Effect of Extraction Procedure Substitution on Automated Tacrolimus, Sirolimus, and Cyclosporine Assays.

BACKGROUND: An erroneously high tacrolimus level was reported to a clinician. A root cause analysis investigation failed to determine the cause of the error. It was suspected that the incorrect preanalytical extraction reagent and procedure was used during testing; however, how this would affect the assayed drug concentration was unclear. Here we investigated the effect of the substitution of sirolimus, tacrolimus, and cyclosporine extraction reagents on assayed drug concentration. METHODS: Tacrolimus, sirolimus, and cyclosporine concentration were measured on the Abbott Architect i2000 analyzer. Each assay requires a preanalytical extraction step, with a distinct reagent. We investigated the effect of the substitution of the extraction reagents and procedure between the 3 assays on the measured drug concentration. Two experiments were performed, one on samples of known drug concentration and one on samples with no drug present. RESULTS: Substituting cyclosporine and sirolimus extraction procedures increased assayed tacrolimus concentrations from 5.6 to 8.47 (+51.25%) and 8.13 (+45.18%) ng/mL, respectively. Extraction procedure substitutions decreased assayed sirolimus from 13.63 to 4.60 (-66.25%) and 8.07 (-40.79%) ng/mL for cyclosporine and tacrolimus. Cyclosporine concentration increased from 274.60 to 391.30 (+42.50%) ng/mL using sirolimus extraction reagents and to 757.30 (+175.78%) ng/mL using tacrolimus extraction reagents. Cross-reactivity was observed between the tacrolimus assay and sirolimus and cyclosporine extraction reagents. CONCLUSIONS: Significant changes, both positive and negative, are observed in assayed drug concentration when incorrect extraction procedures are used in the Abbott i2000 tacrolimus, sirolimus, and cyclosporine assays. Preanalytic extraction procedures should be investigated when performing root cause analysis for erroneous therapeutic drug values.

Screening the possible anti-cancer constituents of Hibiscus rosa-sinensis flower to address mammalian target of rapamycin: an in silico molecular docking, HYDE scoring, dynamic studies, and pharmacokinetic prediction.

One of the most common malignancies diagnosed and the leading cause of death for cancer-stricken women globally is breast cancer. The molecular subtype affects therapy options because it is a complex disorder with multiple subtypes. By concentrating on receptor activation, mTOR (mammalian target of rapamycin) can be employed as a therapeutic target. The goal of this work was to screen a number of inhibitors produced from Hibiscus rosa-sinensis for possible target to inhibit the mTOR and to determine which has the greatest affinity for the receptor. Primarily, the ionization states of the chosen compounds were predicted using the ChemAxon web platform, and their pKa values were estimated. Given the significance of interactions between proteins in the development of drugs, structure-based virtual screening was done using AutoDock Vina. Approximately 120 Hibiscus components and ten approved anti-cancer drugs, including the mTOR inhibitor everolimus, were used in the comparative analysis. By using Lipinski's rule of five to the chosen compounds, the ADMET profile and drug-likeness characteristics were further examined to assess the anti-breast cancer activity. The compounds with the highest ranked binding poses were loaded using the SeeSAR tool and the HYDE scoring to give interactive, desolvation, and visual ΔG estimation for ligand binding affinity assessment. Following, the prospective candidates underwent three replicas of 100 ns long molecular dynamics simulations, preceded with MM-GBSA binding free energy calculation. The stability of the protein-ligand complex was determined using root mean square deviation (RMSD), root mean square fluctuation (RMSF), and protein-ligand interactions. The results demonstrated that the best mTOR binding affinities were found for stigmastadienol (107), lupeol (66), and taraxasterol acetate (111), which all performed well in comparison to the control compounds. Thus, bioactive compounds isolated from Hibiscus rosa-sinensis could serve as lead molecules for the creation of potent and effective mTOR inhibitors for the breast cancer therapy.

Development and Validation of a Bioanalytical LC-MS/MS Method for Simultaneous Determination of Sirolimus in Porcine Whole Blood and Lung Tissue and Pharmacokinetic Application with Coronary Stents.

