A área das Ciências Farmacêuticas busca constantemente por tratamentos mais eficientes, direcionados para alvos específicos, com diminuição da dose necessária e com a minimização dos efeitos adversos. Neste contexto, a área de Nanotecnologia Farmacêutica apresenta grande potencial de aplicabilidade, com resultados bastante promissores para o tratamento de diversas doenças. Os sistemas nanoestruturados têm sido avaliados para a incorporação de fármacos já utilizados em tratamentos administrados formas farmacêuticas convencionais que apresentam problemas farmacocinéticos ou farmacodinâmicos quando administrados. E, também, para a incorporação de novas moléculas com potencial para o tratamento de determinada doença. Neste trabalho de tese, nanocápsulas de núcleo lipídico versáteis contendo metotrexato na forma ácida e éster, bromelina, etanercept e infliximab foram desenvolvidas buscando contornar as limitações e aumentar a eficácia terapêutica desses fármacos. Inicialmente, as propriedades anti-inflamatórias de nanocápsulas de núcleo lipídico revestidas por micelas de polissorbato 80 contendo metotrexato encapsulado foram avaliadas em experimentos in vitro e in vivo, em células mononucleares obtidas a partir do líquido sinovial de pacientes com artrite reumatoide e em ratos Lewis com artrite induzida por adjuvante completo de Freund, respectivamente. As nanocápsulas de núcleo lipídico demonstraram serem altamente eficazes no controle da inflamação, sendo que os efeitos anti-inflamatórios in vivo foram alcançados em doses 75% menores que o metotrexato em solução. Na sequência, o tratamento in vitro da linhagem de células de carcinoma de mama humano, MCF-7, com nanocápsulas de núcleo lipídico multiparede funcionalizadas com bromelina demonstrou uma redução de 160 vezes na concentração necessária para obter o mesmo efeito quando comparada a uma solução de bromelina. A influência das pseudofases aniônicas e catiônicas no mecanismo de distribuição da indometacina, tacrolimus, aciclovir, metotrexato e éster etílico de metotrexato, foram avaliadas aplicando um algoritmo desenvolvido para nanocápsulas de núcleo lipídico. Verificou-se que somente a indometacina sofreu influência da presença de cargas, aumentando a afinidade pela fase dispersa das formulações. Formulações de nanocápsulas de núcleo lipídico multiparede contendo metotrexato na forma ácida e éster encapsulados e/ou funcionalizando a superfície das nanocápsulas foram desenvolvidas e testadas in vitro em linhagens de células tumorais (MCF-7) e em linhagens de células sadias (HaCaT). Essas formulações demonstraram atividade antiproliferativa maior para as MCF-7 (com redução em mais de 50% na viabilidade celular) em comparação com as soluções de metotrexato e éster etílico de metotrexato e esta atividade foi maior para as formulações em que as moléculas foram funcionalizadas na superfície das nanopartículas. A captação das nanopartículas pelas células também foi maior para as formulações funcionalizadas com metotrexato ou éster etílico de metotrexato em comparação com a formulação em que o éster de metotrexato está encapsulado. As três formulações contendo metotrexato na forma ácida ou éster não demonstraram ação antiproliferativa em linhagens de células sadias (HaCaT). Devido à baixa expressão de receptores de folato nessas células, não houve aumento da captação celular em comparação à formulação sem fármaco. Por último, foram desenvolvidas satisfatoriamente formulações de nanocápsulas de núcleo lipídico multiparede funcionalizadas com os anticorpos monoclonais infliximab e etanercept, e contendo éster etílico de metotrexato encapsulado, demonstrando que são adequadas para futuros estudos visando o tratamento da artrite reumatoide. Esse conjunto de resultados demonstra que as nanocápsulas de núcleo lipídico com funcionalização de superfície versátil, sejam revestidas com polissorbato 80 ou multiparede funcionalizadas são um sistema bastante promissor para a administração de fármacos de modo a aumentar sua especificidade e eficácia. / The Pharmaceutical Sciences field is constantly searching for more effective treatments, aiming specific targets, with dose reduction and minimization of side effects. In this context, the Pharmaceutical Nanotechnology field presents great applicability potential, with highly promising results for the treatment of several diseases. Nanostructured systems have been evaluated for the encapsulation of drugs approved for use in conventional pharmaceutical dosage forms that, however, exhibit pharmacokinetic or pharmacodynamics problems when administered, and for the encapsulation of novel molecules with potential to treat a determined disease. In the present thesis, versatile lipid-core nanocapsules containing methotrexate in the acid and ester forms, bromelain, etanercept and infliximab were developed, seeking to circumvent the limitations and increase the therapeutic efficacy of these drugs. Initially, the anti-inflammatory properties of methotrexate-loaded lipid-core nanocapsules coated with polysorbate 80 micelles were evaluated in in vitro and in vivo experiments, using mononuclear cells obtained from the synovial fluid of rheumatoid arthritis patients and Lewis rats with Freund complete adjuvant-induced arthritis. Lipid-core nanocapsules demonstrated to be highly effective in the control of inflammation, and the in vivo anti-inflammatory effects were reached in a dose 75% lower than the methotrexate in solution. In the sequence, the in vitro treatment of a human breast cancer cell line, MCF-7, with bromelina-functionalized multiple-wall lipid-core nanocapsules demonstrated a 160-fold reduction of the concentration required to obtain the same effect when compared with a bromelain solution. The influence of the anionic and cationic pseudo-phases in the distribution mechanism of indomethacin, tacrolimus, acyclovir, methotrexate and methotrexate ethyl ester was evaluated through an algorithm developed for lipid-core nanocapsules. It was verified that only indomethacin underwent influence in the presence of charge, increasing the affinity by the disperse phase of the formulations. Multiple-wall lipid-core nanocapsules formulations containing methotrexate in the acid and ester forms encapsulated and/or functionalizing the surface of the nanoparticles were developed and tested in vitro in tumour MCF-7 cells and in a healthy cell line (HaCaT). These formulations demonstrated higher anti-proliferative activity for the MCF-7 cells (reduction of over 50 % in cellular viability) in comparison with the methotrexate and methotrexate ethyl ester solutions and this activity was higher for the formulations in which the molecules were functionalized in the surface of the nanoparticles. A higher cellular uptake was observed for the formulations functionalized with methotrexate or methotrexate ethyl ester in comparison with the formulations in which the methotrexate ester is encapsulated. The three formulations containing methotrexate in the acid or ester form did not demonstrate anti-proliferative activity in non-tumour cell lines (HaCaT). Since these cells have a small expression of folate receptors, the uptake was not increased in comparison with the formulation without drug. Lastly, formulations of methotrexate ethyl ester-loaded multiwall lipid core nanocapsules functionalized with monoclonal antibodies infliximab and etanercept were successfully developed demonstrating suitability for future studies aiming the treatment of rheumatoid arthritis. These groups of results demonstrate that versatile lipid core nanocapsules, either coated with polysorbate 80 or multiwalled functionalized are a very promising system for the administration of drugs aiming their specificity and efficacy.

