Metabolomic Insights into the Pharmacological and Genetic Inhibition of Cyclooxygenase-2 William T. E. Briggs The cyclooxygenase (COX)-2 inhibitors, or “coxibs,” are excellent anti-inflammatory agents, but their reputation has been tarnished by the adverse cardiovascular (CV) events, including heart failure (HF), with which they are associated. Whilst the risk of HF represents the greatest adverse CV event signal seen with these compounds, it is also perhaps the least well understood and has often been explained away as a consequence of the thrombotic risk with which the coxibs are also associated. One recent hypothesis, put forward by Ahmetaj-Shala et al., suggests that asymmetric dimethylarginine (ADMA) may serve as a mechanistic bridge between COX-2 inhibition and HF. However, the ADMA-COX-2 hypothesis was developed based on findings in a constitutive mouse model of COX-2 knock-out (KO), which is compromised by severe developmental cardio-renal pathology, and pharmacological studies which may not accurately reflect coxib use in clinical practice. Various studies have explored the metabolic changes induced by coxib treatment. However, these studies have been limited in scope and have tended to focus on specific pathways or certain tissues/bio-fluids. This has left large regions of the metabolome, in the context of coxib-treatment, unexplored. Given that metabolic remodelling is a key feature of HF, changes in these metabolites may hold the key to understanding the pathogenesis of coxib-induced HF. L-Carnitine shuttles activated long-chain fatty acids (FAs) across the inner mitochondrial membrane to the mitochondrial matrix, where they are oxidised by β-oxidation. This is especially important in the heart, which derives the majority of its energy from the metabolism of FAs. Changes in carnitine metabolism are also seen in HF. It is therefore biologically plausible that derangements in carnitine metabolism may contribute to the pathogenesis of coxib-induced HF. This thesis employs a combination of targeted and untargeted metabolomic techniques, stable isotope labelling and quantitative reverse transcription polymerase chain reaction (RT-qPCR) to i) profile the metabolic changes induced by celecoxib and rofecoxib, in the mouse; ii) specifically interrogate the effect of celecoxib, rofecoxib and global COX-2 gene deletion on carnitine synthesis, metabolism and shuttling, and iii) explore the advantages and disadvantages of the inducible post-natal global (IPNG) COX-2 KO (COX-2-/-) mouse, an alternative to the constitutive COX-2-/- mouse used by Ahmetaj-Shala et al. The results of this thesis demonstrate that i) celecoxib and rofecoxib have similar metabolomic consequences in the mouse; ii) carnitine metabolism may be affected by celecoxib, rofecoxib and dietary composition, via a peroxisome proliferator-activated receptor-alpha (PPAR-α) mediated effect on hepatic carnitine synthesis and iii) the IPNG COX-2-/- mouse neither exhibits the severe developmental cardio-renal pathology nor the altered ADMA metabolism observed in the constitutive COX-2-/- mouse. These findings contradict those of Ahmetaj-Shala et al., oppose the ADMA-COX-2 hypothesis and highlight a potential role for carnitine metabolism and diet in coxib induced HF.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for systemic control of acute and chronic pain and inflammation (Lin et ah, 2000:1129), but usage problems and side-effects that occur during the post-marketing phase of these drugs are well documented (Thiefin & Beaugerie, 2005:287). Following the demonstration of the value of anti-inflammatory therapy in diseases like rheumatoid arthritis (Boardman & Dudley Hart, 1967:268), new NSAIDs appeared on the market (Dieppe et al., 2004:867), and the indications steadily broadened from inflammatory diseases to almost any painful condition. Studies have indicated that NSAID-associated serious upper gastro-intestinal (GI) adverse events result in 103 000 hospitalisations (Bombardier, 2002:4) and 165 000 deaths per year in the United States. A study in South Africa in 2002 indicated that NSAID utilisation contributed considerably to the total cost of all medicine items from a medicine claim database in the private health care sector (Joubert, 2002:260). The objective of this study was to determine the prevalence and cost of non-steroid anti-inflammatory drugs in a section of the private health care sector, and specifically to determine the prevalence, usage and cost of Coxib (Specific cyclo-oxygenase-2 inhibitor) medicine items before and after the withdrawal of Vioxx® from the market in September 2004 (Merck, 2004). Data from two medicine claim databases for the years 2004, 2005 and 2006 (medicine claim database I) and the years 2005 and 2006 (medicine claim database M), were analysed by means of a retrospective drug utilisation review (DUR) study. The usage of Coxib medicine items was determined, and compared for the periods before and after the withdrawal of Vioxx® in September 2004. It was found that between 9 and 10.5 per cent of prescriptions dispensed through both medicine claim database I and medicine claim database M during the study period were NSAID