Sirolimus is a hydrophobic macrolide compound that has been used for long-term immunosuppressive therapy, prevention of restenosis, and treatment of lymphangioleiomyomatosis. In this study, a simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated for the simultaneous determination of sirolimus in both porcine whole blood and lung tissue. Blood and lung tissue homogenates were deproteinized with acetonitrile and injected into the LC-MS/MS system for analysis using the positive electrospray ionization mode. The drug was separated on a C18 reversed phase column with a gradient mobile phase (ammonium formate buffer (5 mM) with 0.1% formic acid and acetonitrile) at 0.2 mL/min. The selected reaction monitoring transitions of m/z 931.5 → 864.4 and m/z 809.5 → 756.5 were applied for sirolimus and ascomycin (the internal standard, IS), respectively. The method was selective and linear over a concentration range of 0.5-50 ng/mL. The method was validated for sensitivity, accuracy, precision, extraction recovery, matrix effect, and stability in porcine whole blood and lung tissue homogenates, and all values were within acceptable ranges. The method was applied to a pharmacokinetic study to quantitate sirolimus levels in porcine blood and its distribution in lung tissue following the application of stents in the porcine coronary arteries. It enabled the quantification of sirolimus concentration until 2 and 14 days in blood and in lung tissue, respectively. This method would be appropriate for both routine porcine pharmacokinetic and bio-distribution studies of sirolimus formulations.

Long-term stability of 0.1% rapamycin hydrophilic gel in the treatment of facial angiofibromas.

Objectives: In recent years, various formulations containing rapamycin, mainly petrolatum-based, have been tested on facial angiofibromas in tuberous sclerosis. They are often poorly tolerated due to irritation and bleeding. In addition, their effectiveness was insufficient in young adults. The objective of this study was to develop and characterise a hydro-alcoholic gel containing solubilised rapamycin. The stability of the product stored at 4°C was evaluated over 1 year. Methods: Two different 0.1% rapamycin gels were formulated with or without α-tocopherol and urea. Different methods were used to characterise the gels: HPLC, gas chromatography, pH, visual observation and optical microscopy. A physico-chemical and microbiological stability study was also conducted for 1 year at 4°C. Results: Gels were physically and microbiologically stable after 1 year at 4°C: organoleptic characteristics and pH unchanged, no significant decrease in rapamycin was observed, tocopherol droplet size was constant and rheological behaviour was not altered. Conclusions: This study describes a new gel formulation to improve skin penetration using various excipients to promote skin tolerance. This study provides, for the first time, detailed stability data for a hydro-alcoholic rapamycin gel.

Wide electrochemical window of screen-printed electrode for determination of rapamycin using ionic liquid/graphene composites.

A disposable screen-printed carbon electrode (SPCE) modified with an ionic liquid/graphene composite (IL/G) exhibits a wider potential window, excellent conductivity, and specific surface area for the improvement in the voltammetric signal of rapamycin detection. The modified composite was characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and electrochemical impedance spectroscopy (EIS). The electrochemical behavior of rapamycin at the modified SPCE was investigated by cyclic and square wave voltammetry in 60:40 EtOH: 0.1 M LiClO4 at pH 5.0. A high reproducible and well-defined peak with a high peak current were obtained for rapamycin detection at a position potential of + 0.98 V versus Ag/AgCl. Under the optimized conditions, the rapamycin concentration in the range 0.1 to 100 µM (R2 = 0.9986) had a good linear relation with the peak current. The detection limit of this method was 0.03 µM (3SD/slope). The proposed device can selectively detect rapamycin in the presence of commonly interfering compounds. Finally, the proposed method was successfully applied to determine rapamycin in urine and blood samples with excellent recoveries. These devices are disposable and cost-effective and might be used as an alternative tool for detecting rapamycin in biological samples and other biological compounds. Graphical abstract Schematic presentation of wide electrochemical window and disposable screen-printed sensor using ionic liquid/graphene composite for the determination of rapamycin. This composite can enhance the oxidation current and expand the potential for rapamycin detection.

