Synthetic analogues of protein-lipid complexes

Hypercoiling poly(styrene-alt-maleic anhydride) (PSMA) is known to undergo conformational transition in response to environmental stimuli. The association of PSMA with lipid 2-dilauryl-sn-glycero-3-phosphocholine (DLPC) produces polymer-lipid complex analogues to lipoprotein assemblies found in lung surfactant. These complexes represent a new bio-mimetic delivery vehicle with applications in the cosmetic and pharmaceutical industries. The primary aim of this study was to develop a better understanding of PSMA-DLPC association by using physical and spectroscopic techniques. Ternary phase diagrams were constructed to examine the effects of various factors, such as molecular weight, pH and temperature on PSMA-DLPC association. 31P-NMR spectroscopy was used to investigate the polymorphic changes of DLPC upon associating with PSMA. The Langmuir Trough technique and surface tension measurement were used to explore the association behaviour of PSMA both at the interface and in the bulk of solution, as well as its interaction with DLPC membranes. The ultimate aim of this study was to investigate the potential use of PSMA-DLPC complexes to improve the bioavailability and therapeutic efficacy of a range of drugs. Typical compounds of ophthalmic interest range from new drugs such as Pirenzepine, which has attracted clinical interest for the control of myopia progression, to the well-established family of non-steroid anti-inflammatory drugs. These drugs have widely differing structures, sizes, solubility profiles and pH-sensitivities. In order to understand the ways in which these characteristics influence incorporation and release behaviour, the marker molecules Rhodamine B and Oil Red O were chosen. PSMA-DLPC complexes, incorporated with marker molecules and Pirenzepine, were encapsulated in hydrogels of the types used for soft contact lenses. Release studies were conducted to examine if this smart drug delivery system can retain such compounds and deliver them at a slow rate over a prolonged period of time.

A combinatorial approach to the chemical synthesis and biological evaluation of 3,4,5-trisubstiituted furan-2(5H)-ones

Many important natural products contain the furan-2(5H)-one structure. The structure of this molecule lends itself to manipulation using combinatorial techniques due to the presence of more than one site for the attachment of different suhstituents. By developing different reaction schemes at the three sites available for attachment on the furan-2(5H)-one scaffold, combinatorial chemistry techniques can be employed to assemble libraries of novel furan 2(5H)-ones. These libraries can then be entered into various biological screening programmes. This approach will enable a vast diversity or compounds to be examined, in the hope or finding new biologically active Iead structures. The work in this thesis has investigated the potential that combinatorial chemistry has in the quest for new biologically active lead structures based on the furan-2(5H)-one structure. Different reactions were investigated with respect to their suitability for inclusion in a library. Once sets of reactions at the various sites had been established, the viability of these reactions in the assembly of combinatorial libraries was investigated. Purification methods were developed, and the purified products entered into suitable biological screening tests. Results from some of these tests were optimised using structure activity relationships, and the resulting products re-screened. The screening tests performed were for anticancer and antimicrobial activity, cholecystokinin (CCK-B) antagonism and anti-inflammatory activity (in the quest for novel cyclo-oxygenase (COX-2) selective non-steroidal anti-inflammatory drugs). It has been shown that many reactions undergone by the furan-2(5H)-one structure are suitable for the assembly of a combinatorial library. Investigation into the assembly of different libraries has been carried out with initial screening results included. From this work, further investigation into combinatorial library assembly and structure activity relationships of screened reaction products can be undertaken.

