京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第17859号 / 医博第3828号 / 新制||医||1000(附属図書館) / 30679 / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 秀一, 教授 佐藤 俊哉, 教授 戸口田 淳也 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

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Les lymphocytes T Vγ9Vδ2 sont impliqués dans la réponse immunitaire contre de nombreux pathogènes et contre les cellules tumorales. Ils reconnaissent des antigènes solubles, non peptidiques, ayant une faible masse moléculaire, qui sont appelés phosphoantigènes. Dans ce contexte, l’isopentenyl pyrophosphate (IPP) et un métabolite de l’ATP (ApppI) sont des phosphoantigènes qui s’accumulent dans les cellules suite à l’inhibition par les amino-bisphosphonates de l’activité d’une enzyme clé de la voie du mévalonate, la farnesyl pyrophosphate synthase (FPPS). Les bisphosphonates sont utilisés en clinique dans le traitement et la prévention des complications liées aux ostéolyses malignes. Les études pré-cliniques montrent que les bisphosphonates (et en particulier les amino-bisphosphonates) ont également une activité anti-tumorale qui peut être directe et/ou indirecte. Ils peuvent bloquer différentes fonctions des cellules tumorales (adhésion, invasion, prolifération) en inhibant l’activité de la FPPS. Ils agissent aussi sur les cellules endothéliales et inhibent l’angiogenèse tumorale. Les amino-bisphosphonates activent aussi l’activité cytotoxique des lymphocytes T Vγ9Vδ2 vis-à-vis des cellules tumorales. Les mécanismes moléculaires sous-jacents responsables de cette activation sont par contre méconnus. Les travaux réalisés dans cette thèse montrent tout d’abord que les aminobisphosphonates (zolédronate et risédronate) induisent l’accumulation d’IPP et d’ApppI dans différentes lignées humaines de cancer du sein et qu’il existe une corrélation entre le niveau de production d’IPP/ApppI et la capacité des lymphocytes T Vγ9Vδ2 à détruire ces cellules tumorales in vitro. Nous avons ensuite montré que le traitement de souris immunodéficientes NOD-SCID avec des aminobisphosphonates stimule la différenciation des lymphocytes T Vγ9Vδ2 humains à partir des cellules du sang périphérique lorsque celles-ci sont injectées par voie intrapéritonéale aux animaux. Par ailleurs, ces lymphocytes T vont ensuite venir infiltrer des tumeurs mammaires sous-cutanées qui ont été préalablement greffées chez les animaux traités. Cette infiltration lymphocytaire survient uniquement lorsque les tumeurs produisent de l’IPP/ApppI. Il en résulte alors une régression des tumeurs chez les souris NOD-SCID. L’infiltration des tumeurs par les lymphocytes T Vγ9Vδ2 humains s’explique par le fait que l’IPP et l’ApppI stimulent la migration des lymphocytes T. Les lymphocytes T Vγ9Vδ2 interagissent alors avec les cellules tumorales par le biais de mécanismes faisant intervenir ICAM-1, puis les lymphocytes sécrètent des facteurs (interféron γ, perforine) qui sont cytotoxiques pour les cellules tumorales. L’ensemble de nos travaux montre donc que les amino-bisphosphonates pourraient être utilisés en immunothérapie dans le traitement des cancers du sein. / Vγδ9Vδ2 T lymphocytes are involved in the immune response against several pathogens and tumoral cells. They recognize non-peptidic, low molecular weight soluble antigens, so called phosphoantigens. In this context, isopentenyl pyrophosphate (IPP) and a metabolite of ATP (ApppI) are phosphoantigens which accumulate in many cells following the inhibition by amino-bisphosphonates of farnesyl pyrophosphate synthase (FPPS), a key enzyme in the mevalonate pathway. Bisphosphonates are commonly used in the clinic for the treatment and prevention of complications associated with malignant osteolysis. Preclinical studies demonstrate that bisphosphonates (in particular amino-bisphosphonates) also have direct and indirect anti-tumor activity. They are able to block different functions in tumor cells, such as adhesion, invasion and proliferation, by inhibiting FPPS activity. Bisphosphonates also act on endothelial cells and inhibit tumor angiogenesis. Amino-bisphosphonates also activate the cytotoxic activity of Vγδ9Vδ2 T lymphocytes against tumor cells. However, the underlying molecular mechanisms responsible for this activation are still unknown. In the present thesis, we first demonstrate that amino-bisphosphonates (e.g. zoledronate and risedronate) induce the accumulation of IPP and ApppI in different human breast cancer cell lines and that a correlation exists between the levels of IPP/ApppI production and the capacity of Vγδ9Vδ2 T lymphocytes to destroy tumor cells in vitro. We then demonstrate that the treatment of immunodeficient NOD-SCID mice with amino-bisphosphonates stimulates the differentiation of Vγδ9Vδ2 T lymphocytes from human peripheral blood mononuclear cells injected intraperitoneally in animals. Following their expansion, the T lymphocytes are then able to infiltrate subcutaneous mammary tumors, which were grafted in treated animals. This infiltration of γδ T lymphocytes arises only when the tumors produce IPP/ApppI, resulting in the regression of tumor growth in NOD-SCID mice. The infiltration of tumors by human Vγδ9Vδ2 T lymphocytes is explained by the capacity of IPP and ApppI to stimulate the migration of γδ T lymphocytes. Vγδ9Vδ2 T lymphocytes then interact with tumor cells through a mechanism involving ICAM-I and several secretion factors (i.e. interferon γ, perforine) which are cytotoxic for tumor cells. Thus, our work demonstrates that amino-bisphosphonates could be useful for breast cancer immunotherapy.