Accuracy evaluation of automated electrochemiluminescence immunoassay for everolimus and sirolimus compared to liquid chromatography-tandem mass spectrometry.

BACKGROUND: We evaluated the analytical performance of a newly developed electrochemiluminescence immunoassay for everolimus and sirolimus compared to that of liquid chromatography-tandem mass spectrometry (LC-MS/MS). METHODS: According to Clinical and Laboratory Standards Institute guidelines, the analytical performance including precision, recovery, linearity, and carryover was evaluated. For correlation evaluation, the results of Elecsys® analysis of everolimus and sirolimus were compared with those of LC-MS/MS using 120 samples from patients treated with everolimus or sirolimus. RESULTS: The within-run and total imprecision values were as follows: 2.3%-4.5% and 4.5%-6.4% for the everolimus assay; 3.3%-4.8% and 4.7%-8.1% for the sirolimus assay, respectively. The measured concentration was linear over the range of 0.718-27.585 ng/mL for everolimus analysis and 0.789-26.880 ng/mL for sirolimus analysis (all R2  > 0.99). Recovery was 93.5%-105.5% for the everolimus assay and 99.2%-109.1% for the sirolimus assay (except lowest levels). Carryover was -1.09% for the everolimus assay and -0.12% for the sirolimus assay. The results of the two chemiluminescence immunoassays showed acceptable correlations with those of LC-MS/MS (R = 0.9585 and R = 0.9799, respectively). The two immunoassays showed slightly proportional biases compared to LC-MS/MS. CONCLUSION: Elecsys® Everolimus and Sirolimus assays showed acceptable analytical performance in precision, linearity, and correlation compared to LC-MS/MS These methods can be adopted in the clinical laboratory for rapid therapeutic drug monitoring of patients who require treatment with immunosuppressants.

Clinical application of a dried blood spot assay for sirolimus and everolimus in transplant patients.

Background Monitoring of immunosuppressive drugs such as everolimus and sirolimus is important in allograft rejection prevention in transplant patients. Dried blood spots (DBS) sampling gives patients the opportunity to sample a drop of blood from a fingerprick at home, which can be sent to the laboratory by mail. Methods A total of 39 sirolimus and 44 everolimus paired fingerprick DBS and whole blood (WB) samples were obtained from 60 adult transplant patients for method comparison using Passing-Bablok regression. Bias was assessed using Bland-Altman. Two validation limits were pre-defined: limits of analytical acceptance were set at >67% of all paired samples within 20% of the mean of both samples and limits of clinical relevance were set in a multidisciplinary team at >80% of all paired samples within 15% of the mean of both samples. Results For both sirolimus and everolimus, Passing-Bablok regression showed no differences between WB and DBS with slopes of 0.86 (95% CI slope, 0.72-1.02) and 0.96 (95% CI 0.84-1.06), respectively. Only everolimus showed a significant constant bias of 4%. For both sirolimus and everolimus, limits of analytical acceptance were met (76.9% and 81.8%, respectively), but limits or clinical relevance were not met (77.3% and 61.5%, respectively). Conclusions Because pre-defined limits of clinical relevance were not met, this DBS sampling method for sirolimus and everolimus cannot replace WB sampling in our center at this time. However, if the clinical setting is compatible with less strict limits for clinical relevance, this DBS method is suitable for clinical application.

Pharmacokinetic study of sirolimus ophthalmic formulations by consecutive sampling and liquid chromatography-tandem mass spectrometry.

