IntroduÃÃo: Diversos mecanismos tÃm sido propostos para explicar a Osteonecrose dos Maxilares induzida por Bisfosfonatos (OMB), mas nÃo hà consenso acerca do desenvolvimento fisiopatolÃgico dessa condiÃÃo. Objetivo: Avaliar o efeito de alteraÃÃes sistÃmicas e hematolÃgicas que possam interferir no desenvolvimento de OMB. MÃtodos: ApÃs trÃs infusÃes venosas semanais consecutivas de Ãcido ZoledrÃnico (AZ) (0,04, 0,20, 1,00mg/kg) ou salina (controle), ratos Wistar machos (n=6-7) tiveram seus primeiros molares inferiores esquerdos extraÃdos quatro semanas apÃs a Ãltima administraÃÃo. Uma semana apÃs exodontia foi realizada infusÃo extra de AZ ou salina (controle), sendo os animais sacrificados 28 dias apÃs a exodontia. Os animais foram pesados e foi coletado sangue semanalmente (anÃlise de variaÃÃo de peso corpÃreo e anÃlise hematolÃgica, respectivamente), alÃm disso, mandÃbulas, fÃgado, baÃo, rins e estÃmago foram removidos e analisados microscopicamente. Resultados: Obervou0se necrose Ãssea nos animais tratados com 0,20 e 1,00 mg/kg de AZ sob os aspectos radiogrÃficos e histolÃgicos (p<0.0001). Nesses dois grupos houve aumento significativo do nÃmero de leucÃcitos circulantes (p<0.0001) em relaÃÃo ao grupo controle e os Ãndices de anemia (p<0.0001) tambÃm se mostraram superiores. NÃo houve toxicidade hepÃtica e renal, no entanto o baÃo apresentou nÃmero aumentado de deposiÃÃo de pigmentos de hemossiderina nos dois grupos experimentais (p=0.0004), os quais apresentaram tambÃm significante alteraÃÃo inflamatÃria gÃstrica (p=0.0168). ConclusÃo: a OMB està diretamente associada a leucocitose, anemia e provÃvel toxicidade sistÃmica (hematolÃgica e ÃrgÃo-especÃfica). / Introduction: Several mechanisms have been proposed to explain the induced Osteonecrosis of the Jaw Bisphosphonates (OMB), but there is no consensus about the pathophysiological development of this condition. Objective: To evaluate the effect of systemic and hematologic changes that may interfere with the development of OMB. Methods: After three consecutive weekly venous infusions of zoledronic acid (AZ) (0.04, 0.20, 1,00mg / kg) or saline (control), male Wistar rats (n = 6-7) had their first molars left extracted four weeks after the last administration. A week after extraction extra infusion of AZ or saline (control) was performed, and the animals were sacrificed 28 days after the extraction. The animals were weighed and blood was collected weekly (analysis of variance in body weight and hematologic analysis, respectively) furthermore jaws, liver, spleen, kidney and stomach were removed and examined microscopically. Results: Obervou0se bone necrosis in animals treated with 0.20 and 1.00 mg / kg of AZ under the radiographic and histological aspects (p <0.0001). In both groups there was a significant increase in the number of circulating leukocytes (p <0.0001) compared to the control group and the rates of anemia (p <0.0001) were also higher. There was no liver and kidney toxicity, however the spleen showed increased numbers of hemosiderin pigment deposition in both experimental groups (p = 0.0004), which also showed a significant gastric inflammatory changes (p = 0.0168). Conclusion: The OMB is directly associated with leukocytosis, anemia and probable (hematologic and organ-specific) systemic toxicity.

