Introduction: Rheumatoid arthritis (RA) is a multisystem disease associated with early mortality. Methotrexate (MTX) is the first-line therapy in RA but is associated with significant adverse events and response is not universal. There is, therefore, a need to identify early those patients with RA unlikely to respond or develop toxicity to MTX. One of the major influences on drug response is adherence and MTX can cause a range of side effects known to impact on adherence such as pneumonitis (MTX-P). The gold standard measurement of adherence would be direct detection of MTX or its metabolites in a biochemical assay. Currently, there are no reliable markers that predict response to MTX but some studies have suggested measurement of MTX levels may predict response. Previous studies have suggested that MTX-P may occur in individuals genetically predisposed to the disease. The aims of this research are to i) develop an assay to measure MTX levels; ii) test the ability of the assay to measure adherence; iii) investigate if MTX levels are associated with response; and iv) conduct a genome wide association study (GWAS) investigating MTX-P. Methods: An assay to measure MTX and 7-OH-MTX in urine and plasma was developed using HPLC-SRM-MS and the assay was used to measure levels in a cohort of RA patients to develop a pharmacokinetic model. Simulations of the model were used to determine the ability of the assay to monitor adherence and the model was validated in a separate cohort of patients with RA. An observational study of RA patients was used to measure MTX and 7-OH-MTX levels to investigate if levels are associated with response. Finally, a GWAS investigating MTX-P was conducted. Results: Results of the pharmacokinetic model demonstrated that MTX is the preferred analyte to monitor adherence. The model was validated in a separate cohort of patients with RA demonstrating the ability of the assay to measure adherence. MTX levels were not associated with disease response in this cohort. A GWAS of MTX-P demonstrated three SNPs associated with disease (p <5 x 10-5) with subsequent bioinformatics analysis showing a potential functional role for rs7514182.ConclusionAdherence to MTX may be a significant barrier to patients achieving full response to therapy. The development of a direct test to detect adherence based on measuring MTX levels using HPLC-SRM-MS has been developed in urine and blood. The assay was shown to be accurate in several domains from EMA guidelines and was validated in a separate cohort of patients. Finally, this program of work has investigated genetic markers associated with MTX-P. The results demonstrated a potential SNP associated with disease which demonstrates a functional role in the development of pulmonary fibrosis.

BACKGROUND: Epiretinal membranes are a common disease that can either be idiopathic, meaning no cause can be detected this is usually caused by aging, or secondary which is caused by injury disease or surgery. Despite current treatment methods, there are still persistence of this disease in some rare cases. Methotrexate although traditionally used to treat cancer and rheumatoid arthritis has been being explored as a treatment option in the field of ophthalmology for use against proliferative and migrating cellular diseases coupled with inflammation. Methotrexate has been reported in a few ocular diseases to reduce or stop cell migration and proliferation due to this finding a case study was conducted with this recurrent ERM patient to test its effectiveness against this disease. PURPOSE: To investigate a potential new treatment method for recurrent epiretinal membranes. After a visually significant epiretinal membrane develops there would be an epiretinal membrane (ERM) peel performed. Traditionally if there is recurrence of epiretinal membranes post ERM peeling the patient will be treated with an internal limiting membrane (ILM) peel. For most cases, this will resolve the issue. In the rare instances where an ILM peel doesn’t resolve recurrence, like in this case, we sought to test whether a series of methotrexate injections could help prevent ERM re-proliferation. CASE REPORT: Reporting on a case of a 65-year-old woman with a recurrent recalcitrant epiretinal membrane. This membrane was treated with a pars plana vitrectomy and ERM peeling. The membrane grew back and was met with an ILM peel in hopes of resolution. With continuing recurrence, the patient was treated with another ERM and ILM peel and 12 weekly intravitreal methotrexate (MTX) injections. METHODS: A patient with persistent recurrent epiretinal membranes underwent three surgeries in an attempt to cure the ERM. At every clinical visit, best corrected distance visual acuity was assessed with a Snellen Vision Test and the retina was imaged using optical coherence tomography. Measurements were taken using the machines built in analysis technology to measure retinal thickness and retinal volume at each visit. These were graphed alongside visual acuity to determine complimenting trends. RESULTS: At the first visit the patient began treatment at a visual acuity of 20/200 and a central macular thickness of 676. Seven months after the final methotrexate injection the patient was at a visual acuity of 20/80 and a central macular thickness of 328. The overall results were that visual acuity and central macular thickness significantly improved without ERM recurrence at seven months after treatment. CONCLUSION: When an ERM is significantly impacting the patient’s visual acuity surgery is usually performed in the form of an ERM peel or ILM peel. Although treatment of recurrent epiretinal membranes is well maintained by these procedures there are a small percentage of cases where recurrence is still found post ILM surgery. This case represents the first documented use of MTX to treat recurrent ERM and it suggests great potential for its use in otherwise treatment resistant cases. More research is required to better understand the true potential of this treatment option as well as associated risks.