Sirolimus is regarded as one of the most effective immunosuppressants receiving extensive attention over the years, for which the ocular application needs further development in clinical keratoplasty. In order to study the transcorneal absorption effect of ophthalmic administration, there was a need to study the pharmacokinetics of drugs in aqueous humor. In this work, a validated and reliable HPLC-ESI-MS/MS method was established to study the pharmacokinetics of sirolimus nanoformulations in rabbit aqueous humor. The analysis conditions were as follows. Ascomycin was chosen as internal standard. After a simple precipitation extraction procedure, the aqueous humor samples were separated on a XBridge C18 column (4.6 mm × 150 mm, 3.5 µm, Waters Co., USA) with a mobile phase comprised of water (0.1% formic acid and 5 mM ammonium formate) and methanol (0.1% formic acid) at the ratio of 10:90 (v/v). The mass analysis was achieved by positive ionization with multiple reaction monitoring (MRM) mode. The highest response ion pairs m/z at 931.5→864.5 were chosen for sirolimus. The validated results showed that the calibration range was 0.3-100.6 ng/mL with r = 0.9997 (n = 6). The R.S.D. values of the intra- and inter-day precision were less than 11% and the average accuracy values were between 94.73%-100.20%. Besides, for reducing the consumption of rabbits and the variation of the data, we designed a consecutive sampling method in pharmacokinetic study, with only seven rabbits consumed for each formulation. In conclusion, the developed analysis method was more reliable and practical than previously reported experiments. Meanwhile, the validated method was successfully applied to study the pharmacokinetics of sirolimus micelle and sirolimus nanosuspension after ophthalmic administration.

Recent advances in analytical methods for the therapeutic drug monitoring of immunosuppressive drugs.

Therapeutic drug monitoring (TDM) of immunosuppressive drugs (ISDs) with a narrow therapeutic index is an increasingly popular tool for minimizing drug toxicity while maximizing the prevention of graft loss and organ rejection. This review focuses on trends regarding analytical methods for the TDM of ISDs since 2011. The five most commonly prescribed immunosuppressive medications are critically reviewed: cyclosporine A, tacrolimus, sirolimus (rapamycin), everolimus, and mycophenolic acid. This review introduces the general background of TDM and ISDs and presents the recent developments in using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immunoassays for the TDM of ISDs. Finally, a future perspective for these analytical methods is briefly discussed.

Very thin strut biodegradable polymer everolimus-eluting and sirolimus-eluting stents versus durable polymer zotarolimus-eluting stents in allcomers with coronary artery disease (BIO-RESORT): a three-arm, randomised, non-inferiority trial.

BACKGROUND: In patients with coronary artery disease, treated with durable polymer-coated drug-eluting stents, the life-long presence of the polymer might delay arterial healing. Novel very thin strut biodegradable polymer stents, which leave only a bare metal stent after polymer resorption, might improve long-term outcome. We investigated in allcomers the safety and efficacy of three stents eluting either everolimus, sirolimus, or zotarolimus, often clinically used but never compared, of which the biodegradable polymer everolimus-eluting stent was never before assessed in allcomers. METHODS: The large-scale, investigator-initiated, multicentre, assessor and patient blinded, three-arm, randomised, BIO-RESORT non-inferiority trial was done at four clinical sites in the Netherlands. All-comer patients were aged 18 years or older, capable of providing informed consent, and required a percutaneous coronary intervention with drug-eluting stent implantation according to clinical guidelines or the operators' judgment. Exclusion criteria were: participation in another randomised drug or device study before reaching the primary endpoint of that study; planned surgery necessitating interruption of dual antiplatelet therapy within the first 6 months; known intolerance to components of the investigational product or medication required; uncertainty about the adherence to follow-up procedures or an assumed life expectancy of less than 1 year; or known pregnancy. Web-based computer-generated allocation sequences randomly assigned patients (1:1:1) to treatment with very thin strut biodegradable polymer everolimus-eluting or sirolimus-eluting stents (which differ substantially in type, amount, distribution, and resorption speed of their respective coating), or thin strut durable polymer zotarolimus-eluting stents. The primary endpoint was a composite of safety (cardiac death or target vessel-related myocardial infarction) and efficacy (target vessel revascularisation) at 12 months of follow up with a very thin strut biodegradable polymer of either everolimus-eluting or sirolimus-eluting stents, compared with durable polymer zotarolimus-eluting stents, analysed by intention to treat (non-inferiority margin 3·5%). This trial was registered with ClinicalTrials.gov, number NCT01674803. FINDINGS: From Dec 21, 2012, to Aug 24, 2015, 3514 patients were enrolled and analysed, of whom 2449 (70%) had acute coronary syndromes, which included 1073 (31%) ST-elevation myocardial infarctions. 12 month follow-up of 3490 (99%) patients (three lost to follow-up; 21 withdrawals) was available. The primary endpoint was met by 55 (5%) of 1172 patients assigned to everolimus-eluting stents, 55 (5%) of 1169 assigned to sirolimus-eluting stents and 63 (5%) of 1173 assigned to zotarolimus-eluting stents. Non-inferiority of the everolimus-eluting stents and sirolimus-eluting stents compared with zotarolimus-eluting stents was confirmed (both -0·7% absolute risk difference, 95% CI -2·4 to 1·1; upper limit of one sided 95% CI 0·8%, pnon-inferiority<0·0001). Definite stent thrombosis (defined by the Academic Research Consortium) occurred in four (0·3%) of 1172 patients who were allocated to everolimus-eluting stents, four (0·3%) of 1169 patients who were allocated to sirolimus-eluting stents, and three (0·3%) of 1173 patients who were allocated to zotarolimus-eluting stents (log-rank p=0·70 for both comparisons with zotarolimus-eluting stents). INTERPRETATION: At 12 month follow-up, both very thin strut drug-eluting stents with dissimilar biodegradable polymer coatings (eluting either everolimus or sirolimus) were non-inferior to the durable polymer stent (eluting zotarolimus) in treating allcomers with a high proportion of patients with acute coronary syndromes. The absence of a loss of 1 year safety and efficacy with the use of these two biodegradable polymer-coated stents is a prerequisite before assessing their potential longer-term benefits. FUNDING: Biotronik, Boston Scientific, and Medtronic.

