Estudo do efeito de estatinas e antiplaquetários na evolução do infarto agudo do miocárdio: uma abordagem metabolômica multiplataform / Effect of statins and antiplatelets in the evolution of acute myocardium infarction: a multiplatform metabolomics approach

O infarto agudo do miocárdio (IAM) é a maior causa de mortalidade no mundo. A oclusão coronária determina a necrose completa de cardiomiócitos (células musculares cardíacas) durante as primeiras horas do IAM. Porém, mesmo após a perda de massa de miocárdio viável cessar, a região infartada pode se expandir ou contrair no decorrer das primeiras semanas, afetando o prognóstico dos pacientes. Alguns tratamentos podem auxiliar na recuperação e melhoria do prognóstico desses pacientes, como o uso de estatinas e antiplaquetários, que quando utilizados em conjunto, proporcionam efeitos sinérgicos. O presente estudo investigou e comparou, através da óptica da metabolômica global multiplataforma, tratamentos concomitantes de estatinas (sinvastatina ou rosuvastatina) e antiplaquetários bloqueadores do receptor de ADP (clopidogrel ou ticagrelor), em pacientes que sofreram IAM. Foram coletadas amostras de plasma e urina de cerca 40 pacientes tratados com clopidrogrel e sinvastatina ou ticagrelor e rosuvastatina no Hospital São Paulo em diferentes períodos (basal, 1 mês e 6 meses após IAM). Amostras de plasma (basal e 1 mês) foram analisadas por RPLC-MS nos modos de ionização positivo e negativo, GC-MS e CEMS. Amostras de urina (basal, 1 mês e 6 meses) foram analisadas por RPLC-MS no modo de ionização positivo e HILIC-MS nos modos de ionização positivo e negativo. A abordagem metabolomica global multiplataforma evidenciou alterações no metabolismo de diferentes vias pelos dois tratamentos. Os dois tratamentos proporcionaram um efeito pronunciado no metabolismo de diferentes lipídios, como glicerolipídios, esfingolipídios, glicerofosfolipídios e ácidos graxos, sendo que a combinação rosuvastatina e ticagrelor resultou num efeito mais acentuado. Já o tratamento com clopidogrel e sinvastatina alterou de maneira mais pronunciada o metabolismo de aminoácidos ramificados e de acilcarnitinas de cadeia curta. Observou-se ainda a alteração de possíveis biomarcadores relatados na literatura como associados a problemas cardiovasculares, como hipoxantina, ácido 2-hidroxibutírico, algumas espécies de ceramidas, fosfatidilcolinas e acilcarnitinas de cadeia curta

cute myocardium infarction (AMI) is the main mortality cause in the world. The coronary occlusion determines the complete necrosis of cardiomyocytes (cardiac muscle cells) during the first hours of AMI. However, even after the loss of viable myocardial mass ceases, the infarcted area may still expand or contract during the first weeks after AMI, affecting the patient prognosis. Some treatments may assist patient recovery and improve prognostic, such as statins and antiplatelets which, when combined, provide synergic effects. This study investigated and compared, by untargeted multiplatform metabolomics, simultaneous treatments of statins (simvastatin or rosuvastatin) and ADP receptor antagonist antiplatelets (clopidogrel or ticagrelor) in patients that suffered AMI. Plasma and urine samples from around 40 patients treated with clopidogrel and simvastatin or ticagrelor and rosuvastatin were collected in Hospital Sao Paulo at different time points (basal, 1 month, 6 months after AMI). Plasma samples (basal and 1 month) were analyzed by RPLC-MS in positive and negative ionization modes, GC-MS and CE-MS. Urine samples (basal, 1 month, 6 months) were analyzed by RPLC-MS in positive ionization mode and by HILIC-MS in positive and negative ionization modes. The untargeted multiplatform metabolomics approach has shown that different metabolic pathways have been altered by the two treatments. Both treatments had a profound impact on the metabolism of different lipids, such as glycerolipids, sphingolipids, glycerophospholipids, and fatty acids. However, the combined treatment using rosuvastatin and ticagrelor impacted the most the lipid pathways. On the other hand, clopidogrel and simvastatin treatment affected more intensily the branched chain amino acids and short chain acylcarnitines metabolisms. Reported biomarkers in the literature related to cardiovascular diseases were also observed in this study, such as hypoxanthine, 2-hydroxybutyric acid, some species of ceramides, phosphatidylcholines and short chain acylcarnitines

UPLC-MS/MS method for the simultaneous quantification of pravastatin, fexofenadine, rosuvastatin, and methotrexate in a hepatic uptake model and its application to the possible drug-drug interaction study of triptolide.