Background: Rheumatoid arthritis (RA) is an inflammatory disease associated with an increased risk of cardiovascular morbidity and mortality. Methotrexate is a widely used RA medication that has encouraging results for being protective from RA cardiovascular related complications. In a published meta-analysis, MTX showed a 21% reduction in cardiovascular mortality; however, this meta-analysis had multiple limitations. Arterial stiffness is considered to be one of RA's extra-articular manifestations. As a common medication, could MTX have a beneficial effect on traditional cardiovascular risk factors and arterial stiffness parameters? Aims: The aim of this thesis is to assess the effect of MTX on cardiovascular morbidity and mortality using meta-analysis and a cross sectional and longitudinal study to determine whether MTX therapy is associated with reduced blood pressure, AS parameters (measured by pulse wave velocity (PWV), augmentation index (AIX)) and other traditional cardiovascular risk factors (glucose, lipids) in rheumatoid arthritis patients in Saudi Arabia. Methods: Meta-analysis of cohort study design was conducted using combined reporting guidelines. Studies were selected using a systematic search in five databases. Meta-analysis of the effect estimate of the cohort study design was done using a fixed effect model. Another part of the thesis is a cross sectional and longitudinal study of RA patients attending the rheumatology clinic at the university hospital in Saudi Arabia that were classified into three groups: 'current-MTX', which were patients that took MTX for at least three months; 'no-MTX', which were patients that were not on MTX for at least one year; and 'new-MTX', which were patients that were due to commence MTX directly after recruitment. Arterial stiffness and central 2 blood pressure parameters were assessed in RA patients using a validated non-invasive Mobil-O-Graph device. Other cardiovascular and non-cardiovascular parameters were also assessed during patient recruitment from patient interviews, medical records and the laboratory database. Patients were followed-up with two times. Linear regression analysis was performed; a mixed model for repeated measures was done to evaluate the effect of time on differences in blood pressure and arterial stiffness between groups. Results: Nine studies were included in the meta-analysis, and MTX showed a 46% reduction in cardiovascular events. Non-significant heterogeneity was documented between studies. A total of 353 patients (mean age 49 years, female 89%, median RA duration 10 years) were recruited at baseline (March 2013 to January 2014); with 117 reassessed over 3-6 months of follow-up. Augmentation index of the 'current-MTX' group was reduced compared to the 'no-MTX' group by 1.1 (95% CI -4.7 to 2.6) %; and systolic blood pressure was increased by 2.5 (95% CI -2.3 to 7.4) mmHg in 'current-MTX', but results were not statistically significant. During follow-up, no difference was found between treatment groups or within each individual group in the longitudinal analysis. Conclusion: Methotrexate is associated with reduced cardiovascular events on meta-analysis of cohort studies. In the cross sectional setting and longitudinal analysis, methotrexate did not prove to be beneficial in reducing arterial stiffness and blood pressure parameters and other cardiovascular risk factors in rheumatoid arthritis patients in Saudi Arabia.

