Phosphorus is an essential element for life, observed in all biological systems usually as inorganic phosphate and various organic phosphate esters. Phosphonates are a metabolically stable analogues of natural phosphorus containing compounds and have been used in a variety of industrial and medicinal applications. Bisphosphonates have been found to be especially good inhibitors of enzymes in the isoprenoid biosynthetic pathway. A screen of bisphosphonates against the enzyme squalene synthase (SQS) resulted in the identification of a lead target. The lead compound was resynthesized via a new method along with two other analogues. It was determined that although the lead bisphosphonate had potent and selective activity against SQS and resulted in the reduction of cholesterol, use of this drug in combination with either a statin (lovastatin) or a nitrogenous bisphosphonate (zolendronate) had an even greater impact. Furthermore co-treatment of cells with the lead compound and either lovastatin or zoledronate significantly prevented a reduction of cell viability caused by lovastatin or zoledronate alone. The combination of an SQS inhibitor with either an HMGCR or FDPS inhibitor may be beneficial for reducing cholesterol synthesis while preventing non-sterol isoprenoid depletion. A series of stilbenoid bisphosphonates has been developed to afford potential inhibitors of enzymes in the isoprenoid biosynthetic pathway, a potential target for cancer therapies. Although the new compounds had limited activity against major enzymes in the isoprenoid biosynthetic pathway, it was determined that one of the targets had modest activity in a screen of inhibitors for decaprenyl diphosphate synthase, an enzyme vital to synthesis and maintenance of cell walls in mycobacteria. A second generation synthesis of stilbenoid bisphosphonates that contain a para-substituted electron withdrawing group was initiated, resulting in the identification of a more potent inhibitor of decaprenyl diphosphate synthase. The use of novel bisphosphonate inhibitors could have an impact on treatment of bacterial diseases such as tuberculosis. The development of novel phosphorus containing compounds could provide new inspiration for industrial and medicinal products. The á-trisphosphonic acid esters provide a unique spatial arrangement of three phosphonate groups, and may represent an attractive motif for inhibitors of enzymes that utilize di- or triphosphate substrates. A general route to alkyl derivatives of the parent system has been developed through phosphinylation and subsequent oxidation of tetraethyl alkylbisphosphonates, and the reactivity of these new compounds has been studied in representative reactions that afford additional examples of this functionality. During the course of synthesis of the parent trisphosphonate system, an unusual oxidized bisphosphonate phosphate was discovered, and the methods to synthesize this species have been investigated.

This thesis investigates bisphosphonate adsorption onto plasma sprayed HA (PS-HA) and simulated body fluid-grown HA (SBF-HA) coatings commonly used for orthopaedic implants. Bisphosphonates exhibit high binding affinity for the calcium present in HA and hence can be adsorbed onto HA-coated implants to exploit their beneficial properties for improved bone growth at the implant interface.

Bisphosphonate-induced osteonecrosis of the jaw (BPONJ) has been identified as a severe complication of dental treatment in 1-10% of patients previously treated with intravenous bisphosphonates. The mechanism by which bisphosphonates induce BPONJ is uncertain. It has been noted that necrotic bone from BPONJ sites display signs of bacterial infection that suggests that an immune defect may play a role in the pathophysiology of BPONJ. The purpose of this thesis examined the effect of a potent bisphosphonate, zoledronate, on the innate immune system, specifically, neutrophil function, differentiation and survival with in vitro and in vivo murine models. Zoledronate exposure leads to decreased neutrophil migration, neutrophil NADPH oxidase activity, circulating neutrophil counts, as well as neutrophil survival, however does not appear to affect neutrophil differentiation. We present evidence that bisphosphonates have the potential to depress the immune system in mice and a subset of patients, possibly contributing to the pathogenesis of BPONJ.

Bisphosphonate-induced osteonecrosis of the jaw (BPONJ) has been identified as a severe complication of dental treatment in 1-10% of patients previously treated with intravenous bisphosphonates. The mechanism by which bisphosphonates induce BPONJ is uncertain. It has been noted that necrotic bone from BPONJ sites display signs of bacterial infection that suggests that an immune defect may play a role in the pathophysiology of BPONJ. The purpose of this thesis examined the effect of a potent bisphosphonate, zoledronate, on the innate immune system, specifically, neutrophil function, differentiation and survival with in vitro and in vivo murine models. Zoledronate exposure leads to decreased neutrophil migration, neutrophil NADPH oxidase activity, circulating neutrophil counts, as well as neutrophil survival, however does not appear to affect neutrophil differentiation. We present evidence that bisphosphonates have the potential to depress the immune system in mice and a subset of patients, possibly contributing to the pathogenesis of BPONJ.