A rapid and specific UPLC-MS/MS method with a total run time of 3.5 min was developed for the determination of pravastatin, fexofenadine, rosuvastatin, and methotrexate in rat primary hepatocytes. After protein precipitation with 70% acetonitrile (containing 30% H2 O), these four analytes were separated under gradient conditions with a mobile phase consisting of 0.03% acetic acid (v/v) and methanol at a flow rate of 0.50 mL/min. The linearity, recovery, matrix effect, accuracy, precision, and stability of the method were well validated. We evaluated drug-drug interactions based on these four compounds in freshly suspended hepatocytes. The hepatic uptake of pravastatin, fexofenadine, rosuvastatin, and methotrexate at 4°C was significantly lower than that at 37°C, and the hepatocytes were saturable with increased substrate concentration and culture time, suggesting that the rat primary hepatocyte model was successfully established. Triptolide showed a significant inhibitory effect on the hepatic uptake of these four compounds. In conclusion, this method was successfully employed for the quantification of pravastatin, fexofenadine, rosuvastatin, and methotrexate and was used to verify the rat primary hepatocyte model for Oatp1, Oatp2, Oatp4, and Oat2 transporter studies. Then, we applied this model to explore the effect of triptolide on these four transporters.

Sustainable environment-friendly quantitative determination of three anti-hyperlipidemic statin drugs and ezetimibe in binary mixtures by first derivative Fourier transform infrared (FTIR) spectroscopy.

FTIR spectrometry is considered a sustainable green analytical chemistry procedure. Its use in quantitative analysis of pharmaceutical compounds in their raw resources and in their dosage forms is growing currently. The current research offers an environment-friendly, speedy, cost-effective, reliable and easy method for the simultaneous estimation of anti-hyperlipidemic drugs. No sample preparation was required except for grinding and mixing with KBr for making pellets used for acquisition of the FT-IR spectra. First-derivative FTIR spectroscopy is used to assess quantitatively atorvastatin (ATR), rosuvastatin (RSV) and simvastatin (SMV) in their binary mixtures with ezetimibe (EZT). For the first mixture, EZT and ATR were determined at 1733.18 cm-1 and 1647.74 cm-1, respectively. In the second mixture, the zero-crossing wave numbers selected for the determination of EZT and RSV were 1733.18 cm-1 and 955.69 cm-1, correspondingly. Whereas, the third mixture was quantified at the wavenumbers of 1520.93 and 3569.68 cm-1 for EZT and SMV, respectively. Validation of the procedure has been performed complying with recommendations of the International Conference of Harmonization (ICH) presenting linearity, accuracy, precision, robustness and selectivity. The linear range for all drugs was 2-30 mg/g. It was found that the LOD was 0.607, 0.311, 0.491 and 0.395 mg/g and the LOQ was found to be 1.839, 0.942, 1.490 and 1.190 mg/g for EZT, ATR, RSV, and SMV, correspondingly. The proposed technique was found to be accurate and precise in terms of percentage error and percentage relative standard deviation among intraday and interday measurements. It was also found selective through comparison of the results of standard drugs with results of binary mixtures and of pharmaceutical tablets. It was found robust through making slight variations in the working conditions and the results obtained remained statistically equivalent. The technique was applied effectively for the estimation of the binary mixtures under study in their tablets. Comparing the found outcomes to those of reference derivative UV spectrophotometric methods gave no significant difference between them. Analytical eco-scale and the scale of Green Analytical Procedure Index (GAPI) are the two scales utilized for evaluation of the greenness of the technique and it was found to be excellent green.