A aterosclerose é considerada, hoje, doença inflamatória e com intensa proliferação celular, daí o racional de se usar medicamentos antiproliferativos e com ação anti-inflamatória como o paclitaxel (PTX) e o metotrexato (MTX) no tratamento dessa condição. A nanoemulsão lipídica (LDE), de composição semelhante à da lipoproteína de baixa densidade (LDL), se liga a receptores de LDL após sua injeção endovenosa na corrente sanguínea. Como tais receptores estão superexpressos em células com altas taxas de proliferação, como ocorre no câncer e na aterosclerose, a LDE pode ser usada como veículo para direcionar agentes antiproliferativos a essas células, aumentando a sua eficácia e diminuindo a toxicidade. O paclitaxel é um quimioterápico com ação antiproliferativa usado em vários tipos de câncer e recobrindo stents farmacológicos, trabalhos anteriores, usando coelhos submetidos a uma dieta aterogênica, nos animais tratados com LDE-PTX houve redução de 60% da área lesionada. O metrotexato, além de ser usado em vários esquemas quimioterápicos, possui, também, ação anti-inflamatória, sendo usado em doenças inflamatórias crônicas, como a artrite reumatoide. Em outro estudo envolvendo, coelhos hipercolesterolêmicos, o uso de MTX comercial por 4 semanas demonstrou uma redução de 75% na área de placa aterosclerótica. Esse estudo tem por objetivo avaliar macroscopicamente a eficácia das terapias quimioterápicas combinada, composta de PTX-LDE com MTX-LDE, e monoterapia, apenas com PTX-LDE, na regressão da aterosclerose experimental. No presente trabalho, vinte e oito coelhos machos da raça New Zealand receberam dieta rica em colesterol a 1% durante 8 semanas. Depois desse período, foram divididos em quatro grupos: grupo CONTROLE, que foi sacrificado e as aortas fixadas para análise posterior, grupo DIETA, que apenas teve a ração enriquecida com colesterol a 1% suspensa, PTX, que recebeu tratamento com injeções endovenosas semanais de LDE-paclitaxel na dose de 4 mg/kg por 8 semanas, e PTX+MTX, que recebeu LDE-paclitaxel e LDE-metotrexato na dose de 4 mg/kg/semana por 8 semanas. Foram avaliados perfil hematológico, lipídico, bioquímico, ponderal e o consumo de ração. Após a eutanásia, foram medidas as lesões ateroscleróticas macroscópicas nas aortas dos coelhos. Em seguida, o arco aórtico foi analisado por morfometria e por imuno-histoquímica. Os marcadores inflamatórios foram analisados no plasma, por ELISA e por meio de expressão gênica por Qrt-pcr. Observou-se que não houve diferença no perfil ponderal e no consumo de ração entre os grupos de estudo. Não houve toxicidade hematológica, hepática e renal relacionada ao tratamento. No perfil lipídico, ao final do estudo, as concentrações de colesterol total, não HDL-C e triglicerídeos aumentaram significativamente em todos os grupos. Houve uma marcante regressão na área de placa aterosclerótica nos coelhos tratados com LDE-paclitaxel, da ordem de 64% e mais marcante no grupo LDE-metotrexato de 71%, quando comparados ao grupo CONTROLE. Na comparação com o grupo DIETA, houve, também, regressão, de 49% nos coelhos do grupo PTX e de 59% no grupo PTX+MTX. O tratamento quimioterápico também mostrou ação antiaterosclerótica nos outros parâmetros avaliados, destacando a intensa redução na relação íntima-média das aortas, na expressão proteica de MMP-9 e da redução na expressão gênica de TNF-? em relação ao grupo DIETA. Portanto, o tratamento quimioterápico com PTX e MTX associado à LDE possui potencial para uso clínico em pacientes com doença aterosclerótica, sendo muito eficaz e com boa tolerabilidade / Atherosclerosis is nowadays considered as an inflammatory disease with intense cell proliferation, hence the rationale of using antiproliferative drugs with an anti-inflammatory action such as paclitaxel (PTX) and methotrexate (MTX) in the treatment of this condition. The lipid nanoemulsion (LDE), with a similar composition to low density lipoprotein (LDL) binds to LDL receptors after their intravenous injection into the bloodstream. Since such receptors are overexpressed in cells with high proliferation rates, such as occurs in cancer and atherosclerosis, LDE can be used as a vehicle to direct antiproliferative agents to these cells, increasing their efficacy and reducing toxicity. Paclitaxel is a chemotherapeutic drug with an anti-proliferative action used in various types of cancer and drug-eluting stents. In previous studies using rabbits subjected to an atherogenic diet, animals treated with LDE-PTX had a 60% reduction in the injured area. Methotrexate in addition to being used in various chemotherapy regimens also has an anti-inflammatory action and is used for chronic inflammatory diseases such as rheumatoid arthritis; another study involving hypercholesterolemic rabbits using commercial MTX for 4 weeks showed a 75% reduction of the atherosclerotic plaque area. This study aims to evaluate the effectiveness of combined chemotherapy treatments, composed of PTX-LDE with MTX-LDE, and PTX-LDE in monotherapy, on the regression of experimental atherosclerosis. In this study, twenty eight male New Zealand breed rabbits received a diet enriched with 1% cholesterol for 8 weeks. After that time, they were divided into four groups: the CONTROL group, which was sacrificed and the aortas kept for later analysis, the DIET group, which only had the diet supplemented with 1% cholesterol suspended; the PTX group which received treatment with weekly intravenous injections of LDE paclitaxel, at a dose of 4 mg/kg for 8 weeks and the PTX+MTX group which received PTX - LDE + MTX-LDE at a dose of 4 mg/kg per week for 8 weeks. The hematological, lipid, biochemical, weight and food intake profiles were evaluated. After euthanasia, macroscopic atherosclerotic lesions in the aortas of the rabbits were measured. Then, the aortic arch was analyzed by morphology and immunohistochemistry. The inflammatory markers were analyzed in the plasma by ELISA and gene expression by qRT-PCR. There was no difference in weight profile and feed intake among the study groups. There was no hematological, hepatic or renal toxicity related to treatment. The lipid profiles