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Chemotherapy related neurotoxicity is the decrease in cognitive function observed in patients receiving chemotherapy for breast cancer. For cancers with higher survival rates such as breast cancer, quality of life for patients after treatment cessation is a major concern. In studies performed in our laboratory, we reported cognitive deficiencies in male Swiss-Webster mice on administering 75 mg/kg 5-FU with 3.2 mg/kg MTX and these deficits were significantly greater than groups receiving either drug alone or in another higher dose combination. The probable mechanisms for the reported drug-drug interaction (DDI) between 5-FU and MTX could be either pharmacokinetic (PK) or pharmacological. Since the reported study consists of a combination of two drugs, it is imperative to determine if the PK of either drug was altered. On performing the PK based study we established the nature of the DDI to be PK based. We observed statistically significant changes for PK parameters clearance and apparent volume of distribution. Since, 5-FU and MTX are high clearance drugs, uptake transporters responsible for presenting the drugs to the clearing organs are the limiting factors for their clearance. Therefore, for any PK based interactions observed between 5-FU and MTX in the different dose groups a highly probable mechanism would be interactions at the site of uptake transporters. Based on the physicochemical properties of 5-FU and MTX and the results observed form the PK study, we hypothesized transporter-based interactions to be a probable mechanism for the observed DDI. From the transporter based studies we hypothesized 5-FU probably inhibited the uptake of MTX's transport across the blood brain barrier (BBB). To date the transport of MTX and other similar folates has not been characterized extensively. However, MTX is a very close analogue for reduced folates and therefore shares the transporter reduced folate carrier-1 (Rfc-1) expressed abundantly at the BBB, with endogenous reduced folates. Hence we hypothesized the decreased exposure of MTX in the presence of 5-FU would most probably be as a result of inhibition of uptake transporters such as Rfc-1. Finally, we developed a mathematical PK model for MTX to predict appropriately drug concentrations in the plasma and the brain tissue. The utility of the model was to support the hypothesized interactions responsible for the observed PK data. This models utility is to provide the PK component for the future PK-pharmacodynamic models, which would narrow the gap between the reported cognitive deficits and the PK results reported in this dissertation. / Pharmaceutical Sciences

Bone Cancer Research Trust / The full text will be available at the end of the embargo: 6th October 2027

High-dose methotrexate (HD MTX) is critical for treating pediatric malignancies such as acute lymphoblastic leukemia and neuro-carcinoma. However, its significant toxicity due to drug accumulation poses substantial risks. This retrospective study assesses the impact of demographic factors on MTX toxicity and clearance in pediatric oncology patients. Patient records from Saint Mary Hospital were analyzed, focusing on two MTX administration protocols: a 24-hour infusion followed by alkaline hydration and a 4-hour infusion followed by alkaline hydration. We hypothesize that factors such as age, body surface area (BSA), and body mass index (BMI) are associated with MTX clearance and toxicity. The study found no significant difference in clearance between genders, but females exhibited higher toxicity rates. Ethnicity comparisons showed Caucasians had the fastest clearance, followed by Hispanics, African Americans, and others, with Hispanics experiencing the highest toxicity rates. Patients with a BSA of less than one had a lower risk of delayed clearance, although toxicity levels were similar across BSA groups. BMI analysis indicated that patients with a BMI over 25 were at a higher risk of toxicity. Taken together, these findings suggest the need for personalized treatment plans in pediatric oncology to enhance therapeutic efficacy and reduce adverse effects. Future research should expand the sample size and develop a risk stratification guideline to identify patients suitable for outpatient treatment.