A movimentação dentária ortodôntica ocorre através de dois processos, nos quais o osso alveolar é reabsorvido nas áreas de pressão, enquanto que novo osso é formado na área de tração. O processo de reabsorção óssea ocorre pela ação de células multinucleadas, os osteoclastos. Os bisfosfonatos constituem um grupo de fármacos com propriedade de inibir a reabsorção óssea, foi utilizado no presente estudo com a finalidade de interferir na remodelação óssea induzida ortodonticamente. Para isso, força contínua de 15 cN foi aplicada aos primeiros molares superiores de ratos machos Wistar de 2 1/2 meses, utilizando uma biomecânica com fios superelásticos. Os animais foram divididos aleatoriamente em 4 grupos: 1) O grupo controle constituído por dezoito ratos, os quais foram injetados solução salina por 7 dias antes da instalação da biomecânica passiva, que permaneceu por 3, 10 e 18 dias; 2) Dezoito animais foram tratados com ALN (dose 2,5 mg/Kg) por 7 dias antes da instalação da biomecânica passiva que permaneceu por 3, 10 e 18 dias; 3) Dezoito animais foram tratados com alendronato com a mesma dose citada acima por 7 dias antes da instalação da biomecânica ativa que permaneceu por 3, 10 e 18 dias; 4) Dezoito animais foram injetados com solução salina 7 dias antes da instalação da biomecânica ativa que permaneceu por 3, 10 e 18 dias. As maxilas foram fixadas com 4% de formaldeído + 0,1% de glutaraldeído, descalcificadas em EDTA a 4,13% e incluídas em parafina ou resina Spurr. Os cortes foram corados com HE para análise morfológica. Alguns cortes foram submetidos à imuno-histoquímica para detecção de RANKL e OPG. Foi utilizado o método TRAP, marcador de osteoclastos e microscopia eletrônica de transmissão para análise ultraestrutural. Alguns espécimes tiveram a cortical óssea vestibular do primeiro molar superior congelada em nitrogênio líquido para análise da expressão de RANKL por Western Blotting. O ALN inibiu a reabsorção óssea e radicular de todos os grupos tratados. As células clásticas apresentaram-se em estado latente. No grupo da movimentação ortodôntica o osso alveolar foi remodelado e com 18 dias a superfície radicular apresentou-se reabsorvida e o TRAP revelou clastos ativos, achados confirmados pela microscopia eletrônica de transmissão. A expressão de RANKL, molécula ativadora de células clásticas, nao foi inibida pela droga. A expressão de OPG foi aumentada nos animais tratados. Os resultados demonstram que o uso de alendronato sódico na movimentação ortodôntica não interfere no recrutamento dos osteoclasto, ele aparentemente inibe sua ativação, o que pode interferir no processo de remodelação óssea e talvez diminua a quantidade de movimentação dentária. / Orthodontic tooth movement occurs through two processes in which the alveolar bone is resorbed in the pressure areas, whereas new bone is formed in the tension area. The bone resorption occurs by multinucleated cell, the osteoclasts. The bisphosphonates are drugs with capability to inhibit clastic activity were used in the present study in order to interfere with the bone remodeling induced orthodontic. For this continuous force of 15 cN was applied to the first molars of Wistar male rats of 2 1/2 months, using a biomechanical with superelastic wire. The animals were randomly divided into 4 groups: 1) The control group consisted of eighteen mice, which received sterile saline solution saline for 7 days prior to installation of passive biomechanics, which remained for 3, 10 and 18 days; 2) Eighteen animals were treated with ALN (dose 2.5 mg / kg) for 7 days prior to installation of the passive biomechanical to remain for 3, 10 and 18 days; 3) Eighteen animals were treated with alendronate with the same dose quoted above for 7 days prior to the biomechanical installation that remains active for 3, 10 and 18 days; 4) Eighteen animals were injected with sterile saline solution 7 days prior to the biomechanical installation that remains active for 3, 10 and 18 days. The maxillae were fixed with 4% formaldehyde + 0.1% glutaraldehyde, decalcified in EDTA 4.13% and embedded in paraffin or Spurr resin. The specimens were morphologically analyzed in HE stained sections. Some stained sections were used for immunolabeling for RANKL and OPG. The osteoclasts were marked by tartrate-resistant acid phosphatase (TRAP) histochemistry. The ultrathin sections were examined in a trasnmission electron micrsocpe. Some specimens were frozen in liquid nitrogen for protein extraction and Western Blotting protein expression analyzes. The ALN inhibited bone resorption and root of all the treated groups. The clastic cells present in a latent state. In the orthodontic movement group alveolar bone was remodeled with 18 days to root surface presented itself reabsorbed and the TRAP revealed clasts assets, findings confirmed by transmission electron microscopy. Expression of RANKL activating molecule clastic cells was not inhibited by the drug. The OPG expression was increased in treated animals. The results demonstrate that the use of alendronate in the orthodontic movement does not interfere with osteoclast recruitment, it apparently inhibits their activation, which can interfere in the bone remodeling process and may reduce the amount of tooth movement.

