Development, physicochemical evaluation, and in vivo permeation studies of topical formulations containing 0.1% tacrolimus

Abstract The objective of this paper was to develop and evaluate two semi-solid pharmaceutical forms containing 0.1% tacrolimus: cream (CRT01) and gel (GLT01). For the evaluation of physicochemical stability, at times 0, 30, 60 and 90 days, at 23°C and at 40°C, High Performance Liquid Chromatography coupled with a Diode Array Detector (HPLC-DAD) was employed. This method was developed and validated for tacrolimus quantification. The occlusivity test and skin permeation assay were also performed, using an animal model (Wistar rats), and the CRT01 and GLT01 were compared to the 0.1% tacrolimus ointment (PFU01) obtained from the University Pharmacy, Federal University of Rio de Janeiro, Brazil. CRT01 and GLT01 presented a homogeneous aspect and consistency adequate for topical products, along with sensory characteristics above PFU01. They also presented adequate physicochemical stability for 90 days and a lower occlusive effect than PFU01 (p<0.05). CRT01 showed greater affinity for the skin when compared to PFU01 and GLT01, with low systemic absorption. The CRT01 semi-solid formulation was considered the most adequate one to treat patients with atopic dermatitis or other dermatologic inflammatory diseases, promoting rational use of tacrolimus

Algae Metabolites as an Alternative in Prevention and Treatment of Skin Problems Associated with Solar Radiation and Conventional Photo-protection

Abstract The histological structure and biochemistry of the skin is affected by solar radiation having adverse effects ranging from sunburns, premature aging that includes wrinkles, spots, dryness, and loss of collagen to cancer development. The skin has defense mechanisms to prevent damage caused by radiation, but when radiation exposure is excessive these mechanisms are not strong enough to protect the skin. The use of sunscreen is the most common practice of photo- protection. The active ingredients of these cosmetic protective formulations are generally from synthetic origin and have presented several drawbacks at the level of photo-stability, systemic absorption and can generate contact and photo-contact dermatitis. This review illustrates skin solar radiation problems, common sunscreen ingredients limitation and mentions how algae can be an alternative according to studies that have evaluated the photo-protective potential of extracts and compounds isolated by different techniques.

Systemic absortion and adverse effects of topical ocular use of ketorolac tromethamine and sodium diclofenac in New Zealand rabbits for 90 days / Absorção sistêmica e efeitos adversos do uso tópico ocular por 90 dias de cetorolaco de trometamina e diclofenacode sódio em coelhos da raça Nova Zelândia

The effect of the systemic absorption of 0.1% diclofenac sodium (DS) eyedrop was compared to that of 0.5% ketorolac tromethamine (KT) in female New Zealand white rabbits treated on both eyes three times a day for 90 days. The rabbits were divided in three groups of six animals (n= 18): KT group, DS group, and control (Co) group, in which saline (0.9% NaCl) solution was instilled. Water and food consumption were measured daily, clinical examination was performed weekly, and blood samples were collected every 30 days for laboratory examination. The plasma was analyzed for the presence of KT and DS by solid-phase extraction (SPE) associated with mass spectrometry (MS). Systemic absorption of these drugs was confirmed by SPE-MS, allowing their separation and identification in the plasma. At the end of the treatment, the animals were euthanized and necropsied, and no macroscopic or microscopic changes were found. This observation confirmed the laboratory results, which were within normal reference standards for the species. According to the results obtained, it can be concluded that treatment with eyedrops containing KT and DS for 90 days in healthy rabbits does not cause adverse systemic effects.(AU)

Comparou-se o efeito da absorção sistêmica do colírio de diclofenaco de sódio 0,1% (DS) em relação ao de cetorolaco de trometamina 0,5% (CT) em coelhas da raça Nova Zelândia, tratadas nos dois olhos, três vezes ao dia, por 90 dias. As coelhas foram separadas em três grupos de seis animais (n=18): grupo CT, grupo DS e grupo controle (Co), no qual foi instilada solução fisiológica (NaCl 0,9%). Os consumos de água e ração foram mensurados diariamente, os exames clínicos foram realizados semanalmente e o sangue foi coletado a cada 30 dias para realização de exames laboratoriais. O plasma foi analisado para detectar a presença de CT e DS por extração em fase sólida (SPE) associada à espectrometria de massas (MS). A absorção sistêmica desses fármacos foi confirmada por SPE-MS, permitindo sua separação e identificação no plasma. Ao final do tratamento, os animais foram eutanasiados e necropsiados, e não foram encontradas alterações macroscópicas ou microscópicas. Essa observação confirmou os resultados laboratoriais, que estavam dentro dos padrões de referência para a espécie. De acordo com os resultados obtidos, pode-se concluir que o tratamento com colírio contendo KT e DS, por 90 dias, em coelhos saudáveis, não causa efeitos adversos sistêmicos.(AU)

