Novo sistema de aplicação de drogas em cirurgias oftalmológicas

The topical corneal application of antimitotic mitomycin-C (MMC) during refractive surgery is still characterized by a lack of standardization and considerable empirism. For this reason the creation of a system capable of reliable drug delivery represents a beneficial innovation for patients submitted to these procedures. Objective: Elaborate a new MMC delivery system during the transoperatory period of photorefractive keratectomy (PRK) followed by patent application. Methods: The project consists of an in vitro experimental study to create an MMC (0.02%) release system. The drug was impregnated in sterile Whatman® 41 paper filter discs with a diameter of 8 mm. After drying, the discs were applied to antibiogram plates seeded with Staphylococcus epidermidis (American Type Culture Collection ATCC 12228), followed by the addition of a drop of sterile water. At the end of 1 minute, the discs were removed and the plates incubated for 48 hours at 35oC. Mean drop volume in the collyrium flasks was measured using analytical balance weighing. The inhibition halo (mm) was correlated with the MMC impregnated into the disc. After completion of the invention design a patent application was lodged at the National Institute of Industrial Property. Results: The correspondence between MMC-produced inhibition halos indicated that a dose of 16μg was ideal for impregnating into the discs. The mean drop volume obtained from the collyrium flasks was 37.7 μL. A minute after the application of one drop of balanced saline solution, the system released an adequate concentration for PRK surgery. Conclusion: A new MMC delivery system was created for transoperatory application in photorefractive keratectomy (PRK). Publication of the patent application (number PI 0704739-8) gives the authors exclusive intellectual property rights. The study was sponsored by Ophthalmos Indústria e Comércio de Produtos Farmacêuticos S.A. (São Paulo-SP, Brazil) and received the indispensable scientific contribution of researchers from the fields of Pharmacy, Medicine, Biology, Statistics and Law, characterizing the work as multidisciplinary, in accordance with norms established by the Postgraduate Health Sciences Program of the Federal University of Rio Grande do Norte (UFRN)

Uso do 5-fluorouracil no intra-operatório da cirurgia do pterígio

Objetivo: Avaliar a efetividade e as complicações com a aplicação do 5- fluorouracil (5-FLU) no intra-operatório da cirurgia do pterígio. Método: Foram avaliados 28 olhos de 26 indivíduos quanto ao tipo e tamanho do pterígio, cirurgias prévias e a resposta ao tratamento cirúrgico (no 7º , 21º , 60º e 90º dia de pós-operatório). Logo após a exerese do pterígio, aplicou-se 5-FLU (25 mg/ml) no leito cirúrgico, durante cinco minutos; a seguir, realizou-se a técnica de deslizamento de retalho conjuntival. Resultados: A maioria dos pacientes tinha mais de 50 anos de idade e apresentava pterígio primário (70,0%), grau II (60,7%), do tipo involutivo (60,7%). No pós-operatório observaram-se: isquemia (10,7%), deiscência da conjuntiva (7,1%), ceratite (3,5%), conjuntivite (3,5%) e recidiva da lesão em 1 olho (3,5%).Conclusão: O 5-FLU se mostrou droga segura e efetiva na prevenção das recidivas, podendo ser usado como coadjuvante no tratamento do pterígio para prevenir recidivas.

Exposição de fibroblastos da cápsula de Tenon normal de portadores de pterígio ao 5-fluorouracil e à mitomicina C

OBJETIVO: Avaliar a atividade proliferativa dos fibroblastos da cápsula de Tenon normal, proveniente de portadores de pterígios primários e recidivados. MÉTODOS: Foi realizado estudo prospectivo, aleatório, avaliando-se fragmentos da cápsula de Tenon normal, removidos de 20 portadores de pterígios primários e 21, recidivados. A taxa de proliferação foi avaliada em fibroblastos de terceira passagem, quando as culturas foram expostas a agentes antimitóticos: mitomicina C e 5-fluorouracil. Os dados obtidos foram submetidos à análise estatística. RESULTADOS: Dentre os 41 espécimes cultivados, apenas 1 de pterígio primário e 2 de recidivado proliferaram. Quando expostos a mitomicina C e ao 5-fluorouracil não houve diferença estatisticamente significativa quanto a inibição de proliferação celular. CONCLUSÃO: Desta forma, in vitro, ambos os antimitóticos estudados têm a mesma eficácia na inibição da proliferação sobre os fibroblastos de cápsulas de Tenon normal.

Carcinoma espinocelular de conjuntiva com evolução para exenteração: relato de caso

Portador de carcinoma espinocelular da conjuntiva teve a lesão removida, com recorrência em outra localização. O paciente recebeu ciclos de 5-Fluoruracila como tratamento adjuvante à remoção cirúrgica, apresentando evolução desfavorável que chegou à exenteração orbitária. São feitos comentários quanto ao uso de antimitóticos no tratamento destas lesões.