Ionic and neutral hydrogels for dermal and ophthalmic applications

This thesis is concerned with the use of ionic and neutral hydrogels in dermal and ocular applications with particular reference to controlled release applications. The work consists of three interconnected themes.The first area of study is the use of skin adhesive bioelectrode hydrogels as ground plate electrodes for ophthalmic iontophoresis applications. The work provides a basis of understanding the relative contributions made by ionic monomers (such as sodium s-(acrylamide)-2-methyl propane sulphonate and acrylic acid-bis-(3-sulfopropyl-ester, potassium salt) and neutral monomers (such as acryloymorpholine, N,N-dimethylacrylamide and N-vinyl pyrrolidone) to adhesion, rheology and impedance of bioelectrode gels. The general advantage of neutral monomers, which have been used to successfully replace ionic monomers, is that they enable more effective control of independent anion and cation species (for example potassium chloride and sodium chloride) unlike ionic monomers where polymerisation produces an immobile polyanion thus limiting cation mobility. Secondly, release from a completely neutral hydrogel under the influence of mechanical shaking was studied for the case of crosslinked polyvinyl alcohol (PVA) containing low concentration of linear soluble PVA in a contact lens application. The soluble PVA was observed to be eluting by reptation from the lens matrix due to the mechanical action of the eyelid. This process was studied in an in vitro model, which in this research was used as a basis for developing a lens made with enhanced release polymer. The third area of work is related to the factors that control drug release (in particular non-steroidal anti-inflammatory drugs) from a hydrogel matrix. This links both electrotherapy applications, such as transcutaneous electrical nerve stimulation, in which the passive diffusion from the gel could be used in conjunction with enhanced transmission across the dermal surface with passive diffusion from a contact lens matrix and the development of therapeutic contact lenses.

Development of an enantiomer selective microsampling assay for the quantification of ketorolac suitable for paediatric pharmacokinetic studies

Background: Ketorolac, a potent nonsteroidal anti-inflammatory drug used for pain control in children, exists as a racemate of inactive R (+) and active S (-) enantiomers. Aim: To develop a microsampling assay for the enantioselective analysis of ketorolac in children. Methods: Ketorolac enantiomers were extracted from 50 µl of plasma by liquid–liquid extraction and separated on a ChiralPak AD-RH. Detection was by a TSQ quantum triple quadrupole mass spectrometer with an electrospray ionisation source operating in a positive ion mode. Five children (age 13.8 (1.6) years, weight 52.7 (7.2) kg), were administered intravenous ketorolac 0.5 mg/kg (maximum 10 mg) and blood samples were taken at 0, 0.25, 0.5, 1, 2, 4, 6, 8 and 12 h post administration. CL, VD and t1/2 were calculated based on non-compartmental methods. Results: The standard curves for R (+) and S (-) ketorolac were linear in the range 0–2000 ng/ml. The LLOQs of the method were 0.15 ng on column and 0.31 ng on column for R (+) and S (-) ketorolac, respectively. The median (range) VD and CL of R (+) and S (-) ketorolac were 0.12 l/kg (0.07–0.17), 0.017 l/h/kg (0.12–0.29) and 0.17 (0.09–0.31) l/kg, 0.049 (0.02–0.1) l/h/kg, p = 0.043), respectively. The median (range) elimination half-life (t1/2) of the R (+) and S (-) ketorolac was 5.0 h (2.5–5.8) and 3.1 h (1.8–4.4), p = 0.043), respectively. Conclusion: The development of a simple, rapid and reliable ketorolac assay suitable for paediatric PK studies is reported. Copyright © 2013 John Wiley & Sons, Ltd.

Apoptosis induction in gastric mucous cells in vitro:lesser potency of Helicobacter pylori than Escherichia coli lipopolysaccharide, but positive interaction with ibuprofen

Non-steroidal anti-inflammatory drugs (NSAIDs) cause peptic ulcer disease, but whether they interact with Helicobacter pylori to promote damage is controversial. Moreover, the reported induction of apoptosis in gastric cells by H. pylori lipopolysaccharide (LPS) (10-9 g /ml) contrasts with studies showing low immunological potency of this LPS. Therefore, the effects of LPS from H. pylori NCTC 11637 and Escherichia coli 0111:B4 on apoptosis in a primary culture of guinea-pig gastric mucous cells were investigated in the presence and absence of the NSAID, ibuprofen. Cell loss was estimated by a crystal violet assay, and apoptosis determined from caspase activity and from condensation and fragmentation of nuclei. Exposure to E. coli LPS for 24 h caused cell loss and enhanced apoptotic activity at concentrations ≥ 10-9 g/ml, but similar effects were only obtained with H. pylori LPS at concentrations ≥10-6 g/ml. Although ibuprofen (250 μM) caused cell loss and apoptosis, addition of either E. coli or H. pylori LPSs further enhanced these effects. In conclusion, LPS and ibuprofen interact to enhance gastric cell loss and apoptosis. In such interactions, E. coli LPS is more potent than that of H. pylori. The low potency of H. pylori LPS may contribute to a chronic low-grade gastritis that can be enhanced by the use of NSAIDs. © W. S. Maney & Son Ltd.

