Effects of Cytochrome P450 Enzyme Inhibitors on the Pharmacokinetics of Nonsteroidal Anti-inflammatory Drugs and Venlafaxine

Cytochrome P450 (CYP) enzymes play a pivotal role in the metabolism of many drugs. Inhibition of CYP enzymes usually increases the plasma concentrations of their substrate drugs and can thus alter the safety and efficacy of these drugs. The metabolism of many widely used nonsteroidal antiinflammatory drugs (NSAIDs) as well as the metabolism of the antidepressant venlafaxine is nown to be catalyzed by CYP enzymes. In the present studies, the effect of CYP inhibition on the armacokinetics and pharmacodynamics of NSAIDs and venlafaxine was studied in clinical trials with healthy volunteers and with a crossover design, by using different antifungal agents as CYP inhibitors. The results of these studies demonstrate that the inhibition of CYP enzymes leads to increased concentrations of NSAIDs. In most cases, the exposure to ibuprofen, diclofenac, etoricoxib, and meloxicam was increased 1.5to 2 fold when they were used concomitantly with antifungal agents. CYP2D6 inhibitor, terbinafine, substantially increased the concentration of parent venlafaxine, whereas the concentration of active moiety of venlafaxine (parent drug plus active metabolite) was only slightly increased. Voriconazole, an inhibitor of the minor metabolic pathway of venlafaxine, produced only minor changes in the pharmacokinetics of venlafaxine. These studies show that an evident increase in the concentrations of NSAIDs may be expected, if they are used concomitantly with CYP inhibitors. However, as NSAIDs are generally well tolerated, use of single doses of NSAIDs concomitantly with CYP inhibitors is not likely to adversely affect patient safety, whereas clinical relevance of longterm concomitant use of NSAIDs with CYP inhibitors needs further investigation. CYP2D6 inhibitors considerably affect the pharmacokinetics of venlafaxine, but the clinical significance of this interaction remains unclear.

Kipulääkkeiden käyttö kissalla : kirjallisuuskatsaus : [osa] 1 : tulehduskipulääkkeet

Summary: Use of analgesics in cats : a literature study : [part] 1 : non-steroidal anti-iflammatory drugs

Negative Regulation of Receptor Tyrosine Kinases by T-cell Protein Tyrosine Phosphatase

Protein tyrosine phosphorylation controls a wide array of cellular responses such as growth, migration, proliferation, differentiation, metabolism and cytoskeletal organisation. Tyrosine phosphorylation is a dynamic process involving the competing activities of protein tyrosine kinases and protein tyrosine phosphatases. The protein tyrosine kinases are further divided into non-receptor- and receptor tyrosine kinases. The latter are transmembrane glycoproteins activated by the binding of specific ligands, mostly growth factors, to their extracellular domain, transmitting different signals to the cell. Growth factor receptors such as the epidermal growth factor receptor, vascular endothelial growth factor receptor 2 and platelet-derived growth factor receptor β, belong to the receptor tyrosine kinases, the signalling of which is often disturbed in various diseases, including cancer. This has led to the development of receptor tyrosine kinase antagonists for use as anti-cancer drugs. As the receptor tyrosine kinases, also the protein tyrosine phosphatases can be divided into receptor- and non-receptor types. The protein tyrosine phosphatases have attained much less attention than the receptor tyrosine kinases partly because they were identified later. However, accumulating evidence shows that the protein tyrosine phosphatases have important roles as specific and active regulators of tyrosine phosphorylation in cells and of physiological processes. Consequently, the protein tyrosine phosphatases are receiving arising interest as novel drug targets. The aim of this work was to elucidate the negative regulation of receptor tyrosine kinases by one non-receptor protein tyrosine phosphatase, T-cell protein tyrosine phosphatase TCPTP. The results show that TCPTP activated by cell adhesion receptor integrin α1 functions as a negative regulator of the epidermal growth factor receptor. It was also found that TCPTP affects vascular endothelial growth factor receptor 2 signalling and angiogenesis. Lastly, a High-throughput screen with 64,280 compounds was performed to identify novel TCPTP activators, resulting in identification of one small molecule compound capable of exerting similar effects on TCPTP signalling as integrin α1. This compound is shown to downregulate signalling of epidermal growth factor receptor and platelet-derived growth factor receptor β, as well as to inhibit cell proliferation and angiogenesis. Our results suggest that a suitable small-molecule TCPTP activator could be utilized in the development of novel anti-cancer drugs.