of all the groups at the end of the study showed significantly increased concentrations of total cholesterol, non-HDL-C and triglyceride levels. There was a marked regression of 64% in the atherosclerotic plaque area, in the LDE-paclitaxel treated rabbits, and an even more striking 71% in the LDE-methotraxate group compared to the CONTROL group. There was also regression when compared to the DIET group, 49% in rabbits from the PTX group and 59% in the PTX+MTX group. The chemotherapy also showed an anti-atherosclerotic action in the other evaluated parameters, especially notable were the intense reduction in the intima-media ratio of the aortas in protein expression of MMP-9 and the reduction in gene expression of TNF-alpha compared to the DIET group. Therefore, chemotherapy with PTX and MTX associated with LDE, has potential for clinical use in patients with atherosclerotic disease, as it is very effective and well tolerated

O glioblastoma multiforme (GBM) é uma doença grave e sem tratamento eficaz, especialmente pelos agentes terapêuticos disponíveis causarem reações adversas importantes nas doses terapêuticas. O metotrexato (MTX) é um fármaco citotóxico utilizado para tratar diversas neoplasias, no entanto, sua utilização é limitada pela baixa biodisponibilidade e reações adversas. A nanotecnologia tem sido utilizada para aumentar a eficácia dos medicamentos antitumorais, com o intuito de direcioná-los para o sítio de ação e reduzir os efeitos adversos. Nesse sentido, realizamos ensaios com nanocápsulas lipídicas de MTX (LNC MTX) para avaliar os mecanismos de captação em linhagens celulares de glioblastoma e micróglia, além investigar a eficácia terapêutica da LNC MTX em ensaios in vitro e in vivo. Inicialmente, ensaios de microscopia de fluorescência, empregando bloqueadores farmacológicos específicos para transportes de membrana, mostraram que as LNC MTX marcadas com Rodamina B penetram em células tumorais GL261 por endocitose, dependente de caveolinas, e em células de micróglia da linhagem BV2 por fagocitose e macropinocitose. Os tratamentos com LNC MTX ou solução de MTX (em concentrações correspondentes) em células GL261 inibiram a proliferação; aumentaram a fragmentação de DNA, mas, somente as LNC induziram a morte celular por necrose e diminuíram o número de células na fase G1/G0 do ciclo celular. Na linhagem celular BV2, os tratamentos com LNC MTX ou solução de MTX inibiram a proliferação, reduziram a quantidade de células na fase G1/G0 do ciclo celular, aumentaram a fragmentação de DNA e induziram morte celular por apoptose e apoptose tardia. Os ensaios in vivo de microscopia intravital mostraram que a LNC MTX atravessa a barreira Hematoencefálica (BHE) de camundongos fêmea C57Bl/6 após administração intravenosa ou oral, sem danificar a sua estrutura. O tamanho do glioblastoma in vivo foi reduzido em animais tratados com LNC MTX por via oral em relação aos animais tratados com salina. Esta redução não foi detectada em animais tratados com solução de MTX. Em conjunto, os dados obtidos mostram que a LNC MTX penetram em células de glioma e da glia e causam toxicidade, atravessam a BHE in vivo e sugerem que a nanoencapsulação do MTX pode ser uma estratégia importante para o tratamento do glioblastoma. / Glioblastoma multiforme (GBM) is a serious disease and no effective treatment is availabe, especially because the drugs cause significant adverse reactions in therapeutic doses. Methotrexate (MTX) is a cytotoxic drug used to treat many neoplasms, however, their use is limited by the low bioavailability and adverse reactions. Nanotechnology has been used to increase the effectiveness of antitumor drugs in order to direct them to the site of action and to reduce adverse effects. Accordingly, we carried out an experimental approach with MTX lipid nanocapsules (MTX LNC) to evaluate the uptake mechanisms in glioblastoma and microglia cell lines, and the therapeutic efficacy of MTX LNC in vitro and in vivo systems. Initially, fluorescence microscopy assays employing specific pharmacological blockers for membrane transport showed that the MTX LNC stained with Rhodamine B penetrated into GL261 tumor cells by caveolae-mediated endocytosis, and in BV2 microglia cells by phagocytosis and macropinocytosis. Treatment with MTX solution or MTX LNC (at corresponding concentrations) on GL261 cells inhibited the proliferation; increased DNA fragmentation, but only the LNC induced cell death by necrosis and decreased the number of cells in the G1/G0 phase of the cell cycle. In BV2 cells, treatment with MTX solution or MTX LNC inhibited proliferation, reduced number of cells in the G1/G0 phase of the cell cycle, increased DNA fragmentation and cell death, induced by apoptosis and late apoptosis. Intravital microscopy study showed that the MTX LNC across the Blood-Brain Barrier (BBB) of C57BL/6 female mice after intravenous or oral administrations, without damaging its structure. The area of glioblastoma in vivo was reduced in animals oral treated with MTX LNC comparing to saline treated mice. This reduction was not observed in animals treated with MTX solution. Together, the data herein obtained show that MTX LNC penetrate the cell membrane and cause cell toxicity on glioma and neurons lineage, cross the BBB and suggest that the nanoencapsulation of MTX can be an important strategy for the treatment of glioblastoma.