High permeability of the anionic form restricts accumulation of indomethacin by cultured gastric surface epithelial cells exposed to low apical pH

The 'ion-trapping' hypothesis suggests that the intracellular concentration of acidic non-steroidal anti-inflammatory drugs in gastric epithelial cells could be much higher than in the gastric lumen, and that such accumulation could contribute to their gastrotoxicity. Our aim was to examine the effect of the pH of the apical medium on the apical to basal transfer of the acidic drug indomethacin (pK a 4.5) across a gastric mucous epithelial cell monolayer, and to determine whether indomethacin accumulated in cells exposed to a low apical pH. Guinea-pig gastric mucous epithelial cells were grown on porous membrane culture inserts (Transwells®) for 72 h. Transfer and accumulation of [ 14C] indomethacin were assessed by scintillation counting. Transfer of [ 3H]mannitol and measurement of trans-epithelial electrical resistance were used to assess integrity of the monolayer. Distribution of [ 14C] urea was used to estimate the intracellular volume of the monolayer. The monolayer was not disrupted by exposure of the apical face to media of pH ≥ 3, or by indomethacin. Transfer of indomethacin (12 μM) to the basal medium increased with decreasing apical medium pH. The apparent permeability of the undissociated acid was estimated to be five times that of the anion. The intracellular concentration of indomethacin was respectively 5.3, 4.1 and 4.3 times that in the apical medium at pH 5.5, 4.5 and 3.0. In conclusion, this study represents the first direct demonstration that indomethacin accumulates in gastric epithelial cells exposed to low apical pH. However, accumulation of indomethacin was moderate and the predictions of the ion-trapping hypothesis were not met, probably due to the substantial permeability of anionic indomethacin across membranes. © 2006 Elsevier B.V. All rights reserved.

Um dermatam sulfato antitrombótico do camarão Litopenaeus vanammei inibe a inflamação e angiogênese

Inflammation is combined of a vascular and a cellular reaction, resulting in different cells and tissue responses, both the intravascular and extravascular environment. As the inflammatory process occurs, coagulation proteases, in particular thrombin (FIIa), are able to initiate various cellular responses in vascular biology and therefore is often observed activation of other biological systems, leading to complications during an event inflammatory, such as thrombosis and angiogenesis. Thus, antagonists molecules of these events are interesting models for the development of novel anti-inflammatory drugs. Thereby, it is worth stressing the glycosaminoglycans (GAGs), which are able to interact with several proteins involved in important biological processes, including inflammation and coagulation. Therefore, this study aimed to evaluate the anti-inflammatory, antithrombotic and anti-angiogenic potentials, as well anticoagulant of a dermatan sulfate-like GAG (DS) extracted from the Litopenaeus vannamei cephalotorax. The compound was obtained after proteolysis and purification by ion-exchange chromatography. After total digestion by DS-like compounds digesting lyases (chondroitinase ABC), the DS-like nature was revealed, and then called DSL. The shrimp compound showed reduced anticoagulant effect by the aPTT assay, but high anti-IIa activity, directly and through heparin cofactor II. On inflammation, the compound had a significant inhibitory effect with the reduction of proinflammatory cytokines. Potential Inhibitory were reported in the antithrombotic and anti-angiogenic assay, the latter being dose dependent. As for anti-hemostatic activity, the polysaccharides did not induced significant bleeding effect. Thus, the results shown by the shrimp DS-like compound indicate this glycosaminoglycan as a biotechnology target with prospects for the development of new multipotent drugs.