prescriptions. NSAID medicine items on medicine claim database I represented between 3.9 % (R25 942 986) and 2.9 % (R8 073 034) of the total cost of all medicine items claimed from 2004 to 2006. NSAIDs represented 3.1 % (R58 290 412) and 2.8 % (R57 752 267) of the cost of all medicine items claimed through medicine claim database M during 2005 and 2006 respectively, indicating similar trends in the two medicine claim databases. The prevalence of Coxibs on medicine claim database I decreased from almost 20 % (47 938) in 2004 to 8.4 % (13 276) in 2005, but showed an increase again to 10.9 % (12 355) in 2006. The prevalence of both cyclo-oxygenase (COX) inhibitors, and Coxibs demonstrated a change during 1 September 2004 to 31 December 2004 when COX-inhibitors showed an increase in use, while Coxibs showed and almost equal but opposite trend with a decrease in use. This could possibly be related to perceptions of providers and public with regard to Coxibs and their related safety after the withdrawal of Vioxx® on 30 September 2004 (Merck, 2004) and other Coxibs such as Bextra® (FDA, 2005) in 2005 in USA. It is concluded that most patients who were using Coxibs before the withdrawal of Vioxx®, substituted Coxibs for COX-inhibitors, that are known for their possible gastro-intestinal side-effects. Recommendations for future research regarding NSAID use were also made, and included an investigation of the usage of Coxibs in different age groups, as well as the combination of NSAIDs with gastro-protective medicines in long-term use. / Thesis (M.Pharm. (Pharmacy Practice))--North-West University, Potchefstroom Campus, 2009.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for systemic control of acute and chronic pain and inflammation (Lin et ah, 2000:1129), but usage problems and side-effects that occur during the post-marketing phase of these drugs are well documented (Thiefin & Beaugerie, 2005:287). Following the demonstration of the value of anti-inflammatory therapy in diseases like rheumatoid arthritis (Boardman & Dudley Hart, 1967:268), new NSAIDs appeared on the market (Dieppe et al., 2004:867), and the indications steadily broadened from inflammatory diseases to almost any painful condition. Studies have indicated that NSAID-associated serious upper gastro-intestinal (GI) adverse events result in 103 000 hospitalisations (Bombardier, 2002:4) and 165 000 deaths per year in the United States. A study in South Africa in 2002 indicated that NSAID utilisation contributed considerably to the total cost of all medicine items from a medicine claim database in the private health care sector (Joubert, 2002:260). The objective of this study was to determine the prevalence and cost of non-steroid anti-inflammatory drugs in a section of the private health care sector, and specifically to determine the prevalence, usage and cost of Coxib (Specific cyclo-oxygenase-2 inhibitor) medicine items before and after the withdrawal of Vioxx® from the market in September 2004 (Merck, 2004). Data from two medicine claim databases for the years 2004, 2005 and 2006 (medicine claim database I) and the years 2005 and 2006 (medicine claim database M), were analysed by means of a retrospective drug utilisation review (DUR) study. The usage of Coxib medicine items was determined, and compared for the periods before and after the withdrawal of Vioxx® in September 2004. It was found that between 9 and 10.5 per cent of prescriptions dispensed through both medicine claim database I and medicine claim database M during the study period were NSAID prescriptions. NSAID medicine items on medicine claim database I represented between 3.9 % (R25 942 986) and 2.9 % (R8 073 034) of the total cost of all medicine items claimed from 2004 to 2006. NSAIDs represented 3.1 % (R58 290 412) and 2.8 % (R57 752 267) of the cost of all medicine items claimed through medicine claim database M during 2005 and 2006 respectively, indicating similar trends in the two medicine claim databases. The prevalence of Coxibs on medicine claim database I decreased from almost 20 % (47 938) in 2004 to 8.4 % (13 276) in 2005, but showed an increase again to 10.9 % (12 355) in 2006. The prevalence of both cyclo-oxygenase (COX) inhibitors, and Coxibs demonstrated a change during 1 September 2004 to 31 December 2004 when COX-inhibitors showed an increase in use, while Coxibs showed and almost equal but opposite trend with a decrease in use. This could possibly be related to perceptions of providers and public with regard to Coxibs and their related safety after the withdrawal of Vioxx® on 30 September 2004 (Merck, 2004) and other Coxibs such as Bextra® (FDA, 2005) in 2005 in USA. It is concluded that most patients who were using Coxibs before the withdrawal of Vioxx®, substituted Coxibs for COX-inhibitors, that are known for their possible gastro-intestinal side-effects. Recommendations for future research regarding NSAID use were also made, and included an investigation of the usage of Coxibs in different age groups, as well as the combination of NSAIDs with gastro-protective medicines in long-term use. / Thesis (M.Pharm. (Pharmacy Practice))--North-West University, Potchefstroom Campus, 2009.

