in Hradec Králové Student: Johana Urbanová Supervisor: PharmDr. Jana Mandíková, Ph.D. = 38.02 μM), lowest indometacin μM

Non-steroidal anti-inflammatory drugs (NSAID) are a class of medications used in the treatment of pain, inflammation, and illness. These medications are common, affordable, and easy to access. For these reasons, NSAIDs are commonly used by athletes of all backgrounds for treating injuries and as ergogenic aids. However, despite these behaviors, NSAIDs have well-documented side effects and the efficacious nature of these medications has been brought into question. Despite this, many athletes continue to use these medications frequently and indiscriminately. It is not known why athletes use these medications in light of their questionable effectiveness and cited adverse effects. Therefore, this study was designed to (1) further investigate the prevalence of NSAID use in collegiate-level athletes, (2) investigate attitudes and behaviors toward the use of NSAIDs cross-tabulated by sport, gender, and competition level, and (3) investigate athletes' general knowledge of NSAIDs. Subjects for this study included 79 student-athletes (44 male; 25 female) attending Portland State University (PSU). The majority of the athletes started taking NSAIDs before high school (72% of the males and 64% of the females). Thirty-three percent of males and 32% of females reported that they had been taking NSAIDs within the past week. High in-season use of NSAIDs was reported by 52% of the male athletes and 48% of the female athletes, whereas off-season use was reported by 21% and 12% of the males and females, respectively. Cited reasons for NSAID use both in-season and off-season were relief of pain due to injury, prevention, recovery, soreness, and tightness. In total, 83% of males and 76% of females reported obtaining NSAIDs primarily through means other than health-care professionals. With regard to dosage, athletes reported taking NSAIDs based on product directions, instructions of an athletic trainer or perceived pain levels. An overwhelming majority of athletes (83% male; 76% female) were not aware of any side-effects from taking NSAIDs In summary, this study revealed a pattern of high NSAID use in athletes competing in-season compared to a high prevalence of low NSAID use in athletes off-season. It also revealed a high prevalence of non-prescription NSAID use. Additionally, there was a high prevalence of self-purchasing of NSAIDs, combined with self-medication and a long history of NSAID use. This study also revealed a general lack of knowledge about NSAIDs.

Inhibition of cyclooxygenase (COX)-2 increases cardiovascular deaths. Identifying a biomarker of COX-2 is desirable but difficult, since COX-1 and COX-2 ordinarily catalyze formation of an identical product, prostaglandin H2. When acetylated by aspirin, however, COX-2 (but not COX-1) can form 15(R)-HETE, which is metabolized to aspirin-triggered lipoxin (ATL), 15-epi-lipoxin A4. Here we have used COX-1- and COX-2-knockout mice to establish whether plasma ATL could be used as a biomarker of vascular COX-2 in vivo. Vascular COX-2 was low but increased by LPS (10 mg/kg; i.p). Aspirin (10 mg/kg; i.v.) inhibited COX-1, measured as blood thromboxane and COX-2, measured as lung PGE2. Aspirin also increased the levels of ATL in the lungs of LPS-treated wild-type C57Bl6 mice (vehicle: 25.5±9.3 ng/ml; 100 mg/kg: 112.0±7.4 ng/ml; P<0.05). Despite this, ATL was unchanged in plasma after LPS and aspirin. This was true in wild-type as well as COX-1−/− and COX-2−/− mice. Thus, in mice in which COX-2 has been induced by LPS treatment, aspirin triggers detectable 15-epi-lipoxin A4 in lung tissue, but not in plasma. This important study is the first to demonstrate that while ATL can be measured in tissue, plasma ATL is not a biomarker of vascular COX-2 expression.—Kirkby, N. S., Chan, M. V., Lundberg, M. H., Massey, K. A., Edmands, W. M. B., MacKenzie, L. S., Holmes, E., Nicolaou, A., Warner, T. D., Mitchell, J. A. Aspirin-triggered 15-epi-lipoxin A4 predicts cyclooxygenase-2 in the lungs of LPS-treated mice but not in the circulation: implications for a clinical test.