O uso da LDE como veículo para quimioterápicos tem mostrado ser uma boa estratégia para aumentar a eficácia terapêutica dos mesmos. Nesse estudo, a LDE foi empregada como veículo para um derivado lipofílico do metotrexato (MTX), o di-dodecil metotrexato, que foi obtido com rendimento elevado através de reação de esterificação do MTX. O aumento na lipofilicidade do derivado possibilitou incorporação na LDE com rendimento e estabilidade elevados. O IC50 de LDE-di-dodecil MTX foi cerca de 100 vezes menor em relação ao MTX comercial, sua captação celular mais elevada nas linhagens leucêmicas estudadas e sua toxicidade animal reduzida, mostrando que a LDE é um veículo promissor para este fármaco. / The use of LDE as vehicle to drugs is a great strategy to improve the therapeutic index and reduce the side effects. In this study LDE was used as vehicle to di-dodecyl methotrexate, a lipophilic derivative of MTX, obtained through an esterification reaction with a high yield. The increased lipophilicity of the derivative allowed a high association to LDE and good stability. The IC50 of LDE-di-dodecyl MTX was lower than that of the MTX and the uptake was higher in leukemic cells. The MTX toxicity in mice was reduced after association to LDE, showing that LDE is a promising vehicle to this drug.

O uso da LDE como veículo para quimioterápicos tem mostrado ser uma boa estratégia para aumentar a eficácia terapêutica dos mesmos. Nesse estudo, a LDE foi empregada como veículo para um derivado lipofílico do metotrexato (MTX), o di-dodecil metotrexato, que foi obtido com rendimento elevado através de reação de esterificação do MTX. O aumento na lipofilicidade do derivado possibilitou incorporação na LDE com rendimento e estabilidade elevados. O IC50 de LDE-di-dodecil MTX foi cerca de 100 vezes menor em relação ao MTX comercial, sua captação celular mais elevada nas linhagens leucêmicas estudadas e sua toxicidade animal reduzida, mostrando que a LDE é um veículo promissor para este fármaco. / The use of LDE as vehicle to drugs is a great strategy to improve the therapeutic index and reduce the side effects. In this study LDE was used as vehicle to di-dodecyl methotrexate, a lipophilic derivative of MTX, obtained through an esterification reaction with a high yield. The increased lipophilicity of the derivative allowed a high association to LDE and good stability. The IC50 of LDE-di-dodecyl MTX was lower than that of the MTX and the uptake was higher in leukemic cells. The MTX toxicity in mice was reduced after association to LDE, showing that LDE is a promising vehicle to this drug.

Aprofundamos o estudo dos efeitos farmacológicos da dexametasona, indometacina e tenoxicam e de uma droga citostática (metotrexato), assim como a análise dos mecanismos envolvidos na estimulação flogógena causada por agentes de intensidades fraca, média e forte. Para isso. empregamos o teste edemogênico, que demonstrou ser os fármacos antiinflamatórios não esteróides (tenoxicam e indometacina), os de maior potência na inibição da exsudação plasmática induzida pela paracoccidioidina. Todavia, com agentes flogógenos de intensidade média (carragenina), o metotrexato foi o medicamento mais potente; enquanto que com a placa microbiana dental (agente forte), a dexametasona e a indometacina foram os de maior potência antiinflamatória. Por meio da análise histomorfométrica relativa dos granulomas induzidos por esse último agente, verificamos, até 14 dias, a maior potência antiinflamatória apresentada pelo tenoxicam, comparativamente à da indometacina - em relação à inibição da região central de necrose supurativa-, demonstrando efeito