52 research outputs found

    Application of quality-by-design for adopting an environmentally green fluorogenic determination of benoxinate hydrochloride in eye drops and artificial aqueous humour

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    Abstract Herein, a sensitive and selective spectrofluorimetric method has been developed for the determination of the ocular local anesthetic benoxinate hydrochloride (BEN-HCl) in eye drops and artificial aqueous humour. The proposed method is based on the interaction of fluorescamine with the primary amino group of BEN-HCl at room temperature. Following the excitation of the reaction product at 393¬†nm, the emitted relative fluorescence intensity (RFI) was measured at 483¬†nm. The key experimental parameters were carefully examined and optimized by adopting an analytical quality-by-design approach. The method used a two-level full factorial design (24 FFD) to obtain the optimum RFI of the reaction product. The calibration curve was linear at the range of 0.10‚Äď1.0¬†őľg/mL of BEN-HCl with sensitivity down to 0.015¬†őľg/mL. The method was applied for analyzing the BEN-HCl eye drops and could also assess its spiked levels in artificial aqueous humour with high % recoveries (98.74‚Äď101.37%) and low SD values (‚ȧ‚ÄČ1.11). To investigate the green profile of the proposed method, a greenness assessment was performed with the aid of the Analytical Eco-Scale Assessment (ESA) and GAPI. The developed method obtained a very high ESA rating score in addition to being sensitive, affordable, and environmentally sustainable. The proposed method was validated according to ICH guidelines

    In Vivo Oxygen Uptake into the Human Cornea

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    PURPOSE. We provide a new procedure to quantify in situ corneal oxygen uptake using the micropolarographic Clark electrode. METHODS. Traditionally, upon placing a membrane-covered Clark microelectrode onto a human cornea, the resulting polarographic signal is interpreted as the oxygen partial pressure at the anterior corneal surface. However, the Clark electrode operates at a limiting current. Hence, oxygen flux is directly detected rather than partial pressure. We corrected this misunderstanding and devised a new analysis to quantify oxygen uptake into the cornea. The proposed analysis is applied to new polarographic data for 10 human subjects during open-eye oxygen uptake. RESULTS. Average open-eye corneal oxygen uptake over 10 subjects is approximately 11 lL/(cm 2 h), approximately five times larger than the average reported by researchers who invoke the original mathematical analysis. Application of the classical interpretation scheme to our experimental data also garners uptake values that are approximately a factor of three to five times smaller than those obtained with our new procedure. CONCLUSIONS. The classical procedure originally developed by Fatt and colleagues misinterprets the behavior of the Clark microelectrode. We corrected the analysis of the in situ polarographic technique to provide a simple yet rigorous procedure for analyzing both previous data in the literature and those newly obtained. Our proposed interpretation scheme thus provides a reliable tool for in vivo assessment of corneal oxygen uptake. (Invest Ophthalmol Vis Sci. 2012;53:6331-6337


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    Objective: The main purpose of the study was to develop niosomal in situ gel of prednisolone sodium phosphate (PSP) with increased bioavailability (enhanced permeation) and sustained action (drug retention at the target site). Methods: Using different ratios of span 60 and cholesterol (chol), niosomes were prepared by thin film hydration method and optimized by evaluating different parameters like drug content, entrapment efficiency, particle size and in vitro drug diffusion study. The niosomal pellets were further incorporated in in situ gel, prepared by the cold method and further optimized by parameters like gelling parameters, mucoadhesive strength and in vitro, in vivo drug release study. Results: The optimized niosomal formulation containing span 60 and chol in equal proportion (1:1) showed better drug content (DC) i.e. 86.3¬Ī0.39% and entrapment efficiency (EE) i.e. 83.4¬Ī0.22 with vesicle size of 465¬Ī0.24 nm. The in vitro drug diffusion study indicated t90 value of 490 min thus proving sustained action of the formulation. The optimized in situ gel containing poloxamer 407 (P407) and poloxamer 188 (P188) in the ratio of 1:2.7 showed gelation temperature at 37 ‚ĀįC (physiological temperature of the body) and t90 value of 10 h thus depicting sustained action. The increased area under curve (AUC) value by 1.75 folds proved increased bioavailability of the drug. Conclusion: Thus sustained drug delivery with increased bioavailability was designed for prednisolone sodium phosphate for the treatment of ocular inflammation

    A High Throughput Assay for the Detection of Stimulator of Interferon Genes (STING) Agonists