Dissertation submitted in partial compliance with the requirements for a Masters Degree in Technology: Chiropractic, Durban University of Technology, 2009. / Headaches are one of the most common clinical conditions in medicine, and 80% of these are tension-type headaches (TTH). TTH has a greater socioeconomic impact than any other type of headache due to its prevalence. Within the TTH category, episodic TTH are more prevalent than chronic TTH. The mainstay in the treatment of TTH are simple analgesics and NSAIDs. Unless contraindicated, NSAIDs are often the most effective treatment for ETTH. However patients suffering with TTH tend to relate their headaches to increased muscle stiffness in the neck and shoulders and thus the non-pharmacological treatment of ETTH could be directed at the associated musculoskeletal components of ETTH. It is therefore proposed that the Kinesio Taping® Method may have an effect in the treatment of the muscular component of ETTH. Method: This study was a prospective randomised clinical trial with two intervention groups (n=16) aimed at determining the relative effectiveness of a NSAID and the Kinesio Taping® Method in the treatment of ETTHs. The patients were treated at 5 consultations over a 3 week period. Feedback was obtained using the: NRS – 101, the CMCC Neck Disability Index and a Headache Diary. Results: The Headache Diary showed a reduction in the presence and number, mean duration and pain intensity of ETTH in both groups. These treatment effects were sustained after the cessation of treatment with the exception of mean pain intensity in the Kinesio Taping® Method group. The mean NRS score decreased in both groups but at a slightly faster rate in the Kinesio Taping® Method group. The CMCC showed an improvement in the functional ability of the patients in both groups. Conclusion: There seems to be no significant difference in the relative effectiveness of the treatment modalities. We can thus state that the overall short-term reduction in symptomatology supports the use of NSAIDs or Kinesio Taping® Method in the treatment of ETTH.

Dissertation submitted in partial compliance with the requirements for the Masters Degree in Technology: Chiropractic, Durban University of Technology, 2010. / The 1 year overall prevalence of Episodic Tension-Type Headache (ETTH) is 38.3%; with lifetime prevalence at 46% for TTH. Little literature exists to support the effectiveness of spinal manipulation in the treatment of ETTH. Therefore aim of this study was to determine the relative effectiveness of cervical spine manipulation and a Nonsteroidal Anti-inflammatory drug (NSAID) (Ibuprofen®) in the treatment of ETTH. Method: This study was a prospective randomised clinical trial with two intervention groups (N=32, n1=16 and n2=16). The allocation of participants to the two groups was completed by means of simple randomization. Group one were treated using cervical spine manipulation. Group two were treated using Ibuprofen. Subjective measurements included the Numerical Rating Scale 101 Questionnaire (NRS-101), Short Form McGill Pain Questionnaire (SF-MPQ), CMCC Neck Disability Index (CMCC) and Headache Diary. A p value <0.05 was considered as statistically significant. Results: The subjective measurements of the NRS-101, SF-MPQ and CMCC showed a significant time effect in both treatment groups. Several of the subjective Headaches Diary outcomes followed this trend with significant time effect in both groups. There was a significant treatment effect for the NRS-101. Several subject outcomes from the Headache Diary showed a significant treatment effect in favour of manipulation, namely frequency and duration of headaches. Conclusion: The findings in this study have shown that cervical spine manipulation is more effective than Ibuprofen® for the treatment of ETTH in terms of several subjective outcomes namely: pain intensity (NRS-101), and the frequency and the duration of headache per day.

Prostanoids are oxygenated derivatives of arachidonic acid with a wide range of physiological effects in vertebrates including modulation of inflammation and innate immune responses. Nonsteroidal anti-inflammatory drugs (NSAIDs) act through inhibition of cyclooxygenase (COX) conversion of arachidonic acid to prostanoids. In order to better understand the potential of environmental NSAIDS for interruption of normal levels COX products in fishes, we developed an LC/MS/MS-based approach for tissue analysis of 7 prostanoids. Initial studies examining muscle, gut and gill demonstrated that prostaglandin E2 (PGE2) was the most abundant of the measured prostanoids in all tissues and that gill tissue had the highest and most consistent concentrations of PGE2. After short-term 48-h laboratory exposures to concentrations of 5, 25, 50 and 100 ppb ibuprofen, 50.0ppb and 100.0 ppb exposure concentrations resulted in significant reduction of gill tissue PGE2 concentration by approximately 30% and 80% respectively. The lower exposures did not result in significant reductions when compared to unexposed controls. Measured tissue concentrations of ibuprofen indicated that this NSAID had little potential for bioaccumulation (BCF 1.3) and the IC50 of ibuprofen for inhibition of PGE2 production in gill tissue was calculated to be 0.4 µM. Short-term laboratory exposure to ibuprofen did not result in significant alteration of concentrations of PGE2 at environmentally relevant concentrations.