semelhante ao da dexametasona; não obstante, nesse período experimental, em relação à inibição da densidade do volume do tecido granulomatoso, os fármacos mais potentes terem sido a indometacina e a dexametasona. Após 14 dias, foi constatada diferença não significativa (p>0.05) entre o efeito apresentado pelo tenoxicam e o da indometacina. O acentuado efeito apresentado pelos NSAIDs em relação à inibição da densidade de volume dos macrófagos, semelhante ao do metotrexato, sugeriu que os NSAIDs inibiram a proliferação das células progenitoras mielóides dos monócitos/macrófagos. Também agiram tanto aumentando (21 dias) como inibindo (28 dias) a densidade de volume ocupada pelas fibras colágenas; enquanto que a dexametasona apresentou efeito contrário. Tais resultados indicaram que no processo inflamatório induzido por agentes flogógenos de intensidade forte (PMD), estimulores da acentuada produção de LTs e PGs, o emprego de antiinflamatórios esteróides e não esteróides foi vantajosa em relação ao fármaco citostátco. / Was studied comparatively the mechanisms of action and the antiinflammatory effect presented by dexamethasone, tenoxican, indomethacin and methotrexate in acute and chronic inflammation induced by agent flogogenous of minim, media and elevated intensity. With the employ of edemogenic test was verified that the effect presented by nonsteroid antiinflammatory (tenoxican and indomethacin), in relation to inhibition of the plasmatic exsudation induced by paracoccidioidin, was more potent than other medicaments tested. Meanwhile, in the inhibition of the acute inflammation caused by carrageenan and dental microbian plaque, methotrexate, dexamethasone and indomethacin were the most potent pharmacs, respectively. The injection of the dental microbian plaque in the air pouch model induced two experimental granulomas susceptive to the antiinflammatory effects presented for the pharmacs tested, which were utilized in the determination of the weights and of the volume density occupied by structures presents in the periods experimental of 7, 14, 21 and 28 days. With relation the inhibition of the differential mid and dry weights, methotrexate and dexamethasone followed by indomethacin and tenoxican were the most effective pharmacs in decrescent order of potency. The histomorphometric studies of the volume density of the granulomatous tissue revealed that indomethacin reduced this structure comparatively to the tenoxican at 14 days. After this experimental period tenoxican presented the most potent anti-inflammatory effect; however, without significant statistical difference to indomethacin. Tenoxican too presented elevated inhibitory effect of the volume density of the region of supurative necrose (similar to dexamethasone) particularly along to first week. NSAIDs also showed in relation the inhibition of volume density of the collagen effect stimulator (21 days) and inhibitor (28 days), while that dexamethasone revealed contrary effect. The accentuate inhibitory effect of the volume density of macrophages presented by NSAIDs was similar to methotrexate, indicating that these medicaments possibly presented anti-mitotic effect to the progenitors myeloid cells of monocytes/macrophages. These results indicated that in the inflammatory process induced by flogogenous of strong intensity, stimulators of increased production of LTS and PGs, the administration of steroids and non-steroids anti-inflammatory was advantageous in relation to methotrexate.