This thesis investigates bisphosphonate adsorption onto plasma sprayed HA (PS-HA) and simulated body fluid-grown HA (SBF-HA) coatings commonly used for orthopaedic implants. Bisphosphonates exhibit high binding affinity for the calcium present in HA and hence can be adsorbed onto HA-coated implants to exploit their beneficial properties for improved bone growth at the implant interface.

Abstract Bisphosphonates (BPs) are used in the treatment of osteoporosis. However, their effects, especially long-term effects, on bone and bone healing are not fully known. Clodronate (dichloromethylene bisphosphonic acid) is a first-generation BP. The thesis was based on two animal experiments. The first, with 199 rats on long-term clodronate treatment, was divided into four separate substudies. The effects of long-term administration of clodronate to rats were investigated after 32 weeks of treatment. The effects on the femoral shaft, femoral neck and vertebra in normal, non-osteoporotic skeleton were described in two publications. The evaluations were made by biomechanical, densitometric, histological, hematological and electron-microscopic investigations. Fracture healing was investigated in rats after 24 weeks of clodronate treatment. The tibia was fractured, and the effects of treatments were evaluated at 4 and 8 weeks after the fracture. Radiographs and densitometric pQCT in the evaluation of experimental fracture healing were compared. In the other experiment with 30 mice, a mouse immobilisation osteoporosis model for further studies was investigated. Long-term administration of clodronate at therapeutic dosage had no harmful impacts but rather some beneficial effects on normal, non-osteoporotic bone. However, long-term high-dose clodronate treatment resulted in a decrement of tibial length but did not have any other significant or adverse effects. In the evaluation of fracture healing, pQCT proved to be better than radiographs in differentiating the total mineralised cross-sectional area of callus and the area of compact bone. Clodronate treatment does not seem to prolong the fracture healing process, even when administered on a long-term basis before the fracture. Clodronate increased the size of callus, but had only a minor effect on its biomechanical properties. Three weeks of hind limb immobilization caused local osteopenia in the tibia when compared to its contralateral leg. In conclusion, this thesis suggests that long-term administration of clodronate at therapeutic dosage has no harmful, but rather some beneficial effects on normal, non-osteoporotic bone. However, a fivefold dose of clodronate causes a slight decrease in the growth of tibial length. Healing of fractures during or after clodronate treatment is not inhibited.