The spindle assembly checkpoint as a drug target - Novel small-molecule inhibitors of Aurora kinases

Cell division (mitosis) is a fundamental process in the life cycle of a cell. Equal distribution of chromosomes between the daughter cells is essential for the viability and well-being of an organism: loss of fidelity of cell division is a contributing factor in human cancer and also gives rise to miscarriages and genetic birth defects. For maintaining the proper chromosome number, a cell must carefully monitor cell division in order to detect and correct mistakes before they are translated into chromosomal imbalance. For this purpose an evolutionarily conserved mechanism termed the spindle assembly checkpoint (SAC) has evolved. The SAC comprises a complex network of proteins that relay and amplify mitosis-regulating signals created by assemblages called kinetochores (KTs). Importantly, minor defects in SAC signaling can cause loss or gain of individual chromosomes (aneuploidy) which promotes tumorigenesis while complete failure of SAC results in cell death. The latter event has raised interest in discovery of low molecular weight (LMW) compounds targeting the SAC that could be developed into new anti-cancer therapeutics. In this study, we performed a cell-based, phenotypic high-throughput screen (HTS) to identify novel LMW compounds that inhibit SAC function and result in loss of cancer cell viability. Altogether, we screened 65 000 compounds and identified eight that forced the cells prematurely out of mitosis. The flavonoids fisetin and eupatorin, as well as the synthetic compounds termed SACi2 and SACi4, were characterized in more detail utilizing versatile cell-based and biochemical assays. To identify the molecular targets of these SAC-suppressing compounds, we investigated the conditions in which SAC activity became abrogated. Eupatorin, SACi2 and SACi4 preferentially abolished the tensionsensitive arm of the SAC, whereas fisetin lowered also the SAC activity evoked by lack of attachments between microtubules (MTs) and KTs. Consistent with the abrogation of SAC in response to low tension, our data indicate that all four compounds inhibited the activity of Aurora B kinase. This essential mitotic protein is required for correction of erratic MT-KT attachments, normal SAC signaling and execution of cytokinesis. Furthermore, eupatorin, SACi2 and SACi4 also inhibited Aurora A kinase that controls the centrosome maturation and separation and formation of the mitotic spindle apparatus. In line with the established profound mitotic roles of Aurora kinases, these small compounds perturbed SAC function, caused spindle abnormalities, such as multi- and monopolarity and fragmentation of centrosomes, and resulted in polyploidy due to defects in cytokinesis. Moreover, the compounds dramatically reduced viability of cancer cells. Taken together, using a cell-based HTS we were able to identify new LMW compounds targeting the SAC. We demonstrated for the first time a novel function for flavonoids as cellular inhibitors of Aurora kinases. Collectively, our data support the concept that loss of mitotic fidelity due to a non-functional SAC can reduce the viability of cancer cells, a phenomenon that may possess therapeutic value and fuel development of new anti-cancer drugs.

Identification and application of bile metabolites to assess the exposure of fish to pharmaceuticals in the environment

Thousands of tons of pharmaceuticals are consumed yearly worldwide. Due to the continuous and increasing consumption and their incomplete elimination in wastewater treatment plants (WWTP), pharmaceuticals and their metabolites can be detected in receiving waters, although at low concentrations (ng to low μg/L). As bioactive molecules the presence of pharmaceuticals in the aquatic environment must be considered potentially hazardous for the aquatic organisms. In this thesis, the biotransformation and excretion of pharmaceuticals in fish was studied. The main biotransformation pathways of three anti‐inflammatory drugs, diclofenac, naproxen and ibuprofen, in rainbow trout were glucuronidation and taurine conjugation of the parent compounds and their phase I metabolites. The same metabolites were present in fish bile in aquatic exposures as in fish dosed with intraperitoneal injection. Higher bioconcentration factor in bile (BCFbile) was found for ibuprofen when compared to diclofenac and naproxen. Laboratory exposure studies were followed by a study of uptake of pharmaceuticals in a wild fish population living in lake contaminated with WWTP effluents. Of the analyzed 17 pharmaceuticals and six phase I metabolites, only diclofenac, naproxen and ibuprofen was present in bream and roach bile. It was shown, that diclofenac, naproxen and ibuprofen excreted by the liver can be found in rainbow trout and in two native fish species living in the receiving waters. In the bream and roach bile, the concentrations of diclofenac, naproxen and ibuprofen were roughly 1000 times higher than those found in the lake water, while in the laboratory exposures, the bioconcentration of the compounds and their metabolites in rainbow trout bile were at the same level as in wild fish or an order of magnitude higher. Thus, the parent compounds and their metabolites in fish bile can be used as a reliable biomarker to monitor the exposure of fish to environmental pharmaceuticals present in water receiving discharges from WWTPs.