A major effort to develop anticancer drugs through both empiric screening and rational design of new compounds has been under way for over 30 years (Katzung, 2004). In recent years, research and development in the field of sitespecific drug therapy has progressed significantly. Safe and non-toxic formulations of cytotoxic drugs based on polymers with their improved sitespecific delivery and effective activation to biologically active cytotoxic compounds at the targeted tumours have become a promising approach to cancer therapy. Drug delivery systems based on polymer micelles, coated microand nanoparticles, liposomes and various pro-drug systems including watersoluble polymer–drug conjugates and immunoconjugates have been prepared and extensively studied as novel drug delivery systems designed for cancer chemotherapy. Amongst these drug delivery systems that enable specific drug delivery and release, water-soluble polymer–drug conjugates rank among the most promising, versatile and efficient systems. This dissertation reviews the preclinical testing and pharmacokinetic study of D85, a novel water-soluble macromolecular pro-drug that is a polymer with pHcontrolled methotrexate (MTX) release with potential for treatment of cancer in humans (Ulbrich&Subr, 2004). As MTX is also indicated in low doses for the treatment of chronic inflammatory conditions, the polymer was further tested in an acute inflammatory model to determine whether the polymer would be more effective than MTX in controlling inflammation. The objective of this study was to compare the potency and efficacy of D85 to MTX. D85, a MTX conjugated polymeric lead compound, was designed and synthesised as a potential anti-neoplastic and anti-inflammatory agent. It was initially tested in vitro on three different cancer cell lines where selective toxicity towards the cancer cell cultures compared to primary cell cultures and greater toxicity than MTX was observed. The initial in vitro tests showed very promising results with D85 demonstrating approximately 300 times greater cytotoxicity than MTX against a colon cancer cell line (COLO 320 DM). This high cytotoxic effect warranted further investigation in an in vivo colon cancer tumour model. An induced murine tumour model of COLO 320 DM was successfully developed in nude mice, and the anti-tumour efficacy of D85 tested in this model. The maximum tolerated dose of D85 was established by carrying out an in vivo dose ranging toxicity test in BALB/c mice. The anti-inflammatory effects of D85 were also determined using the carrageenan-induced paw oedema model in rats where carrageenan was injected into a footpad of a rat causing acute oedema, which was measured using a water displacement plethysmometer. D85 was found to exacerbate the inflammatory response. Finally, the pharmacokinetic parameters of MTX and D85 were assessed using a LC/MS/MS method specifically developed and validated to determine low concentrations of MTX in small volumes of plasma. This new method made use of online solid phase extraction and sample cleanup on 2μl injections of diluted plasma allowing an entire pharmacokinetics study to be completed on an individual rat. Fairly similar pharmacokinetics were determined from both compounds. Copyright / Dissertation (MSc)--University of Pretoria, 2010. / Pharmacology / unrestricted

The activity of dihydrofolate reductase, a key enzyme in the de novo biosynthesis of thymidylate, purines, and some amino acids, had previously been found to be increased in a methotrexate-resistant mouse leukemia (L5178Y) cell line as compared to the activity in the parental methotrexate-sensitive cell line. The increased activity was composed of two forms of the enzyme, one of which, form 2, was highly insensitive to inhibition by methotrexate. Both forms were purified to near homogeneity and using the antibodies prepared against them, it could be demonstrated that the two forms are antigenically distinct. The increased dihydrofolate reductase activity present in the methotrexate resistant cells resulted from an overproduction of both forms of the enzyme due to the presence of abundant mRNA coding for these enzymes. An increase in the dihydrofolate reductase gene copy number could be demonstrated in the resistant cells. mRNA coding for form 1 and form 2 enzymes was greatly enriched by polysome immunoprecipitation and complimentary DNA (cDNA) was synthesized in vitro from these enriched mRNA molecules. Evidence was found for the presence of methotrexate-insensitive forms of dihydroflate reductase in the blast cells of three out of eight acute myelogenous leukemia patients, and in two (distinct from the above) of the eight patients the activity was significantly increased. In contrast to the overproduction of dihydrofolate reductase protein in the methotrexate-resistant mouse cell line, increased enzyme activity in a methotrexate-resistant human promyelocytic leukemia (HL-60) cell line could not be correlated with an increase in the enzyme protein. Furthermore, the amounts of dihydrofolate reductase mRNA and gene-dosages were similar in the parental metho-trexate-sensitive and methotrexate-resistant cells. The enzyme from the resistant cells differed significantly in some of its physical and kinetic properties from that present in the parental cells. An increase in dihydrofolate reductase activity resulting from a modification of the enzyme rather than gene amplification has not to date been reported in the literature and may present a novel mechanism of resistance to methotrexate. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

O infarto agudo do miocárdio (IAM) é a principal causa de mortalidade mundial. O IAM é acompanhado de remodelamento cardíaco, caracterizado por alterações gênicas, moleculares e celulares, com consequentes alterações no tamanho, forma e função do coração, e resultante disfunção ventricular e insuficiência cardíaca. Evidências experimentais e clínicas indicam que a prevenção ou o tratamento do remodelamento cardíaco beneficiam a função ventricular. A LDE é uma nanopartícula lipídica, com estrutura semelhante à lipoproteína de baixa densidade (LDL). A LDE tem a capacidade de se concentrar em células com superexpressão de receptores de LDL, como em processos proliferativos e inflamatórios, sendo utilizada com um direcionador de fármacos a sítios específicos. Nosso laboratório demonstrou que o tratamento com metotrexato (MTX), um fármaco antiproliferativo e imunossupressor, associado à LDE reduziu acentuadamente as lesões ateroscleróticas na aorta de coelhos submetidos à dieta hipercolesterolêmica. Esses resultados nos levaram à hipótese de que a LDE-MTX possa ser utilizada para minimizar o processo inflamatório pós-IAM, determinante para o remodelamento cardíaco, e seus efeitos deletérios. O objetivo do trabalho foi investigar o efeito do tratamento da LDE-MTX sobre o remodelamento