Combined image guided monitoring the pharmacokinetics of rapamycin loaded human serum albumin nanoparticles with a split luciferase reporter.

Imaging guided techniques have been increasingly employed to investigate the pharmacokinetics (PK) and biodistribution of nanoparticle based drug delivery systems. In most cases, however, the PK profiles of drugs could vary significantly from those of drug delivery carriers upon administration in the blood circulation, which complicates the interpretation of image findings. Herein we applied a genetically encoded luciferase reporter in conjunction with near infrared (NIR) fluorophores to investigate the respective PK profiles of a drug and its carrier in a biodegradable drug delivery system. In this system, a prototype hydrophobic agent, rapamycin (Rapa), was encapsulated into human serum albumin (HSA) to form HSA Rapa nanoparticles, which were then labeled with Cy5 fluorophore to facilitate the fluorescence imaging of HSA carrier. Meanwhile, we employed transgenetic HN12 cells that were modified with a split luciferase reporter, whose bioluminescence function is regulated by Rapa, to reflect the PK profile of the encapsulated agent. It was interesting to discover that there existed an obvious inconsistency of PK behaviors between HSA carrier and rapamycin in vitro and in vivo through near infrared fluorescence imaging (NIFRI) and bioluminescence imaging (BLI) after treatment with Cy5 labeled HSA Rapa. Nevertheless, HSA Rapa nanoparticles manifested favorable in vivo PK and tumor suppression efficacy in a follow-up therapeutic study. The developed strategy of combining a molecular reporter and a fluorophore in this study could be extended to other drug delivery systems to provide profound insights for non-invasive real-time evaluation of PK profiles of drug-loaded nanoparticles in pre-clinical studies.

Therapeutic drug monitoring of immunosuppressants by liquid chromatography-mass spectrometry.

Immunosuppressant medications allow the transplantation of tens of thousands of allografts per year and consequently have great potential to decrease patient morbidity and mortality. However, some medications have great risk associated with over- and under-dosing leading to adverse effects or allograft rejection, respectively. This necessitates immunosuppressant therapeutic drug monitoring accomplished by immunoassay or liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The former's accuracy can be hindered by metabolites of immunosuppressant medications, antibodies against these medications and heterophilic antibodies. Although LC-MS/MS has superior specificity which allows it to be less susceptible to interference, this methodology lacks standardization and the necessary throughput. Recent developments in LC-MS/MS quantitation, however, include patient-friendly sample submission as dried blood spots, higher sample throughput and commercialization. Here we critically review recent LC-MS/MS publications (January 2010 to July 2015) on the quantitation of cyclosporine A, tacrolimus, sirolimus and everolimus.