Os bisfosfonatos (BFs) têm sido amplamente utilizados para o tratamento de doenças do metabolismo ósseo, principalmente na prevenção de metástases ósseas e na prevenção e tratamento da osteoporose. No entanto, existem efeitos colaterais indesejáveis sendo um deles, a indução da Osteonecrose dos Maxilares (Medication-Related Osteonecrosis of the Jaws - MRONJ), uma complicação de difícil tratamento e solução. Até o presente momento, não foi definida a fisiopatologia da MRONJ e nem estabelecido protocolo de tratamento eficaz para esta doença. Diversas terapias vem sendo descritas na literatura para o tratamento da MRONJ dentre elas a laserfototerapia. Assim sendo, os objetivos deste estudo foram: inicialmente avaliar in vitro o efeito de diferentes concentrações dos BFs mais empregados na atualidade (alendronato e ácido zoledrônico) na viabilidade de células envolvidas na reparação de lesões MRONJ (osteoblastos e fibroblastos); secundariamente estudar o efeito da fototerapia com laser de baixa potência sobre estas células previamente induzidas pelos BFs. Foram utilizados osteoblastos-símile da linhagem OSTEO 1 e fibroblastos de mucosa bucal humana da linhagem FMM1. Após terem sido submetidos aos testes de citotoxicidade expondo as células as concentrações de 1?M, 10?M e 100?M de alendronato sódico e 3?M, 5?M e 10?M de ácido zoledrônico por 24 horas, os grupos testes foram irradiados com laser de diodo no modo contínuo, puntual e de contato (InGaAIP, 660nm, 30mW, spot 0,028cm2 ) com duas densidades de energia diferentes 5J/cm2 (4,5s) e 10J/cm2 (9s). Duas irradiações com intervalo de 6 horas entre cada uma delas foram executadas. A viabilidade celular foi determinada utilizando o ensaio de redução do MTT, e a atividade de fosfatase alcalina dos osteoblastos foi avaliada utilizando ensaio de ponto final. Os resultados obtidos foram submetidos à análise estatística ANOVA 1 critério complementado por Tukey (p<0,05). Foi possível concluir que: as concentrações de 100?M e 10?M do alendronato sódico foram tóxicas para os osteoblastos e fibroblastos em cultura. As concentrações de 3?M, 5?M e 10?M do ácido zoledrônico foram tóxicas para os osteoblastos e fibroblastos a longo prazo (48h e 72h). A atividade da fosfatase alcalina nos osteoblastos foi afetada por todas as concentrações de ácido zoledrônico testadas (3?M, 5?M e 10?M). Nos parâmetros aqui aplicados a LPT não teve efeito sobre a atividade da fosfatase alcalina das células tratadas com o alendronato de sódio ou ácido zoledrônico. E a laserfototerapia de baixa potência nos parâmetros utilizados nesse estudo não foi capaz de reverter a toxicidade dos bisfosfonatos testados, independentemente das concentrações destas substâncias / Bisphosphonates (BPs) have been widely used for treating bone metabolism diseases, especially for prevention of bone metastasis and osteoporosis. However, there are undesirable side effects and one of them, the induction of Osteonecrosis of the Jaw (Medication-Related Osteonecrosis of the Jaws - MRONJ), a complication of difficult treatment and solution. Until now the pathophysiology and an effective treatment protocol for MRONJ have not been established. Various therapies have been described in the literature for the treatment of MRONJ including laserphototherapy. The objectives of this study were to evaluate the effect of different concentrations of two BPs used today (alendronate and zoledronic acid) on the viability of cells involved in the repair of MRONJ lesions (osteoblasts and fibroblasts); and to study the effect of phototherapy with low power laser on these cells previously treated with referred BPs. After being submitted to cytotoxicity testing by exposing the cells to concentrations of 1?M, 10?M and 100?M of sodium alendronate and 3?M, 5?M and 10?M of zoledronic acid for 24 hours, the test groups were irradiated with diode laser in continuous mode, punctual and contact (InGaAIP, 660nm, 30mW, spot 0,028cm2) with two different energy densities 5 J/cm2 (4,5s) and 10J/cm2 (9s). Two irradiations with an interval of 6 hours between each of them were performed. Cell viability was determined using the MTT reduction assay, and the alkaline phosphatase activity of osteoblasts was evaluated using the end point assay. The results were submitted to statistical analysis using ANOVA 1 criteria complemented by Tukey (p <0.05). It was possible to conclude that: concentrations of 100?M and 10?M of alendronate were toxic to osteoblasts and fibroblasts in culture. The concentrations of 3?M, 5?M and 10?M of zoledronic acid was toxic to osteoblasts and fibroblasts in long-term (48h and 72h). The activity of alkaline phosphatase in osteoblasts was affected by all the zoledronic acid concentrations tested (3?M, 5?M and 10?M). In the LPT parameters applied here had no effect on alkaline phosphatase activity of the cells treated with the sodium alendronate or zoledronic acid. And low power laserphototherapy, in the parameters used in this study, was unable to reverse the toxicity of bisphosphonates tested, irrespective of the concentrations of these substances.