Metalofármacos baseados em rutênio têm se mostrado promissores com relação à atividade anticancerígena frente a diversos tipos de tumores. Nosso grupo de pesquisa dedica-se ao estudo de compostos contendo o centro dimetálico de valência mista Ru2(II,III) coordenado a ligantes derivados de Faines (Fármacos anti-inflamatórios não esteroides), tendo demostrando o potencial desses complexos frente a glioma. O entendimento do modo de ação destes complexos requer o estudo de suas interações com biomoléculas presentes no meio biológico. Neste cenário, o presente trabalho teve por objetivo investigar a interação de três complexos de dirutênio-Faines, ou RuFaines, [Ru2(ibp)4Cl], [Ru2(ceto)4Cl] e [Ru2(npx)4(H2O)2]PF6 (ibp = ibuprofenato, ceto = cetoprofenato e npx = naproxenato), e também do precursor [Ru2(O2CCH3)4Cl], RuAc, com as principais proteínas presentes no soro humano, transferrina e albumina. Os complexos foram sintetizados e caracterizados conforme metodologias desenvolvidas no grupo. A interação destes complexos com a transferrina, em suas formas apo e holo, e com a albumina foi avaliada por técnicas como espectroscopia eletrônica, dicroísmo circular, fluorescência, e realizaram-se estudos de ultrafiltração com análise do aduto formado por ICP-OES e espectrometria de massas. Além disso, fez-se um estudo de captação celular dos complexos RuFaines por células de glioma humano da linhagem U-87. Os resultados demonstraram que os complexos de dirutênio-Faines interagem com ambas as proteínas séricas (transferrina (apo e holo) e albumina), de modo semelhante, mas que é distinto daquele observado para o complexo RuAc. A presença de íons Fe(III) nos sítios específicos da transferrina não afetou a interação dos complexos RuFaines, enquanto que um comportamento diferente foi observado para o RuAc. Verificou-se que todas as proteínas avaliadas (albumina, apo-transferrina e holo-transferrina) apresentam capacidades similares de retenção dos complexos (~ 70% da quantidade de Ru adicionada inicialmente), independentemente da natureza do ligante carboxilato coordenado. Estudos de captação celular mostraram que a interação dos complexos RuFaines com a transferrina não contribuiu para modificar a capacidade de entrada desses complexos na célula, em comparação com os metalofármacos livres. Em alguns casos, inclusive, a formação de aduto com a apo-transferrina teve um efeito contrário, diminuindo a captação de rutênio. Dessa forma, concluiu-se que o ciclo da transferrina provavelmente não é a principal rota de entrada nas células para os complexos estudados. / Ruthenium metallodrugs have shown promising antitumor activity against to several tumor types. Our research group is dedicated to study compounds containing the mixed-valence Ru2(II,III) dimetallic center coordinated to NSAIDs (nonsteroidal anti-inflammatory drugs) derived ligands, and have demonstrated the potential of these complexes against glioma. The understanding of the mode of action of these complexes requires the study of their interactions with biomolecules present in biological environment. In this scenario, the present work aimed to investigate the interaction of three complexes of diruthenium-NSAIDs, or RuNSAIDs, [Ru2(ibp)4Cl], [Ru2(ceto)4Cl] and [Ru2(npx)4(H2O)2]PF6 (ibp = ibuprofenate, ceto = ketoprofenate, npx = naproxenate), and also of the precursor [Ru2(O2CCH3)4Cl], RuAc, with the major proteins present in the human serum, transferrin and albumin. The complexes were synthesized and characterized according to methods developed in our group. The interaction of these complexes with transferrin, in the two forms apo and holo, and with albumin was evaluated by techniques as electronic spectroscopy, circular dichroism, fluorescence, and ultrafiltration studies accompanied by analysis of adducts by ICP-OES and mass spectrometry. Moreover, cellular uptake studies of the RuNSAIDs complexes by U-87 human glioma cells line were performed. The results demonstrated that the diruthenium-NSAIDs complexes interact with both proteins (transferrin (apo and holo) and albumin), in a similar way, but that is distinct of that observed for the RuAc complex. The presence of Fe(III) ions in transferrin specific binding sites did not affect the interaction of the RuNSAID complexes with the protein, while a different behavior was shown by RuAc. All the proteins studied here (albumin, apo-transferrin and holo-transferrin) showed similar capabilities for retention of the complexes (~ 70 % of the initial amount of Ru added), independently of the nature of the coordinated carboxylate ligand. Cellular uptake studies showed that the interaction of the RuNSAIDs complexes with transferrin did not contribute to modify the internalization capacity of these complexes, in comparison with the free metallodrugs. In some cases, the adduct formation with apo-transferrin showed an opposite effect, leading to the decrease of ruthenium uptake. The findings led to the conclusion that transferrin cycle probably is not the main entry way to the cells for the studied complexes.