No presente estudo avaliamos a influência do Metotrexato (MTX-3mg/kg/semana) e do Corticosteróide (CE- prednisona 1,05 mg/kg/semana), ministrados de forma isolada, associados entre si, ou associados ao Ácido Folínico (AF- 0,25 mg/kg/semana) e ao Ácido Zoledrônico (ZOL- 0,1mg/kg dose única) respectivamente, na osseointegração de implantes de titânio realizados 6 semanas após o início da administração das drogas, em coelhos NZW. Os animais (2,67 ± 0,67 Kg) foram tratados durante um total de 18 semanas com solução salina (CTL; n=6), MTX (n=6), CE (n=8), MTX-CE (n=6), MTX-AF (n=6) e CE-ZOL (n=6). Foram realizadas análises de densitometria mineral óssea (DMO) na tíbia e na coluna antes e após os tratamentos para avaliação da variação da DMO (Delta DMO). Os resultados revelaram perda de osso cortical nos grupos tratados com CE (Delta DMO tíbia: 0,018 ± 0,010 vs CTL: 0,040 ± 0,011 p= 0,001; Delta DMO coluna: 0,004± 0,011 vs CTL: 0,055 ± 0,009 p= 0,009). A adição de ZOL ao CE reverteu a perda de DMO (Delta DMO tíbia: 0,027 ± 0,003 p= 0,002 em relação ao CE; Delta DMO coluna: 0,043 ± 0,011 p= 0,02 em relação ao CE). O tratamento com MTX não promoveu alteração da DMO. As análises histomorfométricas das tíbias com e sem o implante, foram realizadas 12 semanas após a colocação dos implantes, e os resultados mostraram uma redução de espessura cortical e de tecido ósseo em função do tratamento com CE sendo que a adição de ZOL reverteu também esse parâmetro. Da mesma forma, o percentual de contato entre osso e o implante foi alterado pelo tratamento com CE (CE: 25,98% vs. CTL: 42,40%; p= 0,013) e o ZOL reverteu esse efeito (CE- ZOL: 38,95% vs. CE: 25,98%; p= 0,014). Em conjunto, nossos resultados mostraram que a terapia com MTX não alterou a osseointegração de implantes de titânio, enquanto que o CE promoveu uma diminuição de contato entre osso e implante que foi revertida pela administração de ZOL, comprovando a eficácia terapêutica dessa associação. / In this study we evaluated the influence of Methotrexate (MTX- 3mg/kg/week) and Corticosteroid (CE- prednisona 1, 05 mg/kg/week), alone and in association with Folinic Acid (FA- 0, 25 mg/kg/week) and Zoledronic Acid (ZOL-0,1mg/kg/week) respectively, on osseointegration of titanium dental implants performed 6 weeks after treatment started in adult male NZW rabbits. Six animals in each group and eight in CE, were treated with the drugs during 18 weeks with exception of ZOL, which was infused as a single dose, at the moment of surgery. Dual-energy X-ray absorptiometryies were performed before and after treatment to determine bone mineral density alterations (Delta BMD). After 12 weeks post-implant placement, the animals were sacrificed for histomorphometric analysis. The BMD was significantly reduced by treatment with CE (Delta tibia: 0.018±0.010 vs CTL: 0.040±0.011; Delta lumbar: 0.004±0.011 vs CTL: 0.055±0.009), with recovery of BMD after ZOL administration (Delta tibia: 0,027± 0,003 vs CE: 0,018±0,010; Delta lumbar: 0.043±0.011 vs CE: 0.004±0.011). The BMD was not altered by MTX treatment or MTX-FA. The histomorphometric analysis revealed cortical bone loss and reduction in bone tissue in tibia in animals treated with CE, and these parameters were reversed by ZOL. Similarly, the percentage of bone to implant contact was reduced in the group treated with CE (CE: 25.98% vs. CTL: 42.40%; p=0.013), and reverted by ZOL (CE+ZOL: 38.95% vs. CE: 25.98%; p=0.014).The MTX and MTX-FA treatments did not alter osseointegration of the implant (35.09% and 35.92% respectively). Together our results showed that therapy with MTX did not interfere on osseointegration of titanium implants, while the CE reduced the bone to implant contact. ZOL administration reversed this effect, showing the therapeutic importance of this association.