Os bisfosfonatos são drogas capazes de inibir a reabsorção óssea por meio de seu efeito direto sobre as células ósseas, interferindo na dinâmica dos tecidos mineralizados. O alendronato (ALN), um tipo de bisfosfonato nitrogenado, foi utilizado com o objetivo de investigar os seus efeitos sobre os tecidos dentários e periodontais após luxação lateral de molares com as raízes em desenvolvimento. Ratos Wistar com 21 dias de idade tiveram os segundos molares superiores luxados lateralmente. Doses diárias de 2,5 mg / kg de ALN começaram no dia seguinte à luxação; os controles receberam solução salina estéril. As maxilas foram fixadas, descalcificadas e incluídas em parafina ou em resina Spurr 7, 14 e 21 dias pós-luxação. Os cortes foram corados com H & E, incubados por histoquímica TRAP e imuno marcados para osteopontina (OPN), bem como para análise ultraestrutural. Após 21 dias, o ápice dos molares luxados sem ALN estava aberto e desorganizado, coberto por uma camada irregular de cemento celular. Os molares luxados dos animais tratados com ALN apresentaram alguns locais de anquilose, bem como lacunas de reabsorção na superfície do cemento. Os osteoclastos TRAP positivos foram mais numerosos no grupo ALN, apesar de sua aparência latente e sua localização, afastados das trabéculas ósseas, em relação aos controles, achado que foi confirmado com a análise ultraestrutural. A imunomarcação de OPN revelou uma linha grossa imunopositiva na dentina, que deve ter surgido a partir do momento da luxação, enquanto que as amostras tratadas com ALN não apresentaram alterações na dentina. Os resultados indicam que o alendronato inibe algumas alterações na dentina e na formação do cemento, induzidas pelo trauma dental de luxação. / Bisphosphonates are drugs that inhibit bone resorption through its direct effect on bone cells, interfering with the dynamics of mineralized tissues. Alendronate (ALN), a nitrogenated bisphosphonate, was used in order to investigate their effects on dental and periodontal tissues after lateral dislocation of molars with developing roots. Twenty one days old Wistar rats had their second molars laterally l. Daily doses of 2.5 mg / kg ALN started the day following the dislocation, while controls received saline solution. The maxillae were fixed, decalcified and embedded in paraffin or in Spurr resin after 7, 14 and 21 days post-dislocation. The sections were stained with H & E, incubated for TRAP, immunolabeled for osteopontin (OPN), and ultrastructurally analyzed by transmission electron microscopy. After 21 days, the apex of the luxated molar without ALN was open and disorganized, covered by an irregular layer of cellular cementum. The luxated molar from ALN-treated animals showed some areas of ankylosis and resorption lacunae on the cementum surface. TRAP-positive osteoclasts were more numerous in the ALN group, despite their latent appearance compared to controls, a finding that was ultrastructurally confirmed. OPN immunostaining revealed a thick immunopositive line in dentin, which must be resultant from the moment of dislocation, while the samples treated with ALN showed no changes in dentin. The results indicate that alendronate inhibits some changes in dentin and cementum formation induced by dental trauma of lateral luxation.

