The Role of Cytochrome P450 3A Inducers and Inhibitors in the Metabolism and the Effects of Oxycodone

Oxycodone is an opioid used in the treatment of moderate or severe pain. It is principally metabolized in the liver by cytochrome P450 3A (CYP3A) enzymes whereas approximately 10% is metabolized by CYP2D6. Little is known about the interactions between oxycodone and other drugs, herbals and nutritional substances. In this work the effects of CYP3A inducers rifampicin and St. John’s wort and CYP3A inhibitors voriconazole, grapefruit juice, ritonavir and lopinavir/ritonavir were investigated on the pharmacokinetics and pharmacodynamics of oxycodone. All studies were randomized, balanced, placebo-controlled crossover clinical studies in healthy volunteers. The plasma concentrations of oxycodone and its metabolites were determined for 48 hours and pharmacodynamic parameters were recorded for 12 hours in each study. Pharmacokinetic parameters were calculated by noncompartmental methods. Rifampicin decreased the plasma concentrations, analgesic effects, and oral bioavailability of oral oxycodone. St. John’s wort reduced the concentrations of oxycodone and diminished the self-reported drug effect. Voriconazole increased the exposure to oral oxycodone by 3.6-fold whereas grapefruit juice, which inhibits predominantly the intestinal CYP3A, elevated the mean concentrations of oxycodone by 1.7-fold. Ritonavir and lopinavir/ritonavir increased the mean AUC of oxycodone by 3.0- and 2.6-fold, respectively, and prolonged its elimination half-life. In spite of increased oxycodone plasma concentrations during concomitant administration of CYP3A inhibitors, the analgesic effects were not increased. These studies show that the induction or inhibition of CYP3A alters the pharmacokinetics and pharmacologic effects of oxycodone. The exposure to oxycodone decreased after induction and increased after inhibition of CYP3A. As a conclusion, the clinicians should avoid concomitant administration of CYP3A inducers or inhibitors and oral oxycodone. If this is not possible, they should be prepared to interactions leading to impaired analgesia after CYP3A inducers or increased adverse effects after CYP3A inhibitors and oral oxycodone.

Observations on the Pharmacokinetics and Pharmacodynamics of Dexmedetomidine. Clinical Studies on Healthy Volunteers and Intensive Care Patients

Patients treated in intensive care units require sedation and analgesia. However, sedative drugs also have potential adverse effects, and there is no single ideal sedativeanalgesic drug for these patients. Dexmedetomidine is an apha2-adrenoceptor agonist licenced for sedation of intensive care patients and patients undergoing surgery and other invasive procedures. Several routes of parenteral administration (intravenous, intramuscular, subcutaneous and intranasal) have been utilized. In the present series of studies, the pharmacokinetics and pharmacodynamics of intranasally administered dexmedetomidine as well as the gastrointestinal effects of intravenous dexmedetomidine were determined in healthy volunteers. Pharmacokinetics of dexmedetomidine during long lasting, high-dose infusions were characterized in intensive care patients. The bioavailability of intranasal dexmedetomidine was relatively good (65%), but interindividual variation was large. Dexmedetomidine significantly inhibited gastric emptying and gastrointestinal transit. In intensive care patients, the elimination half-life of dexmedetomidine was somewhat longer than reported for infusions of shorter duration and in less ill patients or healthy volunteers. Dexmedetomidine appeared to have linear pharmacokinetics up to the studied dose rate of 2.5 μg/kg/h. Dexmedetomidine clearance was decreasing with age and its volume of distribution was increased in hypoalbuminaemic patients, resulting in a longer elimination half-life and context-sensitive half-time. Intranasally administered dexmedetomidine was efficacious and well tolerated, making it appropriate for clinical situations requiring light sedation. The clinical significance of the gastrointestinal inhibitory effects of dexmedetomidine should be further evaluated in intensive care patients. The possibility of potentially altered potency and effect duration should be taken into account when administering dexmedetomidine to elderly or hypoalbuminaemic patients.