cardíaco em ratos submetidos ao IAM. Ratos machos Wistar (300-400g) foram submetidos ao modelo cirúrgico de IAM ou à cirurgia fictícia (SHAM). Os grupos foram divididos entre: SHAM (solução fisiológica), IAM-LDE, IAMMTXc (metotrexato comercial), IAM-LDE-MTX. Os animais foram tratados uma vez por semana na dose de 1 mg/kg intraperitonealmente, por 6 semanas. Após 24 horas do IAM e ao final do seguimento, foi realizado o ecocardiograma. O coração, o pulmão, o fígado e os rins foram coletados para obtenção do peso relativo dos órgãos. O tamanho do IAM foi estimado pela média dos tamanhos dos IAM externo e interno. A avaliação da necrose dos miócitos, processo inflamatório, diâmetro dos miócitos e fibrose miocárdica nas regiões subendocárdica (SE) e intersticial (INT) foi realizada na região remota ao IAM. Marcadores de estresse oxidativo, inflamação, fibrose, angiogênese e os receptores de lipoproteínas foram quantificados por PCR em tempo real. O tratamento com LDE-MTX diminuiu a dilatação do VE, hipertrofia cardíaca, volumes sistólicos e diastólicos, espessura do septo interventricular e da parede posterior e massa do VE, comparado aos grupos IAM-LDE e IAM-MTXc. Além disso, houve uma melhora de aproximadamente 40% da função sistólica do VE em relação aos demais grupos IAM. O tratamento com LDE-MTX não alterou a função diastólica. O peso relativo do coração e do pulmão foi menor no grupo IAM-LDE-MTX quando comparado ao IAM-LDE. Na histomorfometria, houve diminuição no tamanho do infarto no grupo IAM-LDE-MTX quando comparado com IAMLDE. A necrose, infiltrado inflamatório e fração de volume do colágeno nas regiões INT e SE foram menores no IAM-LDE-MTX em relação aos grupos IAM-LDE e IAM-MTXc, assim como o diâmetro dos miócitos. A expressão gênica dos marcadores de estresse oxidativo e fibrose foi menor no grupo IAM-LDE-MTX quando comparado ao grupo IAM-MTXc. Com relação à inflamação, o grupo IAM-LDE-MTX apresentou menor expressão do gene para TNF-alfa quando comparado aos grupos IAM-MTXc e SHAM. No que se refere ao receptor de lipoproteína, nos grupos IAM-LDE-MTX houve menor expressão do gene para receptor de lipoproteína de baixa densidade (LDLR) quando comparado ao grupo tratado com MTXc. Não foi observada toxicidade em nenhum grupo. Os resultados deste estudo indicam que o tratamento com LDE-MTX melhora significantemente a função cardíaca e atenua o remodelamento cardíaco em modelo experimental cirúrgico para IAM / Acute myocardial infarction (AMI) is the main cause of worldwide mortality. AMI is accompanied by cardiac remodeling, characterized by genetic, molecular and cellular alterations, with consequent changes in the size, shape and function of the heart, resulting in ventricular dysfunction and heart failure. Experimental and clinical evidence indicate that prevention or treatment of cardiac remodeling benefits the ventricular function. LDE is a lipid nanoparticle with a structure similar to low density lipoprotein (LDL). LDE has the ability to concentrate in cells with overexpression of LDL receptors, such as in proliferative and inflammatory processes, and is used with a drugtargeting agent at specific sites. Our laboratory demonstrated that the treatment with methotrexate (MTX), an antiproliferative and immunosuppressive drug, associated to LDE markedly reduced atherosclerotic lesions in aorta of rabbits submitted to the hypercholesterolemic diet. These results led us to the hypothesis that LDEMTX could be used to minimize the post-AMI inflammatory process, determinant for cardiac remodeling, and their deleterious effects. The aim of this study was to investigate the effect of LDE-MTX treatment on cardiac remodeling in rats submitted to AMI. Male Wistar rats (300-400g) were submitted to the surgical model of AMI or Sham surgery. The groups were divided into: SHAM (saline solution), AMI-LDE, AMI-MTXc (commercial methotrexate), AMI-LDE-MTX. The animals were treated once a week at a dose of 1 mg/kg intraperitoneally, for 6 weeks. After 24 hours of AMI and at the end of the follow-up, the echocardiogram was performed. The heart, lung, liver and kidneys were collected to obtain the relative weight of the organs. Infaction size was estimated by mean extern and intern size of IAM. Evaluation of myocyte necrosis, inflammatory process, myocyte diameter and myocardial fibrosis in the subendocardial (SE) and interstitial (INT) areas was performed in remote area from AMI. Markers of oxidative stress, inflammation, fibrosis, angiogenesis and lipoprotein receptors were quantified by quantitative real-time PCR. Treatment with LDE-MTX decreased LV dilation, cardiac hypertrophy, systolic and diastolic volumes, interventricular septum and posterior wall thickness and LV mass, compared to AMI-LDE and AMI-MTXc groups. In addition, there was an improvement of approximately 40% of LV systolic function compared to other AMI groups. Treatment with LDE-MTX did not alter diastolic function. The relative weight of the heart and lung were lower in the AMI-LDE-MTX group when compared to the AMI-LDE. In morphometry, infarct size decreased in the AMI-LDE-MTX group when compared to AMI-LDE. Necrosis, inflammatory infiltrate and collagen volume fraction in the INT and SE regions were lower in AMI-LDEMTX than in AMI-LDE and AMI-MTXc groups, as well as the myocyte diameter. The gene expression of oxidative stress and fibrosis markers were lower in the AMI-LDE-MTX group when compared to the AMI-MTXc. Regarding inflammation, the AMI-LDE-MTX group had lower expression of the TNF-alfa gene when compared to the AMI-MTXc group and the SHAM group. As regards the lipoprotein receptor, in the AMI-LDE-MTX there was lower expression of the gene for low-density lipoprotein (LDLR) receptor compared to MTXc treated animals. No toxicity was observed in any groups. The results of this study indicate that treatment with LDE-MTX significantly improves cardiac function and attenuates cardiac remodeling in an experimental surgical model for AMI