A osteonecrose dos maxilares associada ao uso dos Bisfosfonatos é uma exposição óssea que persiste por mais de 8 semanas na cavidade oral em pacientes sob tratamento com bisfosfonatos e que não foram submetidos a radioterapia de cabeça e pescoço. Explicar o motivo do desenvolvimento destas lesões ósseas principalmente nos maxilares e desvendar a sua patofisiologia ainda é necessário. Por isso, o reparo ósseo em um modelo animal de osteonecrose dos maxilares associada ao uso de bisfosfonatos em ratos Wistar (Rattus norvegicus, albinus) foi avaliado através de análise microscópica e molecular. A amostra foi composta por 48 ratos machos, com 12 semanas de vida e peso aproximado de 300 gramas, que foram submetidos a administração de Ácido Zoledrônico, 0,6 mg/kg a cada 28 dias com um total de 5 doses. Os animais foram dividos em quatro grupos, cada um composto por 12 animais; 2 grupos de tratamento AZ e AZ-experimental (AZ-exp) e 2 grupos controles CO e CO-experimental (CO-exp) com administração de cloreto de sódio 0,9% no mesmo volume e frequencia do Ácido Zoledrônico. As soluções foram administradas por via intraperitoneal. O grupo AZ-exp e o CO-exp foram submetidos a exodontias dos molares superiores direitos e a realização de defeito ósseo no fêmur esquerdo 45 dias após a primeira aplicação das soluções. A eutanásia dos animais ocorreu após 150 dias do início do experimento. A avaliação histológica foi realizada através de análises qualitativa e quantitativa, verificou a presença de sequestros ósseos, áreas de osteonecrose e áreas de osso total por meio de estudos de microscopia óptica pela coloração Hematoxilina e Eosina. Análise quantitativa da expressão do RNAm de proteínas envolvidas no processo de reparo ósseo pelo método de reação em cadeia da polimerase em tempo real (RealTimePCR) também foi realizada para avaliação dos osteoclastos (RANK, RANKL e OPG), osteoblastos (ALPL e OCN) osteócitos (DMP-1 e PHEX) e vascularização (VEGF). Apenas o grupo com administração de AZ e realização de exodontias apresentou sequestros ósseos e áreas significativamente maiores de osteonecrose em comparação aos animais com exodontia e sem a administração de AZ. Não foram encontradas áreas de osteonecrose na análise do fêmur. A avaliação do osso total apresentou maior quantidade de osso nos animais submetidos a procedimentos cirúrgicos e administração de AZ, porém não houve significância estatística. Na análise molecular, a administração de AZ e a realização das exodontias e do defeito ósseo, provocou alteração na expressão de marcadores para osteoclastos. A administração do AZ associada à realização dos procedimentos cirúrgicos aumentou a expressão de marcadores iniciais da osteoblastogênese e diminuiu a expressão tardia dos osteoblastos. A expressão de marcadores de osteócitos foi menor após a administração de AZ e exodontias na maxila, no entanto, no fêmur, houve aumento na expressão destes marcadores. A realização das exodontias e de defeitos ósseos no fêmur aumentou a expressão de VEGF nos grupos com e sem administração de AZ. Estes resultados evidenciam a interferência do Ácido Zoledrônico no reparo ósseo dos alvéolos e dos defeitos ósseos nos fêmures, causando incidência de osteonecrose na maxila, atraso na remodelação óssea, e ausência de lesões de osteonecrose no fêmur. A presença de lesões de osteonecrose na maxila pode ser consequência do atraso na remodelação óssea, evidenciado pela alteração na expressão de marcadores para osteclastos e osteoblastos, e as mesmas ocasionam a diminuição da expressão de proteínas pelos osteócitos. / Bisphosphonate-related osteonecrosis of the jaws is a bone exposure persisting for more than 8 weeks in the oral cavity in patients receiving bisphosphonates and who did not undergo radiation therapy for head and neck. Explain the reason for these development bone lesions mainly in the jaws and unveil its pathophysiology is still needed. Therefore, the bone repair in an animal model of Bisphosphonate-related osteonecrosis of the jaws in Wistar rats (Rattus norvegicus, Albinus) was evaluated by microscopic and molecular analysis. The sample was composed of 48 male rats, with 12 weeks old and weighing approximately of 300 grams, who underwent the administration of zoledronic acid, 0.6 mg / kg every 28 days with a total of 5 doses. The animals were divided into four groups, each consisting of 12 animals; 2 treatment groups AZ and AZ-experimental (AZ-exp) and 2 control groups CO and CO-experimental (CO-exp) with sodium chloride administration 0.9% in the same volume and frequency of zoledronic acid. All solutions were administered intraperitoneally. The AZ-exp group and the CO-exp group underwent extractions rights upper molars and the bone defect was performed in the left femur 45 days after the first application of the solutions. Euthanasia of animals occurred after 150 days from the beginning of the experiment. Histological evaluation was performed through quantitative and qualitative analysis checked the presence of bone sequestration, osteonecrosis area and whole bone areas by means of optical microscopy by hematoxylin-eosin staining. Quantitative analysis of mRNA expression of proteins involved in bone repair by polymerase chain reaction method in real time (RealTimePCR) was also performed: osteoclasts (RANK, RANKL and OPG), osteoblasts (ALPL and OCN) osteocytes (PMD-1 and PHEX) and vascularization (VEGF). Only the group with administration of AZ and performing tooth extractions presented bone sequestration and significantly larger areas of osteonecrosis when compared to animals with extraction and without AZ administration. There were no areas of osteonecrosis of the femoral analysis. The evaluation of the whole bone showed more bone in animals undergoing surgeries and AZ administration but there was no statistical significance. Molecular analysis, AZ administration and the performance of tooth extractions and bone defect changed the expression of markers for osteoclasts. The AZ administration associated with surgical procedures increased the expression of early markers of osteoblastogenesis and decreased the late expression of osteoblasts. The expression of osteocytes markers was lower after AZ administration and maxillary tooth extractions; however, there was an increase in the expression of these markers in the femur. The performance of tooth extractions and bone defects in the femur increased VEGF expression in animals with and without administration of AZ. These results show the interference of zoledronic acid on bone repair of the sockets and bone defects in the femurs, causing incidence of osteonecrosis of the upper jaw, delayed bone remodeling, and no femoral osteonecrosis injuries. The presence of osteonecrosis injuries in the upper jaw can be the consequence of the delay on bone remodeling, evidenced by the change in the markers expression for osteoclasts and osteoblasts, and they cause the decreased in protein expression by osteocytes.