Lifestyle Risk Factors for Serrated Colorectal Polyps: a Systematic Review and Meta-Analysis

Background & Aims: Certain subsets of colorectal serrated polyps (SP) have malignant potential. Weperformed a systematic review and meta-analysis to investigate the association between modifiablelifestyle factors and risk for SPs. 
Methods: We conducted a systematic search of Medline, Embase, and Web of Science, forobservational or interventional studies that contained the terms risk or risk factor, and serrated orhyperplastic, and polyps or adenomas, and colorectal (or synonymous terms), published by March2016. Titles and abstracts of identified articles were independently reviewed by at least 2 reviewers.Adjusted relative risks (RR) and 95% CIs were combined using random effects meta-analyses toassess the risk of SP, when possible. 
Results: We identified 43 studies of SP risk associated with 7 different lifestyle factors: smoking,alcohol, body fatness, diet, physical activity, medication and/or hormone replacement therapy.When we compared the highest and lowest categories of exposure, factors we found to significantlyincrease risk for SP included tobacco smoking (RR, 2.47; 95% CI, 2.12–2.87), alcohol intake (RR, 1.33;95% CI, 1.17–1.52), body mass index (RR, 1.40; 95% CI, 1.22–1.61), and high intake of fat or meat.Direct associations for smoking and alcohol, but not body fat, tended to be stronger for sessileserrated adenomas/polyps than hyperplastic polyps. In contrast, factors we found to significantlydecrease risks for SP included use of non-steroidal anti-inflammatory drugs (RR, 0.77; 95% CI, 0.65–0.92) or aspirin (RR, 0.81; 95% CI, 0.67–0.99), as well as high intake of folate, calcium, or fiber. Nosignificant associations were detected between SP risk and physical activity or hormone replacementtherapy. 
Conclusions: Several lifestyle factors, most notably smoking and alcohol, are associated with SP risk.These findings enhance our understanding of mechanisms of SP development and indicate that riskof serrated pathway colorectal neoplasms could be reduced with lifestyle changes.

Alveolite

A etiologia da alveolite é desconhecida. Considera-se não haver uma causa específica, mas sim uma associação de factores inerentes ao seu aparecimento. O diagnóstico é realizado, geralmente, entre o 2º e 5º dia após exodontia, sendo bastante claro. Manifesta-se por uma dor aguda e pulsátil, mal controlada com analgésicos, e de apresentação clínica uma inflamação da mucosa em torno do alvéolo, com parcial ou total perda do coágulo, apresentando-se este vazio ou com tecido necrótico e/ou restos alimentares. Pode ainda haver uma exposição óssea. Diversas são as taxas de incidência, variando consoante os factores de risco predisponentes da doença. São estes a idade, género, ciclo menstrual e toma de contraceptivos orais (nas mulheres), trauma cirúrgico e experiência do médico, a indicação da extração, bem como características do dente a ser extraído, ainda as técnicas e anestesia usadas, bem como remanescentes ósseos e/ou radiculares, uso de retalhos e suturas, patologias sistémicas, medicação e cuidados pós-operatórios do doente. Todos estes podem ter impacto no desenvolvimento desta condição, devendo o médico dentista eliminá-los e reduzi-los ao máximo. A prevenção aplica-se a medidas não farmacológicas, como redução de factores de risco, em combinação a terapia farmacológica, de modo a favorecer uma correta cicatrização. Recurso a soluções antissépticas, medicação tópica (intra-alveolar) e prescrição sistémica de antibióticos e anti-inflamatórios não esteroides são algumas dessas medidas. Ao nível do tratamento, vários métodos e materiais estão disponíveis no mercado. Por ser uma condição que o próprio organismo “combate”, o objectivo terapêutico passa basicamente pela redução dos sintomas debilitantes do paciente e controlo bacteriano. A opção é individual, uma vez que não existe nenhum tratamento com características ideais, sendo os resultados na literatura bastante discrepantes. Limpeza do alvéolo, medicação intra-alveolar (tópica) e/ou sistémica, bem como terapia com laser de baixa intensidade, são algumas opções. A administração antibiótica deve ser reservada para casos especiais, não devendo ser abordada como método de rotina. Analgésicos são uma opção, podendo ser aconselhada consoante o quadro clínico doloroso. Também o reforço para uma higiene oral rigorosa, com irrigação do alvéolo para evitar detritos e impactação alimentar (no caso de não existirem obtundantes intra-alveolares) devem ser preocupação do médico dentista. Deve haver um seguimento regular do paciente, especialmente se aplicados medicamentos tópicos, para avaliação e renovação (se necessário) do curativo até recuperação. O médico dentista deve saber identificar um caso de alveolite, encontrando-se informado e consciente das várias opções preventivas e terapêuticas. Estudos mais claros e objectivos são necessários na procura de critérios de diagnóstico genéricos da doença, bem como terapêuticas preventivas e de tratamentos com taxas de sucesso altas e suportadas por evidência científica. Para que, desta forma, seja elaborado um protocolo universal a seguir na prática clínica.