Timing and placement of cattle manure and/or gliricidia affects cotton and sunflower nutrient accumulation and biomass productivity

ABSTRACT Organic fertilizers are a viable alternative to increase oilseed productivity in family agriculture systems. The study aimed to evaluate the effects of timing and placement of cattle manure and/or gliricidia (Gliricidia sepium Jacq. Walp) prunings on cotton (Gossipium hirsutum L.) and sunflower (Helianthus annuus L.) nutrient accumulation and biomass productivity. Experiments were carried out in 2010 and 2011 in Taperoá, Paraíba, Brazil. The organic fertilization treatments were: GI - gliricidia incorporated before planting; GS - gliricidia applied on surface 45 days after planting (DAP); MI + GI - manure and gliricidia incorporated before planting; MI + GS - manure incorporated before planting and gliricídia applied on the surface 45 DAP; MI - manure incorporated before planting; and T - with no organic fertilization. In 2010, treatment MI + GS increased N, P, and K accumulation in cotton (12 and 7 kg ha-1) as well as in sunflower (20 and 29 kg ha-1). In 2011, GI and GS treatments resulted in higher N, P, K accumulations in both crops. The highest cotton productivity in 2010 was obtained with MI + GS treatment (198 kg ha-1) and in 2011 with GS treatment (594 kg ha-1). For sunflower, MI + GS treatment yielded the highest productivity in 2010 (466 kg ha-1) and GI treatment in 2011 (3542 kg ha-1). GI and MI + GS treatments increased total biomass productivity for cotton and sunflower. The treatment that combined both cattle manure incorporated into the soil before planting and gliricidia applied on the surface 45 days after planting was the most viable management strategy.

Pharmacokinetics of monoclonal antibodies and Fc-fusion proteins

There are many factors that can influence the pharmacokinetics (PK) of a mAb or Fc-fusion molecule with the primary determinant being FcRn-mediated recycling. Through Fab or Fc engineering, IgG-FcRn interaction can be used to generate a variety of therapeutic antibodies with significantly enhanced half-life or ability to remove unwanted antigen from circulation. Glycosylation of a mAb or Fc-fusion protein can have a significant impact on the PK of these molecules. mAb charge can be important and variants with pI values of 1-2 unit difference are likely to impact PK with lower pI values being favorable for a longer half-life. Most mAbs display target mediated drug disposition (TMDD), which can have significant consequences on the study designs of preclinical and clinical studies. The PK of mAb can also be influenced by anti-drug antibody (ADA) response and off-target binding, which require careful consideration during the discovery stage. mAbs are primarily absorbed through the lymphatics via convection and can be conveniently administered by the subcutaneous (sc) route in large doses/volumes with co-formulation of hyaluronidase. The human PK of a mAb can be reasonably estimated using cynomolgus monkey data and allometric scaling methods.

Bioacessibilidade dos polifenóis do jatobá-do-cerrado (Hymenaea Stigonocarpa Mart. ) e seus efeitos em genes relacionados à absorção de glicose em células Caco-2 / Bioaccessibility of polyphenols from jatobá-do-cerrado (Hymenaea stigonocarpa Mart. ) and its effects on genes related to glucose uptake in Caco-2 cells