Differential Regulation of c-FLIP Isoforms Through Post-translational Modifications

Cells are constantly responding to signals from the surrounding tissues and the environment. To dispose of infected and potentially dangerous cells, to ensure the optimal execution of developmental processes and to maintain tissue homeostasis, a multicellular organism needs to tightly control both the number and the quality of its cells. Apoptosis is a form of active cellular self-destruction that enables an organism to regulate its cell number by deleting damaged or potentially dangerous cells. Apoptosis can be induced by death ligands, which bind to death receptors on the cell surface. Ligation of the receptors leads to the formation of an intracellular death inducing signaling complex (DISC). One of the DISC components is caspase-8, a protease that triggers the caspase cascade and is thereby a key initiator of programmed cell death. The activation of caspase-8 is controlled by the cellular FLICE-inhibitory proteins (c-FLIPs). Consequently, sensitivity towards receptor-mediated apoptosis is determined by the amount of c-FLIP, and the c-FLIP levels are actively regulated for example during erythroid differentiation of K562 erythroleukemia cells and by hyperthermia in Jurkat leukemia cells. The aim of my thesis was to investigate how c-FLIP is regulated during these processes. We found that c-FLIP isoforms are short-lived proteins, although c-FLIPS had an even shorter half-life than c-FLIPL. In both experimental models, increased death receptor sensitivity correlated with induced ubiquitylation and consequent proteasomal degradation of c-FLIP. Furthermore, we elucidated how phosphorylation regulates the biological functions and the turnover of c-FLIP, thereby contributing to death receptor sensitivity. We mapped the first phosphorylation sites on c-FLIP and dissected how their phosphorylation affects c-FLIP. Moreover, we demonstrated that phosphorylation of serine 193, a phosphorylated residue common to all c-FLIPs, is primarily mediated by the classical PKC. Furthermore, we discovered a novel connection between the phosphorylation and ubiquitylation of c-FLIP: phosphorylation of S193 protects c-FLIP from ubiquitylation. Surprisingly, although all c-FLIP isoforms are phosphorylated on this conserved residue, the biological outcome is different for the long and short isoforms, since S193 specifically prolongs the half-lives of the short c-FLIP isoforms, but not c-FLIPL. To summarize, we show that c-FLIP proteins are modified by ubiquitylation and phosphorylation, and that the biological outcomes of these modifications are isoform-specifically determined.

Kloorifenoleja sisältävien jätevesien AOP-käsittely

Kirjallisuustyössä tutkittiin tehostetun hapetuksen menetelmiä (engl. Advanced Oxidation Processes, AOPs) kloorifenolien käsittelyssä. Tutkittava aine valittiin US EPA:n (United States Environmental Protection Agency) ympäristölle haitallisten aineiden listalta. Työssä tutkitut AOP-menetelmät olivat otsonointi kasvatetussa pH:ssa, O3/H2O2-prosessi, fotolyyttinen otsonointi (O3/UV), H2O2/UV-prosessi ja Fenton-prosessi (H2O2+Fe2+). AOP-käsittelyssä OH-radikaalien oletetaan pääosin aiheuttavan epäpuhtauksien hapettumisen. Kirjallisuustyössä tutkittiin OH-radikaaleihin vaikuttavien parametrien, kuten pH:n, lämpötilan sekä hapettimien ja hapetettavan aineen konsentraatioiden vaikutusta kloorifenolien hapetusprosessissa. Työn tarkoituksena oli selvittää tehokkain AOP-käsittely kloorifenoleja sisältävien jätevesien käsittelylle. AOP-käsittelyjen tehokkuutta verrattiin hajoamisnopeusvakioiden, puoliintumisaikojen sekä hapettimen kemikaalikulutuksen ja kustannuksen perusteella. Fenton-prosessin ja otsonoinnin pH:ssa 9 todettiin olevan tehokkaimpia menetelmiä kloorifenolien hapetuksessa. Fenton-prosessin hapetusnopeus oli tehokkaampaa 4-CP:n ja 2,4-DCP:n hapetuksessa, kun taas otsonointi pH:ssa 9 hapetti nopeammin 2,3,4,6-TeCP:n ja 2,4,6-TCP:n. Kustannustehokkuuden perusteella Fenton-prosessi oli otsonointia tehokkaampi. Parhaan menetelmän valinta kloorifenoleiden poistamiseksi oli vaikeaa, sillä useissa tutkimuksissa oli tutkittu vain yhtä menetelmää. Lisäksi eri tutkimuksissa käytetyt prosessiolosuhteet olivat erilaiset, joka hankaloitti tutkimusten vertailua. Lopullinen AOP-menetelmän valinta tulisikin suorittaa vasta laboratoriotutkimusten jälkeen.

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.