Determination of lovastatin, mevastatin, rosuvastatin and simvastatin with HPLC by means of gradient elution.

A novel HPLC method with UV detection was developed and validated in skin penetration (in vitro) studies to identify and quantify lovastatin, mevastatin, rosuvastatin and simvastatin. A Venusil XBP C18 (2), 150 x 4.6 mm, 5 µm column (Agela Technologies, Newark, DE) was used with gradient elution (start at 45 % acetonitrile and increase linearly to 90 % after 1 min; hold at 90 % until 6 min and then re-equilibrate at start conditions) and the mobile phase consisted of (A) Milli-Q ® water and 0.1% orthophosphoric acid, and (B) HPLC grade acetonitrile. The flow rate was set at 1 ml/min, 240 nm UV detection and an injection volume of 10 µl. Linearity was obtained over a range of 0.50-200.00 µg/ml and correlation coefficients ranging from 0.998-1.000 were obtained. Average recovery ranged from 95.9-100.6 %. The LOD and LOQ values obtained from the slope of a calibration curve and the standard deviation of the response ranged from 0.0138-0.0860 µg/ml and 0.0419-0.2615 µg/ml, respectively, where lovastatin and simvastatin could be detected at a concentration similar to the other statins, but could only be quantified at a higher concentration than the remaining statins. The specificity of the method was proved as accurate and quantification of statins was found, even within the incorporation of other compounds.

Developing spectral numerical factor technique for the determination of amlodipine besylate and the latest generation of statins in their new pharmaceutical combination.

Spectral numerical factor techniques have been developed for quantification of a new pharmaceutical combination. Five simple, fast and accurate spectrophotometric methods using spectral factor manipulation have been tested and validated so as to determine quantitatively a new fixed-dose combination tablet containing both amlodipine besylate (ADB) and rosuvastatin calcium (ROS). Namely, these methods are induced dual wavelength, absorbance correction, absorbance subtraction, amplitude correction and advanced amplitude subtraction. Their corresponding spectral factors are equality, absorption, absorbance, amplitude and unity factor, respectively. Calibration curves of ADB and ROS were set up for all the previously mentioned methods under the optimum conditions in a concentration range of 3.0-21.0 µg/mL with correlation coefficients ≥0.9990. Selectivity was calculated by analyzing laboratory-prepared synthetic blends of the cited drugs. A comparative study between these methods and the reported ones has been carried out statistically forming a judgment that the difference between both results is totally insignificant. Actually, the suggested spectral factor technique is so effective for use in routine laboratory essay owing to its simplicity, rapidity and precision. Generally, the proposed factor procedures have the ability to run smoothly without any interference from additives that may deteriorate analysis efficiency of a pharmaceutical combination.

Evaluating the discriminatory power of a dissolution assay for rosuvastatin calcium capsules: Solid-state properties and dissolution media

We propose to evaluate the dissolution properties of rosuvastatin calcium (ROSC) capsules in different media to characterize the discriminatory power of the assay method. Dissolution assays were performed in media with different pH, and including the surfactant sodium dodecyl sulfate (SDS). Several immediate-release formulations were manufactured using the commercial raw material characterized as amorphous solid. The hydrophobic adjutant magnesium stearate was employed in some formulations due to its negative effect in the wettability and dissolution efficacy of solid dosages. These formulations showed the lower dissolution efficacy values in media without surfactant; however, when SDS was added to the medium, the dissolution efficacy increased, and the discriminatory power was lost. In spite of micellar solubilization does not increase the ROSC solubility, it modifies the discriminatory power of the assay method, increasing the wettability of the powder mixtures. The crystalline form M of ROSC was recrystallized in our laboratory, and it showed lower solubility in water than amorphous solid. However, its dissolution properties were not influenced by SDS. These results are important to develop dissolution assays for other hydrophilic drugs with increased water solubility, once that dissolution media with surfactants increase the wettability of the formulations, leading to an overrated dissolution rate.

Transfer of rosuvastatin into breast milk: liquid chromatography-mass spectrometry methodology and clinical recommendations.