<b>Background:</b> Comparative assessments of the vascular and upper gastrointestinal risks of different regimens of non-steroidal anti-inflammatory drugs (NSAIDs) are required. <b>Methods:</b> Meta-analyses were conducted, using individual participant data where possible, of placebo-controlled trials of a selective cyclo-oxygenase [COX]-2 inhibitor ('coxib') or traditional NSAID, or randomised trials of a coxib versus traditional NSAIDs. A prespecified subdivision of traditional NSAID regimens of those with antiplatelet activity (mainly naproxen) and those without (mainly diclofenac) was made. Primary outcomes were major vascular events (MVEs; nonfatal myocardial infarction, nonfatal stroke or vascular death) and upper gastrointestinal complications (UGICs; perforation, obstruction or bleed). <b>Findings:</b> Searches identified 788 trials: 200 comparisons of a coxib vs placebo (88,604 participants, mean follow-up 0.60 years), 206 comparisons of a traditional NSAID vs placebo (43,482 participants, 0.46 years) and 149 comparisons of a coxib vs traditional NSAID (137,466 participants, mean follow-up 0.95 years). Compared to placebo, allocation to a coxib increased the risk of MVEs (rate ratio 1.38, 95&percnt; CI 1.14-1.66), vascular mortality (1.58, 1.11-2.24) and UGICs (1.81, 1.17-2.81). Overall, in the population studied, coxibs were associated with three additional major vascular events (one fatal) and two (rarely fatal) upper gastrointestinal complications per 1000 person-years exposure. There was no evidence of heterogeneity by duration of follow-up, coxib type, dose (other than for celecoxib), or patient characteristics, for the primary outcomes. The risk of MVEs for traditional NSAIDs without antiplatelet activity (mostly diclofenac 75mg bd or ibuprofen 800mg tds) were comparable to coxibs (1.40, 1.15-1.72); but the risk of UGICs (1.98, 1.39-2.84) was significantly greater. For traditional NSAIDs with antiplatelet activity (mostly naproxen 500mg bd) there were no significant excess of MVEs (0.84, 0.66-1.08), but UGICs were substantially increased (4.06, 2.85-5.78). Both coxibs and traditional NSAIDs increased risk of hospitalisation for heart failure by about two-fold. <b>Interpretation:</b> The vascular and upper gastrointestinal risks of coxibs and high-dose tNSAID regimens can be predicted, allowing the choice of analgesia to be tailored for particular patients.

Submitted by ALEXANDRE BORGES null (alex.brgs@hotmail.com) on 2016-06-28T19:11:21Z No. of bitstreams: 1 ESTUDOS DE MODELAGEM MOLECULAR DE LIGNANAS EM COMPLEXOS COM CICLOOXIGENASES-1 E 2.pdf: 4325586 bytes, checksum: 2f6ab56677aea7746bd28dad4b24ea23 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-06-29T17:41:12Z (GMT) No. of bitstreams: 1 borges_a_dr_ilha.pdf: 4325586 bytes, checksum: 2f6ab56677aea7746bd28dad4b24ea23 (MD5) / Made available in DSpace on 2016-06-29T17:41:12Z (GMT). No. of bitstreams: 1 borges_a_dr_ilha.pdf: 4325586 bytes, checksum: 2f6ab56677aea7746bd28dad4b24ea23 (MD5) Previous issue date: 2016-05-11 / Os inibidores seletivos da ciclooxigenase-2 (COX-2), como o rofecoxibe (2) e o celecoxibe (1), formam uma importante classe de medicamentos anti-inflamatórios desenvolvidos a partir da descoberta das duas isoformas das ciclooxigenases (COX-1 e COX-2) na década de 1979. A isoforma 1 esta relacionada com a citoproteção gástrica, agregação plaquetária e função renal e a isoforma 2 relacionada a processos inflamatórios. Estes inibidores seletivos apesar de não apresentarem os efeitos colaterais (ulceras e gastrites) dos anti-inflamatórios não esteroidais (AINEs) clássicos por inibirem apenas a COX-2, apresentam grave risco cardiovascular, o que motivou à retirada do rofecoxibe do mercado. Porém, por ser um eficiente inibidor seletivo da COX-2 a estrutura do rofecoxibe tornou-se referência no estudo de novas substâncias capazes de inibir seletivamente a COX-2. Dentre as ferramentas utilizadas na busca destas novas estruturas está a modelagem molecular através de programas como o GOLD 5.1, que foi utilizado neste trabalho. O uso do GOLD 5.1 possibilitou o estudo do comportamento das estruturas avaliadas em ligação com as ciclooxigenases. O objetivo foi obtenção de estruturas com comportamento semelhante ao rofecoxibe (em relação às COXs) como potenciais candidatos ao desenvolvimento de novos