Nitrogen-containing bisphophonates (N-BPs) are a blockbuster class of drugs for the treatment of common metabolic bone diseases.  Recently, N-BPs have been shown to inhibit FPP synthase and/or isopentenyl diphosphate (IPP) isomerase (both enzymes in the mevalonate pathway), thereby preventing the synthesis of the isoprenoid lipids farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP), which are vital substrates for protein prenylation.  By preventing the synthesis of FPP and GGPP, N-BPs prevent the prenylation of small GTPases and inhibit osteoclast function. This study demonstrates conclusively that the major pharmacological target of N-BPs is FPP synthase.  Furthermore, minor structural modifications to the N-BPs that govern <i>n vivo </i>potency have a marked effect on potency for inhibition of FPP synthase <i>in vitro.  </i>Non-N-BPs, such as clodronate and etidronate, did not inhibit FPP synthase, consistent with other studies suggesting that the non-N-BPs and N-BPs act by different molecular mechanisms. Clinically, N-BPs have been shown to exhibit marked differences in efficacy between patients and may lie resident in the skeleton for many years.  J774 macrophage-like cells resistant to the effects of N-BP (J774-RES) were generated to study the possible cellular mechanisms underlying clinical resistance to treatment.  The J774-RES cells accumulated N-BP to a lesser extent than parental cells and also exhibited increased expression of the MCSF receptor, although further studies are required to clarify the exact mechanism of the resistance of J774-RES cells. Finally, N-BPs have been shown to induce the proliferation of the major subset (V<span style='font-family:Symbol'>g9V<span style='font-family:Symbol'>d2) of <span style='font-family:Symbol'>g,<span style='font-family:Symbol'>d-T cells in humans, attributed to an agnostic effect on the <span style='font-family:Symbol'>g,<span style='font-family:Symbol'>d-T cell receptor (TCR).  The findings of this study indicate that the N-BPs act indirectly, by inhibiting FPP synthase and causing the accumulation of IPP, a known agonist of the <span style='font-family:Symbol'>g,<span style='font-family:Symbol'>d-TCR.  Furthermore, this proliferative effect of N-BPs could be abrogated by statins, possibly indicating a means of preventing the acute-phase response, the major side effect to intravenously-administrated N-BPs.

Products of the isoprenoid biosynthetic pathway are involved in diverse biological functions. For example, the isoprenoid diphosphate, farnesyl diphosphate (FPP), is used for synthesis of squalene, a precursor of cholesterol. In addition, FPP and geranylgeranyl diphosphate (GGPP) are used for protein prenylation, which is a post-translational modification of certain proteins required for their proper membrane localization and function. Enzymes within the isoprenoid biosynthetic pathway have been inhibited successfully by drugs that are now used clinically, including statins and nitrogenous bisphosphonates (NBPs). Statins and NBPs are inhibitors of isoprenoid biosynthetic enzymes, due to their structural resemblance to substrates within the pathway. The bisphosphonate core resembles the diphosphate portion of isoprenoid diphosphate intermediates within the isoprenoid biosynthetic pathway. It is hypothesized that distinct isoprenoid biosynthetic enzymes can be inhibited by bisphosphonates in a manner dependent upon the overall bisphosphonate structure. Along with our collaborators, we have developed novel bisphosphonate inhibitors of multiple isoprenoid biosynthetic enzymes. Potent in vitro inhibitors of squalene synthase (SQS) were identified and evaluated in HepG2 liver cells. A lead inhibitor of squalene synthase was combined with a statin and a nitrogenous bisphosphonate, and focus was placed on these combinations as potential novel mechanisms to reduce cholesterol synthesis while minimizing impairment of non-sterol synthesis. Specifically, it was found that the lead SQS inhibitor prevents lovastatin-mediated impairment of protein farnesylation but not geranylgeranylation. Also, the lead SQS inhibitor prevented both zoledronate-induced impairment of protein farnesylation and geranylgeranylation. Novel bisphosphonates were also identified as inhibitors of geranylgeranyl diphosphate synthase (GGDPS) and protein prenylation in K562 leukemia cells. A novel cellular consequence of GGPP depletion was also established. In PC3 cells, zoledronate and digeranyl bisphosphonate (DGBP; a lead inhibitor of GGDPS) were determined to induce autophagy as measured by accumulation of the autophagic marker LC3-II. GGPP depletion was implicated as the cause of autophagic induction in this system. Specifically, results suggest that impairment of proteins geranylgeranylated by geranylgeranyl transferase II is responsible for the induction of autophagy. Mycobacterium isoprenoid biosynthetic enzymes were also evaluated as inhibitory targets for bisphosphonates. Novel inhibitors of Mycobacteria tuberculosis omega-E,Z-FPP synthase and decaprenyl diphosphate synthase were identified. A lead inhibitor of decaprenyl diphosphate synthase was also evaluated in Mycobacterium smegmatis, which was utilized as a surrogate model. The lead inhibitor was found to have no effect on M. smegmatis growth; however it enhanced growth inhibition mediated by ethambutol. This effect was prevented by addition of exogenous decaprenyl diphosphate, suggesting that the growth inhibition was due to decaprenyl diphosphate depletion. Decaprenyl diphosphate was also found to prevent the growth inhibitory effect of SQ109, a novel anti-mycobacterial drug in clinical development with an unknown mechanism of action.