Introdução: O diabetes mellitus (DM) está associado a complicações que comprometem a qualidade de vida e a sobrevida dos indivíduos. Além disso, acarreta elevados custos para o controle metabólico e o tratamento de suas complicações, sendo assim caracterizado como um problema de saúde pública. A regulação da digestão e da absorção intestinal dos carboidratos, com vista a manter a homeostase da glicose plasmática, constituem importantes estratégias de proteção em condições clínicas como o diabetes tipo 2 (DM2), obesidade e síndrome metabólica. Os compostos fenólicos compreendem um grupo complexo de fitoquímicos bioativos presentes nos vegetais. Estudos in vitro e in vivo têm demonstrado que os compostos fenólicos inibem a atividade de carbohidrases (α-amilase e α-glicosidase) e o transporte intestinal de glicose mediado pelos transportadores SGLT1 e GLUT2. O cerrado brasileiro compreende uma larga biodiversidade, porém, apesar de muitas espécies terem sido identificadas, o seu potencial nutritivo e funcional ainda é pouco conhecido. Dentre estas espécies nativas é destacado o jatobá-do-cerrado. O jatobá-do-cerrado é uma leguminosa nativa brasileira, cuja a polpa farinácea que envolve suas sementes apresenta quantidades significativas de compostos fenólicos, podendo ter um potencial efeito sobre o metabolismo da glicose. Objetivos: Verificar os efeitos dos compostos fenólicos da farinha de jatobá-do-cerrado na digestão de carboidratos e na captação de glicose em células intestinais Caco-2. Metodologia: Os compostos fenólicos da farinha de jatobá foram obtidos por extração sequencial com as soluções de etanol (60%) e acetona (70%). Em seguida, o extrato foi digerido utilizando enzimas (α-amilase, pepsina e pancreatina) em pH fisiológico. Os compostos fenólicos presentes no extrato antes e após a digestão foram identificados por cromatografia líquida de ultra performance - espectrômetro de massas (UPLC-MS/MS). Foi avaliada a capacidade de inibição dos extratos de jatobá digeridos em relação à atividade das enzimas α-amilase e α-glicosidase. Células intestinais Caco-2 foram incubadas com diferentes concentrações (0,05 mg/mL - 0,1 mg/mL) de extratos de farinha de jatobá digeridos em diferentes tempos (30 min, 2h e 12 h) para a avaliação da captação de glicose e da expressão gênica dos transportadores de glicose SGLT1 e GLUT2. Resultados: 44 compostos fenólicos foram identificados, dentre eles, a principal classe presente são os flavonoides. Compostos como o ácido cafeico, o kaempferol, quercetina-3- rutinosideo e a quercetrina estavam presentes no extrato antes da digestão. O conteúdo de compostos fenólicos do extrato foi reduzido após a digestão, entretanto o mesmo ainda apresentou compostos de relevância biológica como o ácido p-cumárico, ácido 3-o-feruloilquinico, theaflavina, crisina e grandinina que já apresentaram efeito positivo sobre o metabolismo da glicose in vitro em outros trabalhos. Os extratos fenólicos de jatobá após a digestão in vitro inibiram significativamente a atividade das enzimas α-amilase (76 e 91%) e α- glicosidase (53 e 77%). Os extratos também demonstraram inibir significativamente tanto a captação de glicose independente de sódio quanto a expressão gênica dos transportadores de glicose SGLT1 e GLUT2 de maneira dose-dependente. Conclusão: Este é o primeiro trabalho que identificou os compostos fenólicos presentes na farinha de jatobá. A partir do exposto, podemos concluir que a farinha de jatobá apresenta potencial benefício a saúde devido ao seu conteúdo de compostos fenólicos e a capacidade destes compostos de regular a digestão e a absorção de carboidratos in vitro

Introduction: Diabetes mellitus (DM) is associated with complications that decrease the quality of life and survival of individuals. In addition, it entails high costs for metabolic control and treatment of its complications, thus being characterized as a public health problem. The regulation of digestion and intestinal absorption of carbohydrates to maintain plasma glucose homeostasis are important strategies for protection in chronic diseases such as type 2 diabetes (DM2), obesity and metabolic syndrome. Phenolic compounds are a complex group of chemical substances present in plants. In vitro and in vivo studies have shown that phenolic compounds are able to inhibit the activity of carbohydrases (α-amylase and α-glycosidase) and the intestinal transport of glucose mediated by SGLT1 and GLUT2 transporters. Brazilian Cerrado present a large biodiversity, but although many species have been identified, its nutritional and functional potential is still little known. Among these native species is the jatobá-docerrado. Jatobá-do-cerrado is a brazilian native legume, whose farinaceous pulp that surrounds its seeds presents significant amounts of phenolic compounds and may have a potential effect on glucose metabolism. Objectives: To verify the effects of phenolic compounds from jatobá-do-cerrado flour in the digestion of carbohydrates and uptake of glucose in Caco-2 intestinal cells. Methods: Phenolic compounds of jatobá flour were obtained by sequential extraction with olutions of ethanol (60%) and acetone (70%). The extract was digested using enzymes (α-amylase, pepsin and pancreatin) at physiological pH. The phenolic compounds present in the extract before and after the digestion were identified by liquid chromatography of ultra-performance - mass spectrometer (UPLCMS / MS). The ability of inhibition of the extracts of jatobá digested in relation to the activity of α-amylase and α-glycosidase enzymes was evaluated. Caco-2 intestinal cells were incubated with different concentrations of jatobá flour extracts (0.1 mg / mL - 0.05 mg / mL) for different time (30 min, 2 h and 12 h) to the evaluation of facilitated uptake (sodium-free buffer) and gene expression of SGLT1 and GLUT2 glucose transporters. Results: 44 phenolic compounds have been identified, among them a major class present are flavonoids. Compounds such as caffeic acid, quercetin-3-rutinoside and quercetrine were present in the extract before in vitro digestion. The content of phenolic compounds of the extract after digestion was reduced. However, the extract presents compounds with biological activity such as p-coumaric acid, 3-o-feruloylquinic acid , theaflavin, chrysin and grandinine, which already presented positive effects on glucose metabolism in vitro in other studies. Phenolic extracts of jatobá after in vitro digestion inhibited the activity of α-amylase (76 and 91%) and α-glycosidase (53 and 77%). The extracts also shown to inhibit both glucose uptake and gene expression of glucose transporters SGLT1 and GLUT2 in a dose-dependent manner. Conclusion: This is the first work that identified the phenolic compounds present in jatobá flour. Thus, we can conclude that the jatobá flour presents potential health benefit by modulate digestion and the absorption of carbohydrates in vitro

