Enhanced pharmacological efficacy of sumatriptan due to modification of its physicochemical properties by inclusion in selected cyclodextrins

The study focused on the pharmacological action of sumatriptan, in particular its antiallodynic and antihyperalgesic properties, as an effect of cyclodextrinic inclusion of sumatriptan, resulting in changes of its physicochemical qualities such as dissolution and permeability through artificial biological membranes, which had previously been examined in vitro in a gastro-intestinal model. The inclusion of sumatriptan into β-cyclodextrin and 2-hydroxylpropylo-β-cyclodextrin by kneading was confirmed with the use of spectral (fourier-transform infrared spectroscopy (FT-IR); solid state nuclear magnetic resonance spectroscopy with magic angle spinning condition, 1H and 13C MAS NMR) and thermal (differential scanning calorimetry (DSC)) methods. A precise indication of the domains of sumatriptan responsible for its interaction with cyclodextrin cavities was possible due to a theoretical approach to the analysis of experimental spectra. A high-performance liquid chromatography with a diode-array detector method (HPLC-DAD) was employed to determine changes in the concentration of sumatriptan during dissolution and permeability experiments. The inclusion of sumatriptan in complex with cyclodextrins was found to significantly modify its dissolution profiles by increasing the concentration of sumatriptan in complexed form in an acceptor solution compared to in its free form. Following complexation, sumatriptan manifested an enhanced ability to permeate through artificial biological membranes in a gastro-intestinal model for both cyclodextrins at all pH values. As a consequence of the greater permeability of sumatriptan and its increased dissolution from the complexes, an improved pharmacological response was observed when cyclodextrin complexes were applied

Enhanced separation in ambient mass spectrometry imaging:towards quantification of pharmaceuticals

In the pharmaceutical industry, the development and application of good separation methods is important to study the distribution and the effect of a drug candidate. Mass spectrometry imaging is a technique that is often applied to study the distribution of a drug candidate. This technique is not always able to separate the drug candidate from other molecules that are present in the sample. Therefore, we need better separation techniques in addition to mass spectrometry imaging. This PhD research investigates novel technological developments as possible tools for the pharmaceutical industry to use. The techniques presented in this thesis allow for better separation of molecules that are structurally alike. Because these techniques separate the drug candidate better from other molecules in the sample, their addition to mass spectrometry imaging is used for quantification of two drug candidates in the last chapter of this thesis

Human Antimicrobial Peptides and Proteins

As the key components of innate immunity, human host defense antimicrobial peptides and proteins (AMPs) play a critical role in warding off invading microbial pathogens. In addition, AMPs can possess other biological functions such as apoptosis, wound healing, and immune modulation. This article provides an overview on the identification, activity, 3D structure, and mechanism of action of human AMPs selected from the antimicrobial peptide database. Over 100 such peptides have been identified from a variety of tissues and epithelial surfaces, including skin, eyes, ears, mouths, gut, immune, nervous and urinary systems. These peptides vary from 10 to 150 amino acids with a net charge between −3 and +20 and a hydrophobic content below 60%. The sequence diversity enables human AMPs to adopt various 3D structures and to attack pathogens by different mechanisms. While α-defensin HD-6 can self-assemble on the bacterial surface into nanonets to entangle bacteria, both HNP-1 and β-defensin hBD-3 are able to block cell wall biosynthesis by binding to lipid II. Lysozyme is well-characterized to cleave bacterial cell wall polysaccharides but can also kill bacteria by a non-catalytic mechanism. The two hydrophobic domains in the long amphipathic α-helix of human cathelicidin LL-37 lays the basis for binding and disrupting the curved anionic bacterial membrane surfaces by forming pores or via the carpet model. Furthermore, dermcidin may serve as ion channel by forming a long helix-bundle structure. In addition, the C-type lectin RegIIIα can initially recognize bacterial peptidoglycans followed by pore formation in the membrane. Finally, histatin 5 and GAPDH(2-32) can enter microbial cells to exert their effects. It appears that granulysin enters cells and kills intracellular pathogens with the aid of pore-forming perforin. This arsenal of human defense proteins not only keeps us healthy but also inspires the development of a new generation of personalized medicine to combat drug-resistant superbugs, fungi, viruses, parasites, or cancer. Alternatively, multiple factors (e.g., albumin, arginine, butyrate, calcium, cyclic AMP, isoleucine, short-chain fatty acids, UV B light, vitamin D, and zinc) are able to induce the expression of antimicrobial peptides, opening new avenues to the development of anti-infectious drugs

Electrochemistry meets mass spectrometry:a combined analytical platform for characterization of novel gut microbiota-produced metabolites in fecal samples

The human gut microbiota is a microbial community inhabiting the human gastrointestinal tract, which has the capacity to influence host health and disease. One of the ways of doing so is through the microbial metabolization of food components and drugs and the subsequent production of bioactive metabolites. Gut microbiota-produced metabolites play an important role in several disorders, including brain disorders such as Attention-Deficit/Hyperactivity Disorder (ADHD). Thus, the study of fecal metabolites is emerging as a powerful tool to identify microbiota-based non-invasive biomarkers. In this thesis, mass spectrometry-based methods were employed to analyze the possible interference of gut microbiota metabolization with the psychostimulant drug, and the main treatment available for ADHD, methylphenidate (MPH). The results showed how MPH is spontaneously degraded in conditions resembling its site of absorption, independent of the presence or absence of gut microbiota. Furthermore, the thesis proposes a combined liquid chromatography-electrochemical detection-mass spectroscopy platform for the analysis of the fecal metabolome. This optimized approach may prove to be a significant improvement in the field, which remains limited by the challenges of metabolite identification

Complex interaction of sensory and motor signs and symptoms in chronic CRPS.