Background: Bisphosphonate related osteonecrosis of the jaws (BRONJ) is a severe but extremely rare complication of prolonged treatment with bisphosphonates. Improper treatment or misdiagnosis can have serious repercussions. Objective: is to measure the awareness of Ontario dentists about BRONJ and to identify any gaps in their knowledge of the condition and its treatment. Material and Methods: A survey was sent to a random sample of dentists in Ontario, Canada. Information about their awareness of bisphosphonates, and their awareness of an established BRONJ guideline was collected. Results: 60% of responding Ontario dentists had good knowledge of BRONJ, only 23% followed the guideline when surgical treatment was indicated. However, about 50% of responding Ontario dentists are not comfortable treating BRONJ patients. Conclusion: The finding reveals that Ontario dentists have moderate knowledge about BRONJ, which suggest greater educational efforts should be made to promote their knowledge.

Background: Bisphosphonate related osteonecrosis of the jaws (BRONJ) is a severe but extremely rare complication of prolonged treatment with bisphosphonates. Improper treatment or misdiagnosis can have serious repercussions. Objective: is to measure the awareness of Ontario dentists about BRONJ and to identify any gaps in their knowledge of the condition and its treatment. Material and Methods: A survey was sent to a random sample of dentists in Ontario, Canada. Information about their awareness of bisphosphonates, and their awareness of an established BRONJ guideline was collected. Results: 60% of responding Ontario dentists had good knowledge of BRONJ, only 23% followed the guideline when surgical treatment was indicated. However, about 50% of responding Ontario dentists are not comfortable treating BRONJ patients. Conclusion: The finding reveals that Ontario dentists have moderate knowledge about BRONJ, which suggest greater educational efforts should be made to promote their knowledge.

La synthèse de nanoparticules (NPs) d’or fonctionnalisées en phase aqueuse est encore aujourd’hui un enjeu majeur de la recherche dans le domaine des nanomatériaux. Depuis les travaux de J. Turkevich de 1951, la synthèse utilisant le citrate comme ligand et agent réducteur est la méthode de choix pour obtenir des NPs d'or. Cependant cette synthèse nécessite une étape supplémentaire de modification de surface par échange de ligand, pour pouvoir accrocher des molécules d’intérêt. Afin de simplifier la procédure, notre projet propose de synthétiser en une seule étape des NPs qui possèdent un groupement permettant une post-fonctionnalisation. La nouvelle voie de synthèse fait intervenir des composés bifonctionnels de la famille des 1-hydroxy-1,1-méthylène bisphosphonates (HMBP). Ainsi la base conjuguée de l'acide (1-hydroxy-1-phosphonopent-4-ènyl) phosphonique (HMBPène), qui possède une fonction éthylénique terminale nous a permis d'obtenir des dispersions de nanosphères de tailles contrôlées et nous avons pu rationaliser le mécanisme de synthèse utilisant ce type de molécules. Nous avons ensuite évalué plusieurs modalités de post-fonctionnalisation de notre nanoplateforme et validé une approche par chimie « Click » la via cycloaddition de composés tétrazine. En utilisant une nouvelle classe de HMBP couplés à une chaine polyéthylène glycol, des NPs stables en milieu physiologique ont pu être synthétisées selon le même modèle. Elles offrent également des possibilités de post-fonctionnalisation par couplage carbodiimide, que nous avons illustré par le couplage d'un fluorophore. Nous développons en dernière partie les résultats préliminaires sur deux types NPs d'or synthétisées à l'aide des HMBP pour des applications thérapeutiques. / In the ever growing fields of nanoscience the control of the synthesis of gold nanoparticles (GNPs) owing to their large variety of applications has emerged as an important domain. Among all methodologies Turkevich-Frens synthesis using citrates that act as ligand and reducing agent remains a method of choice for the obtaining of water soluble GNPs. Nevertheless, in post-synthesis, citrates are often exchanged with other ligands to enhanced stabilization and allow further functionalisation. In our work we present a new class of bi-functional molecules (1-hydroxy-1,1-methylene bisphosphonates HMBP) that can both reduce Au(III) and act as an efficient stabilizer of the formed GNPs in water. The first size controlled GNPs “one pot” synthesis was achieved by using an alkene conjugated HMBP, the (1-hydroxy-1-phosphonopent-4-enyl)phosphonic acid (HMBPene). We moreover, rationalized the mechanism of the GNPs synthesis using this type of molecule. We then, evaluated several methodologies for the post-functionalization of our nanoplateform and developed a « Click » chemistry approach to nanoparticle coating by tetrazine cycloaddition. Other nanoplatforms were synthesized using pegylated hydroxyl methylene bisphosphonates. This new class of bisphosphonate coated GNPs showed an improved stability in biological media and brought reactive groups available for post-functionalization as well, illustrated by the coupling of a fluorescent dye. The last part of this was dedicated to our latest results on GNPs synthesis for biomedical applications with HMBP compounds.