Die Osteoporose ist eine Erkrankung, die durch verminderte Dichte und erhöhte Fragilität des Knochens gekennzeichnet ist. Sie zählt zu den häufigsten Erkrankungen weltweit und geht mit erheblicher Einschränkung der Lebensqualität und erhöhter Mortalität einher. Eine Behandlungsmöglichkeit dieses schwerwiegenden Krankheitsbilds ist die Therapie mit Bisphosphonaten. Diese hemmen mit ihren antiresorptiven Eigenschaften den Knochenabbau und fördern vermutlich gleichzeitig den Knochenaufbau. Obwohl schon lange die Wirkungsweise der Bisphosphonate erforscht wird, ist noch nicht sicher geklärt, wie beispielsweise osteoanabole oder antitumoröse Effekte vermittelt werden. Einen Erklärungsansatz bietet der MEK5/ Erk5-Signalweg. Diesem werden unter anderem antiangiogenetische, antiinflammatorische und antiproliferative Eigenschaften zugesprochen. In früheren Studien konnte gezeigt werden, dass Statine Erk5 und Erk5-abhängige Gene aktivieren können. Statine wiederum inhibieren ebenfalls den Mevalonatstoffwechsel, jedoch weiter upstream als Bisphosphonate. Da Statine zudem osteoanabole Effekte aufweisen, lag die These nahe, dass auch Bisphosphonate ihre Wirkung über den MEK5/Erk5-Signalweg vermitteln könnten. Die These konnte im Rahmen dieser Arbeit bestätigt werden: Stickstoffhaltige, nicht aber stickstofffreie Bisphosphonate aktivieren Erk5 sowohl in Endothelzellen als auch in Osteoblasten. Es gilt jedoch zu bedenken, dass eine Weiterentwicklung der Substanzen mit verbesserter Aufnahme in die Zelle zur Vermeidung von Apoptose-Induktion anzustreben ist. Des Weiteren konnte gezeigt werden, dass ein Knock-down der FDPS, dem Angriffspunkt der Bisphosphonate im Mevalonatstoffwechsel, ebenfalls eine Erk5-Phosphorylierung zur Folge hat. Durch Inhibition der FDPS wird die Prenylierung kleiner G-Proteine wie Cdc42 unterbunden, was eine veränderte Funktion der Proteine zur Folge hat. Ein Knock-down von Cdc42 mittels siRNA führt wiederum zu einer Aktivierung von Erk5. Auf diese Weise wurde nicht nur ein neuer Wirkungsweg der Bisphosphonate identifiziert, sondern auch ein möglicher Aktivierungsmechanismus der MEK5/ Erk5-Signalkaskade aufgedeckt. Erk5 wandert nach seiner Aktivierung in den Zellkern und beeinflusst dort die Genexpression. Im Rahmen dieser Arbeit wurden knochenrelevante Gene identifiziert, die durch Zoledronat-Stimulation induziert werden konnten. Zu diesen zählen INPP4B, das die Osteoklastogenese inhibiert, und PTHLH sowie FOSL1, welche die Osteoblastogenese fördern. Somit kann davon ausgegangen werden, dass die Aktivierung des MEK5/ Erk5-Signalwegs durch Zoledronat eine Geninduktion zur Folge hat, die sowohl die osteoanabole Wirkung unterstützt, als auch die katabolen Effekte hemmt. Auf diese Weise konnte neben den bereits bekannten Wirkungswegen der Bisphosphonate ein neuer identifiziert werden, der auch einen möglichen Ansatz für weitere, bisher ungeklärte Effekte von Bisphosphonaten darstellt. / Osteoporosis is a disease characterised by decreased bone density and increased fragility. It is one of the most frequent diseases worldwide and leads to a considerable reduction in quality of life and increased mortality. One option to treat this severe illness is a therapy based on bisphosphonates. These drugs inhibit bone resorption while possibly stimulating bone formation. Although a lot of research has been done to elucidate the cellular mechanisms of bisphosphonates, it remains unclear how they might mediate for instance osteoanabolic or antitumor effects. One possible explanation offers the MEK5/ Erk5 signal cascade which has been implicated in the regulation of anti-angiogenetic, anti-inflammatory and anti-proliferative effects. Previous studies have shown that statins can activate Erk5 as well as Erk5-dependent genes. Moreover, statins inhibit, like bisphosphonates, the mevalonate pathway further upstream. In addition, statins show osteoanabolic effects which led to the assumption that bisphosphonates might mediate some of their effects by the MEK5/ Erk5 signal cascade as well. Within this thesis, we confirmed this assumption by revealing that nitrogen-containing, but not non-nitrogen-containing bisphosphonates activate Erk5 in endothelial cells as well as osteoblasts. But we have to be aware of the need to optimize the substances to gain a better uptake into the cells and to avoid apoptosis. Furthermore, it has been shown that a knock-down of the FDPS, which is the molecular target of bisphosphonates in the mevalonate pathway, induced a phosphorylation of Erk5 as well. Inhibition of FDPS suppresses the prenylation of small G-proteins such as Cdc42 which leads to a modified function of the proteins. A knock-down of Cdc42 by siRNA in turn leads to activation of Erk5. Thus, it has not only been identified a new molecular target of bisphosphonates, but also a possible activation mechanism of the MEK5/ Erk5 signal cascade. After its activation, Erk5 migrates to the nucleus to influence gene expression. Within this thesis we identified zoledronic acid to induce bone relevant genes such as INPP4B which inhibits osteoclastogenesis and PTHLH or FOSL1 which support osteoblastogenesis. Thus, the activation of the MEK5/ Erk5 signal cascade by zoledronic acid induces gene induction which might support anabolic and inhibit catabolic effects in bone. Like this, a new molecular mechanism of action has been identified which might represent a new approach to understand effects of bisphosphonates that have not been explained thus far.