Evaluation of a LC-MS method for everolimus preclinical determination in brain by using [(13)C2D4]RAD001 internal standard.

Isotopic internal standards are increasingly frequent in LC-MS analysis to control biological matrix effects in the quantitation of immunosuppressant drugs, such as everolimus (RAD001). Here we present the evaluation of a LC-MS method, exploiting [(13)C2D4]RAD001 as internal standard, for preclinical determination of RAD001 in mice brain tissue. Samples were purified by solid phase extraction. Brain and blood were collected from vehicle-treated and RAD001-treated mice. The QTOF MS detector was set to select RAD001 ammonium adducts (m/z 975.6152) and [(13)C2D4]RAD001 (m/z 981.6481). Two different UHPLC columns were preliminarily tested. The method showed linear behavior between 4 and 100ng/mL (r(2)=0.99943) and linearity was preserved in the presence of blood (r(2)=0.99107) and brain (r(2)=0.99098) matrix components. Intra-day and inter-day precision (3-19%) and accuracy (82-109%) were comparable between standards and spiked blood and brain samples. As resulting from recovery comparison (82-98%), [(13)C2D4]RAD001 compensated ion suppression phenomena maintaining method performance over a wide range of consecutive analytical runs. The comparison with a HPLC-UV method showed reliability of the method with good correlation between blood (r(2)=0.94319) and brain (r(2)=0.97773) samples and acceptable biases (<15%). This validation suggests that the investigated method could be useful for the preclinical monitoring of RAD001 brain therapeutic concentrations in animal models.

Long-term evaluation of analytical methods used in sirolimus therapeutic drug monitoring.

Results of therapeutic monitoring of sirolimus blood concentrations are assay and laboratory dependent. This study compared performance over time of the IMx microparticle enzyme immunoassay (MEIA), Architect chemiluminescent microparticle immunoassay (CMIA), and liquid chromatography with mass spectrometric detection (LC/MS/MS) as part of a proficiency testing scheme. Pooled samples from sirolimus-treated patients and whole-blood samples spiked with known quantities of sirolimus were assayed monthly between 2004 and 2012. When results of pooled patient samples were compared with LC/MS/MS, the MEIA assay showed an overall mean percent bias of -2.3% ± 11.2% that, although initially positive, became increasingly negative from 2007 through 2009. The CMIA, which replaced the MEIA assay, had a mean percent bias of 21.9% ± 12.3%, remaining stable from 2007 through 2012. Similarly, for spiked samples, the MEIA showed an increasingly negative bias over time vs. LC/MS/MS, whereas CMIA maintained a stable positive bias. Based on comparison of immunoassay measurements on individual patient samples, CMIA values were more than 25% higher than MEIA values. These results highlight the importance of continued proficiency testing and regular monitoring of sirolimus assay performance. Clinicians must be aware of the methodology used and adjust target levels accordingly to avoid potential effects on efficacy and toxicity.

Efeitos de compostos naturais, sintéticos e da fototerapia antifúngica sobre Candida tropicalis resistente ao fluconazol / Effects of natural compounds, synthetic and herbal medicine on Candida tropicalis antifungal fluconazole-resistant