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Cancerous solid tumors can migrate and lead to metastatic bone disease. Drugs prescribed to reduce bone resorption from metastasis, such as zoledronic acid and the RANKL-inhibitory antibody Denosumab, cause side effects such as osteonecrosis of the jaw when delivered systemically. This project used two biocompatible materials, acrylic bone cement (PMMA) and poly(lactic-co-glycolic acid) (PLGA), to incorporate and sustain release of anti-resorptive agents. Results showed similar mechanical properties for acrylic bone cements loaded up to 6.6% drug by weight. Results showed sustained zoledronic acid release for 8 weeks from both systems, with PMMA releasing up to 22% of loaded drug and PLGA films releasing over 95%. The antibody release rate was lower, with the majority of antibody still inside the PLGA films after 8 weeks. In vitro bioactivity remained above 50% for zoledronic acid eluted from both materials at early, middle, and late time points. This study sheds light on the behavior of these biocompatible polymers at high drug weight percent loadings compared to previous studies. PLGA demonstrated superior release kinetics but inferior bioactivity of eluted drug. By incorporating anti-resorptive drugs into locally implantable materials, this work could lead to a treatment offering improved quality of life for cancer patients.

Active targeting with controlled delivery of therapeutic agents to bone is an ideal approach for treatment of several bone diseases. Since bisphosphonates (BPs) are known to have high affinity to bone mineral and are being widely used in treatment of osteoporosis, they are well-suited for drug targeting to bone. For this purpose, bifunctional hydrazine-bisphosphonates (HBPs) with spacers of various lengths and lipophilicity were synthesized and studied. Crystal growth inhibition assays demonstrated that the HBPs with shorter spacers bound more strongly to bone mineral, hydroxyapatite (HA), than did alendronate. HBPs were also demonstrated to be non-toxic to MC3T3-E1 pre-osteoblasts. The targeted delivery of the HBP-conjugated model drug, 4-nitrobenzaldehyde, was demonstrated through hydrolysis of the hydrazone linkage at the low pH of bone resorption and wound healing sites. In another series of experiments, a method to orient proteins on HA surfaces was developed to improve protein bioactivity. Enhanced green fluorescent protein (EGFP) and β-lactamase were used as model proteins. These proteins have a Ser or Thr at their N-terminus, which was oxidized to obtain a single aldehyde group that was subsequently used for bonding HBPs of various length and lipophilicity through formation of a hydrazone bond. The amount of protein immobilized through various HBPs was determined and found not to be exclusively dependent on the length of HBPs. The enzymatic activity of HBP-immobilized β-lactamase, measured with cefazolin as substrate, was found to be higher than β-lactamase that was simply adsorbed on HA. In a third set of studies, HBPs were evaluated for delivering parathyroid hormone (PTH) to bone mineral to enhance cell responses for bone formation. PTH was oxidized and conjugated to HBPs, followed by targeting to bone wafers. In vitro bioassays demonstrated that HBP-targeted PTH stimulated greater synthesis of cAMP in pre-osteoblasts compared to surfaces with simply adsorbed PTH. HBPs were also found to have similar pro-apoptotic activity to widely used alendronate. Overall, HBPs can be used for drug delivery to bone and oriented immobilization of proteins and peptides, with or without anti-osteoclastic action, for a variety of applications including bone tissue engineering.