Spontaneous pain, hyperalgesia as well as sensory abnormalities, autonomic, trophic, and motor disturbances are key features of Complex Regional Pain Syndrome (CRPS). This study was conceived to comprehensively characterize the interaction of these symptoms in 118 patients with chronic upper limb CRPS (duration of disease: 43±23 months). Disease-related stress, depression, and the degree of accompanying motor disability were likewise assessed. Stress and depression were measured by Posttraumatic Stress Symptoms Score and Center for Epidemiological Studies Depression Test. Motor disability of the affected hand was determined by Sequential Occupational Dexterity Assessment and Michigan Hand Questionnaire. Sensory changes were assessed by Quantitative Sensory Testing according to the standards of the German Research Network on Neuropathic Pain. Almost two-thirds of all patients exhibited spontaneous pain at rest. Hand force as well as hand motor function were found to be substantially impaired. Results of Quantitative Sensory Testing revealed a distinct pattern of generalized bilateral sensory loss and hyperalgesia, most prominently to blunt pressure. Patients reported substantial motor complaints confirmed by the objective motor disability testings. Interestingly, patients displayed clinically relevant levels of stress and depression. We conclude that chronic CRPS is characterized by a combination of ongoing pain, pain-related disability, stress and depression, potentially triggered by peripheral nerve/tissue damage and ensuing sensory loss. In order to consolidate the different dimensions of disturbances in chronic CRPS, we developed a model based on interaction analysis suggesting a complex hierarchical interaction of peripheral (injury/sensory loss) and central factors (pain/disability/stress/depression) predicting motor dysfunction and hyperalgesia

Multivalently binding antagonists for the neurokinin-1 receptor

The goal of this thesis was to investigate the neurokinin-1 receptor (NK1R) as a potential target site for multivalent receptor blockade. Since the NK1R is peripherally expressed on various cell types of the human body and its expression increases when an immune response occurs, this receptor seems to offer great potential for selective and specific antagonistic surface receptor targeting with a minimizing effect of inflammation and pain. In the experiments described in Chapter 3, it was found that the inflammatory factor IL-1β has a direct influence on NK1R expression levels in human U87 MG glioblastoma and MDA-MB-231 breast cancer cell lines and primary bovine chondrocytes in 2D cell culture. In addition, it could be demonstrated in 2D cell cultures that IL-1β and substance P together contribute to the regulation of the chondrocytes´ surrounding extracellular matrix (ECM) behavior by the selective regulation of matrix-metalloproteinases´ (MMPs) gene expression. In this context, it could be shown that MMP-13 in particular is regulated in a time and concentration-dependent manner by substance P and can be antagonized by the specific NK1R antagonist spantide I, which allows to assume that this intracellular signaling pathway is triggered via NK1Rs. This aspect is intriguing since the literature also mentions that MMP-13 has an impact on the progression of arthritis. In Chapter 4, fluorescent PEGylated quantum dots (QDs) were used for ligand coupling to the surface of nanoparticles and to study their interactions with NK1R positive cells. The introduction of thiol groups into cysteine-free peptide ligands is a common strategy for coupling well water-soluble ligands to maleimide functionalized nanoparticles such as QDs. In nanoparticle uptake experiments with receptor positive CHO-NK1R cells, a high unspecific nanoparticle binding for amino-PEG modified QDs was examined. However, in FACS displacement experiments with high concentrations of free competing antagonists it was shown that nanoparticle binding was inhibited. This indicated receptor mediated nanoparticle binding. Besides PEGylated QDs, branched 8-arm PEGs were used in Chapter 5 for multivalent cell interaction studies. In contrast to QDs, PEGs are classified as nontoxic biomaterials; additionally, they do not interfere with luminescence-based calcium assays. It was shown that there is a gain in affinity for 8armPEG-20k-spantide I due to multivalent receptor binding. In Chapter 6, a small molecular weight antagonist, aprepitant, was modified to make it amenable for further PEG coupling, either to branched PEGs or PEG-coated nanoparticles. It could be shown that chemical modification of the triazole group of aprepitant is possible by using a strong deprotonation reagent and a tert-Butyl-(3-bromopropyl)carbamate as an alkyl linker with Bocprotected amine functionality. Another new strategy for site-specific multivalent nanoparticle targeting is presented in the final chapter, Chapter 7. This strategy is based on an enzyme driven activation mechanism of ligands which are immobilized on the surface of nanoparticles. For these studies, the ACE driven angiotensin I to angiotensin II conversion was used and the successful conversion was checked by AT1 receptor binding studies. The results of these experiments have shown that enzymatically processed angiotensin I coated nanoparticles are able to selectively bind to AT1R positive mesangial cells