Determinants of self-medication with NSAIDs in a Portuguese community pharmacy

Background: Non-steroid anti-inflammatory drugs (NSAIDs) are a widely used therapeutic group in the world, and particularly in the Portuguese population. Objective: To compare NSAID’s use by prescription and self-medication acquisition and to determine the pattern of indication of NSAIDs, their usage profile and possible implications for patients’ safety. Methods: A cross-sectional design was used where individuals presenting at a community pharmacy requesting NSAIDs during the study period (one month) were invited to answer a face-to-face interview where socio-demographic characteristics, the indication pattern and previous experience of side effects were assessed. A follow-up interview was performed one week later to assess the incidence of adverse effects. The study was ethically approved. Results: A sample of 130 NSAIDs users was recruited, comprising mostly women (n=87; 66.9%), actively employed (n=77; 59.2%) and presenting a mean age of 49.5 years old (SD=20.49). An equal proportion of individuals acquired NSAIDs by self-medication and with medical prescription (n=65; 50%). Over 4/5 of patients (n=57; 87.7%) acquiring NSAIDs without a prescription were self-medicated by their own initiative, and only 10.8% (n=7) had been advised by the pharmacist. The most commonly acquired active substances were ibuprofen and diclofenac. Self-medicated users more frequently resorted to topical NSAIDs following short term treatments. The major underlying condition motivating NSAIDs sought were musculoskeletal disorders (45.0%), regardless of the regimen. An important proportion of prevalent users of NSAIDs reported previous experience of adverse effects (11.3%). One week after initiating NSAID therapy, a small proportion of patients reported incidence of adverse effects. Conclusion: Self-medication with NSAIDs is sought for numerous medical conditions. Reported adverse effects (prevalent and incident) confirm the need for a more rational use of NSAIDs and ongoing pharmacovigilance.

Ibuprofen Inhibits Overexpression of Tumor-Related Rac1b through SRSF1

Abstract: The serrated pathway to colorectal tumor formation involves oncogenic mutations in the BRAF gene, which are sufficient for initiation of hyperplastic growth but not for tumor progression. A previous analysis of colorectal tumors revealed that overexpression of splice variant Rac1b occurs in around 80% of tumors with mutant BRAF and both events proved to cooperate in tumor cell survival. Patients with inflamed human colonic mucosa also have increased expression of Rac1b as well as mice with experimentally induced colitis. The increase of Rac1b in the mouse model was specifically prevented by the nonsteroidal anti-inflammatory drug ibuprofen. Purpose: The objective of our study is to understand the molecular regulation of Rac1b alternative splicing event and how it contributes to tumorigenesis. Experimental description: HT29 colorectal cell line was used as model to test several signaling pathways after 48h of treatment with ibuprofen. For this we analyzed the proteins of interest by Western Blot and the transcript levels by RT-PCR. Results: Mechanistic studies in cultured HT29 colorectal tumor cells revealed that ibuprofen inhibited Rac1b expression in a cyclooxygenase inhibition–independent manner and targets directly the alternative splicing event. Here, we provide evidence that ibuprofen leads to a decrease in expression of SRSF1, a splicing factor that we previously identified to promote Rac1b alternative splicing. Together, our results suggest that stromal cues, namely, inflammation, can trigger changes in Rac1b expression in the colon and identify ibuprofen as a highly specific and efficient inhibitor of Rac1b overexpression in colorectal tumors. Conclusions: Our data identify an additional cyclooxygenase–independent action of ibuprofen and suggest it may be beneficial in the treatment of patients with the subtype of BRAF-mutated serrated colorectal tumors.