A candidíase é uma infecção oportunista provocada por diversas espécies de fungos do gênero Candida, frequentemente encontrados integrando a microbiota, da superfície cutânea, no trato gastrointestinal e cavidades mucosas do ser humano desde o seu nascimento. A incidência das infecções fúngicas sistêmicas têm aumentado consideravelmente nas últimas décadas em função do grande número de pacientes com SIDA, a grande quantidade de transplantes e condições crônicas como o câncer, a terapia prolongada com imunossupressores e o uso de agentes corticosteroides. Além disso, a exposição prolongada aos antifúngicos azólicos promove a seleção de patógenos resistentes. No presente estudo avaliou-se a atividade antifúngica do complexo Rutênio-pirocatecol (RPC) frente a um isolado clinico de Candida tropicalis resistente ao fluconazol. A metodologia empregada para os testes de susceptibilidade foi de acordo com o documento M27-A3 do National Committee for Clinical Laboratory Standards (NCCLS, 2008). Esplenócitos de camundongos Balb/c foram obtidos de forma asséptica para avaliar a citotoxicidade do composto para células de mamíferos. O estresse oxidativo promovido pelo composto foi avaliado através da reação ao ácido tiobarbitúrico (TBARS) e ensaios de fluorescência com a sonda diclorodihidrofluoresceína diacetato (DCFH2DA). O Calcofluor White foi empregado para avaliar a integridade da parede celular. A análise ultraestrutural foi realizada através da microscopia eletrônica de varredura e transmissão. Os resultados encontrados para os testes de atividade antifúngica foram analisados através do teste estatístico ANOVA e pós-teste Dunnett. Os resultados encontrados para os testes de atividade antifúngica do RPC mostraram uma Concentração Inibitória de 50% (IC50) de 20,3 μM, enquanto em esplesnócitos a concentração efetiva de 50% foi de 325 μM mostrando um índice de seletividade igual a 16...

Candidiasis is an opportunistic infection caused by several species of fungi of the genus Candida, often found is the microbiota, on the skin, gastrointestinal tract and mucous cavities of the human beings birth. The incidence of systemic fungal infections have increased considerably in recent decades due to the large number of AIDS patients, the large number of transplants and chronic conditions such as cancer, prolonged therapy promotes the selection of resistant pathogen with immunosuppressant and corticosteroid agents. Also prolonged exposure azole antifungals to make them strong candidates for patients resistance. In the present study we evaluated the antifungal activity of Ruthenium-pyrocatechol complex (RPC) against a clinical isolate of Candida tropicalis resistant to fluconazole. The methodology for susceptibility testing was in accordance with the M27-A3 document of there National Committee for Clinical Laboratory Standards (NCCLS, 2008). Splenocytes from Balb/c mice were obtained aseptically to evaluate the cytotoxicity of the compound to mammalian cells. Oxidative stress caused by the compound was assessed by reaction to thiobarbituric acid (TBARS) and fluorescence assays with the probe diclorodihidrofluoresceína diacetate (DCFH2DA). The Calcofluor White was used to evaluate the integrity of the cell wall. The ultrastructural analysis was performed by scanning and transmission electron microscopy. The results for the antifungal activity tests were analyzed using ANOVA and pos-test Dunnett test statistic. The results for the tests of antifungal activity of the RPC showed a 50% inhibitory concentration (IC50) of 20.3 μM while in splenocytes the 50% effective concentration was 325 μM showing a selectivity index of 16...

Dual drug-eluting stents coated with multilayers of hydrophobic heparin and sirolimus.

Polymer coatings for stents are considered one of the key factors that lead to adverse cardiac events after coronary arterial stenting. This study presents a dual drug-eluting stent (DES) that is coated with multilayers of Duraflo heparin and sirolimus but containing no other organic polymers. The hydrophobic Duraflo heparin coating was used to improve the hemocompatibility of the stent and serve as a drug reservoir for the controlled release of sirolimus, thus avoiding inflammatory reactions induced by the conventional polymers. The Duraflo heparin and sirolimus were coated layer-by-layer onto the stent surface using a homemade spray-coating device. The drug loading amount can be easily controlled by adjusting the numbers of layers applied and the concentration of the drug solution, indicating the developed coating process is reproducible and well-controlled. After balloon expansion, the coating did not crack or peel off, which demonstrates that the sirolimus/Duraflo heparin coating layers tightly adhere to the stent surface. The activated partial thromboplastin time (APTT) assay showed that the Duraflo heparin coating significantly prolonged the APTT from 27.3 ± 0.3 s to 69.7 ± 6.2 s, demonstrating the anticoagulant ability of the coated stents. The dual DES exhibited a nearly linear sustained-release profile of Duraflo heparin and an initial burst release followed by a slow release of sirolimus. Less than 15% of heparin was released from the DES within 14 days, indicating the stent can maintain its antithrombotic surface for a long time. Because of the layer-by-layer structure, the most outer layer of Duraflo heparin coating may act as a diffusion barrier to retard sirolimus release from the stent. These results confirm that the dual DESs enable simultaneous delivery of antithrombotic and antiproliferative drugs and have potential for the treatment of coronary artery disease.