Metalofármacos baseados em rutênio têm se mostrado promissores com relação à atividade anticancerígena frente a diversos tipos de tumores. Nosso grupo de pesquisa dedica-se ao estudo de compostos contendo o centro dimetálico de valência mista Ru2(II,III) coordenado a ligantes derivados de Faines (Fármacos anti-inflamatórios não esteroides), tendo demostrando o potencial desses complexos frente a glioma. O entendimento do modo de ação destes complexos requer o estudo de suas interações com biomoléculas presentes no meio biológico. Neste cenário, o presente trabalho teve por objetivo investigar a interação de três complexos de dirutênio-Faines, ou RuFaines, [Ru2(ibp)4Cl], [Ru2(ceto)4Cl] e [Ru2(npx)4(H2O)2]PF6 (ibp = ibuprofenato, ceto = cetoprofenato e npx = naproxenato), e também do precursor [Ru2(O2CCH3)4Cl], RuAc, com as principais proteínas presentes no soro humano, transferrina e albumina. Os complexos foram sintetizados e caracterizados conforme metodologias desenvolvidas no grupo. A interação destes complexos com a transferrina, em suas formas apo e holo, e com a albumina foi avaliada por técnicas como espectroscopia eletrônica, dicroísmo circular, fluorescência, e realizaram-se estudos de ultrafiltração com análise do aduto formado por ICP-OES e espectrometria de massas. Além disso, fez-se um estudo de captação celular dos complexos RuFaines por células de glioma humano da linhagem U-87. Os resultados demonstraram que os complexos de dirutênio-Faines interagem com ambas as proteínas séricas (transferrina (apo e holo) e albumina), de modo semelhante, mas que é distinto daquele observado para o complexo RuAc. A presença de íons Fe(III) nos sítios específicos da transferrina não afetou a interação dos complexos RuFaines, enquanto que um comportamento diferente foi observado para o RuAc. Verificou-se que todas as proteínas avaliadas (albumina, apo-transferrina e holo-transferrina) apresentam capacidades similares de retenção dos complexos (~ 70% da quantidade de Ru adicionada inicialmente), independentemente da natureza do ligante carboxilato coordenado. Estudos de captação celular mostraram que a interação dos complexos RuFaines com a transferrina não contribuiu para modificar a capacidade de entrada desses complexos na célula, em comparação com os metalofármacos livres. Em alguns casos, inclusive, a formação de aduto com a apo-transferrina teve um efeito contrário, diminuindo a captação de rutênio. Dessa forma, concluiu-se que o ciclo da transferrina provavelmente não é a principal rota de entrada nas células para os complexos estudados. / Ruthenium metallodrugs have shown promising antitumor activity against to several tumor types. Our research group is dedicated to study compounds containing the mixed-valence Ru2(II,III) dimetallic center coordinated to NSAIDs (nonsteroidal anti-inflammatory drugs) derived ligands, and have demonstrated the potential of these complexes against glioma. The understanding of the mode of action of these complexes requires the study of their interactions with biomolecules present in biological environment. In this scenario, the present work aimed to investigate the interaction of three complexes of diruthenium-NSAIDs, or RuNSAIDs, [Ru2(ibp)4Cl], [Ru2(ceto)4Cl] and [Ru2(npx)4(H2O)2]PF6 (ibp = ibuprofenate, ceto = ketoprofenate, npx = naproxenate), and also of the precursor [Ru2(O2CCH3)4Cl], RuAc, with the major proteins present in the human serum, transferrin and albumin. The complexes were synthesized and characterized according to methods developed in our group. The interaction of these complexes with transferrin, in the two forms apo and holo, and with albumin was evaluated by techniques as electronic spectroscopy, circular dichroism, fluorescence, and ultrafiltration studies accompanied by analysis of adducts by ICP-OES and mass spectrometry. Moreover, cellular uptake studies of the RuNSAIDs complexes by U-87 human glioma cells line were performed. The results demonstrated that the diruthenium-NSAIDs complexes interact with both proteins (transferrin (apo and holo) and albumin), in a similar way, but that is distinct of that observed for the RuAc complex. The presence of Fe(III) ions in transferrin specific binding sites did not affect the interaction of the RuNSAID complexes with the protein, while a different behavior was shown by RuAc. All the proteins studied here (albumin, apo-transferrin and holo-transferrin) showed similar capabilities for retention of the complexes (~ 70 % of the initial amount of Ru added), independently of the nature of the coordinated carboxylate ligand. Cellular uptake studies showed that the interaction of the RuNSAIDs complexes with transferrin did not contribute to modify the internalization capacity of these complexes, in comparison with the free metallodrugs. In some cases, the adduct formation with apo-transferrin showed an opposite effect, leading to the decrease of ruthenium uptake. The findings led to the conclusion that transferrin cycle probably is not the main entry way to the cells for the studied complexes.