A Artrite Reumatóide (AR) é uma artropatia autoimune multifatorial com etiologia desconhecida que afeta aproximadamente 1% da população adulta. A estratégia padrão para o tratamento da AR consiste na administração de baixas doses de Metotrexato (MTX), cujo efeito anti-inflamatório está relacionado com a manutenção dos níveis elevados de adenosina (ADO) extracelular. No entanto, uma parte considerável dos pacientes com AR é refratária ao tratamento com MTX e o mecanismo pelo qual este fenômeno ocorre ainda não está totalmente esclarecido. Neste contexto, o presente estudo descreveu que a eficácia terapêutica ao MTX está associada com a expressão em células Tregs da ectoenzima CD39, cuja função biológica é a geração de ADO extracelular via metabolização do ATP. Especificamente, através da realização de um estudo longitudinal, observamos que pacientes respondedores ao MTX (R-MTX) apresentam uma expansão de células Tregs circulantes expressando CD39 após o tratamento com MTX. Por outro lado, identificamos que pacientes não respondedores ao MTX (UR-MTX) possuem uma redução da expressão de CD39 em células Tregs, o que culmina em um comprometimento das suas funções supressoras. Ainda, demonstramos que a expressão de CD39 em células Tregs é um biomarcador apto em predizer a resposta terapêutica ao MTX, visto que pacientes UR-MTX apresentam uma expressão reduzida de CD39 em Tregs mesmo antes do início do tratamento com MTX. Posteriormente, nós investigamos as bases moleculares que acarretam na expressão reduzida de CD39 observada em células Tregs de pacientes URMTX. Demonstramos que a estimulação com TGF-? tanto em células Tregs isoladas quanto diferenciadas in vitro aumenta a expressão de CD39 através da ativação sequencial da seguinte plataforma molecular: receptores de TGF-? (TGFBRII e TGFBRI), transdutor de sinal SMAD2, fator de transcrição CREB, de modo dependente da atividade de p38. Uma vez identificada a via envolvida com a indução da expressão de CD39, demonstramos que células Tregs diferenciadas de indivíduos que apresentam uma expressão reduzida de CD39 são incapazes de induzir a expressão desta ectoenzima através da estimulação com TGF-?. Por fim, transpondo nossos achados para pacientes com AR, observamos que pacientes UR-MTX apresentam uma redução nos níveis de RNAm para TGFBRII e CREB bem como também uma redução das proteínas fosforiladas SMAD2 e CREB em células CD4+ e Tregs, sugerindo que o comprometimento na cascata de sinalização de TGF-?, envolvida com a indução da expressão de CD39 em células Tregs, está associado com a resistência ao MTX. / Rheumatoid arthritis (RA) is an autoimmune multifactorial arthropathy with unknown etiology that affects approximately 1% of the adult population. The standard strategy for RA treatment comprises the administration of low doses of methotrexate (MTX), whose antiinflammatory effects are associated with maintenance of high levels of extracellular adenosine (ADO). However, a considerable proportion of RA patients is resistant to MTX treatment and the mechanisms underlying this phenomenon occurs is poorly understood. Within this context, the present study showed that therapeutic efficacy of MTX is associated with expression on Treg cells of the ectoenzyme CD39, whose function is related to the generation of extracellular ADO by ATP metabolism. Specifically, we conducted a longitudinal study and observed that responsive patients to MTX (R-MTX) exhibit an increase in the frequency of circulating Treg cells expressing CD39 after MTX treatment. On the other hand, we found that non-responsive patients to MTX (UR-MTX) have a reduction of CD39 expression on Treg cells, which culminates in an impairment of Treg function. Furthermore, these findings indicate that CD39 expression on Treg cells is a biomarker for therapeutic response to MTX, since UR-MTX patients had a depressed CD39 expression on Treg cells even before MTX treatment. Subsequently, the present study investigated the molecular mechanisms that would cause the reduction of CD39 expression on Treg cells from UR-MTX patients. For this, we demonstrated that TGF-? stimulation increases CD39 expression in isolated and in vitro differentiated Treg cells through participation/activation of the following molecules: receptors of TGF-?, TGFBRII and TGFBRI, signal transducer SMAD2 and transcription factor CREB, through p38 activity dependent-manner. Once identified these molecules involved with CD39 induction, we demonstrated that differentiated Treg cells from healthy individuals with an intrinsic reduction of CD39 expression on circulating Treg cells are unable to increase CD39 expression by TGF-? stimulation. Transposing our findings to RA patients, we found that UR-MTX patients exhibit a reduction of mRNA for TGFBRII and CREB as well as reduction on levels of phospho-SMAD2 and phospho-CREB in CD4+ and Treg cells, suggesting that an impairment in TGF-? signaling pathway, related to induction of CD39 expression on Treg cells, is associated with MTX resistance.