Bisphosphonates (BPs) are stable analogues of pyrophosphate widely used for the treatment of bone diseases characterised by increased bone resorption. Studies over the years have shown that the pharmacological potencies of BPs are dependent both on their binding affinities for bone mineral and on their inhibitory actions on osteoclasts. In addition, potential effects on other cell types present locally in the environment of skeletal tissues have been reported. The present study systematically evaluated the relative mineral-binding affinities of individual BPs of clinically relevance in mixtures of these compounds and the changes with elution pH by using column chromatography with ceramic hydroxyapatite and fluoroapatite combined with mass spectrometric identification and quantitation of the individual BPs. The results indicate that pH has a profound effect on the ionisation of the phosphonate and R2 functional groups, with BPs having greater affinities at lower pH as shown by increased retention times. Moreover, two other approaches, namely using Langmuir adsorption isotherms and competition assays based on fluorescent BP, have been developed to assess the mineral-binding capacities and dissociation constants of BPs. These results suggest that there are substantial differences among BPs in their binding to hydroxyapatite. From the cellular aspect of my study, I present evidence for the anti-apoptotic effects of BPs in osteocytes and osteoblasts. However, the study of prosurvival signalling pathways involved in these cells needs to be optimised. The work described in this thesis provides novel insights into the physiological and biological mechanisms of BP action. My project has provided a better knowledge of the physicochemical properties of BPs, which are highly relevant to their differential distributions within bone, their biological potencies, and their durations of action. Additionally, the cell culture studies may provide new information on the cellular effects of BPs on osteocytes and osteoblasts.

Fragility fractures caused by loss of bone mass due to postmenopausal osteoporosis represent a growing morbidity worldwide. Bisphosphonates are first-line medications for fracture treatment and prevention. In the first phase, we updated a Cochrane systematic review of randomized controlled trials on alendronate, assessing its efficacy for five types of fracture prevention, quality of life, and various safety outcomes. In the second phase, we combined indirect and direct evidence to perform a network meta-analysis including alendronate and nine other bisphosphonates evaluating the comparative efficacy and safety of these treatments. Overall, 58 studies were included in the review and 83 studies in the network. Most evidence was of moderate to high quality. Alendronate and zoledronic acid were effective for preventing the most types of fractures, while off-label and unapproved bisphosphonates showed poor efficacy. More evidence is required to evaluate long-term treatment and rare adverse events.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / The bisphosphonate tiludronic acid (TIL) presents anti-resorptive and anti-inflammatory properties and it has not been evaluated in the association periodontitis-diabetes mellitus (DM) to date. The purpose of this study was to evaluate the effects of local administration of TIL on experimental periodontitis (EP) in rats with streptozotocin (STZ)-induced DM. On day 1, thirty two rats received STZ injection. The animals were divided into groups (n=8): DM/C (Control), DM/EP, DM/EP/TIL1 and DM/EP/TIL3. In groups EP, a ligature was placed around the cervical area of mandibular first molars at day 8. In groups DM/EP/TIL1 and DM/EP/TIL3, TIL solutions of 1 and 3 mg/kg body weight, respectively, were injected into the buccal gingival margin of mandibular first molars every other day. Animals were euthanized at day 18. Histomorphometric analyses were performed. Data were statistically analyzed (p<0.05). Group DM/EP/TIL3 presented reduced alveolar bone loss and attachment loss when compared with group DM/EP (p<0.05). Within the limits of this study, it can be concluded that i) the local administration of TIL solutions presented a protective effect on tissue destruction in EP in diabetic rats and ii) the dosage of TIL may influence its effects. / O bisfosfonato Ãcido tiludrÃnico (TIL) apresenta propriedades antirreabsortivas e anti-inflamatÃrias e ainda nÃo foi estudado na associaÃÃo