inibidores seletivos para a COX-2. O estudo foi realizado com 480 estruturas modeladas a partir de lignanas naturais como a hinoquinina, cubebina, deoxipodofilotoxina e podofilotoxina, que apresentam atividade anti-inflamatória in vivo ou in vitro, além de semelhanças estruturais com o rofecoxibe. A deoxipodofilotoxina por apresentar seletividade para a COX-2 em ensaio in vitro também foi utilizada como estrutura de referência além do rofecoxibe. Os resultados observados a partir da simulação molecular permitiram concluir que embora tanto o rofecoxibe como a deoxipodofilotoxina (3) inibam seletivamente a COX-2 in vitro, o fazem de modo diferente. Em relação a COX-2 as duas estruturas ocupam a mesma região do sítio ativo, mas o rofecoxibe apresenta interações mais fortes com o bolso hidrofílico desta isoforma (condição necessária para a inibição seletiva para os coxibes). Já para a COX-1 enquanto o rofecoxibe ocupa a porção superior do canal hidrofóbico (sítio ativo) como os demais AINEs, a deoxipodofilotoxina ocupa uma região vizinha. Pelos resultados obtidos é possível sugerir que tanto a maior flexibilidade das estruturas como a presença do anel lactônico, são importantes para um comportamento análogo ao rofecoxibe ou à deoxipodofilotoxina. Com relação à interação com o bolso hidrofílico da COX-2, os resultados sugerem que a presença de grupos aceptores de prótons menos volumosos nas posições C3 e C4, C3’ e C4’ ou C4 levam a resultados melhores que grupos aceptores de maior volume. A presença de grupos doadores de prótons apesar de permitirem forte interação com o bolso hidrofílico da COX-2 leva a resultados globais insatisfatórios, pois formam interações fortes com o resíduo Arg120 do sítio ativo da COX-1, interação considerada importante para a inibição não seletiva. Resultado semelhante à deoxipodofilotoxina foi observado apenas para a estrutura 17. As estruturas 37, 188, 266, 267, 348 e a hinoquinina (4) apresentam resultados semelhantes ao rofecoxibe, para as duas isoformas. Deste modo permite-se sugerir a partir dos resultados obtidos neste estudo que a hinoquinina (4) e as estruturas 17, 37, 188, 266, 267 e 348 apresentam-se como possíveis protótipos de fármacos que atuem como inibidores seletivos para a COX-2. / The selective inhibitors of the cyclooxygenase-2 (COX-2) as rofecoxib (2) and celecoxib (1), form an important class of anti-inflammatory drugs developed from the discovery of two isoforms of cyclooxygenases (COX-1 and COX-2) in the late 1979. Isoform 1 is related to the gastric cytoprotection, platelet and renal function and isoform 2 related to inflammatory processes. These selective inhibitors although they did not side effects (ulcers and gastritis) of the classic NSAIDs to inhibit only COX-2, have severe cardiovascular risk, which led to the withdrawal of rofecoxib from the market. However, to be an effective selective COX-2 to rofecoxib structure has a reference in the study of new substances capable of selectively inhibiting COX-2. Among the tools used in the search of these new structures is by molecular modeling program such as GOLD 5.1, which was used in this work. Using GOLD 5.1 made it possible to study the behavior of structures evaluated in binding with the cyclooxygenases. With the objective of obtaining structures with similar behavior to rofecoxib (regarding behavior with COX) as potential candidates for the development of new selective inhibitors for COX-2. The study was conducted with 480 structures modeled from natural lignans as hinokinin, cubebin, deoxypodophyllotoxin and podophyllotoxin, which have anti-inflammatory activity in vivo or in vitro as well as structural similarities with rofecoxib. The deoxypodophyllotoxin for presenting selectivity for COX-2 in the in vitro assay was also used as a reference structure beyond rofecoxib. The results observed from the molecular simulation showed that although both rofecoxib (2) as deoxypodophyllotoxin (3) selectively inhibit COX-2 in vitro, they do differently. In relation to COX-2 the two structures occupy the same region of the active site, but rofecoxib has stronger interactions with the hydrophilic pocket of this isoform (a necessary condition for the selective inhibition for coxibs). As for the COX-1 while rofecoxib occupies the upper portion of the hydrophobic channel (active site) like other