O Metotrexato (MTX) é utilizado em altas doses no tratamento de neoplasias e em baixas doses como antiinflamatório. O objetivo deste estudo foi avaliar a reparação da fratura de côndilo mandibular e a simetria facial em ratos tratados com MTX. Foram utilizados 100 ratos, Wistar, machos que foram submetidos a procedimento cirúrgico utilizando modelo experimental de fratura de côndilo do lado direito. Os ratos foram distribuídos em quatro grupos e receberam os seguintes tratamentos: Controle - soro (um mL/semana); Dexametasona - dexametasona (0,15 mg/Kg); MTX Baixa dose - MTX (três mg/kg/semana); MTX Alta dose - MTX (30 mg/Kg). Os períodos de sacrifício foram de um dia, sete, 15, 30 e 90 dias de pós-operatório (n=cinco). O peso dos animais foi documentado. Foi realizada coleta de sangue para análise bioquímica de proteínas totais e fosfatase alcalina. Foi realizado exame radiográfico das cabeças em norma axial para análise cefalométrica. Foram realizadas mensurações lineares da maxila e mandíbula, bem como angulares do desvio mandibular. As amostras foram processadas histologicamente sendo obtidas lâminas com cortes no sentido coronal. Os dados quantitativos foram submetidos a análises estatísticas (=0,05). Os animais recuperaram peso ao longo do tempo, exceto no grupo MTX Alta dose. Os níveis séricos de proteínas totais mostraram aumento nos períodos iniciais e os de fosfatase alcalina queda nos períodos de formação do calo ósseo no tratamento com MTX. Houve redução no comprimento mandibular com alterações também na maxila e desvio progressivo da mandíbula em relação à base do crânio no grupo MTX Alta dose. A análise histológica revelou que houve reparo da fratura, pela formação de calo ósseo, e das estruturas da articulação sendo que no grupo MTX Alta dose ocorreu um retardo neste processo, havendo desvio do côndilo e em um espécime houve anquilose fibrosa. A histomorfometria revelou que a área de neoformação óssea foi menor no grupo MTX Alta dose. Foi concluído que o tratamento com MTX em alta dose teve efeito deletério na simetria facial de ratos submetidos à fratura do processo condilar e prejudicou a formação do calo ósseo e o reparo da articulação temporomandibular, com possível indução de anquilose fibrosa. / low doses as an antiinflammatory. The aim of this study was to evaluate the healing of the mandibular condyle fracture and facial symmetry in rats treated with MTX. 100 Wistar male rats undergone surgery using an experimental model of mandibular condyle fracture of the right side. The rats were distributed in four groups and received the following treatments: Control Saline (1 ml/week); Dexamethasone dexamethasone (0,15mg/kg); MTX Low dose MTX (3 mg/kg/week); MTX High dose MTX (30 mg/kg). Animals were sacrificed at one, seven, 15, 30 and 90 days postoperatively (n=5). Animals body weight were recorded. Blood has been taken to provide the biochemical analysis of total proteins and alkaline phosphatase. Radiographic axial exams from the heads were provided to cephalometric analysis. Linear measures of jaw and mandible, as well as angular measures of the mandibular deviation were done. The samples were histologically processed and coronal sections were obtained. Quantitative data were submitted to statistical analysis (=0,05). The animals regained body weight over the time, except in MTX High dose group. Total protein serum levels demonstrated the increase in initial periods and the alkaline phosphatase levels showed decrease in the periods of bone callus formation. There was reduction in the mandibular length and also changes in the jaw and progressive deviation in the mandible in relation to the skull basis in the MTX High dose group. The histological analysis revealed that there were repair of the bone and temporomandibular joint although in the MTX High dose group there was a delay in this process, in wich there was deviation of the condyle and one specimen was fibrous ankylosis. Histomorphometry revealed that the new bone formation area was lower in MTX High dose group. It was concluded that treatment with high dose MTX had a deleterious effect on facial symmetry of rats submitted to fracture of the condylar process and damaged the bone callus formation and repair of the temporomandibular joint, with possible induction of fibrous ankylosis.