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    The innate immune system includes a menagerie of different cell types, each with a different role in the process of monitoring the body for invaders and presenting gathered debris (antigen) to the adaptive immune system. Somatic cells have intracellular receptors for the same purpose. Cancer cells, however, have avoided these methods of detection despite, in many cases, the tumor’s immunogenic traits. Immuno-oncology is a field dedicated to the immunological traits of tumors, more recently finding ways of instigating an immune response against tumors. In this regard, STING, a receptor of cyclic dinucleotides (CDN), has come to the forefront of immuno-oncology. Activation of STING has been shown to induce profound CD8 T cell-led immune reaction against immunologically suppressed tumors, as well as a memory response upon rechallenge and abscopal response. Most importantly, its simplistic method of activation (intratumoral administration of CDN, with current research formulations exploring the IV route) has scientists clamoring for a CDN mimetic or molecule that likewise activates STING. Currently, numerous pharmaceutical companies have synthesized STING agonists, with some deviating from the canonical CDN formulation. These non-CDN molecules represent the next generation of STING agonists. This thesis set out to develop an efficient screen to discover next generation STING agonists within a commercial compound deck

    Development and characterization of nanostructured lipid carriers for enantiomeric excess (S75:R25) of bupivacaine

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    Orientadores: Eneida de Paula, M√°rcia Cristina BreitkreitzDisserta√ß√£o (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: O baixo tempo de a√ß√£o e toxicidade sist√™mica dos anest√©sicos locais (AL) limitam sua aplica√ß√£o cl√≠nica. Bupivaca√≠na √© o AL mais frequentemente usado em procedimentos cir√ļrgicos, no mundo. A descoberta que o seu enanti√īmero S(-) √© menos t√≥xico que o R(+), levou a introdu√ß√£o no mercado de produtos com excesso enantiom√©rico (bupivaca√≠na S75:R25, aqui representada por BVCS75). Por√©m, o tempo de a√ß√£o da bupivaca√≠na ainda √© curto; para superar isto, BVCS75 foi encapsulada em carreadores lip√≠dicos nanoestruturados (NLC). Este trabalho apresenta o desenvolvimento de uma formula√ß√£o usando m√©todos quimiom√©tricos de planejamento experimental para estudo dos componentes da formula√ß√£o e espectroscopia Raman de imagem, para estudar a miscibilidade dos lip√≠dios s√≥lidos e l√≠quidos. A formula√ß√£o selecionada de carreadores lip√≠dicos nanoestruturados contendo bupivaca√≠na S75:R25 (NLCBVC) mostrou-se f√≠sicoquimicamente est√°vel, por 12 meses √† temperatura ambiente, em rela√ß√£o ao tamanho de part√≠culas, √≠ndice de polidispers√£o (PDI), potencial Zeta e efici√™ncia de encapsula√ß√£o. A caracteriza√ß√£o por DSC, XDR e MET confirmou a encapsula√ß√£o da BVCS75 na matriz lip√≠dica, sem mudan√ßas na estrutura das nanopart√≠culas. No estudo de libera√ß√£o in vitro as NLC prolongaram o tempo de libera√ß√£o do AL para 28 h, e no teste de efeito anest√©sico in vivo o tempo de a√ß√£o duplicou em rela√ß√£o √† BVCS75 livre. Al√©m de melhorar o tempo de a√ß√£o, n√£o foi encontrada diferen√ßa estat√≠stica significativa no bloqueio do nervo ci√°tico de ratos entre NLCBVC 0,125% e BVCS75 0,5%. Assim, a formula√ß√£o permite a redu√ß√£o da dose de anest√©sico, diminuindo a toxicidade sist√©mica da bupivaca√≠na, e abrindo novas possibilidades para futuras aplica√ß√Ķes cl√≠nicasAbstract: The short time of action and systemic toxicity of local anesthetics limit their clinical application. Bupivacaine is the most frequently used local anesthetic in surgical procedures worldwide. The discovery that its S(-) enantiomeric form is less toxic than the R(+) form led to the introduction of products with enantiomeric excess (S75:R25 bupivacaine) in the market. Nevertheless, the time of action of bupivacaine is still short; to overcome that, S75:R25 bupivacaine (BVCS75) was encapsulated in nanostructured lipid carriers (NLC). In this work, we report the development of a formulation, using Chemometric tools (experimental design) to study the formulation factors, and Raman mapping associated with classical Least Squares (CLS) to study the miscibility of solid and liquid lipids. The selected formulation of nanostructured lipid carriers containing bupivacaine S75:R25 (NLCBVC) was found stable for 12 months under ambient temperature, regarding particle size, polydispersion, Zeta potential and encapsulation efficiency. Its characterization by DSC, XDR and TEM confirmed the encapsulation of BVCS75 in the lipid matrix, without changing the nanoparticles structure. Encapsulation of BVCS75 in the nanoparticles prolonged its in vitro release for up to 28 h. The in vivo analgesic effect elicited by NLCBVC was twice that of free BVCS75. Besides improving the time of action, no statistical difference in the blockage of the sciatic nerve of rats was found between 0.125% NLCBVC and 0.5% free BVCS75. Therefore, the formulation allows a reduction in the required anesthesia dose, decreasing the systemic toxicity of bupivacaine, and opening up new possibilities for different clinical applicationsMestradoBioquimicaMestre em Biologia Funcional e Molecular2014/14457-5G.H.R.SFAPESPCNP