A case study of validation and sample analysis for the determination of zotarolimus in various tissues.

BACKGROUND: Biodistribution of drug and drug candidates offers critical information regarding drug disposition in target and nontarget tissues. Concentrations of therapeutic agents in target tissue provide the foundation for efficacy, while concentrations in nontarget tissue pose potential safety risks. Analysis of tissue samples can sometimes provide important information during the development of a drug candidate, especially at early stages when radio labeled drug material is not readily available. RESULTS: Determining the appropriate approach to allow for accurate, precise and reproducible drug concentration measurements from tissue samples requires addressing issues not present in routinely analyzed biological matrices, that is, plasma. We present here the issues encountered and techniques applied during the development, validation and application of a method for the determination of zotarolimus in a variety of tissues. CONCLUSION: When well controlled, analysis of tissue samples can be performed in a manner similar to liquid biological matrices.

Levels of Sirolimus in saliva vs. blood--the rationale of topical oral use for oral malignancy.

BACKGROUND: Sirolimus is a potent blocker of mammalian target of Rapamycin (MTOR), with anti proliferative activity. Its potential for the management of oral cancer has been suggested. Our aim was to establish an analytical method for determining sirolimus levels in human saliva and to calculate the blood vs. saliva ratio in individuals using sirolimus chronically in order to evaluate the total oral tissue exposure. PATIENTS AND METHODS: Chemiluminescent microparticle immunoassay technology (CMIA) was used to determine the blood and saliva levels of sirolimus in four transplant patients chronically-treated with sirolimus. RESULTS: An analytical method for determining sirolimus levels in human saliva was established. We demonstrated that saliva levels were on average six times lower than blood levels. CONCLUSION: The specific sensitive analytical method showed that the saliva levels of sirolimus are significantly lower than blood levels, thus reinforcing the rationale for the use of topical oral sirolimus to enhance availability, efficacy and safety for treating oral malignancies.

Bioanalytical method validation of rapamycin in ocular matrix by QTRAP LC-MS/MS: application to rabbit anterior tissue distribution by topical administration of rapamycin nanomicellar formulation.

A novel, fast and sensitive 3200 QTRAP LC-MS/MS method was validated for rapamycin analysis in the rabbit eye following 0.2% administration of nanomicellar eye drop formulation. The LC-MS/MS technique was developed with electrospray ionization (ESI) in positive mode. Rapamycin was extracted from individual eye tissues and fluids by a simple protein precipitation method. Samples were reconstituted in 200µL of 80% of acetonitrile in water containing 0.05% formic acid. Twenty microliter of the sample was injected on LC-MS/MS. Chromatographic separations was achieved on reversed phase C 8 Xterra column, 50mm×4.6mm, 5µm. Multiple reactions monitoring (MRM) transition m/z 936.6/409.3 for rapamycin and 734.4/576.5 for erythromycin were employed as internal standard. The calibration curves were linear r(2)>0.9998 over the concentration range from 2.3ng/mL to 1000.0ng/mL. Rapamycin was found to be stable in ocular tissue homogenates for 6weeks at a refrigerated -80°C and -20°C temperatures. Rapamycin concentration was found to be 2260.7±507.1 (mean±S.D.)ng/g tissue and 585.5±80.1 (mean±S.D.)ng/g tissue in the cornea and iris ciliary muscle, respectively. This method has two advantages. First, a volatile base was used in the extraction procedure, which is easy to evaporate and generate consistent results. Second, the sodium adduct is employed that was stable in non-ammoniated mobile phase. The method demonstrates that absorption of rapamycin by a topical application of 0.2% rapamycin nanomicellar formulation generates therapeutically effective concentrations in the anterior segment of the eye.