Metastatic and primary bone cancers are devastating diseases of paediatric and adult humans because of the morbidity associated with bone pain. Controlling bone pain from multiple metastatic sites can be difficult in end-stage cancers using conventional therapies. Bone-seeking radiopharmaceuticals have been successful in this area as radiation can be delivered with moderate selectivity to the target. Unfortunately, targeted radiotherapy using radiopharmaceuticals have been relegated to palliative therapy as myelosuppression largely limits the radiation dose to sub-therapeutic levels. Efforts to overcome this therapeutic limitation include autologous bone marrow transplants in combination with chemotherapy-radiosensitization and the development of new radiopharmaceuticals. Development work using laboratory rodent models has been complicated by dosimetric limitations because of size and inherent problems with human xenografted tumour models in rodents. To address this need we studied naturally occurring canine osteosarcoma as a translational model for human bone cancer. Central to our hypothesis was that naturally occurring canine osteosarcoma would serve as an investigational model for comparing the pharmacokinetics (biodistribution), dosimetry, toxicity, and therapeutic effect of 153Sm-EDTMP, 188Re-HEDP, 186Re-HEDP, and a novel ligand, polyethyleneiminomethyl phosphonic acid (PEI-MP). Data collected from existing radiopharmaceuticals was then compared to PEI-MP labelled with 99mTc, 153Sm, and 186Re. This innovative and unique study allowed for the evaluation of various radiopharmaceuticals in a naturally occurring animal model of bone cancer, documenting the pharmacokinetics and dosimetry of a novel radiolabelled-ligand (PEI-MP). Benefits resulting from the successful completion of the study would allow more rapid transfer of rodent preclinical data into a naturally occurring cancer model more resembling to the human diseases and would thus more likely identify problems with pharmacokinetics and toxicity before proceeding to expensive clinical trials. The expected outcomes of the study were originally formulated based on limited previous published data in dogs. For instance, no data exists describing the pharmacokinetics or toxicity of 188Re-HEDP and 186Re-HEDP in the dog. The study was conducted in two phases. The first phase deals with the evaluation of 153Sm-EDTMP, 188Re-HEDP, and 186Re-HEDP in the dog model. Phase-two was the development of a novel ligand (PEI-MP) in the dog model of osteosarcoma, which has the characteristics of an ideal ligand. Pharmacokinetic results for 153Sm-EDTMP in normal dogs (n=4) for blood were similar to published reports for dogs and human. When compared statistically to human data the majority of results were the same, lending credence to the hypothesis that dogs could serve as models for human radiopharmaceutical research. Normal dogs and the osteosarcoma dog did differ from human pharmacokinetics in the urine elimination phase (t½-â). This can most likely be explained by the tumour burden in the human research populations or due to the fact that most humans were aged and likely to have some renal disease. Certainly, the trend in dogs with osteosarcoma was to have a prolonged urine elimination phase (t½-â) compared to normal dogs which supported the hypothesis that the biodistribution and pharmacokinetics results from dogs were similar to human published data. Statistical comparisons were also made between normal dogs receiving 188Re-HEDP and 153Sm-EDTMP. The prolonged urine elimination phase (t½-â) and increased blood retention of 188Re-HEDP was most likely a reflection of prolonged bone washout and soft issue retention. This could be attributed to the differences between the antiresorptive capability of bisphosphonate ligands e.g., EDTMP (lexidronam) with a greater than 100-fold antiresorptive capability than HEDP (etidronate). Additional observational biodistribution studies using macro- and micro-autoradiography techniques were also performed in canine tissue and Sprague-Dawley rats. Results from the studies showed heterogeneous uptake within tumours in dogs. In rats, localization of 153Sm-EDTMP in red marrow areas would lead to a high radiation dose to blood producing elements. In addition, high uptake was documented at the metaphyseal growth plate confirming the likelihood of a delay or cessation of growth if 153Sm-EDTMP were used in growing children. Phase-one of the clinical trial in dogs with naturally occurring osteosarcoma identified only mild toxicity at the dosage rate of 37 MBq/kg (1 mCi/kg) for both 153Sm-EDTMP and188Re-HEDP. In addition, a pilot trial was conducted in dogs receiving 153Sm-EDTMP which also received a carboplatin infusion at the