O Metotrexato (MTX) é um fármaco utilizado como anti-inflamatório no tratamento da artrite reumatóide (AR). O risco de doença cardiovascular em pacientes com AR é menor quando tratados com MTX. Apesar dessa evidência, há poucos relatos da utilização de MTX para o tratamento da aterosclerose. Foi desenvolvida em nosso laboratório uma nanopartícula rica em colesterol (LDE), a qual é reconhecida pelos receptores da lipoproteína de baixa densidade (LDLr) após injeção na corrente sangüínea. A LDE concentra-se em células com hiperexpressão de LDLr, em processos proliferativos como a aterosclerose. Dessa maneira, a LDE pode ser utilizada como veículo para o direcionamento de fármacos contra essas células. A molécula de MTX foi latenciada e a modificação do fármaco aumentou a sua incorporação à LDE. A proposta desse estudo foi avaliar a eficácia de MTX associado à LDE (LDE-MTX) no tratamento da aterosclerose em coelhos além de investigar o efeito desse complexo na expressão de genes inflamatórios que participam do processo aterogênico. Para realização do estudo foram utilizados quatro grupos de 10 coelhos (raça New Zealand) cada, sendo que todos foram submetidos a uma dieta rica em colesterol por 8 semanas. Após as primeiras 4 semanas de dieta, os grupos foram tratados com LDE-MTX (grupo LDE-MTX), MTX comercial (grupo MTX comercial), LDE (grupo controle LDE) ou solução salina (grupo controle Salina), via endovenosa por 4 semanas. O grupo LDE-MTX não apresentou toxicidade ao longo do tratamento de acordo com os parâmetros utilizados, enquanto que o grupo MTX comercial apresentou uma queda acentuada de eritrócitos ao final do tratamento (p<0,001). A análise morfométrica macroscópica mostrou que os grupos LDE-MTX e MTX comercial reduziram as lesões ateroscleróticas quando comparados ao grupo controle Salina (66 e 76%, respectivamente) (p<0,001). Por microscopia, a camada íntima do arco aórtico e torácico foi reduzida nos grupos LDE-MTX (67% e 75%) e MTX comercial (81% e 92%, respectivamente) quando comparados com os mesmos fragmentos do grupo controle Salina (p<0,05). A presença de macrófagos na camada íntima dos grupos LDE-MTX e MTX comercial foi reduzida em 59% e 57% (p<0,001) no arco aórtico e 37% e 38% na região da aorta torácica (p=0,016) em relação ao grupo controle Salina, respectivamente. A porcentagem de MMP-9 no arco aórtico foi reduzida em 48% em ambos os grupos tratados (p=0,0003), enquanto que na aorta torácica LDE-MTX e MTX comercial diminuíram 54% e 66% (p<0,0001) respectivamente, em relação ao grupo controle Salina. Na região da aorta abdominal, a redução de MMP-9 também foi observada nos grupos LDE-MTX (68%) e MTX comercial (70%) (p=0,016). Quando se comparou ao grupo controle LDE, os dois grupos tratados tiveram porcentagens semelhantes de redução em todas as análises morfométricas. Nos estudos de expressão gênica in vivo, 5 genes inflamatórios se mostraram hipoexpressos (TNF-&#945;, MCP-1, IL-1&#946;, IL-18 e MMP-12) e um gene (IL-10) apresentou aumento de expressão no arco aórtico de coelhos tratados com LDE-MTX em relação ao grupo controle. No experimento in vitro, 5 dos 10 genes (TNF-&#945;, VAP-1, IL-1&#946;, CXCL2 e TLR2) avaliados tiveram redução da expressão e 1 (TGF-&#946;1) se mostrou hiperexpresso na linhagem de endotélio humana (HUVEC) tratada com as duas formulações de MTX. Os resultados indicam que tanto a LDE-MTX quanto MTX comercial reduzem acentuadamente as lesões ateroscleróticas em coelhos e parecem minimizar a resposta inflamatória na doença aterosclerótica. Contudo, LDE-MTX apresentou uma tolerabilidade maior ao tratamento, pois não apresentou toxicidade hematológica em comparação com MTX comercial. / Methotrexate (MTX) is the most frequently used drug for rheumatoid arthritis treatment. The incidence of vascular disease in these patients is lower when treated with MTX. However, few studies have been done using MTX for atherosclerosis treatment. In previous studies, we showed that, after injection into blood stream, a cholesterol-rich nanoparticle (LDE) binds to low density lipoprotein receptors (LDLr) and concentrates in tissues with intense cell proliferation such as atherosclerosis. LDE may thus carry drugs directed against those tissues reducing the toxicity of chemotherapeutic agents. For stable association with LDE, a lipophylic methotrexate derivative was used. The purpose of this study was to test MTX associated to LDE (LDE-MTX) in rabbits with atherosclerosis and investigate their anti-inflammatory effects on inflammatory mediators. Atherosclerosis was induced in rabbits by cholesterol rich