Indiana University-Purdue University Indianapolis (IUPUI) / The biodegradation of the hybrid layer of dental restorations is due in part to the degradation of the demineralized collagen by matrix metalloproteinases (MMPs). During the bonding procedure, phosphoric acid/acidic primers activate MMPs that degrade denuded type I collagen. As a result, the hybrid layer loses its integrity overtime, leading to the failure of the resin composite restoration. This study aimed to evaluate doxycycline (DOX) for its effects on preventing the degradation of the hybrid layer through the modification of the dental adhesive with aluminosilicate clay nanotubes (HNT) loaded with doxycycline. Doxycycline was encapsulated into HNT at three distinct concentrations (10%, 20% and 30% DOX, w/v). The increases in the concentration of doxycycline significantly increased the amount of doxycycline that was encapsulated into HNT and the drug loading into the HNT. Conversely, the encapsulation efficiency was significantly decreased with the increases in concentration of doxycycline. The modified adhesives were fabricated by incorporation of DOX-encapsulated HNT into a commercially available dental adhesive (Adper Scotchbond Multi-Purpose, SBMP). The degree of conversion (DC), Knoop microhardness, doxycycline release profiles, the biological activity (antibacterial and anti-MMP activity), and cytocompatibility of the modified adhesives were investigated. There were no statistically significant differences (p > 0.05) in DC and Knoop microhardness compared to the control (SBMP). None of the adhesive eluates was cytotoxic to the human dental pulp stem cells. Although higher concentrations of doxycycline led to a higher release of doxycycline from the modified adhesives, the differences were not significant (p = 0.259) among the groups (10%, 20% and 30% DOX). A significant growth inhibition of S. mutans and L. casei by direct contact illustrated successful encapsulation of doxycycline into the modified adhesives. Doxycycline released from the modified adhesives did not inhibit the growth of both cariogenic bacteria but inhibited MMP-1 activity. The results suggested that subantimicrobial levels of doxycycline were gradually released. The immediate microtensile bond strengths were not significantly different from those of the control (SBMP), suggesting no negative effect of doxycycline on dentin bonding (only 10% DOX were investigated). The long-term resin-dentin bond durability should be evaluated.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18316号 / 農博第2041号 / 新制||農||1020(附属図書館) / 学位論文||H26||N4823(農学部図書室) / 31174 / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 保川 清, 教授 安達 修二, 教授 伏木 亨 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19017号 / 農博第2095号 / 新制||農||1029(附属図書館) / 学位論文||H27||N4899(農学部図書室) / 31968 / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 保川 清, 教授 安達 修二, 教授 入江 一浩 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19587号 / 医博第4094号 / 新制||医||1014(附属図書館) / 32623 / 京都大学大学院医学研究科医学専攻 / (主査)教授 松原 和夫, 教授 妹尾 浩, 教授 浅野 雅秀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Despite the development of therapeutic interventions to prevent mortality following myocardial infarction (MI), there is a significant long-term risk of developing heart failure (HF). Injectable collagen hydrogels have demonstrated considerable promise as a therapeutic solution to reduce adverse ventricle remodeling associated with the development of HF post-MI. Matrix metalloproteinases (MMPs) are proteolytic enzymes involved in the degradation of the structural components of the extracellular matrix (ECM). The activation of MMPs following MI is an essential step in the cardiac repair process. However, uncontrolled enzymatic activity during this time has been associated with the formation of adverse fibrosis. Given the role of the proteases in tissue remodeling, MMPs may be a potential biomarker to predict the development of HF. This thesis work seeks to examine the effect of a novel hydrogel matrix therapy on cardiac tissue post-MI using broad-spectrum MMP-targeted radiotracer, [18F]BR3531. In Study 1, serial positron emission tomography (PET) imaging was performed to elucidate the spatial and temporal binding of [18F]BR351 post-MI using a murine model. Imaging was performed by administering [18F]BR351 at time points corresponding with periods of peak MMP activation post-MI. In vivo PET imaging and in vitro autoradiography demonstrated decreased [18F]BR351 binding in the infarct region. In Study 2, the model was used to evaluate the efficacy of a therapeutic collagen hydrogel to attenuate tissue remodeling. The groups that received the matrix treatment exhibited improved [18F]BR351 uptake in the infarct region. However, conflicting results between in vivo imaging and in vitro autoradiography, and immunohistochemistry using MMP2 and MMP9 indicate that [18F]BR351 may not be suited for MMP imaging in mouse models of MI.

