RESUMEN
Non-invasive visualisation of the expression of hypoxia-related proteins, such as carbonic anhydrase IX (CA IX), by positron emission tomography (PET) could provide important information on the oxygenation status of tumours. Since betulinic acid derivatives bind specifically to CA IX the aim of the study was the development betulinic acid-based 68Ga-labelled PET tracers and to evaluate the hypoxia detecting properties in vitro and in vivo. The binding of betulinic acid (B-DOTA) and betulinyl-3-sulfamate (BS-DOTA) was assessed in two rat tumour cell lines (AT1 prostate and Walker-256 mammary carcinomas). AT1 cells express CA IX in a hypoxia-dependent manner whereas Walker-256 cells, expressing almost no CA IX in wildtype, were transfected with the rat Car9 gene. In vivo measurements were carried out in a small animal PET/CT in AT1 tumours in rats breathing room air, 8% or 100% O2. In AT1 cells hypoxia-induced overexpression of CA IX led to a stronger binding of BS-DOTA but not of B-DOTA. The BS-DOTA binding correlated linearly with the CA IX protein expression and could be blocked by an excess of unlabelled tracer. In the transfected Walker-256 cells no specific binding of either of the tracers was seen. In vivo the intratumoral accumulation of BS-DOTA was increased in animals kept under inspiratory hypoxia and reduced by hyperoxia. Therefore, betulinyl-3-sulfamate could be used as a PET tracer of CA IX expression in tumours and to provide information about the oxygenation status. However, accumulation data indicated that binding not only depends on hypoxia-induce CA IX expression but also on the tumour-line-specific basal expression and on the initial oxygenation status of the tumour.
Asunto(s)
Ácido Betulínico , Tomografía Computarizada por Tomografía de Emisión de Positrones , Masculino , Animales , Ratas , Anhidrasa Carbónica IX/genética , Anhidrasa Carbónica IX/metabolismo , Tomografía de Emisión de Positrones/métodos , Antígenos de Neoplasias/metabolismo , Hipoxia/diagnóstico por imagenRESUMEN
The effective treatment of diseases of the inner ear is currently an unmet medical need. Local controlled drug delivery to the cochlea is challenging due to the hidden location, small volume and high sensitivity of this organ. A local intracochlear delivery of drugs would avoid the problems of intratympanic (extracochlear) drug application, but is more invasive. The requirements for such a delivery system include a small size and appropriate flexibility. The delivery device must be rigid enough for surgical handling but also flexible to avoid traumatizing cochlear structures. We developed biodegradable dexamethasone loaded PLGA extrudates for the controlled intracochlear release. In order to achieve the desired flexibility, Polyethylene glycol (PEG) was used as a plasticizer. In addition to the drug release, the extrudates were characterized in vitro by differential scanning calorimetry (DSC) and texture analysis. Simulation of the pharmacokinetics of the inner ear support the expectation that a constant perilymph drug level is obtained after few hours and retained over several weeks. Ex vivo implantation of the extrudates into a guinea pig cochlea indicate that PEG containing extrudates have the desired balance between mechanical strength and flexibility for direct implantation into the cochlea. The location of the implant was visualized by computer tomography. In summary, we postulate that intracochlear administration of drug releasing biodegradable implants is a new and promising approach to achieve local drug delivery to the cochlea for an extended time.
RESUMEN
The tumor microenvironment is characterized by hypoxia, acidosis as well as other metabolic and biochemical alterations. Its role in cancer progression is increasingly appreciated especially on invasive capacity and the formation of metastasis. The effect of acidosis on metastasis formation of two rat carcinoma cell lines was studied in the animal model. In order to analyze the pH dependency of different steps of metastasis formation, invasiveness, cell adhesion and migration of AT-1 prostate cancer cells as well as possible underlying cell signaling pathways were studied in vitro. Acidosis significantly increased the formation of lung metastases of both tumor cell lines in vivo. In vitro, extracellular acidosis neither enhanced invasiveness nor affected cell adhesion to a plastic or to an endothelial layer. However, cellular motility was markedly elevated at pH 6.6 and this effect was sustained even when extracellular pH was switched back to pH 7.4. When analyzing the underlying mechanism, a prominent role of ROS in the induction of migration was observed. Signaling through the MAP kinases ERK1/2 and p38 as well as Src family kinases was not involved. Thus, cancer cells in an acidic microenvironment can acquire enhanced motility, which is sustained even if the tumor cells leave their acidic microenvironment e.g. by entering the blood stream. This increase depended on elevated ROS production and may contribute to the augmented formation of metastases of acidosis-primed tumor cells in vivo.