diet during eight weeks. After 4 weeks from the introduction of the atherogenic diet, 4 groups of 10 animals were treated with LDE-MTX (LDE-MTX group), commercial MTX (commercial MTX Group), LDE (LDE control group) and saline solulion (Saline control group). MTX dose in both preparations was 4mg/kg/week during 4 additional weeks. LDE-MTX group showed superior tolerability with pronouncedly lesser hematologic toxicity in comparison to commercial MTX (p< 0.001). By morphometric analysis, both LDE-MTX and commercial MTX treatment groups showed a pronounced reduction of lesion area compared with Saline control group (66-76% respectively) (p<0001). By microscopy, intimal width at aortic arch and thoracic segments was reduced by 67% and 75% in LDE-MTX group compared to Saline control group, respectively (p<0.05). Commercial MTX group showed a reduction of 81% and 92% at aortic arch and thoracic segments compared to Saline control group, respectively (p<0.05). Presence of macrophages in intima layer at aortic arch was reduced by 59% and 57% (p<0.001) in LDE-MTX and commercial MTX groups while at thoracic segments was diminished by 37% and 38% (p=0.016) compared to Saline control group, respectively. MMP-9 percentage was diminished by 48% in both treated groups at aortic arch (p=0.0003) while at thoracic segment, LDE-MTX and commercial MTX reduced by 54% and 66% (p<0.0001) compared to Saline control group, respectively. Furthermore, MMP-9 percentage also diminished at abdominal segment in LDE-MTX group (68%) and commercial MTX (70%) when compared to Saline control group (p=0,016). When compared to LDE control group, both treated groups had similar percentage reduction in all morphometric analysis. In vivo studies, the expression of 5 inflammatory genes was downregulated (TNF-&#945;, MCP-1, IL-1&#946;, IL-18 and MMP-12) and 1 was upregulated (IL-10) when rabbits with induced atherosclerosis were treated with LDE-MTX. Besides, 5 inflammatory genes were downregulated (TNF-&#945;, VAP-1, IL-1&#946;, CXCL2 e TLR2) and 1 was upregulated (TGF-&#946;1) when human endothelial cell line (HUVEC) was treated with both MTX preparations. Therefore, MTX can be an effective drug for atherosclerosis treatment and associated to LDE, side effects of this chemotherapeutic agent can be minimized.

O Metotrexato (MTX) é um fármaco utilizado como anti-inflamatório no tratamento da artrite reumatóide (AR). O risco de doença cardiovascular em pacientes com AR é menor quando tratados com MTX. Apesar dessa evidência, há poucos relatos da utilização de MTX para o tratamento da aterosclerose. Foi desenvolvida em nosso laboratório uma nanopartícula rica em colesterol (LDE), a qual é reconhecida pelos receptores da lipoproteína de baixa densidade (LDLr) após injeção na corrente sangüínea. A LDE concentra-se em células com hiperexpressão de LDLr, em processos proliferativos como a aterosclerose. Dessa maneira, a LDE pode ser utilizada como veículo para o direcionamento de fármacos contra essas células. A molécula de MTX foi latenciada e a modificação do fármaco aumentou a sua incorporação à LDE. A proposta desse estudo foi avaliar a eficácia de MTX associado à LDE (LDE-MTX) no tratamento da aterosclerose em coelhos além de investigar o efeito desse complexo na expressão de genes inflamatórios que participam do processo aterogênico. Para realização do estudo foram utilizados quatro grupos de 10 coelhos (raça New Zealand) cada, sendo que todos foram submetidos a uma dieta rica em colesterol por 8 semanas. Após as primeiras 4 semanas de dieta, os grupos foram tratados com LDE-MTX (grupo LDE-MTX), MTX comercial (grupo MTX comercial), LDE (grupo controle LDE) ou solução salina (grupo controle Salina), via endovenosa por 4 semanas. O grupo LDE-MTX não apresentou toxicidade ao longo do tratamento de acordo com os parâmetros utilizados, enquanto que o grupo MTX comercial apresentou uma queda acentuada de eritrócitos ao final do tratamento (p<0,001). A análise morfométrica macroscópica mostrou que os grupos LDE-MTX e MTX comercial reduziram as lesões ateroscleróticas quando comparados ao grupo controle Salina (66 e 76%, respectivamente) (p<0,001). Por microscopia, a camada íntima do arco aórtico e torácico foi reduzida nos grupos LDE-MTX (67% e 75%) e MTX comercial (81% e 92%, respectivamente) quando comparados com os mesmos fragmentos do grupo controle Salina (p<0,05). A presença de macrófagos na camada íntima dos grupos LDE-MTX e MTX comercial foi reduzida em 59% e 57% (p<0,001) no arco aórtico e 37% e 38% na região da aorta torácica (p=0,016) em relação ao grupo controle