Segurança e efetividade no tratamento do pterígio usando infiltração de 5-fluoruracila no intraoperatório

OBJETIVO: Avaliar a segurança e a efetividade do uso do 5-fluoruracila (5-FU) como tratamento adjuvante do pterígio, aplicado sob a forma de infiltração subconjuntival, no período intraoperatório. MÉTODOS: Foram avaliados prospectivamente 125 indivíduos (125 olhos) portadores de pterígio. Os indivíduos foram operados segundo a técnica de retalho de deslizamento e receberam, ao final do procedimento, injeção subconjuntival de 0,2 mL de 5-FU (25 mg/mL). Foram anotados os dados do paciente como idade, sexo, profissão, características da lesão (primário ou recidivado, tamanho, carnoso ou involutivo) e feito seguimento pós-operatório, aos 7, 21, 60 e 180 dias. Os dados foram submetidos à avaliação estatística. RESULTADOS: Não foram observados casos de complicação decorrente do uso do 5-FU em injeção no intraoperatório do pterígio. A taxa de recidiva geral observada aos 180 dias de pós-operatório foi de 35,8%, sendo de 35,7% para os pterígios primários e de 36,4% para os recidivados. CONCLUSÃO: A aplicação do 5-FU no período intraoperatório sob a forma de infiltração subconjuntival é segura. Entretanto, ainda resulta em altas taxas de recidiva e novos estudos devem ser realizados a fim de conhecer a concentração/dose ideal que permitirá menores chances de recidiva da lesão.

Structural Basis for Interaction of Inhibitors with Cyclin-Dependent Kinase 2

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Antitumor activity of extracts from Peperomia elongata

The cytotoxic potential of ethanol extracts from Peperomia elongata H. B. & K. (Piperaceae) were evaluated against human cancer cell lines by the MTT method. The samples considered cytotoxic were tested for antimitotic activity with the sea urchin egg development test and for hemolytic activity using mice erythrocytes. The extracts from leaves (hexane), stems (ethanol, hexane, hexane: AcOEt, AcOEt, and MeOH: H2O insoluble), and roots (R4) presented potential cytotoxic action. The stems extracts showed the highest toxicity in all tumor cell lines tested, with an IC50 <= 9.0 mu g/mL for ethanol extract, IC50 <= 11.6 mu g/mL for MeOH:H2O insoluble, IC50 <= 7.3 mu g/mL for hexane extract, IC50 <= 11.4 mu g/mL for hexane: AcOEt, and IC50 <= 16.2 mu g/mL for AcOEt extract. All extracts considered cytotoxic for tumoral cell lines presented antimitotic activity. The samples from roots (R4) and stems (ethanol, MeOH: H2O insoluble, and hexane extract from leaves) were found to possess lytic activity in mice erythrocytes but in higher doses (> 125 mu g/mL). Further studies for the isolation and identification of the active principles of these extracts should be undertaken.

Exposição de culturas de fibroblastos de pterígios primários e recidivados à mitomicina c e ao 5-fluorouracil

Purpose: To evaluate the proliferation of the fibroblasts from primary and recurrent pterygium in culture of cells, after the exposure to mitomycin C and to 5-fluorouracil. Methods: Cultures of fibroblasts from primary and recurrent pterygiuns had been carried through. The cells of the third passage were exposed to mitomycin C 0.4% during 3 minutes and to 5-fluorouracil 25mg/ml that was kept in the nutritional medium. After 3, 6, 12 and 18 days of the exposure, cell countings were made in hemocytometer, in triplicate, with a control not treated with the drugs for each day. The data were submitted to statistical analysis.Results: Mitomicyn C had homogeneous behavior in the primary and recurrent groups, which inhibited the cellular proliferation since the first counting day. However, with 5-fluorouracil, the proliferation inhibition was verified only after the third day of evaluation (in the second counting day) in the recurrent group, and since the first counting day in the primary group. At the end of the experimental period, 5-fluorouracil and mitomycin C were equally efficient in the inhibition of the fibroblasts from primary and recurrent pterygium proliferation. In the controls not exposed, the cell's numbers were increasing during the experimental time.Conclusion: Mitomycin and 5-fluorouracil are equally able to inhibit fibroblasts proliferation from primary and recurrent pterygium Tenon's capsule in cell culture.

Effect of topical application of 5-FU on the corneoscleral limbus of rabbits

• Aim: To observe the effects of topical application of 5-fluorouracil (5-FU) on the rabbits corneal limbus. • Methods: 5-FU was applied topically to the corneoscleral region of rabbits eyes. The animals were sacrificed immediately or 7, 15, 30 and 60 days later, and tissues were submitted to histological examination. • Results: All animals presented corneoscleral deepithelization close to the site of application during the immediate post-operative period, whereas on the 4th postoperative day ulceration was no longer present. Histological examination showed absence of epithelium and slight edema. After one week (G2), 2 animals presented epithelial defects, thickened epithelium with larger basal cells and loose chromatin, and slight subepithelial edema. The remaining groups showed no alterations. • Conclusion: The 5-FU topically on the corneoscleral limbus postpone the epitelization.

