Background Middle ear (intratympanic; IT) administration is a promising therapeutic method as it offers the possibility of achieving high inner ear drug concentrations while maintaining low levels in the systemic compartment, thus minimizing the risk of systemic side-effects and drug-drug interactions. The issue of premature elimination through the Eustachian tube may be reduced by stabilizing drug solutions with a hydrogel. However, this raises the secondary issue of conductive hearing loss. Aim This study aimed to investigate the properties of a chitosan-based particulate hydrogel formulation when used as a drug carrier for IT administration in an in vivo model of ototoxicity. Material and methods Two particulate chitosan-based IT delivery systems, Thio-25 and Thio-40, were investigated in albino guinea pigs (n=94). Both contained the hearing protecting drug candidate sodium thiosulfate but differed in terms of the concentration of chitosan gel particles (25% versus 40%). The safety of the two systems was explored in vivo. The most promising system was thereafter investigated in guinea pigs subjected to a single intravenous injection of the anticancer drug cisplatin (8 mg/kg b.w.), which has ototoxic side-effects. Hearing status was evaluated with acoustically-evoked frequency-specific auditory brainstem response (ABR) and hair cell counting. Finally, in vivo magnetic resonance imaging (MRI) was used to study the distribution and elimination of the chitosan-based system from the middle ear cavity; this was analyzed and compared to a hyaluronan-based system. Results Both chitosan-based IT delivery systems caused ABR threshold elevations (p<0.05) that remained after 10 days (p<0.05) without evidence of hair cell loss, although the elevations induced by Thio-25 were significantly lower than Thio-40 (p<0.05). Thio-25 significantly reduced cisplatin-induced ABR threshold elevations (p<0.05) and the loss of outer hair cells (p<0.05). IT injection of the chitosan- and hyaluronan-based systems filled up most of the middle ear space. There were no significant differences in terms of the distribution and elimination of the two systems. Conclusion Particulate chitosan is a promising drug carrier for IT administration. Future studies should attempt to demonstrate whether the physical properties of this technique allow for a reduced injection volume and thereby a less pronounced loss of conductive hearing.
The knowledge of the three-dimensional structures of proteins and polypeptides is essential to understand their functions. The work shown in this thesis has two objectives. The first one is to develop a new analytical method based on maximum entropy (ME) theory to analyze NMR experimental data such as NOEs and J-couplings in order to reconstitute φ,ψ Ramachandran plots of flexible biomolecules. Two model systems have been used, the flexible polypeptide motilin and the disaccharide α-D-Mannosep-(1-2)-α-D-Mannosep-O-Me (M2M). The experimental data was defined as constraints that were combined with prior information (priors) which were the φ,ψ distributions obtained from either a coil library, the Protein DataBank or Molecular Dynamics Simulations. ME theory was utilized to formulate φ,ψ distributions (posteriors) that are least committed to the priors and in full agreement with the experimental data. Reparamerization of the Karplus relation was necessary to obtain realistic distributions for the M2M. Clear structural propensities were found in motilin with a nascent α-helix in the central part (residues Y7-E17), a left handed 31 helix in the C-terminus (R18-G21) and an extended conformation in the N-terminus. The contribution of each residue to the thermodynamic entropy (segmental entropy) was calculated from the posteriors and compared favorably to the segmental entropies estimated from 15N-relaxation data. For M2M the dominating conformation of the glycosidic linkage was found to be at φH=-40° ψH=33°, which is governed by the exo-anomeric effect. Another minor conformation with a negative ψH angle was discovered in M2M. The ratio between both populations is about 3:1. The second part of the thesis is a structural study of a DNA-binding protein, the C repressor of the P2 bacteriophage (P2 C). P2 C represses the lytic genes of the P2 bacteriophage, thereby directing the P2 lifecycle toward the lysogenic lifemode. The crystal and solution structures of P2 C have been solved by X-ray crystallography and NMR, respectively. Both structures revealed a homodimeric protein with five rigid α-helices made up by residues 5-66 and a β-strand conformation in residues 69-76 in each monomer. 15N-relaxation data showed that the C-terminus (residues 85-99) is highly flexible and fully unstructured. A model representing the P2 C-DNA complex was built based on the structure and available biochemical data. In the model, P2 C binds DNA cooperatively and two homodimeric P2 C molecules are close enough to interact and bind one direct DNA repeat each.