Biocerâmica de fosfato de cálcio nanoestruturada micro-macroporosa em grânulos de absorção rápida no preenchimento de defeito crítico em rádio de coelhos (Oryctolagus cuniculus) / Bioceramics of calcium phosphate nano-structured micro-macro porous granules rapidly absorbed in filling critical radial defect in rabbits (Oryctolagus cuniculus)

O objetivo do presente trabalho foi avaliar, por radiografia, histologia e densitometria óssea, o efeito da HA/ßTCP em grânulos de absorção rápida em defeito ósseo crítico em rádio de coelhos. Foram utilizados 35 coelhos machos, da raça Nova Zelândia, e realizou-se um defeito crítico nos rádios direito e esquerdo. Os animais foram distribuídos em GI, enxerto autólogo e GII, HA/ßTCP em grânulos de absorção rápida. Avaliações radiográficas foram feitas antes da cirurgia, após, aos oito, 15, 30, 45 e 60 dias e avaliações histológicas e de densitometria. Verificou-se diferença significativa ao se comparar a densidade mineral óssea obtida ao longo do tempo de estudo. Observou-se formação de rede vascular entre os poros da biocerâmica desde o primeiro tempo de avaliação, (oito dias). Foram observados tecido ósseo primário e trabéculas em tecido ósseo preexistente a partir de 30 dias da implantação. Aos 60 dias, constatou-se presença de matriz óssea em segmentos ósseos preexistentes, caracterizando a formação óssea centrípeta. A biocerâmica HA/ßTCP nanoestruturada micro-macroporosa em grânulos de absorção rápida não causa alterações microscópicas indicativas de rejeição, permite a invasão e a multiplicação celular, bem como propicia a regeneração óssea, constituindo um implante apropriado para preenchimento de falhas ósseas críticas.(AU)

The objective of this study was to evaluate the effect of HA/ ßTCP on rapid absorption granules in rabbit radiography, histology, and bone densitometry. Thirty - five male rabbits of the New Zealand breed were used and a critical defect was performed on the right and left radios. The animals were distributed in GI, autologous graft and GII HA / ßTCP in rapidly absorbed granules. Radiographic, histological, and densitometry evaluations were performed before surgery, then after eight, 15, 30, 45 and 60 days. A significant difference was found when comparing the bone mineral density obtained over the study time. Formation of vascular network between the bioceramic pores was observed by the first evaluation time, (eight days). Primary bone tissue and trabeculae were observed from preexisting bone tissue after 30 days of implantation. At 60 days, the presence of bone matrix was observed from the preexisting bone segments, characterizing the centripetal bone formation. The micro-macroporous nanocomposite HA / ßTCP of rapidly absorbing granules do not cause microscopic changes indicative of rejection, allows invasion, cell multiplication, and promotes bone regeneration, constituting an appropriate implant for filling of critical bone failures.(AU)

Seropharmacological study on osteogenic effects of post-absorption ingredients of an osteoprotective herbal formula / 中国结合医学杂志

