality improvement in paediatric anaesthesia