Methotrexate (MTX) is an antimetabolite anticancer agent that is used in treatment of multiple cancers, such as acute lymphoblastic leukaemia and osteosarcoma. A lack of selective tumour toxicity is one of the major problems associated with MTX chemotherapy, especially when given at high doses, as in high dose MTX (HDMTX) therapy. MTX causes various toxicity problems including life-threatening nephrotoxicity, haematological toxicity and neurotoxicity. Overcoming this toxicity is of great importance and has been attempted in various ways, not least via the design of prodrugs. The concept of tumour protease, and specifically matrix metalloproteinase (MMP), activated prodrugs was the focus of the work described in this thesis. This concept relies upon attachment of an MMP-sensitive peptide sequence to a specific site in a drug structure, so as to inactive it. The activity of the parent drug is restored once it is activated by the MMPs in the tumour microenvironment. In this work, different MMP-sensitive peptide sequences linked to MTX were synthesised, resulting in 63 MTX prodrugs. The MMP-mediated activation of these conjugates in tumour tissues (specifically HT1080 homogenates) ex vivo was assessed and the results were compared to the activation of these conjugates in various normal tissues specifically liver, kidney and lung. Specific criteria were established for the selection of promising conjugates for more detailed study. From 7 promising compounds, compound 75 was identified as the lead prodrug, demonstrating selective MMP activation, as indicated by inhibition of its activation by broad spectrum MMP inhibitor ilomastat. The pharmacokinetics of compound 75 was studied in tumour (HT1080) xenograft-bearing mice and the results were compared to those obtained from administration of equimolar doses of conventional MTX. Compound 75 led to enhanced tumour concentrations of MTX, with reduced exposure to normal tissues in vivo compared to conventional MTX therapy. Furthermore, the efficacy of equimolar doses of compound 75 and directly dosed MTX in reduction of HT1080 volume were compared. Superior antitumour activity was observed with compound 75 compared to MTX treatment. Compound 75 is the first example of an MMP-activated prodrug to be reported with enhanced therapeutic index, as evidenced by a full in vivo pharmacokinetic analysis and normal tissue metabolism data. The data presented in thesis support the concept of MMP-activated prodrug development, and form a strong foundation upon which to develop a clinically-useful MTX prodrug, with the potential to enhance efficacy and reduce toxicity to the patient. / Libyan government / The full text will be available after the extended embargo: 5th March 2027

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Several studies have reported that administration of cannabinoid receptor agonists in inflammatory/autoimmune and CNS injury models resulted in significant attenuation of clinical disease. The beneficial effects correlated with the observed reduction of inflammatory mediators and peripheral immune cell infiltration into the site of inflammation. Previous studies from our laboratories demonstrated that administration of cannabinoid type 2 receptor agonist attenuated disease score and improved recovery in two murine models of neuroinflammation; spinal cord injury (SCI) and experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis. The goal of the current investigation was to evaluate the mechanisms through which administration of selective cannabinoid-2 receptor (CB2R) agonists modify inflammatory responses and help to improve function in SCI and EAE. In SCI, an acute neuroinflammatory disorder, administration of CB2R agonist at 1 h and 24 h following contusion injury to the cord resulted in improved recovery of motor function and bladder function (the ability to spontaneously void) compared to control animals. Evaluation of inflammatory mediators at 48h demonstrated a dramatic reduction in the expression of the chemokines CXCL9, 10, 11 and cytokines IL-23 and its receptor in CB2R agonist-treated cords. There was also a reduction in the expression of toll-like receptors (TLR1, TLR4, TLR6, and TLR7), which correlated with a decreased number of immunoreactive microglia. Interestingly, at seven days post injury, CB2R agonist-treated injured cords showed a significant reduction in both hematopoietic and myeloid cell infiltration. In EAE, a chronic neuroinflammatory disorder, our laboratories demonstrated previously that administration of a CB2R agonist led to lower disease scores and improved recovery. In this study, we observed reduced numbers of infiltrating hematopoietic and myeloid cells into the spinal cord and brain of CB2 agonist-treated mice. This reduction was observed at the peak of disease (day 17) and the effect was maintained at the chronic stage of disease (day 30). Evaluation of molecules associated with cell migration showed decreased levels of the adhesion molecule VCAM-1 and matrix metalloproteinases MMP-2 and 9 at peak of EAE in treated mice. The decrease in VCAM-1 correlates with our previous observation of decreased leukocyte rolling and adhesion to brain microvasculature. However, the reduction in MMP-2/9 expression suggests that CB2R agonists may also affect leukocyte transmigration into the perivascular space and further infiltration into the CNS parenchyma. This process requires both chemokine cues and the gelatinases MMP2/9. Animals deficient in these MMPs show leukocyte accumulation in the perivascular space and are resistant to EAE. There are no reports in the literature on possible CB2R agonist effects on gelatinases in myeloid cells. Although both MMP-2 and -9 are produced by antigen-presenting cells and act on similar substrates, MMP-9 appears to play a crucial role in EAE. Therefore, we decided to examine the effects of CB2 signaling on MMP-9 expression in myeloid cells, focusing on myeloid bone marrow-derived dendritic cells (BMDC). Activation of bone marrow-derived macrophages, dendritic cells, and primary microglia with the cytokine cocktail TNFα, IL-1ß, IL-6, containing PGE2, which mimicked an inflammatory milieu, resulted in expression of high levels of MMP-9. Treatment with CB2R agonists reduced MMP-9 in all three cell types. Since migration of DC to various sites is required for their activation and for the initiation of adaptive immune responses, we evaluated the effects of CB2R agonists on migration. The reduced levels of MMP-9 correlated with reduced migration of DC to the draining lymph nodes in vivo, as well as reduced migration vitro in the matrigel migration assay. The effect on MMP-9 expression was mediated through CB2R, resulting in reduction in cAMP levels, subsequent decrease in ERK activation, and reduced binding of c-Fos and c-Jun to the AP-1 site in the MMP-9 promoter. We postulate that, by dampening production of MMP-9 and subsequent MMP-9-dependent DC migration, cannabinoids contribute to resolve acute inflammation and to reestablish homeostasis. Selective CB2R agonists might be valuable future therapeutic agents for the treatment of chronic inflammatory conditions by targeting activated immune cells including DC. / Physiology