Die Bisphosphonat- assoziierte Kiefernekrose (BONJ) gewinnt seit ihrer Erstbeschreibung 2003 durch die steigenden Verschreibungszahlen der Bisphosphonate stetig an Bedeutung. Da eine effektive Therapie der BONJ nach wie vor sehr schwierig ist, liegt das Hauptaugenmerk in der Prävention und frühzeitigen Erkennung der Erkrankung. Die vorliegende Arbeit beschäftigt sich daher vor allem mit dem Stellenwert der Dental- CT in der Diagnostik und Früherkennung der BONJ. Dafür wurden die nativen Dental- CT- Aufnahmen von 99 Patienten unter Bisphosphonattherapie retrospektiv nach im Vorfeld klar definierten qualitativen, quantitativen und halbquantitativen Kriterien neu beurteilt. Entgegen der Literatur und den aktuellen Empfehlungen der AWMF erwies sich die CT zur Früherkennung und stadiengerechten Dokumentation der BONJ als hervorragend geeignet. Bereits unspezifische klinische Entzündungszeichen können auf ein Frühstadium der BONJ hindeuten. Das radiologische Hauptsymptom der Hypersklerosierung stellt dabei ein Durchgangsstadium der BONJ dar. Mit Fortschreiten der Erkrankung kommt es zu einem zunehmend heterogenen Erscheinungsbild mit lokaler Hypersklerosierung, grobwabiger Spongiosastruktur, Osteolysen und Sequestern. Es ist erstmals gelungen die wichtigsten radiologischen Zeichen einer BONJ zu objektivieren und die große biologische Variabilität exakt abzubilden. Bemerkenswert war zudem ein signifikant hochgradigerer Befall der Mandibula durch die BONJ. Die Dental- CT ist damit für die Betreuung der betroffenen Patienten die Methode der 1. Wahl.

The isoprenoid biosynthetic pathway is targeted in the treatment of several diseases, including hypercholesteremia and bone related disorders. Farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) are isoprenoid biosynthetic pathway intermediates that are utilized during post-translational modification of proteins termed farnesylation and geranylgeranylation, respectively, together known as prenylation. The Ras and Rho GTPase family members are examples of proteins that are prenylated. Prenylation is essential for proper membrane localization and function of these small GTPases. Activating mutations or over-expression of these proteins promote oncogenic events, such as increased proliferation and migration. Studies have demonstrated that farnesyl transferase inhibitors and geranylgeranyl transferase inhibitors possess anti-cancer effects in humans and animal models of cancer, respectively. An alternative way to impair protein prenylation is through the depletion of FPP and GGPP. Statins and nitrogenous bisphosphonates (NBPs) deplete FPP and GGPP leading to impaired protein prenylation by inhibiting HMG-CoA Reductase (HMGCR) and FPP synthase (FDPS), respectively. These drugs have been shown to induce apoptosis, inhibit cancer cell migration, and induce cell cycle arrest. The anti-cancer effects of statins and NBPs can be prevented by GGPP addition, suggesting that GGPP depletion may be the mechanism by which these agents interfere with cancer cell survival. We and our collaborators have developed bisphosphonate inhibitors of GGPP synthase (GGDPS), an enzyme that produces GGPP from the substrates FPP and isopentenyl pyrophosphate. The goal of this research was to identify novel GGDPS inhibitors and to assess the effects of specific inhibition of GGDPS on cancer cell survival and function. Two aromatic bisphosphonates were identified as potent inhibitors of GGDPS in enzyme and cellular assays. Apoptosis hallmarks such as PARP cleavage and DNA fragmentation demonstrated that GGDPS inhibition induces apoptosis in K562 chronic myeloid leukemia cells through GGPP depletion and FPP accumulation. Isobologram analysis and enhanced impairment of protein geranylgeranylation showed that GGDPS inhibition is synergistic with the inhibition of HMGCR. Migration assays, transwell assay and large scale digital cell analysis system microscopy, demonstrated that GGDPS inhibition interferes with MDA-MB-231 breast cancer cell migration. Increased LC3-II expression showed that FDPS and GGDPS inhibition induces autophagy in PC3 prostate and MDA-MB-231 breast cancer cells. Inhibition of autophagy enhances the toxic effects of GGDPS inhibition as measured by MTT assay. Propidium iodine staining of DNA and immunostaining of cell cycle proteins such as p27 did not show significant effects of GGDPS inhibition on cell cycle progression. Importantly, exogenous addition of GGPP prevented most of the effects observed with GGDPS inhibition, suggesting specific inhibition of GGDPS by our bisphosphonate inhibitors. The data obtained herein suggest that GGDPS can be targeted to interfere with the progression of cancer cells.