INTRODUCTION: Rosuvastatin reduces concentrations of total cholesterol (TC) and is used for the management of hypercholesterolemia and prevention of acute coronary syndromes. There are no published reports estimating infant exposure to rosuvastatin through breast milk. PURPOSE: The aims of this study were to quantify concentrations of rosuvastatin in human milk and plasma from a lactating woman taking rosuvastatin and to investigate potential infant exposure. MATERIALS AND METHODS: A 38-year-old breastfeeding mother was commenced on rosuvastatin 20 mg daily for secondary prevention of an acute coronary syndrome. Eight maternal breast milk samples and a single plasma sample were collected over a 24-hour period. The samples were quantified using a sensitive liquid chromatography-mass spectrometry (LC-MS/MS) method. RESULTS: The average concentration of rosuvastatin in breast milk was 30.84 ng/mL, and a peak concentration of 58.59 ng/mL occurred at 17 hours after oral administration. Although the milk-to-plasma (M/P) ratio was 16.49 at 14 hours, the theoretical infant dosage (TID) and relative infant dose (RID) were 0.005 mg/kg/day and 1.50%, respectively. CONCLUSION: The findings suggest that only small amounts of rosuvastatin pass into breast milk. Should the maternal condition necessitate treatment, consideration could be given to the use of rosuvastatin during breastfeeding provided the infant is monitored.

High performance liquid chromatographic method validation for determination of rosuvastatin calcium in tablet dosage forms.

A precise, sensitive and quick High Performance Liquid Chromatographic (HPLC) method for the determination of rosuvastatin calcium in bulk and tablet dosage forms has been validated. The chromatographic scheme involved: Sil-20A auto sampler, LC-20A pump, SPD-20A UV/visible detector with separation attained by C18 column at 40ºC temperature through a mobile phase of acetonitrile and buffer (50:50) at a flow rate of 1.0ml/min. The method is precise (%RSD for intra-day and inter-day extended between 1.06-1.54% and 0.103-1.78%) and linear (r2=0.9997). Limit of detection and quantification (LOD & LOQ) of the adopted method were 0.78 and 1.56µg/ml. The proposed HPLC method was established to be sensitive, precise and swift that can be proficiently adopted in quality control/quality assurance laboratories for predictable investigation of the bulk and oral solid dosage forms of rosuvastatin calcium.

Development and validation of a hydrophilic interaction liquid chromatography method for the quantitation of impurities in fixed-dose combination tablets containing rosuvastatin and metformin.

A hydrophilic interaction liquid chromatography method with diode array detection (HILIC-DAD) was developed and validated for the simultaneous determination of impurities in extended-release fixed-dose combination tablets containing rosuvastatin and metformin in a ratio 1:100. The analytes were separated by hydrophilic interaction liquid chromatography using an XBridge®-HILIC analytical column under isocratic elution. The mobile phase was composed of ammonium formate at 150 mM containing 0.05% diethylamine (pH 8.5)/acetonitrile, 4/96 (v/v) and pumped at a flow rate of 0.5 mL min-1. Method validation was performed according to ICH guidelines. The calibration curves for rosuvastatin, metformin and their seven impurities showed good linearity (r > 0.994) within the calibration ranges tested. The intra- and inter-day R.S.D. values were less than 4.5%, while the relative percentage error Er was less than 2.7% for all compounds. Accelerated stability studies performed under various stress conditions including hydrolysis, oxidation and heat proved the selectivity of the procedure. A run time of less than 25 min for each sample made it possible to analyze a large number of samples per day. The method is the first reported application of HILIC for the analysis of impurities in fixed-dose combination tablets containing rosuvastatin and metformin and it can be used for the quality control of these drugs.

Simultaneous determination of rosuvastatin and propranolol in their binary mixture by synchronous spectrofluorimetry.