Salina, respectivamente. A porcentagem de MMP-9 no arco aórtico foi reduzida em 48% em ambos os grupos tratados (p=0,0003), enquanto que na aorta torácica LDE-MTX e MTX comercial diminuíram 54% e 66% (p<0,0001) respectivamente, em relação ao grupo controle Salina. Na região da aorta abdominal, a redução de MMP-9 também foi observada nos grupos LDE-MTX (68%) e MTX comercial (70%) (p=0,016). Quando se comparou ao grupo controle LDE, os dois grupos tratados tiveram porcentagens semelhantes de redução em todas as análises morfométricas. Nos estudos de expressão gênica in vivo, 5 genes inflamatórios se mostraram hipoexpressos (TNF-&#945;, MCP-1, IL-1&#946;, IL-18 e MMP-12) e um gene (IL-10) apresentou aumento de expressão no arco aórtico de coelhos tratados com LDE-MTX em relação ao grupo controle. No experimento in vitro, 5 dos 10 genes (TNF-&#945;, VAP-1, IL-1&#946;, CXCL2 e TLR2) avaliados tiveram redução da expressão e 1 (TGF-&#946;1) se mostrou hiperexpresso na linhagem de endotélio humana (HUVEC) tratada com as duas formulações de MTX. Os resultados indicam que tanto a LDE-MTX quanto MTX comercial reduzem acentuadamente as lesões ateroscleróticas em coelhos e parecem minimizar a resposta inflamatória na doença aterosclerótica. Contudo, LDE-MTX apresentou uma tolerabilidade maior ao tratamento, pois não apresentou toxicidade hematológica em comparação com MTX comercial. / Methotrexate (MTX) is the most frequently used drug for rheumatoid arthritis treatment. The incidence of vascular disease in these patients is lower when treated with MTX. However, few studies have been done using MTX for atherosclerosis treatment. In previous studies, we showed that, after injection into blood stream, a cholesterol-rich nanoparticle (LDE) binds to low density lipoprotein receptors (LDLr) and concentrates in tissues with intense cell proliferation such as atherosclerosis. LDE may thus carry drugs directed against those tissues reducing the toxicity of chemotherapeutic agents. For stable association with LDE, a lipophylic methotrexate derivative was used. The purpose of this study was to test MTX associated to LDE (LDE-MTX) in rabbits with atherosclerosis and investigate their anti-inflammatory effects on inflammatory mediators. Atherosclerosis was induced in rabbits by cholesterol rich diet during eight weeks. After 4 weeks from the introduction of the atherogenic diet, 4 groups of 10 animals were treated with LDE-MTX (LDE-MTX group), commercial MTX (commercial MTX Group), LDE (LDE control group) and saline solulion (Saline control group). MTX dose in both preparations was 4mg/kg/week during 4 additional weeks. LDE-MTX group showed superior tolerability with pronouncedly lesser hematologic toxicity in comparison to commercial MTX (p< 0.001). By morphometric analysis, both LDE-MTX and commercial MTX treatment groups showed a pronounced reduction of lesion area compared with Saline control group (66-76% respectively) (p<0001). By microscopy, intimal width at aortic arch and thoracic segments was reduced by 67% and 75% in LDE-MTX group compared to Saline control group, respectively (p<0.05). Commercial MTX group showed a reduction of 81% and 92% at aortic arch and thoracic segments compared to Saline control group, respectively (p<0.05). Presence of macrophages in intima layer at aortic arch was reduced by 59% and 57% (p<0.001) in LDE-MTX and commercial MTX groups while at thoracic segments was diminished by 37% and 38% (p=0.016) compared to Saline control group, respectively. MMP-9 percentage was diminished by 48% in both treated groups at aortic arch (p=0.0003) while at thoracic segment, LDE-MTX and commercial MTX reduced by 54% and 66% (p<0.0001) compared to Saline control group, respectively. Furthermore, MMP-9 percentage also diminished at abdominal segment in LDE-MTX group (68%) and commercial MTX (70%) when compared to Saline control group (p=0,016). When compared to LDE control group, both treated groups had similar percentage reduction in all morphometric analysis. In vivo studies, the expression of 5 inflammatory genes was downregulated (TNF-&#945;, MCP-1, IL-1&#946;, IL-18 and MMP-12) and 1 was upregulated (IL-10) when rabbits with induced atherosclerosis were treated with LDE-MTX. Besides, 5 inflammatory genes were downregulated (TNF-&#945;, VAP-1, IL-1&#946;, CXCL2 e TLR2) and 1 was upregulated (TGF-&#946;1) when human endothelial cell line (HUVEC) was treated with both MTX preparations. Therefore, MTX can be an effective drug for atherosclerosis treatment and associated to LDE, side effects of this chemotherapeutic agent can be minimized.