NSAIDs, the deoxypodophyllotoxin occupies a neighboring region. From the results it is possible to suggest that the greater flexibility of the structures such as the presence of the lactone ring, are important for a similar behavior to rofecoxib or deoxipodofilotoxina. With respect to the interaction with the hydrophilic pocket COX-2, the results suggest that the presence of acceptors groups less bulky protons in posítions C3 and C4, C3 ' and C4' and C4 lead to better results than acceptors groups of larger volume. The presence of proton donors groups despite allowing strong interaction with the hydrophilic pocket COX-2 lead to poor overall results, since they form strong interactions with Arg120 residue of COX-1 active site, considered important interaction for inhibiting non-selective. Results similar to deoxipodofilotoxina was only observed for structure 17. Structures 37, 188, 266, 267, 348 and hinokinin (4) show results similar to rofecoxib for the two isoforSA. Thus it allows suggest from the results obtained in this study hinokinin (4) and structures 17, 37, 188, 266, 267 and 348 are shown as possible prototype drugs that act as selective inhibitors for COX-2.

Orientador: Rosangela da Silva de Laurentiz / Resumo: Os inibidores seletivos da ciclooxigenase-2 (COX-2), como o rofecoxibe (2) e o celecoxibe (1), formam uma importante classe de medicamentos anti-inflamatórios desenvolvidos a partir da descoberta das duas isoformas das ciclooxigenases (COX-1 e COX-2) na década de 1979. A isoforma 1 esta relacionada com a citoproteção gástrica, agregação plaquetária e função renal e a isoforma 2 relacionada a processos inflamatórios. Estes inibidores seletivos apesar de não apresentarem os efeitos colaterais (ulceras e gastrites) dos anti-inflamatórios não esteroidais (AINEs) clássicos por inibirem apenas a COX-2, apresentam grave risco cardiovascular, o que motivou à retirada do rofecoxibe do mercado. Porém, por ser um eficiente inibidor seletivo da COX-2 a estrutura do rofecoxibe tornou-se referência no estudo de novas substâncias capazes de inibir seletivamente a COX-2. Dentre as ferramentas utilizadas na busca destas novas estruturas está a modelagem molecular através de programas como o GOLD 5.1, que foi utilizado neste trabalho. O uso do GOLD 5.1 possibilitou o estudo do comportamento das estruturas avaliadas em ligação com as ciclooxigenases. O objetivo foi obtenção de estruturas com comportamento semelhante ao rofecoxibe (em relação às COXs) como potenciais candidatos ao desenvolvimento de novos inibidores seletivos para a COX-2. O estudo foi realizado com 480 estruturas modeladas a partir de lignanas naturais como a hinoquinina, cubebina, deoxipodofilotoxina e podofilotoxina, que apre... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The selective inhibitors of the cyclooxygenase-2 (COX-2) as rofecoxib (2) and celecoxib (1), form an important class of anti-inflammatory drugs developed from the discovery of two isoforms of cyclooxygenases (COX-1 and COX-2) in the late 1979. Isoform 1 is related to the gastric cytoprotection, platelet and renal function and isoform 2 related to inflammatory processes. These selective inhibitors although they did not side effects (ulcers and gastritis) of the classic NSAIDs to inhibit only COX-2, have severe cardiovascular risk, which led to the withdrawal of rofecoxib from the market. However, to be an effective selective COX-2 to rofecoxib structure has a reference in the study of new substances capable of selectively inhibiting COX-2. Among the tools used in the search of these new structures is by molecular modeling program such as GOLD 5.1, which was used in this work. Using GOLD 5.1 made it possible to study the behavior of structures evaluated in binding with the cyclooxygenases. With the objective of obtaining structures with similar behavior to rofecoxib (regarding behavior with COX) as potential candidates for the development of new selective inhibitors for COX-2. The study was conducted with 480 structures modeled from natural lignans as hinokinin, cubebin, deoxypodophyllotoxin and podophyllotoxin, which have anti-inflammatory activity in vivo or in vitro as well as structural similarities with rofecoxib. The deoxypodophyllotoxin for presenting selectivity for COX... (Complete abstract click electronic access below) / Doutor