    Deconvolution of MycobacteriumMycobacterium tuberculosistuberculosis drug targets using high throughput screening approaches

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    Tuberculosis (TB) is an infectious bacterial disease mainly infecting the pulmonary system of the human body. It affects around 1.5 million people every year, most of whom live in developing countries. The incidence of TB has increased in line with the rise in incidences of Human Immunodeficiency Virus (HIV) infections and Acquired immune deficiency syndrome (AIDS). Due to the pressing concerns of TB, the World Health Organisation (WHO) came up with the Direct Observed Treatment (DOTS) programme. Unfortunately, the development of several resistant strains against first-line drugs and consequently second and third-line drugs have developed. As the current TB drug regimen is inadequate, a good screening strategy, discovery of newer drugs and identification of the mode of action would help in developing better treatment routines and determining bacterial pathways more clearly. Drug discovery follows two major routes, one leading from the drug to the target and the other from target to the drug. Both methods have been applied in this work in order to identify new drugs effective against mycobacteria. Screens performed against a drug library approved by the Food and Drug Administration (FDA) have resulted in some promising hits. Functional characterisation of a putative enoyl CoA hydratase EchA12, which was targeted by florfenicol, revealed a novel lipid chaperone functionality associated with cell wall lipid biosynthesis. Furthermore, a target based phenotypic drug screen of the GSK177 box set against Mtb-PrsA provided further evidence that this enzyme as a viable drug target (Ballell et. al., 2013)

    Drug induced degradation of driver proteins: A novel approach to target MLL-fusions

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    Acute leukaemias in infants are associated with inferior outcomes. The majority of infant acute leukaemia is characterized by balanced chromosomal translocations involving the mixed lineage leukaemia (MLL) gene. Previous work in the department established that the novel formed MLL fusions are the proto-oncoproteins responsible for leukaemia initiation and maintenance so that inhibition of MLL-fusions, in conditional mouse models, resulted in complete disease remission. Therefore, a therapy that inactivates the MLL fusion protein, by protein degradation for example, would offer new hope to these patients. The aim of this study is to identify clinically approved drugs that are capable of inducing degradation of MLL leukaemic fusion proteins. An indicator cell line consisting of a THP1 cell clone expressing firefly luciferase fused to the MLL-AF9 protein, combined with renilla luciferase, was used to screen a collection of 1,280 approved drugs for their ability to induce proteolysis of MLL fusion proteins. 25 drugs lowered the luciferase to renilla ratio, of which 3 were confirmed by western blotting to decrease MLL-fusion protein levels. One drug was taken forward for further analysis. This drug was able to induce the proteolysis of various MLL fusion proteins in human MLL rearranged cell lines and primary patient material. Transcriptome profiling showed shut down of the MLL-fusion signature within 16hrs of addition of the drug. Proteolysis of MLL fusion proteins should also result in a block in self-renewal, as was previously shown in the conditional mouse model. While the drug had no significant impact on the colony formation of normal haematopoietic progenitor cells, it was able to block the colony formation ability of MLL rearranged cell lines. Finally, global alterations in the epigenetic landscape following drug treatment were analysed. This study highlights a new approach to identifying drugs that block driver oncogenes and has identified a potential novel treatment for a major subtype of acute leukaemias in infants

    Synthesis New Liquid Selective Electrodes of Ciprofloxacin Hydrochloride for Determination Ciprofloxacin in Pure form and Pharmaceuticals Preparation.

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    New membrane electrodes for determination of ciprofloxacin hydrochloride were prepared depending on ciprofloxacin hydrochloride - phosphotungstic acid (CFH-PT) as an active material and these electrodes were made with three plasticizers: Di-octylphenylphosphonate(DOPH), Di-butyl phosphate (DBP)Tri-n-butyl phosphate(TBP), in PVC matrix. One of the ciprofloxacin electrodes was gave Nernstian slope equal to 57.21 mV/ decade for DOPH membrane with concentration range from 1.5√ó10-5 to1.0√ó10-1 M, and detection limit equal to 1.5√ó10-6 M .Lifetime was 93 days. Non- Nernstian responses equal to 39.40 and 30.70 mV/ decade for membranes DBP, TBP, respectively. These electrodes were gave concentration range from 1.0√ó 10-5 to 1.0√ó10-2 and from 4.0√ó10-5 to 1.0√ó10-2 M,respectively. Detection limits were 7.0√ó10-6, and 1.7√ó10-6M, respectively. Lifetimes were 5,2 days, respectively. Also selectivity, influence of PH and samples analysis of ciprofloxacin in a pharmaceutical preparations were studied
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