<p>Metotreksat kao antagonista folne kiseline ima &scaron;iroku upotrebu za lečenje brojnih maligniteta, primenjen u visokim dozama i u&nbsp; kombinciji&nbsp; sa leukovorinom. Iako&nbsp; je&nbsp; terapija&nbsp; visokim&nbsp; dozama&nbsp; metotreksata drastično pobolj&scaron;ala&nbsp; prognozu pacijenata sa malignitetom, te&scaron;ki neželjeni efekti terapije predstavljaju stalan klinički problem. Ciljevi istraživanja bili su određivanje serumske koncentracije metotreksata i izračunavanje farmakokinetičkih&nbsp; parametara&nbsp; metotreksata kod dece obolele od malignih&nbsp; bolesti&nbsp; koja su na terapiji visokim dozama metotreksata (2&nbsp; g/m² i&nbsp; 5&nbsp; g/m² ); ispitivanje postojanja uticaja primenjene doze metotreksata, demografskih i kliničkih karakteristika ispitanika&nbsp; na koncentracije i farmakokinetičke parametare. Ispitivano je prisustvo i stepen kliničkih i laboratorijskih znakova toksičnosti metotreksata, kao i uticaj primenjene&nbsp; doze&nbsp; metotreksata&nbsp; i demografskih karakteristika ispitanika&nbsp; na pojavu i stepen toksičnosti . U okviru retrospektivno - prospektivne&nbsp; studije&nbsp; ukjučeno&nbsp; je&nbsp; četrdeset&nbsp; i dva pedijatrijska&nbsp; pacijenta&nbsp; uzrasta od&nbsp; 0,75 do 17,75 godina (medijana 5,75&nbsp; godina). Svi pacijenti&nbsp; su lečeni&nbsp; u&nbsp; Službi&nbsp; za&nbsp; hematologiju i&nbsp; onkologiju&nbsp; Instituta&nbsp; za&nbsp; zdravstvenu za&scaron;titu dece i omladine Vojvodine (Novi Sad, Srbija) u periodu od juna 2004. godine do juna 2012. godine. Trideset i osam ispitanika&nbsp; je lečeno pod dijagnozom akutne limfoblastne leukemije&nbsp; prema dva&nbsp; uzastopna&nbsp; protokola ALL IC - BFM 2002 i ALL IC - BFM 2009 Internacionalne&nbsp; BFM studijske&nbsp; grupe &bdquo;I - BFM - SG&ldquo;&nbsp; (International Berlin -Frankfurt - M&uuml;nster Study Group) za proučavanje i lečenje dečje non-B akutne limfoblastne leukemije. Četvoro je&nbsp; imalo&nbsp; dijagnozu non - Hodgkin limfoma&nbsp; i bili su uključen i u&nbsp; protokol NHL - BFM&nbsp; 95. Istraživanje je obuhvatilo 113 ciklusa terapije metotreksatom (1&ndash; 4 ciklusa po pacijentu) sa 386 izmerenih serumskih koncentracija metotreksata. Raspon primenjenih doza metotreksata kretao se od 800 do 10.000 mg. Koncentracije metotreksata su merene 24, 36 i 42 sata nakon započinjanja infuzije metotreksata, a po potrebi i u dužim vremenskim intervalima. Za izračunavanje farmakokinetičkih parametara kori&scaron;ćen je dvokompartmanskih farmakokinetički&nbsp; model posle obustavljanja&nbsp; intravenske&nbsp; infuzije,&nbsp; gde&nbsp; postoje relacije&nbsp; za&nbsp; farmakokinetičke&nbsp; tačke. Podaci o kliničkim i laboratorijskim znacima toksičnosti metotreksata prikupljani su iz medicinske dokumentacije, a za stepenovanje toksičnosti kori&scaron;ćen je skor sistem - Common Terminology Criteria for&nbsp; Adverse&nbsp; Events (CTCAE), Version 4.0, U.S. Department&nbsp; of&nbsp; health&nbsp; and&nbsp; human services, National Institute of Health, National Cancer Institute. U cilju utvrđivanju uticaja karakteristika ispitanika, primenjene doze i prisustva produžene eliminacije na posmatrane parametre, vr&scaron;eno je poređenje tri grupe&nbsp; pacijenata (doza 2 g/m² bez produžene eliminacije, 5 g/m² bez produžene liminacije i 5 g/m² sa produženom eliminacijom metotreksata). Za celokupnu grupu ispitanika,&nbsp; medijane&nbsp; koncentracije metotreksta&nbsp; bile&nbsp; su 25,82 &mu;mol/l u 24. satu, 0,68 &mu;mol/l u 36. satu i 0,24 &mu;mol/l u 42. satu merenja. Najizraženija interindividualna varijabilnost u koncentracijama metotreksata bila je u 42. satu merenja, dok je&nbsp; intraindividualna varijabilnost bila najizraženija u 36. satu merenja. Medijana&nbsp; klirensa&nbsp;&nbsp; metotreksata&nbsp;&nbsp; bila&nbsp; je 8,32&nbsp;&nbsp; l/h. Farmakokinetički parametri&nbsp; redom bili su:&nbsp; medijana&nbsp; volumena&nbsp; centralnog&nbsp; kompartmana V₁ 28,47&nbsp; l, medijane konstanti k₁₀ 0,206, k₁₂ 0,0245, k₂₁ 0,1114. Najizraženiji uticaj primenjene doze na koncentracije metotreksata pokazan je u 24.&nbsp; satu&nbsp; merenja, dok uticaj doze na klirens&nbsp; metotreksata nije&nbsp; pokazan. Prisustvo produžene eliminacije metotreksata dovodi do smanjenih vrednosti konstanta k₁₀ i k₂₁. Nije pokazana statistički značajna&nbsp; interakcija ispitivanih demografskih karakteristika (uzrast, telesna povr&scaron;ina i pol)&nbsp; i koncentracija metotreksata, kao ni klirensa metotreksata. Pokazana je značajna interakcija između koncentracija metotreksata i nivoa laktat dehidrogenaze, kao i klirensa metotreksata i nivoa kreatinina i laktat dehidrogenaze. Većina ispoljenih&nbsp; toksičnosti bila je umerenog stepena (&lt;3 stepena). Najzastupljeniji klinički znak toksičnosti bio je oralni mukozitis, koji je bio većeg stepena u grupi sa većom primenjenom dozom metotreksata&nbsp; (5g/m²). Najzastupljeniji&nbsp; laboratorijski toksični efekti&nbsp; metotreksata bili su leukopenija i anemija. Najteži stepeni laboratorijskih znakova toksičnosti (leukopenija, anemija, porast&nbsp; AST,&nbsp; ALT i GGT) nalazili su se u grupi sa većom dozom&nbsp; (5 g/m²) i&nbsp; sa produženom eliminacijom metotreksata. Osnov&nbsp; za&nbsp; kliničko&nbsp; vođenje&nbsp; pacijenata&nbsp; na&nbsp; terapiji visokim dozama metotreksata je terapijsko praćenje leka (therapeutic drug monitoring &ndash; TDM) zbog velikih&nbsp; interindividualnih i intraindividualnih&nbsp; varijabilnosti&nbsp; u&nbsp; farmakokinetici&nbsp; leka. Rutinsko praćenje koncentracija metotreksata važno je za identifikaciju pacijenata sa povećanim rizikom od razvoja toksičnosti ,&nbsp; te&nbsp; je TDM&nbsp; standardna&nbsp; praksa&nbsp; za smernice spasavanja leukovorinom, naročito za pacijente za koje se zna da imaju smanjen&nbsp;&nbsp; klirens metotreksata ili druge rizike povezane sa prolongiranim citotoksičnim koncentracijama (bubrežna ili jetrena o&scaron;tećenja, kolekcije tečnosti u &ldquo;trećem prostoru&rdquo;, gastrointestinalna opstrukcija). Veliki&nbsp; broj&nbsp; istraživanja&nbsp; kod pedijatrijskih pacijenata pokazao je vezu između sistemskog izlaganja metotreksatu i&nbsp; efikasnosti&nbsp; i&nbsp; toksiĉnosti&nbsp; metotreksata. Ipak, ne postoji dovoljno&nbsp; informacija o farmakokinetici metotreksata kod dece obolele od akutne limfoblastne leukemije. Takođe, ova istraživanja nisu do sada sprovođena kod dece koja su lečena u na&scaron;oj sredini.