<p><b>OBJECTIVE</b>To further investigate the {ptin vitro} effects of an osteoprotective herbal formula "ELP" (Herba Epimedii, Fructus Ligustri Lucidi and Fructus Psoraleae) using seropharmacological approach.</p><p><b>METHODS</b>Rats were fed with ELP or its individual component herbs for 2 days. The serum containing the postabsorbed ingredients of the herbal items were collected for cell culture using UMR106 cell, RAW264.7 cell and mesenchymal stem cell (MSC) isolated from the bone marrow of the rats. The effects of the herbal-containing serum on cell toxicity were detected by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay; bromodeoxyuridine assay was conducted to measure the cell proliferation of UMR106 cell and MSC; cell activity was measured using colorimetric method, and mRNA expression of runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and osteopontin (OPN) of UMR106 and MSC as well as matrix metalloproteinase 9 (MMP-9), tartrate-resistant acid phosphatase (TRAP) and cathepsin K of RAW264.7 were analyzed using real-time reverse-transcription polymerase chain reaction.</p><p><b>RESULTS</b>ELP and its component serum exhibited no cytotoxic effects on the cells. The ELP-containing serum increased the proliferation of UMR106 cell and MSC by 25.7% and 14.4 %, respectively and the alkaline phosphatase activity of MSC was increased by 42.6%. On the contrary, it inhibited the RAW264.7 cell differentiation by 29.2 %. ELP serum upregulated the Runx2 expression of UMR and MSC by 1.18 fold and 1.27 fold, respectively. It also upregulated ALP and OPN expression in MSC by 1.69- and 2.12-fold, respectively. On the other hand, ELP serum down-regulated MMP-9 and cathepsin K expression of RAW264.7 cell by 0.46- and 0.36-fold, respectively.</p><p><b>CONCLUSIONS</b>The serum of the animals fed with ELP contains active ingredients which are effective in promoting osteogenesis and inhibiting osteoclastogenesis.</p>

The measurement of serum corticosteroid level in atopic dermatitis patients associated with application of topical corticosteroids

PURPOSE: Most of the atopic dermatitis (AD) patients and their parents refuse topical treatment because of concern about generalized side effect due to systemic absorption of topical corticosteroids. Therefore, a large number of studies reported difficulty in properly controlling in AD. However, investigations of the percutaneous absorption of topical corticosteroids are still insufficient. METHODS: One hundred nine patients who visited our atopy clinic and diagnosed as AD by a physician from January 2005 to January 2012 were enrolled. We examined serum corticosteroid (clobetasol propionate, hydrocortisone) level by liquid chromatography (LC) coupled with a tandem mass spectrometric (MS/MS) method. RESULTS: We developed the LC-MS/MS method to determine corticosteroids (clobetasol propionate, hydrocortisone) in sera of AD patients. Also, we confirmed precision, accuracy, limit of detection, limit of quantification, absolute recovery, and relative recovery of the experimental methods. We could not detect clobetasol propionate or hydrocortisone in sera of 109 AD patients using the newly developed LC-MS/MS method. CONCLUSION: Regardless of age, the severity and illness duration of AD, clobetasol and hydrocortisone were not detected in sera. Although there are many other factors of determining systemic absorption of topical medications, our results showed that topical corticosteroids applied for several years in AD patients may be under the limit of detection in their sera by the LC-MS/MS method.

Chromium oxide (51Cr2O3 ) used as biological marker was not absorbed by fish / Óxido de crômio (51Cr 2O3 ), usado como marcador biológico, não é absorvido por peixe

The aim of this study was to evaluate the rate absorption of radio-labeled chromium oxide (51Cr2O3), used as biological marker in nutrition studies with Nile tilapia Oreochromis niloticus. An experimental diet with approximately 58 µCi of specific activity of the element was encapsulated and fed daily to 35 adult Nile tilapia; a group of 35 fish was used as control feeding on a basal diet. At the beginning of the experiment five fish from each group were randomly selected and blood samples were drawn from control (BC) and experimental fish (BE). Fish were then euthanized by anesthetic overdoses and samples of the liver tissue (LT), renal tissue (RT), stomach without content (S), intestine without content (I), gills tissue (GT), muscle tissue (fillet; MT), visceral fat (VF), content of the digestive tract (CTDE) and water aquarium were collected from the experimental fish. The procedure was repeated daily for one week. Simple linear regressions were adjusted - days of collection vs. determination coefficients, and were established for statistical comparisons of the measured activity of 51Cr readings in sampled blood and tissues (logarithmic transformation) for samples of the control and experimental fish. No differences (P>0.05) were detected between samples from BC fish and BE, RT, VF, MT and LT of treated fish, but samples of GT, I, S, CTDE and WA from the tanks holding fish which received the experimental diet differed from control (P<0.05). The experimental results indicate that the trivalent chromium in the form of 51Cr2O3 was not significantly absorbed by the gastrointestinal tract, gills or another possible route of absorption under these experimental conditions and with Nile tilapia. Therefore, this marker was shown to be inert and can be safely used in nutrition studies.