The transdermal delivery of drugs has a lot of advantages above other routes of delivery, such as the avoidance of first-pass hepatic and intestinal metabolism, the non-invasive infusion of drugs, etc. However, the transdermal delivery of drugs, especially hydrophilic drugs, is limited due to the lipophilic nature of the stratum corneum. Methotrexate is a folic acid antagonist with antineoplastic activity and is used for the treatment of psoriasis and Kaposi's sarcoma. The permeation of methotrexate through the skin for systemic use is however limited due to its high molecular weight, the fact that it is mainty dissociated at physiological pH and its hydrophilic nature (Alvarez-Figueroa et al.. 2001). Thus the aim of my study was to enhance the permeation of methotrexate with the use of terpene. Terpenes are lipophilic in nature and have Log P values of around 2-4 (Godwin & Michniak, 1999). These characteristics make them excellent candidates as penetration enhancers. Terpenes are not only used for penetration enhancers, but in a huge number of other products, such as aromatherapeutic oils. For this reason the permeation of the terpenes through human skin and the effect of methotrexate on this permeation were also determined. The following enhancers were used in this study: menthol, menthone. isomenthol, limonene, B-myrcene, a-pinene and 1,8-cineole Five different sets of experiments were done in this study: a) a control experiment with methotrexate in the absence of the terpenes without ethanol; b) a control experiment with methotrexate in the absence of the terpenes with ethanol: c) experiments with methotrexate in the presence of the terpenes; d) control experiments with the terpenes in the absence of methotrexate and e) experiments with tile terpenes in the presence of methotrexate. For this study only human female abdominal skin was used. A saturated solution of methotrexate in water:propylene glycol (50:50) with a pH between 4 and 5 (Vaidyanathan et al., 1985) was used as the model drug and the receptor phase was PBS-buffer (pH=74) and water:ethanol (50:50) for HPLC and GC analysis respectively. The dilfusion apparatus used consisted of Vertical Franz diffusion cells with a capacity of 2 ml and a diffusion area of 1.075 cm2. The cells were placed in a water bath (+- 37 "C) on magnetic stirrers for the duration of the experiment. After the receptor phase was placed in the receptor compartment the cells were equilibrated for an hour before putting 25 ul of a 5% terpene solution in absolute ethanol on the skin in the donor compartment. This was left for half and hour to allow evaporation of the ethanol. The saturated solution of the methotrexate was now placed on the skin in the donor compartment. The experiments for methotrexate stretched over a period of 12 hours and samples were collected every 2 hours. The terpene experiments were performed over a 24-hour period and samples were taken at 2,4,6,12 and 24 hours. The concentration methotrexate permeated was determined by using HPLC-analysis and terpenes by using GC-analysis. The flux (ug/cm2/h), kp(cm/h), lag time (h) and enhancement ratio were calculated to compare the methotrexate permeation in the control and actual experiments. The results showed that a-pinene, B-myrcene and isomenthol enhanced the permeation of methotrexate most, although all the terpenes had an enhancing effect. They produced a 4- fold increase in the flux values of methotrexate. Due to the fact that the terpene experiments were only a semi-quantitative evaluation only the percentage terpenes that permeated was calculated. The experiments were done on all the terpenes except apinene. All the terpenes permeated the skin with menthol having the highest permeation. The results also showed that methotrexate did have an effect on the terpene permeation. Menthone and menthol's permeation was higher in the presence of methotrexate, while the other terpenes had a higher permeation in the absence of methotrexate. The reason for this is not clear. In conclusion, the study revealed that the enhancers used did have an enhancing effect on methotrexate permeation. This could be due to the extraction or disruption of lipids by the terpenes (Zhoa & Singh, 2000) or an increase in diffusivity and partitioning. The terpene experiments also showed that the terpenes do permeate the skin and that methotrexate does have an effect on this permeation. / Thesis (M.Sc.)--North-West University, Potchefstroom Campus, 2004.