Natural Products from Cyanobacteria with Antimicrobial and Antitumor Activity

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Expression of Vascular Endothelial Growth Factor (VEGF) in Macrophages, Fibroblasts, and Endothelial Cells in Pterygium Treated with 5-Fluorouracil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Stabilization of microtubule dynamics by estramustine by binding to a novel site in tubulin: A possible mechanistic basis for its antitumor action

The cellular targets for estramustine, an antitumor drug used in the treatment of hormone-refractory prostate cancer, are believed to be the spindle microtubules responsible for chromosome separation at mitosis. Estramustine only weakly inhibits polymerization of purified tubulin into microtubules by binding to tubulin (Kd, ≈30 μM) at a site distinct from the colchicine or the vinblastine binding sites. However, by video microscopy, we find that estramustine strongly stabilizes growing and shortening dynamics at plus ends of bovine brain microtubules devoid of microtubule-associated proteins at concentrations substantially below those required to inhibit polymerization of the microtubules. Estramustine strongly reduced the rate and extent both of shortening and growing, increased the percentage of time the microtubules spent in an attenuated state, neither growing nor shortening detectably, and reduced the overall dynamicity of the microtubules. Significantly, the combined suppressive effects of vinblastine and estramustine on the rate and extent of shortening and dynamicity were additive. Thus, like the antimitotic mechanisms of action of the antitumor drugs vinblastine and taxol, the antimitotic mechanism of action of estramustine may be due to kinetic stabilization of spindle microtubule dynamics. The results may explain the mechanistic basis for the benefit derived from combined use of estramustine with vinblastine or taxol, two other drugs that target microtubules, in the treatment of hormone-refractory prostate cancer.

Regulation of neuroblast mitosis is determined by PACAP receptor isoform expression

Although neurogenesis in the embryo proceeds in a region- or lineage-specific fashion coincident with neuropeptide expression, a regulatory role for G protein-coupled receptors (GPCR) remains undefined. Pituitary adenylate cyclase activating polypeptide (PACAP) stimulates sympathetic neuroblast proliferation, whereas the peptide inhibits embryonic cortical precursor mitosis. Here, by using ectopic expression strategies, we show that the opposing mitogenic effects of PACAP are determined by expression of PACAP receptor splice isoforms and differential coupling to the phospholipase C (PLC) pathway, as opposed to differences in cellular context. In embryonic day 14 (E14) cortical precursors transfected with the hop receptor variant, but not cells transfected with the short variant, PACAP activates the PLC pathway, increasing intracellular calcium and eliciting translocation of protein kinase C. Ectopic expression of the hop variant in cortical neuroblasts transforms the antimitotic effect of PACAP into a promitogenic signal. Furthermore, PACAP promitogenic effects required PLC pathway function indicated by antagonist U-73122 studies in hop-transfected cortical cells and native sympathetic neuroblasts. These observations highlight the critical role of lineage-specific expression of GPCR variants in determining mitogenic signaling in neural precursors.

Structural basis for specificity and potency of a flavonoid inhibitor of human CDK2, a cell cycle kinase.

The central role of cyclin-dependent kinases (CDKs) in cell cycle regulation makes them a promising target for studying inhibitory molecules that can modify the degree of cell proliferation. The discovery of specific inhibitors of CDKs such as polyhydroxylated flavones has opened the way to investigation and design of antimitotic compounds. A novel flavone, (-)-cis-5,7-dihydroxyphenyl-8-[4-(3-hydroxy-1-methyl)piperidinyl] -4H-1-benzopyran-4-one hydrochloride hemihydrate (L868276), is a potent inhibitor of CDKs. A chlorinated form, flavopiridol, is currently in phase I clinical trials as a drug against breast tumors. We determined the crystal structure of a complex between CDK2 and L868276 at 2.33 angstroms resolution and refined to an Rfactor 20.3%. The aromatic portion of the inhibitor binds to the adenine-binding pocket of CDK2, and the position of the phenyl group of the inhibitor enables the inhibitor to make contacts with the enzyme not observed in the ATP complex structure. The analysis of the position of this phenyl ring not only explains the great differences of kinase inhibition among the flavonoid inhibitors but also explains the specificity of L868276 to inhibit CDK2 and CDC2.

Two distinct oscillators in the rat suprachiasmatic nucleus in vitro.

In the rat suprachiasmatic nucleus slice culture, circadian rhythms in the release of arginine vasopressin and vasoactive intestinal polypeptide were measured simultaneously and longitudinally. The phase relationship between the two peptide rhythms was relatively constant in the culture without a treatment of antimitotic drugs but became diverse by an introduction of antimitotics, which is generally used to reduce the number of glial cells. By monitoring the two rhythms continuously for 6 days, different periods were detected in culture with the antimitotic treatment. Furthermore, N-methyl-D-aspartate shifted the phase of the two peptide rhythms in the same culture differently. These results indicate that the arginine vasopressin and vasoactive intestinal polypeptide release are under control of different circadian oscillators.