periodontite-diabetes mellitus (DM). O objetivo deste estudo foi avaliar os efeitos da administraÃÃo local do TIL na periodontite experimental (PE) em ratos com DM induzido por streptozotocina (STZ). No 1 dia, trinta e dois ratos receberam injeÃÃo de STZ. Os animais foram divididos nos grupos (n = 8): DM/C (Controle), DM/PE, DM/PE/TIL1 e DM/PE/TIL3. Nos grupos PE, uma ligadura foi colocada na Ãrea cervical dos primeiros molares inferiores no 8 dia. Nos grupos DM/PE/TIL1 e DM/PE/TIL3, soluÃÃes de TIL (1 e 3 mg/kg de peso corporal, respectivamente) foram injetadas na margem gengival vestibular dos primeiros molares inferiores em dias alternados. Os animais foram submetidos à eutanÃsia no 18 dia. AnÃlises histomorfomÃtricas foram realizadas. Os dados foram estatisticamente analisados (p<0,05). O grupo DM/PE/TIL3 apresentou perda Ãssea alveolar e perda de inserÃÃo reduzidas quando comparado com o grupo DM/PE (p<0,05). Dentro dos limites deste estudo, pode-se concluir que i) a administraÃÃo local de soluÃÃes de TIL apresentou um efeito protetor na destruiÃÃo tecidual na PE em ratos diabÃticos e ii) a dosagem de TIL pode influenciar seus efeitos.

Οι διαφορετικές θεραπευτικές προσεγγίσεις σε ασθενείς με καρκίνο (χειρουργική αποκατάσταση, ακτινοθεραπεία κ.λπ) μπορεί να έχουν επίδραση στην ποιότητα ζωής τους. Σκοπός. Σκοπός αυτής της μελέτης ήταν η διερεύνηση και καταγραφή, μέσω ερωτηματολογίων, της ποιότητας ζωής σε ασθενείς με καρκίνο, που υποβλήθηκαν σε ακτινοθεραπεία, και πως επηρεάζεται αυτή από τη χορήγηση διφωσφονικών ή ερυθροποιητίνης Ασθενείς-Μέθοδος. Τον πληθυσμό της έρευνας αποτέλεσαν 22 ασθενείς που νοσηλεύτηκαν στη Μονάδα Ακτινοθεραπευτικής Ογκολογίας στο Πανεπιστημιακό Νοσοκομείο του Pioυ από τον Οκτώβρη του 2007 μέχρι τον Ιούλιο του 2008. Οι ασθενείς έπασχαν από κάποια μορφή καρκίνου με μεταστάσεις και υποβλήθηκαν σε συνδυασμένη θεραπεία που περιλάμβανε ακτινοθεραπεία και χορήγηση διφωσφονικών ή ερυθροποιητίνης, ανάλογα με τις κλινικές ενδείξεις. Η συλλογή των δεδομένων έγινε με τη συμπλήρωση ερωτηματολογίων δημογραφικών στοιχείων και κλινικών χαρακτηριστικών. Η αξιολόγηση της κλινικής ανταπόκρισης ελάμβανε χώρα με την καταγραφή της κλίμακας πόνου (0-10), της ποιότητας ζωής (ερωτηματολόγιο EORTC-QΟL C30- κλίμακα φυσικής λειτουργικότητας, 0-100), και της φυσικής κατάστασης (ECOG κλίμακα 0-5). Αποτελέσματα: H σύγκριση των δεδομένων έδειξε ότι δεν υπήρχε στατιστικά σημαντική διαφορά στην καταγραφή του αισθήματος του πόνου και των παραμέτρων της ποιότητας ζωής των ασθενών με τη χορήγηση διφωσφωνικών ή ερυθροποιητίνης κατά τη διάρκεια του διαστήματος που υποβάλλονταν σε ακτινοθεραπεία. Συμπεράσματα: Η χορήγηση διφωσφωνικών ή ερυθροποιητίνης φαίνεται να μην μπορούν να επιφέρουν αλλαγές σε συγκεκριμένες διαστάσεις της ζωής και να έχουν επίδραση στην αναφερόμενη ποιότητα ζωής των ασθενών με καρκίνο. Η βελτίωση της ποιότητας ζωής αυτών των ασθενών αποτελεί μεγάλη πρόκληση για τους επαγγελματίες υγείας. / The different therapeutic approaches in patients with cancer (surgery, radiation, etc.) can affect their quality of life. Purpose: The purpose of this study was to evaluate and record, through questionnaires, the quality of life of patients with cancer undergoing radiation therapy, and how this is affected by treatment with bisphosphonates or erythropoietin Patients-Method. In this study, 22 patients were participated, admitted to the Radiation Oncology Unit at University Hospital of Rio from October 2007 until July 2008. Patients suffering from some form of cancer and metastases and underwent into combined treatment included radiation therapy and bisphosphonates or erythropoietin. All patients filled questionnaires about their demographic and clinical characteristics. The evaluation of clinical response took place by recording patients’ answers about pain scale (0-10), quality of life (QOL questionnaire EORTC-C30-scale physical functioning, 0-100), and physical condition (ECOG scale 0-5). Results: Elaboration of collected data showed no statistically significant difference in the recording feeling of pain and parameters of quality of life in patients treated with bisphosphonates or erythropoietin during the period of radiotherapy. Conclusions: Treatment with bisphosphonates or erythropoietin seems not to be able to make changes in specific parameters of life and to affect the reported quality of life of patients with cancer. Improving quality of life of these patients is a major challenge for health professionals.