The transdermal delivery of drugs has a lot of advantages above other routes of delivery, such as the avoidance of first-pass hepatic and intestinal metabolism, the non-invasive infusion of drugs, etc. However, the transdermal delivery of drugs, especially hydrophilic drugs, is limited due to the lipophilic nature of the stratum corneum. Methotrexate is a folic acid antagonist with antineoplastic activity and is used for the treatment of psoriasis and Kaposi's sarcoma. The permeation of methotrexate through the skin for systemic use is however limited due to its high molecular weight, the fact that it is mainty dissociated at physiological pH and its hydrophilic nature (Alvarez-Figueroa et al.. 2001). Thus the aim of my study was to enhance the permeation of methotrexate with the use of terpene. Terpenes are lipophilic in nature and have Log P values of around 2-4 (Godwin & Michniak, 1999). These characteristics make them excellent candidates as penetration enhancers. Terpenes are not only used for penetration enhancers, but in a huge number of other products, such as aromatherapeutic oils. For this reason the permeation of the terpenes through human skin and the effect of methotrexate on this permeation were also determined. The following enhancers were used in this study: menthol, menthone. isomenthol, limonene, B-myrcene, a-pinene and 1,8-cineole Five different sets of experiments were done in this study: a) a control experiment with methotrexate in the absence of the terpenes without ethanol; b) a control experiment with methotrexate in the absence of the terpenes with ethanol: c) experiments with methotrexate in the presence of the terpenes; d) control experiments with the terpenes in the absence of methotrexate and e) experiments with tile terpenes in the presence of methotrexate. For this study only human female abdominal skin was used. A saturated solution of methotrexate in water:propylene glycol (50:50) with a pH between 4 and 5 (Vaidyanathan et al., 1985) was used as the model drug and the receptor phase was PBS-buffer (pH=74) and water:ethanol (50:50) for HPLC and GC analysis respectively. The dilfusion apparatus used consisted of Vertical Franz diffusion cells with a capacity of 2 ml and a diffusion area of 1.075 cm2. The cells were placed in a water bath (+- 37 "C) on magnetic stirrers for the duration of the experiment. After the receptor phase was placed in the receptor compartment the cells were equilibrated for an hour before putting 25 ul of a 5% terpene solution in absolute ethanol on the skin in the donor compartment. This was left for half and hour to allow evaporation of the ethanol. The saturated solution of the methotrexate was now placed on the skin in the donor compartment. The experiments for methotrexate stretched over a period of 12 hours and samples were collected every 2 hours. The terpene experiments were performed over a 24-hour period and samples were taken at 2,4,6,12 and 24 hours. The concentration methotrexate permeated was determined by using HPLC-analysis and terpenes by using GC-analysis. The flux (ug/cm2/h), kp(cm/h), lag time (h) and enhancement ratio were calculated to compare the methotrexate permeation in the control and actual experiments. The results showed that a-pinene, B-myrcene and isomenthol enhanced the permeation of methotrexate most, although all the terpenes had an enhancing effect. They produced a 4- fold increase in the flux values of methotrexate. Due to the fact that the terpene experiments were only a semi-quantitative evaluation only the percentage terpenes that permeated was calculated. The experiments were done on all the terpenes except apinene. All the terpenes permeated the skin with menthol having the highest permeation. The results also showed that methotrexate did have an effect on the terpene permeation. Menthone and menthol's permeation was higher in the presence of methotrexate, while the other terpenes had a higher permeation in the absence of methotrexate. The reason for this is not clear. In conclusion, the study revealed that the enhancers used did have an enhancing effect on methotrexate permeation. This could be due to the extraction or disruption of lipids by the terpenes (Zhoa & Singh, 2000) or an increase in diffusivity and partitioning. The terpene experiments also showed that the terpenes do permeate the skin and that methotrexate does have an effect on this permeation. / Thesis (M.Sc.)--North-West University, Potchefstroom Campus, 2004.