O objetivo deste estudo foi avaliar a taxa de absorção de radiomarcador óxido de crômio (51Cr2O3), utilizado como marcador biológico em estudos de nutrição, com tilápia-do-nilo Oreochromis niloticus. Uma dieta experimental com cerca de 58µCi de atividade específica do elemento foi encapsulada, e 35 adultos de tilápia foram alimentados diariamente; um grupo de 35 peixes foi usado como controle e alimentado com uma dieta basal. No início do estudo, cinco peixes de cada grupo foram selecionados aleatoriamente, e amostras de sangue foram coletadas dos peixes controle (BC) e experimentais (BE). Os peixes foram sacrificados por overdose de anestésicos, e amostras do tecido do fígado (LT), rins (RT), estômago sem conteúdo (S), intestino sem conteúdo (I), brânquias (GT), tecido muscular (filé; MT), gordura visceral (VF), conteúdo do trato digestivo (CTDE) e água do aquário (WA) foram coletadas somente dos peixes experimentais. O processo foi repetido diariamente durante uma semana. As regressões lineares simples foram ajustadas - dias de coleta versus coeficientes de determinação - e foram estabelecidas para comparações estatísticas da leitura das atividades medidas de 51Cr (transformação logarítmica) nas amostras dos peixes controle e experimentais. Não foram detectadas diferenças (P>0,05) entre as amostras BC dos peixes controle e BE, RT, VF , MT e LT dos peixes experimentais, mas as amostras de GT, I, S, CTDE e WA dos peixes que receberam a dieta experimental apresentaram diferença significativa em relação aos que receberam a dieta controle (P<0,05). Os resultados experimentais indicam que o crômio trivalente na forma de 51Cr2O3 não foi significativamente absorvido pelo trato gastrointestinal, pelas brânquias ou por outra via possível de absorção nessas condições experimentais e com tilápia do Nilo. Portanto, esse marcador demonstrou ser suficientemente inerte, o que torna seguro seu uso em estudos de nutrição.

Papel de inibidores da síntese e absorção do colesterol na modulação de biomarcadores de inflamação e adesão celular in vivo e in vitro / Role of inhibitors of the cholesterol synthesis and absorption on modulation of inflammation and cell adhesion biomarkers in vivo and in vitro