[Truncated abstract] In recent years a better understanding of the biology of PPAR , a nuclear transcription factor, has emerged, leading to a resurgence in targeting PPAR for chemotherapy. The family of synthetic PPAR agonists, the thiazolidinediones (TZDs), and non-steroidal anti-inflammatory drugs (NSAIDs) have been implicated in the inhibition of cell proliferation, apoptosis and cell cycle arrest of androgen-sensitive (LNCaP) and androgen-independent (PC-3 and DU145) prostate cancer cells generating much interest in their use for potential curative cancer therapies. In light of the potential use of TZDs and NSAIDs in prostate cancer prevention and their ability to induce inhibitory effects in vitro and in vivo, the first aim of this project was to undertake a comprehensive study of the effects of ciglitazone (TZD) and indomethacin (NSAID) on the androgen-independent prostate cancer cell line DU145, using standardised concentrations and time-points to compare the effects of TZDs and NSAIDs on cell proliferation, cell cycle and apoptosis. Treating the cells with either 10 µM ciglitazone or 10 µM indomethacin resulted in a time-dependent decrease in DU145 cell proliferation. The anti-proliferative effects were found to be in-part attributed to the slowing of cell progression through the G1/S-phase checkpoint of the cell cycle, and in the case of ciglitazone, apoptosis also played a role in its anti-proliferative effects in this cell line. Interestingly, although indomethacin failed to induce apoptosis, its antiproliferative effects were more potent than ciglitazone. The second aim of this project was to further investigate the underlying mechanisms responsible for the anti-proliferative effects of ciglitazone and indomethacin by evaluating their ability to modulate PPAR mRNA and protein expression, and to induce PPAR transcriptional activity. ... In addition, ligandinduced regulation of secreted frizzled related protein 4 (sFRP4) expression, a Wnt/ - catenin antagonists, was investigated. It was demonstrated that both ciglitazone and indomethacin attenuated Wnt/ -catenin signalling via the down-regulation of total - catenin levels within the cells, inhibition or slowing of the translocation of cytoplasmic -catenin into the nucleus and inhibition of cyclin–D1 expression An inverse relationship between PPAR and -catenin protein levels was also detected, suggesting that PPAR may directly bind to -catenin itself. sFRP4 expression was transiently upregulated by ciglitazone and indomethacin-treatment, suggesting that the antiproliferative effects of the ligands may be mediated in part through regulation of sFRP4 mRNA and protein levels. In summary, the anti-proliferative effects of ciglitazone and indomethacin on the androgen-independent prostate cancer cell line, DU145, described in this thesis are progressive steps in characterising the role of PPAR in prostate cancer cell proliferation. The identification of indomethacin as a more potent PPAR agonist than ciglitazone represents a novel target for the development of preventative strategies for advanced disease, and the relationship between PPAR and the Wnt/ -catenin signalling pathway provide an insight into the mechanisms involved in the anti-proliferative effects of ciglitazone and indomethacin. Further studies into this relationship would advance help identify novel preventative and curative therapeutic strategies for advanced prostate cancer.

Tez (Tıpta Uzmanlık) - Süleyman Demirel Üniversitesi, Tıp Fakültesi, Biyokimya Anabilim Dalı, 2006. / Bibliyografya var.