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 80-96).

Thesis--University of Oklahoma. / Bibliography: leaves 86-90.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-04-07T13:45:11Z No. of bitstreams: 3 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) tese_completa_BIBLIOTECA_10_11_2015.pdf: 1137406 bytes, checksum: 99ca45ce4b42d3ca289fb6979849607a (MD5) tese_completa_BIBLIOTECA_10_11_2015.pdf: 1137406 bytes, checksum: 99ca45ce4b42d3ca289fb6979849607a (MD5) / Made available in DSpace on 2016-04-07T13:45:11Z (GMT). No. of bitstreams: 3 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) tese_completa_BIBLIOTECA_10_11_2015.pdf: 1137406 bytes, checksum: 99ca45ce4b42d3ca289fb6979849607a (MD5) tese_completa_BIBLIOTECA_10_11_2015.pdf: 1137406 bytes, checksum: 99ca45ce4b42d3ca289fb6979849607a (MD5) Previous issue date: 2015-05-26 / Introdução: O advento dos stents convencionais e daqueles liberadores de fármacos aboliu o recolhimento elástico provocado pela angioplastia por balão, porém, estes dispositivos apresentaram como consequência a hiperplasia neointimal, que é responsável pela reestenose angiográfica. O metotrexato, antagonista do folato, inibe a fase S do ciclo de mitose celular, reduzindo a produção de citocinas e outros mediadores inflamatórios que podem estar envolvidos na hiperplasia neointimal. Objetivo: Avaliar a segurança do Metotrexato (MTX) em pacientes com Doença Arterial Coronariana submetidos à Intervenção Coronariana Percutânea (ICP) com stents convencionais e o impacto da droga na reestenose clínica e angiográfica. Método: Estudo clínico de fase II aberto, prospectivo, não randomizado, realizado de setembro de 2011 a maio de 2014. Resultados: Foram recrutados 16 pacientes com indicação de implante de stent, sendo que estes tomaram 5 mg de MTX 15 dias antes e 30 após a ICP. Todos os pacientes foram submetidos à nova angiografia coronariana após 9 meses. A artéria coronária descendente anterior apresentou o maior número de lesões 16 (34%). O diâmetro médio dos stents foi de 3,0 ± 0,4 mm e o comprimento médio foi de 18,1 ± 5,9 mm. Não houve complicações relacionadas à ICP. As complicações do MTX foram menores e com prevalência de 18,7%. Nenhum paciente necessitou de interrupção no uso do medicamento e os sintomas desapareceram ao final do tratamento. A reestenose angiográfica foi 6,2% e a clínica foi zero (ausência de sintoma e de isquemia em cintilografia do miocárdio). Conclusão: O MTX foi seguro e gerou a hipótese de possível efeito benéfico na reestenose após implante de stent convencional. / Introduction: Bare metal stents (BMS) and those releasing drug abolished the elastic recoil caused by Plain Old Balloon Angioplasty (POBA), however, these devices showed the consequence of neointimal hyperplasia, which is responsible for angiographic restenosis. The methotrexate, folate antagonist inhibits S phase of mitotic cell cycle by reducing the production of cytokines and the other inflammatory mediators that may be involved in the neointimal hyperplasia. Objective: To evaluate the safety of Methotrexate (MTX) in patients with coronary artery disease undergoing Percutaneous Coronary Intervention (PCI) with BMS and the impact of drugs in clinical and angiographic restenosis. Methods: Clinical phase II open, prospective, nonrandomized, held from September 2011 to May 2014. Results: We recruited 16 patients with stent implantation indication, and these took 5 mg of MTX 15 days before and 30 after PCI. All patients underwent coronary angiography after nine months. The anterior descending coronary artery had the highest number of lesions 16 (34%). The average diameter of the stents was 3.0 ± 0.4 mm and the average length was 18.1 ± 5.9 mm. There were no complications related to PCI. The MTX complications were minor and with a prevalence of 18.7%. No patient required interruption in the use of the drug, and the symptoms disappeared after the treatment. Angiographic restenosis was 6.2% and the clinic was zero (no symptoms and ischemia on myocardial scintigraphy). Conclusion: MTX was safe and led the hypothesis of possible beneficial effect on restenosis after conventional stent implantation.