O processo inflamatório tem um papel fundamental na gênese e desenvolvimento da aterosclerose, sendo que a disfunção endotelial é considerada um dos estágios iniciais da aterogênese. Por meio da inibição da enzima hidroxi-metil-glutaril coA redutase (HMGCR), as estatinas reduzem a biossíntese do colesterol e a formação de isoprenóides, produtos intermediários da síntese do colesterol que são importantes na modificação pós-transcricional de GTPases pequenas que estão envolvidas na disfunção endotelial e inflamação vascular. A ezetimiba é um inibidor da absorção do colesterol através da inibição da proteína NPC1L1. Com a finalidade de esclarecer os mecanismos moleculares da inibição da síntese e da absorção do colesterol sobre a modulação de biomarcadores inflamatórios e de adesão celular foram utilizados modelos in vitro com células endoteliais (HUVEC) e monócitos (células THP-1), e in vivo com células mononucleares do sangue periférico (CMSP) de indivíduos hipercolesterolêmicos (HC). O efeito das estatinas, atorvastatina e sinvastatina, e da ezetimiba na expressão de RNAm e proteínas de moléculas de adesão endoteliais e moduladores do processo inflamatório, como citocinas e óxido nítrico (NO), foi estudado em células HUVEC. O efeito desses fármacos sobre a expressão de moléculas de adesão monocitárias foi estudado em células THP-1. O efeito da terapia hipolipemiante sobre essas moléculas foi também estudada em CMSP de HC tratados com ezetimiba (10 mg/dia/4 semanas), sinvastatina (10 mg/dia/8 semanas) e sinvastatina combinada com ezetimiba (10 mg de cada/dia/4 semanas). A expressão de RNAm foi avaliada por RT-qPCR. A expressão de moléculas de adesão na superfície de células THP-1 e HUVEC foi estudada por citometria de fluxo. A quantificação de citocinas secretadas no sobrenadante de células HUVEC e no plasma dos HC foi analisada pela tecnologia Milliplex. A quantificação do perfil lipífico, Proteína C reativa ultra-sensível (PCRus) e NO foi realizada por métodos laboratoriais convencionais. O papel do NO na modulação dos marcadores inflamatórios pelas estatinas foi também estudada, usando modelo de células HUVEC com NOS3 silenciado por interferência de RNAm e também por meio do uso do inibidor da síntese do óxido nítrico, L-NAME. Também foi avaliado o efeito de hipolipemiantes na expressão dos microRNAs (miRs) 221, miR-222 e miR-1303 em células HUVEC por meio do stem-loop RT-qPCR. O tratamento com atorvastatina e sinvastatina reduziu a expressão de RNAm e proteínas das moléculas de adesão LSelectina, PSGL-1 e VLA-4, em células THP-1 pré-tratadas com TNFα por 12 h. A ezetimiba reduziu a expressão de L-Selectina apenas no nível transcricional. Em células HUVEC, as estatinas diminuíram a expressão de RNAm de IL1B e SELP, entretanto aumentaram a de VCAM1. A ezetimiba reduziu a expressão de RNAm do IL1B. Entretanto as expressões de SELE, MMP9, IL6 e MMP9 não foram afetadas pelos tratamentos. A expressão das proteínas ICAM-1 e P-Selectina, na superfície de células HUVEC, foi diminuída pelo tratamento com as estatinas, mas não pela ezetimiba. Da mesma forma, a secreção das citocinas IL-6 e MCP-1 foram reduzidas pelas estatinas, entretanto a secreção de IL-8 não foi modificada por nenhum dos tratamentos. A expressão de NOS3 e a liberação de NO em células HUVEC foi aumentada pelas estatinas, porém não foi estimulada pela ezetimiba. Entretanto, os efeitos antiinflamatórios exercidos pelas estatinas foram independentes dessa via devido a que estes efeitos foram mantidos em células HUVEC com NOS3 silenciado por interferência de RNAm. Apesar de que o efeito sobre ICAM-1 e MCP-1 foi atenuado quando as células foram simultaneamente tratadas com L-NAME, os efeitos das estatinas parecem ser independentes da liberação de NO. As estatinas e a ezetimiba reduziram a expressão do miR-221, em células HUVEC. A expressão do miR-222 foi reduzida só pelo tratamento com atorvastatina. A expressão do miR-1303 não foi modulada pelos tratamentos hipolipemiantes. Em pacientes HC, a terapia de associação da sinvastatina e ezetimiba demonstrou melhorar o perfil lipídico de forma mais efetiva que ambas monoterapias. Da mesma forma, o tratamento combinado resultou em maior beneficio pela redução da expressão de RNAm em CMSP e da concentração plasmática das proteínas IL-1 ß, MCP-1, IL-8 e TNFα. A expressão de ICAM1 foi diminuída apenas no nível transcricional, entretanto a expressão de RNAm mas não da proteína do TNFα foi também reduzida pela sinvastatina em monoterapia. Não houve modulação de RNAm ou proteínas de outros marcadores estudados no modelo in vivo. Por outro lado, os efeitos anti-inflamatórios observados nos indivíduos HC foram independentes da modulação de PCRus e NO que não foram modificados pelos tratamentos hipolipemiantes. Neste estudo, foram confirmados os propostos efeitos pleiotrópicos das estatinas em modelos células de monócito e endotélio vascular in vitro e em pacientes HC. Por outro lado, apesar de ser menos potente que as estatinas foi mostrado que a inibição da absorção do colesterol tem também um efeito anti-inflamatório. A redução adicional do colesterol causado pela combinação das terapias hipolipemiantes outorga um maior beneficio cardiovascular em pacientes hipercolesterolêmicos