Methods of genetic diversity creation and functional display for directed evolution experiments

Protein engineering aims to improve the properties of enzymes and affinity reagents by genetic changes. Typical engineered properties are affinity, specificity, stability, expression, and solubility. Because proteins are complex biomolecules, the effects of specific genetic changes are seldom predictable. Consequently, a popular strategy in protein engineering is to create a library of genetic variants of the target molecule, and render the population in a selection process to sort the variants by the desired property. This technique, called directed evolution, is a central tool for trimming protein-based products used in a wide range of applications from laundry detergents to anti-cancer drugs. New methods are continuously needed to generate larger gene repertoires and compatible selection platforms to shorten the development timeline for new biochemicals. In the first study of this thesis, primer extension mutagenesis was revisited to establish higher quality gene variant libraries in Escherichia coli cells. In the second study, recombination was explored as a method to expand the number of screenable enzyme variants. A selection platform was developed to improve antigen binding fragment (Fab) display on filamentous phages in the third article and, in the fourth study, novel design concepts were tested by two differentially randomized recombinant antibody libraries. Finally, in the last study, the performance of the same antibody repertoire was compared in phage display selections as a genetic fusion to different phage capsid proteins and in different antibody formats, Fab vs. single chain variable fragment (ScFv), in order to find out the most suitable display platform for the library at hand. As a result of the studies, a novel gene library construction method, termed selective rolling circle amplification (sRCA), was developed. The method increases mutagenesis frequency close to 100% in the final library and the number of transformants over 100-fold compared to traditional primer extension mutagenesis. In the second study, Cre/loxP recombination was found to be an appropriate tool to resolve the DNA concatemer resulting from error-prone RCA (epRCA) mutagenesis into monomeric circular DNA units for higher efficiency transformation into E. coli. Library selections against antigens of various size in the fourth study demonstrated that diversity placed closer to the antigen binding site of antibodies supports generation of antibodies against haptens and peptides, whereas diversity at more peripheral locations is better suited for targeting proteins. The conclusion from a comparison of the display formats was that truncated capsid protein three (p3Δ) of filamentous phage was superior to the full-length p3 and protein nine (p9) in obtaining a high number of uniquely specific clones. Especially for digoxigenin, a difficult hapten target, the antibody repertoire as ScFv-p3Δ provided the clones with the highest affinity for binding. This thesis on the construction, design, and selection of gene variant libraries contributes to the practical know-how in directed evolution and contains useful information for scientists in the field to support their undertakings.

Diel patterns and tissue-specificity of environmental responses in fish

Humans are profoundly changing aquatic environments through climate change and the release of nutrients and chemicals. To understand the effects of these changes on natural populations, knowledge on individuals’ environmental responses is needed. At the molecular level, the environmental responses are partly mediated by chances in messenger RNA and protein levels. In this thesis I study messenger RNA and protein responses to an assortment of environmental stressors in fish. As daily (diel) rhythms are known to be ubiquitous in different tissues, I particularly focus on diel patterns in the responses. The studied species are the three-spined stickleback (Gasterosteus aculeatus L.) and the Arctic char (Salvelinus alpinus L.), both of which have circumpolar distribution in the Northern hemisphere. In the first two studies, three-spined sticklebacks were exposed to both the non-steroidal anti-inflammatory drug diclofenac and low-oxygen conditions (hypoxia), and their responses measured at separate time points in the liver and gills. The results show how the seemingly unrelated environmental stressors, hypoxia and anti-inflammatory drugs, can have harmful combined effects that differ from the effects of each stressor alone. Moreover, both stressors disturbed natural diel patterns in gene expression. In the third study, I studied the responses of three-spined sticklebacks to two test chemicals: one used in hormonal medicine (17α-ethinyl-oestradiol) and one used as a plasticizer and solvent chemical (di-n-butyl phthalate). The results suggest that the phthalate can affect genes related to spermatogenesis in fish testes, while estrogen-mimicking compounds can lead to numerous disturbances in the endocrine system. In the final study, the temperature-dependence of diel rhythms in messenger RNA levels were evaluated in the liver tissue of the Arctic char, a cold-adapted salmonid. The results show that cold acclimation repressed diel rhythms in gene expression compared to warm-acclimated fish, in which the expression of hundreds of genes was rhythmic, suggesting the circadian clock of the Arctic fish species can be sensitive to temperature. Overall, the results of the thesis indicate that fishes’ responses to abiotic factors interact with their diel rhythms, and more studies on the consequences of these interactions are needed to comprehensively understand human impacts on ecosystems.