</p> / <p>Methotrexate&nbsp; is&nbsp; an&nbsp; antifolate&nbsp; drug&nbsp; widely&nbsp; used&nbsp; for&nbsp; treatment&nbsp; of&nbsp; various malignant&nbsp; tumours.&nbsp; It&nbsp; is&nbsp; used&nbsp; at&nbsp; high&nbsp; doses&nbsp; and&nbsp; in&nbsp; combination&nbsp; with leucovorin rescue.&nbsp; Although&nbsp; high - dose&nbsp; MTX&nbsp; therapy&nbsp; dramatically&nbsp; improves&nbsp; the&nbsp; prognosis&nbsp; of patients with malignancies, severe adverse events are constant clinical concern. The&nbsp; aims&nbsp; of&nbsp; this&nbsp; stydy&nbsp; were&nbsp; to&nbsp; determine&nbsp; the&nbsp; serum&nbsp; concentration&nbsp; of&nbsp; methotrexate&nbsp; and&nbsp; to&nbsp; calculate&nbsp; the&nbsp; pharmacokinetic&nbsp; parameters&nbsp; of&nbsp; methotrexate&nbsp; in children&nbsp; suffering&nbsp; from&nbsp; malignant&nbsp; deseases&nbsp; who&nbsp; are&nbsp; treated&nbsp; with&nbsp; high&nbsp; doses&nbsp; of metotrexate&nbsp; (2&nbsp; g/m² i&nbsp; 5&nbsp; g/m² );&nbsp; furthermore,&nbsp; to&nbsp; investigate&nbsp; the&nbsp; effects&nbsp; of&nbsp; the&nbsp; applied doses of methotrexate, and demographic and clinical characteristics of the examinees on&nbsp;&nbsp; the&nbsp;&nbsp; concentration&nbsp;&nbsp; and&nbsp;&nbsp; pharmacokinetic&nbsp;&nbsp; parameters&nbsp;&nbsp; of&nbsp;&nbsp; the&nbsp;&nbsp; drug.&nbsp;&nbsp; The&nbsp;&nbsp; study investigated the&nbsp;&nbsp; presence&nbsp;&nbsp; and&nbsp;&nbsp; the&nbsp;&nbsp; degree&nbsp;&nbsp; of&nbsp;&nbsp; clinical&nbsp;&nbsp; and&nbsp;&nbsp; laboratory&nbsp;&nbsp; signs&nbsp;&nbsp; of metotrexate&nbsp; toxicity,&nbsp; as&nbsp; well&nbsp; as&nbsp; the&nbsp; effect&nbsp; of&nbsp; the&nbsp; applied&nbsp; doses,&nbsp; and&nbsp; demographic characteristics of the examinees on the appearance and the degree of toxicity. The retrospective - prospective study included 42&nbsp; pediatric patients aged from 0.75&nbsp; to&nbsp; 17.75&nbsp; years&nbsp; (median&nbsp; 5.75&nbsp; years).&nbsp; All&nbsp; patients&nbsp; were&nbsp; threated&nbsp; at&nbsp; the&nbsp; Children and Youth Health Care Institute of Vojvodina (Novi Sad, Serbia), Hemathology and Oncology&nbsp; Section,&nbsp; in&nbsp; the&nbsp; period&nbsp; from&nbsp; June&nbsp; 20 04&nbsp; to&nbsp; June&nbsp; 2012.&nbsp; 38&nbsp; examinees diagnosed as acute lymphoblastic leukemia were treated according to two subsequent protocols,&nbsp; ALL&nbsp; IC - BFM&nbsp; 2002&nbsp; and&nbsp; ALL&nbsp; IC - BFM&nbsp; 2009&nbsp; of&nbsp; the&nbsp; International&nbsp; BFM study&nbsp; group &bdquo;I - BFM - SG&ldquo; (International Berlin - Frankfurt - M&uuml;nster&nbsp; Study&nbsp; Group)&nbsp; for management&nbsp;&nbsp; of&nbsp;&nbsp; childhood&nbsp;&nbsp; non - B&nbsp;&nbsp; acute&nbsp;&nbsp; lymphoblastic&nbsp;&nbsp; leukemia.&nbsp;&nbsp; 4&nbsp;&nbsp; examinees diagnosed&nbsp; as&nbsp; non - Hodgkin&nbsp; lymphoma&nbsp; were&nbsp; treated&nbsp; according&nbsp; to&nbsp; the&nbsp; NHL - BFM&nbsp; 95 protocol. The study included 113 cycles of therapy with methotrexate (1-4 cycles per patient)&nbsp; with&nbsp; 3 86&nbsp; measured&nbsp; serum&nbsp; concentrations&nbsp; of&nbsp; methotrexate.&nbsp; The&nbsp; range&nbsp; of&nbsp; the applied doses was between 800 and 10,000 mg. The&nbsp; concentration&nbsp; of&nbsp; methotrexate&nbsp; was&nbsp; measured&nbsp; 24,&nbsp; 36&nbsp; and&nbsp; 42&nbsp; hours&nbsp; after the initiation of the methotrexate infusion, as well as in longer time intervals when needed.&nbsp; To&nbsp; calculate&nbsp; the&nbsp; pharmacokinetic&nbsp; parameters,&nbsp; the&nbsp; study&nbsp; applied&nbsp; the&nbsp; two - compartment&nbsp; pharmacokinetic&nbsp; model&nbsp; after&nbsp; the&nbsp; termination&nbsp; of&nbsp; intravenous&nbsp; infusion, when&nbsp; relations&nbsp; for&nbsp; pharmacokinetic&nbsp; points&nbsp; existed.&nbsp; Data&nbsp; on&nbsp; clinical&nbsp; and&nbsp; laboratory signs of methotrexate toxicity were collected&nbsp; from medical documentation, and the Common&nbsp; Terminology&nbsp; Criteria&nbsp; for&nbsp; Adverse&nbsp; Events&nbsp; (CTCAE),&nbsp; Version&nbsp; 4.0,&nbsp; U.S. Department&nbsp; of&nbsp; health&nbsp; and&nbsp; human&nbsp; services,&nbsp; National&nbsp; Institute&nbsp; of&nbsp; Health,&nbsp; National Cancer&nbsp; Institute, was&nbsp; used&nbsp; as&nbsp; the&nbsp; score&nbsp; system&nbsp; for&nbsp; toxicity&nbsp; ranking.&nbsp; In&nbsp; order&nbsp; to determine&nbsp; the&nbsp; effects&nbsp; of&nbsp; the examinees&rsquo;&nbsp; characteristics, applied&nbsp; doses&nbsp; and&nbsp; the presence&nbsp; of&nbsp; prolonged&nbsp; elimination on&nbsp; the&nbsp; parameters&nbsp; of&nbsp; interest,&nbsp; three&nbsp; groups&nbsp; of patients were&nbsp; compared (2 g/m² dose without prolonged elimination, 5 g/m² without prolonged elimination and 5 g/m² with prolonged elimination of methotrexate). In the&nbsp; entire&nbsp; group of&nbsp; examinees, the median&nbsp; concentration of methotrexate was&nbsp; 25.82 &mu;mol/l in the 24th hour, 0.68 &mu;mol/l in the 36th&nbsp; hour&nbsp; and 0.24 &mu;mol/l in the 42nd hour of&nbsp; observation. The largest inter - individual variability of methotrexate concentration&nbsp; was&nbsp; observed&nbsp; in&nbsp; the&nbsp; 24th&nbsp; hour&nbsp; while&nbsp; the&nbsp; largest&nbsp; intra - individual variability&nbsp; was&nbsp; recorded&nbsp; in&nbsp; the&nbsp; 36th&nbsp; hour&nbsp; of&nbsp; observation.&nbsp; The&nbsp; median&nbsp; clearance&nbsp; of methotrexate&nbsp; was&nbsp; 8.32l/h.&nbsp; Pharmacokinetic&nbsp; parameters&nbsp; were&nbsp; the&nbsp; following:&nbsp; median volume&nbsp; of&nbsp; the&nbsp; central&nbsp; compartment V₁ 28.47&nbsp; l,&nbsp; median&nbsp; constants k₁₀ 0,206, k₁₂ 0,0245, k₂₁ 0,1114, respectively. The&nbsp;&nbsp; strongest&nbsp;&nbsp; influence&nbsp;&nbsp; of&nbsp;&nbsp; the&nbsp;&nbsp; applied&nbsp;&nbsp; dose&nbsp;&nbsp; on&nbsp;&nbsp; the&nbsp;&nbsp; methotrexate concentration was recorded in the 24th hour of observation while no influence on the methotrexate&nbsp; clearance&nbsp; was&nbsp; found.