<b><i>Background:</i></b> Neuroinflammation triggered by infection or trauma is the cause of central nervous system dysfunction. High-mobility group box 1 protein (HMGB1), released from stressed and dying brain cells, is a potent neuroinflammatory mediator. The proinflammatory functions of HMGB1 are tightly regulated by post-translational redox modifications, and we here investigated detailed neuroinflammatory responses induced by the individual redox isoforms. <b><i>Methods:</i></b> Male Dark Agouti rats received a stereotactic injection of saline, lipopolysaccharide, disulfide HMGB1, or fully reduced HMGB1, and were accessed for blood-brain barrier modifications using magnetic resonance imaging (MRI) and inflammatory responses by immunohistochemistry. <b><i>Results and Conclusions:</i></b> Significant blood-brain barrier disruption appeared 24 h after injection of lipopolysaccharide, disulfide HMGB1, or fully reduced HMGB1 compared to controls, as assessed in post-gadolinium T1-weighted MRI images and confirmed by increased uptake of FITC-conjugated dextran. Immunohistochemistry revealed that both HMGB1 isoforms also induced a local production of IL-1β. Additionally, disulfide HMGB1 increased major histocompatibility complex class II expression and apoptosis. Together, the results demonstrate that extracellular, cerebral HMGB1 causes significant blood-brain barrier disruption in a redox-independent manner and activates several components of neuroinflammation. Blocking HMGB1 might potentially improve clinical outcome in conditions such as stroke and traumatic brain injury.
Hypothesis: Furosemide alters the permeability of the intrastrial fluid–blood barrier. Background: The cochlear sensory cells are protected by the blood-perilymph and intrastrial fluid–blood barriers, which hinder substances, including gadolinium-based contrast agents (GdCAs), to enter the endolymphatic space. High-dose furosemide causes transient shift of hearing thresholds and morphological changes in stria vascularis. Furosemide is also known to enhance drug-induced ototoxicity. Methods: Furosemide (400 mg/kg b.w.) was injected i.v. in Balb/C mice (n = 20). Twenty minutes later, the GdCA gadobutrol, gadopentetic acid, or gadoteric acid was injected i.v. The distribution of GdCA to the perilymphatic and endolymphatic spaces was studied with MRI (9.4 T) for 250 minutes. Results: The perilymphatic and endolymphatic spaces were signal-enhanced in all animals. Gadopentetic acid and gadoteric acid yielded similar signal enhancement in all three scalae, while gadobutrol yielded significantly higher enhancement in scala tympani than scala media ( p = 0.043) and scala vestibuli ( p = 0.043). The signal enhancement reached a plateau but did not decrease during the time of observation. Conclusion: Treatment with a high dose of furosemide before injection of a GdCA resulted in enhancement of the MRI signal in the endolymphatic space as well as the perilymphatic space, which supports our hypothesis that furosemide alters the permeability of the intrastrial fluid–blood barrier.
The trans-vessel wall device (TW-device) is a new endovascular tool for precise and safe delivery of various payloads (cells, viral, modified RNA, chemotherapy, growth factors) in oncology and regenerative medicine. The twofold aim of this study was to assess cell engraftment and tumor growth using the TW-device for endovascular transplantation and to evaluate its ability to directly access solid tumors. We used the VX2 model in the rabbit kidney to compare percutaneously implanted fresh VX2 cells with TW-device injections of cryopreserved VX2 cells. We demonstrated the feasibility of endovascular transplantation (n = 7) of tumor cells, achieving a 57.1% engraftment rate despite cryopreservation, comparable with 70% for percutaneous delivery of fresh cells (n = 10). Re-access using the TW-device was 100% successful (n = 11) with super-selective intratumoral contrast administration without complications. In conclusion, endovascular transplantation of VX2 cells using the TW-device resulted in proliferating cell grafts in the rabbit kidney establishing functional proof that cells indeed survive handling, preparation, and device passage. We also show the TW-device is able to access solid tumor parenchyma allowing precise intraparenchymal administration.This proof-of-concept study open up possibilities for repeated direct parenchymal injections via the endovascular route in any hard to reach organ.