time of the radiopharmaceutical administration followed by another 3 cycles of carboplatin at 3 weekly intervals. No differences in toxicity were noted between the carboplatin group and dogs receiving only 153Sm-EDTMP. As a part of the 188Re-HEDP clinical trial, 3 dogs with osteosarcomas received weekly dose of 188Re-HEDP at 37MBq/kg for 4 weeks in which only mild toxicity was noted. Unfortunately, there was no cessation in growth of the tumours, with all dogs showing progression. The median survival time for both radiopharmaceuticals was 4 months, significantly shorter than the 10-month median survival time for amputation and chemotherapy. Interestingly six dogs that had 99mTc-MDP and 153Sm-EDTMP showed scans of tumours that had consistently lower 99mTc-MDP uptake ratios (normal bone compared to cancerous bone) compared to solely 153Sm-EDTMP. In contrast, this was not evident for uptake ratios between 188Re-HEDP and 99mTc-MDP scans. Once again, this finding highlights the differences between the antiresorptive capabilities of the bisphosphonates ligands. Interestingly, another three dogs were scanned with 99mTc-MDP , 153Sm-EDTMP, and 99mTc-PEI-MP (10-30 kDa) showed a variation in uptake between scans of the same tumours. More importantly, the uptake ratios of 99mTc-MDP and 153Sm-EDTMP scans showed wide variation with a coefficient of variance of 52% and 39% respectively. However, the range in uptake from the 99mTc-PEI-MP (10-30 kDa) scan was narrow with a coefficient of variance of only 6%. This could be attributed to more consistent uptake ratio of the unique ligand PEI-MP and its hypothesized mechanism of action: enhanced permeability and retention (EPR) in tumour vasculature. This requires further investigation with larger groups. In phase-two, the pharmacokinetic result for the novel ligand PEI-MP was initially studied labelled with 99mTc. Various molecular weights were tested in normal dogs and compared to previously published results in baboons. Results from the dog studies were found to be similar to those from the primate study. As in the primate study, molecular weight and charge played a significant role in 99mTc-PEI-MP pharmacokinetics. Increasing the size of the macromolecules and altering their charge resulted in marked changes in their pharmacokinetics and biodistribution. The PEI-MP molecular weight of 10-30 kDa and 20-30 kDa were the most promising and fulfilled the hypothesized criteria of an ideal radiopharmaceutical. In keeping with the aims of the study, the 20-30kDa polymer was considered more desirable because of its faster clearance. However, because of the limitations imposed by the percentage yield of the different molecular weights of the ligand during filtration, we decided to label the 10-30kDa molecular weight MW-fraction with 153Sm. Unexpectedly, the 153Sm-PEI-MP 10-30 kDa had a prolonged urine elimination phase (t½-â) associated with increased liver uptake when compared to 99mTc-PEI-MP10-30 kDa. To explain this, computer modelling for blood plasma (ECCLES) was done which predicted that there would be some chemical dissociation of the 153Sm from the PEI-MP polymer in blood. This is due to interaction between the radiopharmaceutical and citrate, forming 153Sm-citrate. The ECCLES model for blood plasma also predicted that the anionic MW-fraction, PEI-MP 10-30kDa, would be a poor ligand complexed to 166Ho, 212Pb, 213Pb, and 89Sr, but was expected to be effective when complexed to 186Re or 188Re, based on their close proximity to 99mTc on the periodic table. As a preliminary study 186Re was complexed to 20-30 kDa (n=2) and 30-50 kDa (n=1) MW-fractions and tested in dogs. The results were similar to 99mTc-PEI-MP 10-30 kDa. The biodistribution data and pharmacokinetic data were also used to do comparative dosimetry between radiopharmaceuticals. Not surprisingly, the dosimetry data confirmed the high red marrow dose for 153Sm-EDTMP and the increased soft-tissue dose of the radionuclides complexed to HEDP. The radiation dose to the tumour for all radiopharmaceuticals fell within the range of 26Gy to 44Gy. This is well within the range used to treat canine osteosarcoma using external beam radiotherapy. When compared to external beam radiotherapy, the probable lack of tumour response in our clinical trial relates to the heterogenous distribution of the radiopharmaceutical in the tumour and the inherent resistance of osteosarcoma cells to continuous low-dose radiation delivery (CLDR) inherent in radionuclide -particle decay. The study met the majority of outcomes with the exception of labelling PEI-MP with 153Sm. This was due to the interaction of the 153Sm-PEI-MP complex with citrate ions in blood. Rapid deterioration of the Rhenium-188 generator also led to earlier than expected curtailment of the 188Re-HEDP therapeutic trial although sufficient data was available to be used in a comparative study. / Thesis (PhD)--University of Pretoria, 2013. / Internal Medicine / unrestricted