Submitted by JONATHAN RIBEIRO DA SILVA (bucomaxilofacial@outlook.com) on 2018-08-14T01:44:12Z No. of bitstreams: 1 Prevenção da OMIM - TESE -Jonathan.pdf: 29184918 bytes, checksum: 331abcd1df34df63ea22c72ac257eed0 (MD5) / Approved for entry into archive by Ana Paula Rimoli de Oliveira null (anapaula@foa.unesp.br) on 2018-08-14T17:19:39Z (GMT) No. of bitstreams: 1 Prevenção da OMIM - TESE -Jonathan.pdf: 29184918 bytes, checksum: 331abcd1df34df63ea22c72ac257eed0 (MD5) / Made available in DSpace on 2018-08-14T17:19:39Z (GMT). No. of bitstreams: 1 Prevenção da OMIM - TESE -Jonathan.pdf: 29184918 bytes, checksum: 331abcd1df34df63ea22c72ac257eed0 (MD5) Previous issue date: 2018-06-20 / Outra / Objetivo: Avaliar a prevenção da OMIM em ratos em risco de desenvolvimento de osteonecrose na região em que foi realizada a exodontia utilizando apenas coágulo, enxerto de osso xenógeno, e enxerto de β- trifosfato de cálcio (β-TCP). Métodos: Foram utilizados 20 Ratos Wistar machos com 3 meses de idade, pesando 350 – 450g, submetidos a indução da Osteonecrose por uso de ácido zoledrônico (0,04mg/kg) durante 05 semanas. Na 7a semana foi realizado a cirurgia de exodontia dos molares superiores direito e preenchimento do alvéolo com coágulo (controle), enxerto xenógeno (Grupo 2), e β- trifosfato de cálcio (β-TCP) (Grupo 3). A eutanásia foi realizada na 15a semana. Foram realizadas análises morfométrica, estereológica, e imunohistoquímica, onde aplicou-se os testes estatísticos ANOVA e Tukey, considerando-se um nível de significância de 5%. Resultados: Durante a análise macroscópica não houve manifestação clínica da OMIM nos grupos experimentais. A análise quantitativa demonstrou que o Grupo 3 (BTCP) apresentou menor formação de lacunas ósseas e maior formação de tecido ósseo sadio quando comparado com os grupos 1 e 2 (p<0,05). Não houve diferença estatística entre os grupos durante análise de formação de tecido epitelial. Na análise imunohistoquimica, o grupo experimental apresentou maior atividade de remodelação óssea. Conclusão: Os resultados deste trabalho demonstraram que os grupos experimentais apresentaram maior atividade de remodelação óssea, e ausência de manifestação clínica da OMIM. O grupo BTCP ainda demonstrou menor quantidade de lacunas e maior quantidade de osso formado durante analise histológica. No entanto, mais estudos necessitam ser realizados até o desenvolvimento de um protocolo de prevenção desta complicação / Objective: To evaluate the bone formation in rats with osteonecrosis in the region where the extraction was performed using only clot, xenogen bone graft, and calcium β-triphosphate (β-TCP) graft. METHODS: Twenty male Wistar rats weighing 350-450 g were submitted to osteonecrosis induction for the use of zoledronic acid (0.2 mg / kg) for 5 weeks. In the 7th week, the maxillary right molar extraction and filling of the alveolus with clot (control), xenogene graft (Group 2) and calcium β-triphosphate (β-TCP) were performed (Group 3). Euthanasia was performed in the 15th week. Morphometric and stereological analyzes were performed. The ANOVA and Tukey statistical tests were used, considering a level of significance of 5%. Results: During the macroscopic analysis there was no clinical manifestation of the OMIM in the experimental groups. Quantitative analysis showed that Group 3 (BTCP) presented less bone formation and greater formation of healthy bone tissue when compared to groups 1 and 2 (p <0.05). There was no statistical difference between groups during analysis of epithelial tissue formation. In the immunohistochemical analysis no difference was found in the bone remodeling process between the groups. Conclusion: The results of this work were favorable for the use of BTCP for guided bone regeneration and prevention of the clinical manifestation of OMIM in rats. However, more studies need to be performed until the development of a protocol to prevent this complication