&nbsp; The&nbsp; presence&nbsp; of&nbsp; prolonged&nbsp; elimination&nbsp; of methotrexate&nbsp;&nbsp; causes&nbsp;&nbsp; lower&nbsp; constants k₁₀ and&nbsp;&nbsp; k₂₁. There&nbsp;&nbsp; was&nbsp;&nbsp; no&nbsp;&nbsp; statistically significant&nbsp; interaction&nbsp; between&nbsp; the&nbsp; investigated&nbsp; demographic&nbsp; characteristics&nbsp; (age, body&nbsp; surface&nbsp; and&nbsp; gender)&nbsp; and&nbsp; the&nbsp; methotrexate&nbsp; concentration,&nbsp; nor&nbsp; between&nbsp; the demographic&nbsp;&nbsp; characteristics&nbsp;&nbsp; and&nbsp;&nbsp; the&nbsp;&nbsp; methotrexate&nbsp;&nbsp; clearance.&nbsp;&nbsp; A&nbsp;&nbsp; significant interaction was found between methotrexate concentration and lactat dehydrogenase level, as&nbsp;&nbsp; well&nbsp;&nbsp; as&nbsp;&nbsp; between&nbsp;&nbsp; methotrexate&nbsp;&nbsp; clearance&nbsp;&nbsp; and&nbsp;&nbsp; creatinine&nbsp;&nbsp; and&nbsp;&nbsp; lactate dehydrogenase level, respectively. Most of the observed toxicities were of moderate degree (&lt; 3 degrees). Oral mucositis&nbsp; was&nbsp; the&nbsp; most&nbsp; represented&nbsp; clinical&nbsp; sign&nbsp; of&nbsp; toxicity,&nbsp; and&nbsp; it&nbsp; was&nbsp; of&nbsp; higher degree&nbsp; in&nbsp; the&nbsp; group&nbsp; where&nbsp; the&nbsp; applied&nbsp; dose&nbsp; of&nbsp; methotrexate&nbsp; was&nbsp; higher&nbsp; (5&nbsp; g/m² ). Leucopenia&nbsp; and&nbsp; anemia&nbsp; were&nbsp; the&nbsp; most&nbsp; represented&nbsp; laboratory&nbsp; toxic&nbsp; effects.&nbsp; The most severe laboratory signs of toxicity&nbsp; (leucopenia, anemia, increase in AST, ALT and&nbsp; GGT&nbsp; activity)&nbsp; were&nbsp; observed&nbsp; in&nbsp; the&nbsp; group&nbsp; with&nbsp; the&nbsp; higher&nbsp; dose&nbsp; (5&nbsp; g/m² )&nbsp; and prolonged methotrexate elimination. Due to high inter- and&nbsp;&nbsp; intra-individual&nbsp;&nbsp;&nbsp; variability&nbsp; of&nbsp; the drug pharmacokinetics,&nbsp; the&nbsp; basis&nbsp; for&nbsp; the&nbsp; clinical&nbsp; care&nbsp; of&nbsp; patients&nbsp; on&nbsp; high&nbsp; methotrexate dosage&nbsp; therapy&nbsp; is&nbsp; therapeutic&nbsp; drug&nbsp; monitoring &ndash; TDM.&nbsp; Routine&nbsp; monitoring&nbsp; of methotrexate serum concentration is important for the identification of patients with a high&nbsp; risk&nbsp; of&nbsp; toxicity,&nbsp; and&nbsp; thus&nbsp; TDM&nbsp; is&nbsp; used&nbsp; as&nbsp; a&nbsp; standard&nbsp; procedure&nbsp; which&nbsp; provides guidelines&nbsp; for&nbsp; leucovorin&nbsp; rescue,&nbsp; particularly&nbsp; for&nbsp; patients&nbsp; with&nbsp; a&nbsp; lower&nbsp; methotrexate clearance or other risks associated with prolonged cytotoxic concent rations (kidney or liver&nbsp; damage,&nbsp; body&nbsp; fluid&nbsp; accumulation&nbsp; in&nbsp; the&nbsp; &ldquo;third&nbsp; space&rdquo;,&nbsp; gastrointestinal obstruction). Numerous studies involving pediatric patients have documented the link between&nbsp; a&nbsp; systemic&nbsp; methotrexate&nbsp; exposure&nbsp; on&nbsp; one&nbsp; hand,&nbsp; and&nbsp; the&nbsp; efficiency&nbsp; and toxicity of&nbsp; ethotrexate on the other hand. However, there is no sufficient data on the methotrexate&nbsp;&nbsp;&nbsp; pharmacokinetics&nbsp;&nbsp; in&nbsp;&nbsp; children&nbsp;&nbsp; suffering&nbsp;&nbsp; from&nbsp;&nbsp; acute&nbsp;&nbsp; lymphoblastic leukemia.&nbsp;&nbsp; Moreover,&nbsp;&nbsp; this&nbsp;&nbsp; type&nbsp;&nbsp; of&nbsp;&nbsp; research,&nbsp;&nbsp; involving&nbsp;&nbsp; children&nbsp;&nbsp; treated&nbsp;&nbsp; in&nbsp;&nbsp; the geographical region of this study, have not been conducted.</p>

No description available.

Improving the therapeutic efficacy and quality of life of patients by reducing the side effects caused by non-specificity of cytotoxic drugs has been a challenge in cancer treatment. A hypothesis was developed where integrin binding induced conformational change in a drug conjugated to hairpin peptide with an integrin binding ligand can lead to preferential accumulation of drug and reduced collateral damage by decreased premature prodrug activation. A model drug, MTX and a tripeptide ligand, RGD, known to specifically bind tumor overexpressing α v β 3 integrin receptors, were selected to test the hypothesis. A twelve amino acid sequence that has been previously shown to preferentially adopt an anti-parallel beta hairpin conformation in aqueous environment was flanked with MTX and RGD on N and C termini respectively by solid phase peptide synthesis to form a labile link between Arg-Glu specifically cleaved by SGPE, a Streptomyces griseus derived endopeptidase. Adenoviral vector was developed using AdEasy system for β 3 cDNA transfection to overexpress integrin α v β 3 receptor. MTX-α-RGD and MTX-β-hairpin-RGD were characterized using MALDI-TOF (MTX-α-RGD, 782.6(M+H + ); MTX-β-hairpin-RGD, 2272.1(M+H + )). Cell adhesion assay using HUVEC and A549 cells that overexpress α v β 3 showed that RGD conjugated prodrugs recognize and preferentially bind to integrin α v β 3 in RGD dependent manner. In rabbit plasma, MTX-β-hairpin-RGD was found to be 3 times more stable than MTX-α-RGD. In the absence of α v β 3 binding, SGPE mediated hydrolysis rate of MTX-β-hairpin-RGD was 0.7±0.1 ng/hr, that was significantly (P<0.025) lower than that of MTX-α-RGD (1.0±0.1ng/hr), a prodrug without hairpin structure. In presence of α v β 3 over-expressing cells, significant increase (P<0.025) in hydrolysis rate of MTX-β-hairpin-RGD to 1.0±0.1 ng/hr was observed, not significantly (P=0.6) different from that of MTX-α-RGD (1.1±0.1ng/hr). In addition, there was 400% increase in the fluorescence when FRET based quenching was abolished by the binding induced unfolding. These experiments along with docking studies using molecular modeling support the binding induced unfolding. Results from this investigation suggest that drugs conjugated to peptide ligands such as RGD may reduce the dose needed to achieve therapeutic concentrations by preferential recognition and binding to overexpressed integrin markers. Secondly, reduction of premature activation of prodrugs and thus reduced collateral damage may be achieved by making the the drug release to occurs preferentially upon binding to cells expressing specific integrin markers.

No description available.