Utilisés dans le traitement de nombreuses pathologies osseuses bénignes (ostéoporose) et malignes (myélome, métastases osseuses), les biphosphonates ont une action inhibitrice sur la résorption et le remodelage osseuse. Ils permettent d’augmenter la masse et la densité de minéralisation osseuse. Ils présentent une forte affinité pour l'os notamment pour les sites à fort remodelage. Les conséquences moléculaires de leur incorporation, en particulier dans les sites osseux à fort remodelage, restent encore à élucider. Ce travail se propose donc d’étudier à l’échelle moléculaire l’impact des biphosphonates sur la composition et la structure du tissu osseux à partir de deux modèles de forte accumulation : la néoformation osseuse chez le rat et l’ostéonécrose des maxillaires liée aux biphosphonates. Nos résultats indiquent que, dans ces conditions, les biphosphonates sont à l’origine de modifications importantes du contenu minéral du tissu osseux et provoquent des altérations structurales concernant les phases minérale et organique. En particulier, une hyperminéralisation est mise en évidence dans le contexte physiopathologique spécifique de l’ostéonécrose des maxillaires liée aux biphosphonates. Cette altération résulterait de processus de minéralisation secondaire avancés liés à l’inhibition du remodelage continu des maxillaires par les biphosphonates. De plus, les modifications de la structure cristalline observées dans les deux modèles témoignent d’un effet direct des biphosphonates lié à la fixation/interaction à la maille cristalline. Enfin, une perturbation des phénomènes de stabilisation de la matrice collagénique osseuse au cours de la néoformation osseuse est suggérée et peut être attribuée à un effet potentiel des biphosphonates sur les cellules ostéoblastiques. Ce travail permet ainsi d’appréhender les conséquences moléculaires d’une accumulation de biphosphonates sur la qualité osseuse. / Biphosphonates are known for their anti-resorptive effects in benign (osteoporosis) and malignant bone diseases (myeloma, bone metastasis). Their clinical properties are based on the net increase of bone mass and mineral density. Their strong affinity for biominerals allows them a targeting for specific bone remodeling sites and a prolonged storage within bone. Little is known about molecular changes upon bisphosphonates uptake in high turnover bone sites. Therefore, the aim of the present work was to examine intrinsic bone material properties in a high cumulative bisphosphonates context such as newly-formed bone and bisphosphonates-related osteonecrosis of the jaws. Our results show that bisphosphonates could be linked to the overmineralization reported in jaw bones. In addition, bisphosphonates cause ultrastructural disorders of both mineral and collagenic components. These modifications could be attributed to: i) bisphosphonates effects on the duration of mineralization processes, ii) to its mineral binding/interaction consequences, and iii) interestingly to its potential contribution on the osteoblastic function. This work contributes to clarify the molecular impacts of high cumulative bisphosphonates uptake on bone quality.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13437号 / 論医博第2236号 / 新制||医||1054(附属図書館) / (主査)教授 松田 秀一, 教授 中山 健夫, 教授 森本 尚樹 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Hormone ablation therapy is a mainstay in the treatment of breast and prostate cancers. However, aromatase inhibitors (AIs) used in postmenopausal women with breast cancer and androgen-deprivation therapy (ADT) used in men with prostate cancer contribute to substantial bone loss, thereby increasing the risk of osteoporotic fractures. Evidence-based guidelines, therefore, urge oncology practices to screen these patients for bone loss and, if needed, provide treatment to maintain bone health. In addition to lifestyle modification and calcium or vitamin D supplementation, bone protection strategies include treatment with bisphosphonates and denosumab, a monoclonal antibody against RANK ligand. Identification of patients at greater risk for bone loss and fracture and proper interventions can reduce fracture rates. Oncology nurses can play an important role in screening these patients. The purpose of this article is to inform oncology nurses about the effects of cancer treatment on bone health, review current prevention and treatment options for cancer treatment-induced bone loss, and discuss recommendations for identifying high-risk individuals.

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