Les bisphosphonates (BPs) sont des outils thérapeutiques de choix pour le traitement de l'ostéolyse maligne. Toutefois, ils n'ont pas d'effet anti-tumoral et n'améliorent pas la survie des patients. C'est pourquoi nous avons testé leur efficacité en association avec de nouveaux agents pharmacologiques ciblant les cellules responsables de la résorption osseuse, les ostéoclastes.<br />Nous avons ainsi démontré qu'un inhibiteur de cathepsine K (CKI) réduit l'activité des ostéoclastes in vitro, et de ce fait, le développement des métastases osseuses in vivo en agissant indirectement sur les cellules tumorales. De plus, un inhibiteur de tyrosine kinase (Imatinib) ralentit la formation et la progression des métastases osseuses in vivo, en ayant une activité anti-ostéoclastique et anti-tumorale. Toutefois, bien qu'une polythérapie puisse favoriser une synergie d'action entre les médicaments, nos résultats montrent que dans nos conditions d'utilisation, aucune synergie significative entre le CKI, l'Imatinib et le BP zolédronate n'a lieu.

L’évaluation du risque en pharmacovigilance repose sur l’analyse des données issues des bases de notifications spontanées. Lorsqu’un risque est identifié, il est nécessaire de le quantifier. Le risque d’ostéonécrose de la mâchoire sous traitement antirésorptif est connu. Les données de pharmacovigilance sont donc actuellement utilisées afin de le quantifier. La définition de l’ostéonécrose de la mâchoire n’est pas unique, ni stable dans le temps. Cette définition peut donc avoir un impact sur l’estimation du risque.Les objectifs de cette thèse étaient d’évaluer l’impact de l’utilisation d’une définition standardisée sur l’évaluation du nombre de cas retenus dans une base de notifications spontanées, de décrire les notifications spontanées d’ostéonécroses de la mâchoire, et d’évaluer la précision des essais de phase III concernant la définition utilisée pour valider les ostéonécroses de la mâchoire. Pour réaliser ce travail, nous avons réalisés une analyse sur les données contenues dans la Base nationale française de Pharmacovigilance. Cette étude a été complétée par une analyse exhaustive de la littérature.La définition standardisée actuelle d’ostéonécrose de la mâchoire n’est pas adaptée aux données issues d’une base de notifications spontanées. Son utilisation entraîne une sous-estimation du nombre de cas, conduisant à une sous-évaluation du risque d’ostéonécrose de la mâchoire lors de l’utilisation de médicament antirésorptif. Il existe également un manque de transparence concernant la définition utilisée pour valider les cas d’ostéonécroses de la mâchoire, dans les rapports d’essais de phase III du dénosumab. / Risk assessment in pharmacovigilance is based on data extracted from spontaneous reporting databases. When a risk is identified, it should be quantified. Risk of osteonecrosis of the jaw under antiresorptive medications is known. Pharmacovigilance data are therefore used to quantify it. Definition for osteonecrosis of the jaw is neither unique nor stable over time. The used definition could therefore has an impact on risk assessment.Objectives for this thesis were to evaluate the impact of using a standardized definition when evaluating the number of validated cases in a spontaneous reports database, to describe notifications of osteonecrosis of the jaw, and to evaluate accuracy for the definition of osteonecrosis of the jaw during phase III trials involving denosumab. To carry out this work, we made an analysis in the French national pharmacovigilance database. This was completed by a systematic literature review.Current standardized definition for osteonecrosis of the jaw is unsuitable for spontaneous reports database. Its use lead to under-estimation of cases, leading to an under-estimated risk for osteonecrosis of the jaw under antiresorptive medication. There is also a lack of transparency with the used definition to adjudicate osteonecrosis of the jaw in phase III trials involving denosumab.

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.