Simultaneous determination of rosuvastatin calcium and propranolol hydrochloride using the first derivative synchronous spectrofluorimetry was described. This method involves measuring the synchronous fluorescence of both drugs in ethanol using, ∆ λ = 60 nm then the first derivative was recorded and the peak amplitudes were measured at 350 and 374 nm for rosuvastatin calcium and propranolol hydrochloride, respectively. Under the optimum conditions, the linear ranges of rosuvastatin calcium and propranolol hydrochloride were 0.2-2 µg/mL and 0.1-1 µg/mL, respectively. The method was used for quantitative analysis of the drugs in raw materials and pharmaceutical dosage form. The validity of the proposed method was assessed according to an international conference on harmonization (ICH) guidelines.

Analytical Methods for the Determination of Rosuvastatin in Pharmaceutical Formulations and Biological Fluids: A Critical Review.

Rosuvastatin calcium (ROS), ( Figure 1 ) belongs to the "statins" group, which is the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor. This drug is indicated for dyslipidemias treatment and can help to decrease the level of "bad cholesterol" and can consequently reduce the development of atherosclerosis and the risk of heart diseases. ROS was developed by Astra-Zeneca and it was approved in 2003 by the FDA in the United States. In 2015, under the trade name Crestor®, it was the fourth largest selling drug in the United States with sales above $5 billion. This study presents a literature review of analytical methods for the quantification of ROS in pharmaceutical preparations and biological fluids. The major analytical methods described in this study for ROS were spectrophotometry, high-performance liquid chromatography (HPLC) coupled to ultraviolet (UV) detection, and tandem mass spectrometry (LC-MS/MS).

Stability of extemporaneously prepared rosuvastatin oral suspension.

PURPOSE: The stability of an extemporaneously prepared rosuvastatin suspension stored over 30 days under various storage conditions was evaluated. METHODS: Rosuvastatin suspension was extemporaneously prepared using commercial rosuvastatin tablets as the source of active pharmaceutical ingredient. The organoleptic properties, dissolution profile, and stability of the formulation were investigated. For the stability studies, samples of the suspension were stored under 2 storage conditions, room temperature (25 °C and 60% relative humidity) and accelerated stability chambers (40 °C and 75% relative humidity). Viscosity, pH, organoleptic properties, and microbial contamination were evaluated according to the approved specifications. High-performance liquid chromatography was used for the analysis and quantification of rosuvastatin in selected samples. Microbiological investigations were also conducted. RESULTS: The prepared suspension showed acceptable organoleptic properties. It showed complete release of rosuvastatin within 15 minutes. The pH of the suspension was 9.8, which remained unchanged during the stability studies. The microbiological investigations demonstrated that the preparation was free of any microbial contamination. In addition, the suspension showed stability within at least the period of use of a 100-mL rosuvastatin bottle. CONCLUSION: Extemporaneously prepared rosuvastatin 20-mg/mL suspension was stable for 30 days when stored at room temperature.

Spectrophotometric determination of rosuvastatin in pharmaceutical formulations using quinalizarin

ABSTRACT This work presents the development of a methodology based on the formation of a charge transfer complex between quinalizarin and rosuvastatin, allowing for the spectrophotometric determination of rosuvastatin at 579 nm. The factors involved in the sensitivity of the technique were studied (nature and proportion of the solvent, reaction time, pH of aqueous phase and quinalizarin concentration). The proposed spectrophotometric procedures were validated with respect to linearity, ranges, precision, accuracy, detection and quantification limits. Calibration curves of the formed color products showed good linear relationships over the concentration range of 6-15 mg L-1. The proposed method has been successfully applied, which can be confirmed by interference test (comparison between the standard curves and addition of analyte), method precision (RSD 2.3% to 6 mg L-1), and by accuracy (statistically equivalent results between the proposed method and a chromatographic method of reference).

Optimized Hepatocyte-Like Cells with Functional Drug Transporters Directly-Reprogrammed from Mouse Fibroblasts and their Potential in Drug Disposition and Toxicology.