The inflammatory process has a key role in the genesis and development of atherosclerosis and the endothelial disfunction is considered as a first step in atherogenesis. By inhibiting the hydroxyl-methyl-glutaryl coA reductase (HMGCR)m statins reduce the cholesterol synthesis and isoprenoid generation, which are intermediary products of cholesterol synthesis with important role in posttranscriptional modifications of small GTPases that are involved in endothelial disfunction and vascular inflammation. The ezetimibe is an inhibitor of cholesterol absorption by inhibiting the NPC1L1 protein. To clarify the molecular mechanisms of the inhibition of cholesterol synthesis and absorption modulating inflammatory and cell adhesion biomarkers we used in vitro models of endothelial cells (HUVEC) and monocytes (THP-1), and an in vivo model of peripheral blood mononuclear cells (PBMC) from hypercholesterolemic (HC) patients. The effect of the statins, atorvastatin and simvastatin, and the ezetimibe on mRNA and protein expression of endothelial adhesion molecules and modulators of the inflammatory process, as citokynes and nitric oxide (NO), was analyzed in HUVEC. The effect of these drugs on the expression of monocyte adhesion molecules was also studied in THP-1. The influence of hypolipemiant therapy on the adhesion molecules was also analyzed in PBMC from HC treated with ezetimibe (10 mg/day/4-weeks), simvastatin (10 mg/day/8-weeks) and simvastatin combined with ezetimibe (10 mg each/day/4-weeks). The mRNA expression was evaluated by RT-qPCR. The expression of adhesion molecules on the surface of THP-1 and HUVEC cells was analyzed flow cytometry. The citokynes in the supernatants of HUVEC were quantified using the milliplex technology. The Lipid profile, high-sensivity PCR (hsPCR) and NO were determined by conventional laboratory methods. The role of the NO on the statin-modulation of inflammatory markers was also studied using a model with silenced NOS3 by interference of mRNA and by the use of the inhibitor of NO synthesis, L-NAME. The effect of hypolipemiants on the expression of microRNAs (miRs) 221, miR-222 and miR-1303 was also evaluated in HUVEC using the stem-loom RT-qPCR. Atorvastatin and simvastatin reduced the mRNA and protein expression of the adhesion molecules L-Selectin, PSGL-1 and VLA-4 in THP-1 cells pre-treated with TNFα for 12 h. The ezetimibe reduced the L-Selectin expression only at transcriptional level. In HUVEC, statins diminished IL1B and SELP mRNA expression, whereas VCAM1 was increased. The ezetimibe reduced the IL1B mRNA expression. However, SELE, MMP9, IL6 and MMP9 mRNA expressions were not affected by the treatments. The protein expression of ICAM-1 and P-Selectin on the surface of HUVEC was reduced by statins, but not by the ezetimibe. Similarly, IL-6 and MCP-1 secretion were reduced by statins, whereas IL-8 secretion was not modified by the treatments. The NO release and NOS3 expression in HUVEC was increased by the statins, however it was not stimulated by ezetimibe. Moreover, the anti-inflammatory statin effects were independent of this pathway due to statin effects were maintained in HUVEC with silenced NOS3. Although the statin effect on ICAM-1 and MCP-1 were attenuated by L-NAME co treatment, the statin effects seem to be independent of NO release. Statins and ezetimibe reduced miR221 in HUVEC. miR-222 expression was reduced only by atorvastatin. miR-1303 was not affected by the treatments. In HC patients, the improvement of the lipid profile simvastatin combined with ezetimibe was more efficient than both monotherapies. Similarly, the association therapy was better in reducing the mRNA expression in PBMC and plasma concentration of IL-1ß, MCP-1, IL-8 and TNFα. ICAM1 expression was reduced only at transcriptional level, whereas mRNA but not protein expression of TNFα was also reduced by the simvastatin monotherapy. There was no modulation mRNA or protein expression of other studied markers in the in vivo model. Additionally, the anti-inflammatory effects observed in the HC were independent of PCRus or NO modulation, which were not altered by the hypolipemiant treatments. In this study, the proposed plitropic effects of statins were confirmed in monocytes and endothelial cells in vitro and in HC patients. Moreover, although it was less potent than statins, an anti-inflammatory effect was also observed for the inhibition of cholesterol absorption. An additional reduction of the cholesterol caused by combined hypolipemiant therapies gives a greater cardiovascular beneffict in hypercholesterolemic patients

Acute eosinophilic pneumonia related to a mesalazine suppository

It has been well known that mesalazine can cause the interstitial lung disease, such as Bronchiolitis obliterans with organizing pneumonia (BOOP), Non-Specific Interstitial Pneumonia (NSIP), or eosinophilic pneumonia. 5-Aminosalicylic acid (5-ASA), mesalazine, and sulfasalazine are important drugs for treating inflammatory bowel disease. Topical products of these limited systemic absorption and have less frequent side effects, therefore suppository form of these drugs have been used more than systemic drug. Most cases of measalzine-induced lung toxicity develop from systemic use of the drug. A 30-year-old woman had an interstitial lung disease after using mesalazine suppository because of ulcerative colitis. The lung biopsy demonstrated eosinophilic pneumonia combined with BOOP. She was recovered after stopping of mesalazine suppository and treatment with systemic steroid.