Ibuprofen inhibits overexpression of tumor-related Rac1b through SRSF1

Introduction: The serrated pathway to colorectal tumor formation involves oncogenic mutations in the BRAF gene, which are sufficient for initiation of hyperplastic growth but not for tumor progression. A previous analysis of colorectal tumors revealed that overexpression of splice variant Rac1b occurs in around 80% of tumors with mutant BRAF and both events proved to cooperate in tumor cell survival. Patients with inflamed human colonic mucosa also have increased expression of Rac1b as well as mice with experimentally induced colitis. The increase of Rac1b in the mouse model was specifically prevented by the nonsteroidal anti-inflammatory drug ibuprofen.

Estudo comparativo de carragenanas comerciais Kappa, Iota e Lambda no processo inflamatório em ratos :edema intraplantar e pleurisia

The Iota, Kappa and Lambda commercial carrageenans are rarely pure and normally contain varying amounts of the other types of carrageenans. The exact amount of impurity depends on the seaweed source and extraction procedure. Then, different analysis methods have been applied for determination of the main constituents of carrageenans because these three carrageenans are extensively used in food, cosmetic and pharmaceutical industry. The electrophoresis of these compounds proved that the carrageenans are constituted by sulfated polysaccharides. These compounds were characterized by colorimetric methods and was observed that the Lambda carrageenan shown the greater value (33.38%) of sulfate. These polymers were examined by means of 13C NMR spectroscopy and infrared spectra. The polysaccharides consisted mainly of units alternating of sulfated galactoses and anhydrogalactoses. The aim of the study was also to test the inflammatory action of these different polysaccharides. A suitable model of inflammation is acute sterile inflammation of the rat hind limb induced by carrageenan. Paw edema was induced by injecting carrageenans (κ, ι and λ) in saline into the hind paw of a male Wistar rats (175–200 g). The pathway to acute inflammation by carrageenan (kappa, iota and lambda) were expressed as time-edema dependence and measured by paw edema volume. For this purpose, was used an apparatus (pakymeter), which makes it possible to measure the inflammation (swelling of the rat foot) with sufficient accuracy. The results showed that κ-carrageenan (1%) have an edema of 3.7 mm and the paw edema increase was time and dose dependent; the ι-carrageenan (0.2%) caused an edema of 4 mm and the λ-carrageenan (1%) caused an edema of 3.6 mm. Other model was used in this study based in the inflammation of pleura for comparatives studies. Injection of carrageenans into the pleural cavity of rat induced an acute inflammatory response characterized by fluid accumulation in the pleural cavity, a large number of neutrophils and raised NO production. The levels of NO were measured by Griess reactive. The ι-carrageenan caused the greater inflammation, because it has high concentration of nitrite/nitrate (63.478 nmoles/rat), exudato volume (1.52 ml) and PMNs (4902 x 103 cells). Quantitative evaluation of inflammations of rats is a useful and important parameter for the evaluation of the efficacy of anti-inflammatory drugs

Beers versus STOPP criteria in polyharmacy community-dwelling older patients

Objective: To assess potentially inappropriate prescribing (PIP) using Beers (2012 version) and STOPP (2008 version) criteria in polypharmacy, community-dwelling, older patients. Methods: From the information collected in the invoicing data of the prescriptions and the electronic medical records, a sample was selected of 223 ≥ 65-year-old patients who were taking simultaneously 10 or more drugs per day. Beers and STOPP criteria were separately applied, and the results obtained with the two methods were compared. Results: A total of 141 (63.2%) patients presented at least one Beers criterion. The two most frequently observed Beers criteria independent of diagnosis were the use of benzodiazepines and the use of non-COX-2-selective non-steroidal anti-inflammatory drugs. With regard to Beers criteria considering diagnosis, the most frequent were the use of anticholinergic drugs in patients with lower urinary tract symptoms or benign prostatic hyperplasia, and the use of benzodiazepines, antipsychotics, Zolpidem or H2-antihistamines, in patients with dementia or cognitive impairment. A total of 165 (73.9%) patients had at least one PIP according to the STOPP criteria. Duplicate drug classes and long-term use of long-acting benzodiazepines were the two most frequent STOPP criteria. Discussion: Our study identified a high frequency of PIP in poly-medicated community-dwelling older patients. Simultaneous application of Beers and STOPP criteria represents a useful tool to improve prescribing in this population group.

Tratamento de diarreias neonatais em vitelos

Dissertação de Mestrado Integrado em Medicina Veterinária