BACKGROUND/AIMS: To develop a suitable hepatocyte-like cell model that could be a substitute for primary hepatocytes with essential transporter expression and functions. Induced hepatocyte-like (iHep) cells directly reprogrammed from mice fibroblast cells were fully characterized. METHODS: Naïve iHep cells were transfected with nuclear hepatocyte factor 4 alpha (Hnf4α) and treated with selected small molecules. Sandwich cultured configuration was applied. The mRNA and protein expression of transporters were determined by Real Time PCR and confocal. The functional transporters were estimated by drug biliary excretion measurement. The inhibition of bile acid efflux transporters by cholestatic drugs were assessed. RESULTS: The expression and function of p-glycoprotein (P-gp), bile salt efflux pump (Bsep), multidrug resistance-associated protein 2 (Mrp2), Na+-dependent taurocholate cotransporting polypeptide (Ntcp), and organic anion transporter polypedtides (Oatps) in iHep cells were significantly improved after transfection of hepatocyte nuclear factor 4 alpha (Hnf4α) and treatment with selected inducers. In vitro intrinsic biliary clearances (CLb,int) of optimized iHep cells for rosuvastatin, methotrexate, d8-TCA (deuterium-labeled sodium taurocholate acid) and DPDPE ([D-Pen2,5] enkephalin hydrate) correlated well with that of sandwich-cultured primary mouse hepatocytes (SCMHs) (r2 = 0.984). Cholestatic drugs were evaluated and the results were compared well with primary mice hepatocytes. CONCLUSION: The optimized iHep cells expressed functional drug transporters and were comparable to primary mice hepatocytes. This study suggested direct reprogramming could provide a potential alternative to primary hepatocytes for drug candidate hepatobiliary disposition and hepatotoxicity screening.

Simultaneous estimation of rosuvastatin and amlodipine in pharmaceutical formulations using stability indicating HPLC method

A viable cost-effective and isocratic approach employing C-18 column (250 mm × 4.6 mm, 5 µm) based HPLC has been utilized to separate and estimate the drugs, rosuvastatin, amlodipine and their stress induced degradation products, simultaneously in pharmaceutical formulations. Focused on ICH guideline parameters, the efficient separation of both drugs and their degradation products was achieved by optimizing a 30:70 (v/v) solvent mixture of acetonitrile and 0.1 M ammonium acetate buffer (pH 5) as mobile phase. The flow rate of the mobile phase was 1.5 mL/min and all the detections were carried out at 240 nm using UV detector. The method was linear in the concentration range of 1-200 µg/mL for rosuvastatin with 0.996 coefficient of determination value. For amlodipine, linearity was in the range of 0.5-100 µg/ml with 0.994 coefficient of determination value. Both the drugs along with their degradation products were separated in less than twenty minutes. The results of within-day and between-day precision were varied from 0.72 to 1.81% for rosuvastatin and 0.83 to 1.88% for amlodipine. The results show that this ICH validated method can be employed successfully for the routine as well as stability quantification of both the active ingredients simultaneously in pharmaceutical formulations.

Utilizou-se abordagem de viabilidade custo-efetividade e isocrática, baseada em CLAE, empregando coluna C-18 (250 mm x 4,6 mm, 5 µm) para separar e avaliar os fármacos, rosuvastatina, anlodipino e seus produtos de degradação induzida por estresse, simultaneamente, em formulações farmacêuticas. Focada nos parâmetros das diretrizes da ICH, a separação eficiente de ambos os fármacos e de seus produtos de degradação foi obtida por meio da otimização da fase móvel com mistura de solventes 30:70 (v/v), respectivamente, acetonitrila e tampão acetato de amônio O,1 M (pH 5). A velocidade de fluxo da fase móvel foi de 1,5 mL/min e todas as detecções foram realizadas em 240 nm, utilizando detector de UV. O método foi linear no intervalo de concentração de 1-200 µg/mL para a rosuvastatina com coeficiente de determinação 0,996. Para o anlodipino, a linearidade ficou na faixa de 0.5-100 µg/mL, com coeficiente de determinação 0,994. Ambos os fármacos, junto com seus produtos de degradação, foram separados em menos de vinte minutos. Os resultados de precisão intra-dia e inter-dia variaram de 0,72 a 1,81% para a rosuvastatina e de 0,83 a 1,88%, para o anlodipino. Os resultados mostram que este método validado pelo ICH pode ser empregado com sucesso tanto para a rotina quanto para a quantificação simultânea da estabilidade de ambos os ingredientes ativos em formulações farmacêuticas.