Orientador: Horácio Faig Leite / Banca: Paulo Henrique Ferreira Caria / Banca: Jesus Carlos Andreo / Banca: José de Anchieta de Castro e Horta Junior / Banca: Yasmin Rodarte Carvalho / Resumo: Esta pesquisa avaliou o efeito do uso local do alendronato sódico, da hidroxiapatita e da associação alendronato mais hidroxiapatita em diferentes concentrações molares, no processo de reparação de defeitos ósseos em fêmures de ratos. Foi confeccionado no fêmur de 168 ratos (84 machos e 84 fêmeas) um defeito ósseo medindo 2,5mm de diâmetro. Estes animais foram divididos em grupos: controle, amido, alendronato um mol, alendronato dois moles, hidroxiapatita um mol, hidroxiapatita dois moles e alendronato mais hidroxiapatita, de acordo com o material de preenchimento utilizado. Nos animais do grupo controle o defeito ficou preenchido apenas pelo coágulo proveniente do defeito. Os animais foram sacrificados aos sete e 21 dias, quando o fêmur era removido, fixado e descalcificado, para a confecção de lâminas histológicas. Foi realizada análise histológica e histomorfométrica, e os dados obtidos foram submetidos à análise estatística ANOVA. Aos sete dias, observava-se trabéculas ósseas imaturas, contendo grandes osteócitos. Notava-se neoformação óssea em todos os grupos, exceto nos animais machos onde o alendronato se fazia presente. Nos grupos que receberam a hidroxiapatita, visualizava-se as imagens negativas dos grânulos da hidroxiapatita. Aos 21 dias, as trabéculas praticamente fechavam o defeito da maioria dos espécimes estudados. Estatisticamente, houve diferenças entre machos e fêmeas, entre os períodos de observação e com relação ao uso do alendronato. Concluiu-se que a aplicação local do alendronato sódico interferiu negativamente na reparação óssea, que a hidroxiapatita e o alendronato mais a hidroxiapatita não interferiram na reparação e que a reparação óssea foi maior nas fêmeas independentemente do período estudado. / Abstract: This research evaluated the effect of the local use of sodium alendronate, of hydroxyapatite and the association alendronate more hydroxyapatite in different molars concentrations, in the repair of bone defects in femurs of mices. We made in the femur of 168 mices (84 males and 84 females) a bone defect measuring 2,5mm of diameter. We divided these animals in groups: control, starch, alendronate one mol, alendronate two mols, hydroxyapatite one mol, hydroxyapatite two mols and alendronate more hydroxyapatite, in accordance with the material of fulfilment used. In the animals of the control group, the defect was just filled by the clot originating from defect. The animals were sacrificed at seven and 21 days, when the femur was removed, fixed and decalcified, to making histologics laminae. Histological and histomorphometric analyses were performed and the results obtained were submitted to statistical analysis ANOVA. At seven days, it was observed immature bone trabeculae with larges osteocyties. It was noticed bone formation in all groups, exceptin the male animals where the alendronate was present. In the group that received hydroxyapatite, it was visualized negatives images of the hydroxyapatite's granules. At 21 days, the trabeculae practically closed the defect of most studied specimens. Statistically, there were differences between males and females, between the observation periods and with relationship of the alendronate use. It was concluded that the local application of sodium alendronate interfered negatively in bone repair, that the hydroxyapatite and alendronate more hydroxyapatite didn't interfere in the repair and that the bone repair was larger in the females independently of studied period. / Doutor