Drug epidemiology, interactions and pharmacogenetics : A post-mortem database study

Use of adverse drug combinations, abuse of medicinal drugs and substance abuse are considerable social problems that are difficult to study. Prescription database studies might fail to incorporate factors like use of over-the-counter drugs and patient compliance, and spontaneous reporting databases suffer from underreporting. Substance abuse and smoking studies might be impeded by poor participation activity and reliability. The Forensic Toxicology Unit at the University of Helsinki is the only laboratory in Finland that performs forensic toxicology related to cause-of-death investigations comprising the analysis of over 6000 medico-legal cases yearly. The analysis repertoire covers most commonly used drugs and drugs of abuse, and the ensuing database contains also background information and information extracted from the final death certificate. In this thesis, the data stored in this comprehensive post-mortem toxicology database was combined with additional metabolite and genotype analyses that were performed to complete the profile of selected cases. The incidence of drug combinations possessing serious adverse drug interactions was generally low (0.71%), but it was notable for the two individually studied drugs, a common anticoagulant warfarin (33%) and a new generation antidepressant venlafaxine (46%). Serotonin toxicity and adverse cardiovascular effects were the most prominent possible adverse outcomes. However, the specific role of the suspected adverse drug combinations was rarely recognized in the death certificates. The frequency of bleeds was observed to be elevated when paracetamol and warfarin were used concomitantly. Pharmacogenetic factors did not play a major role in fatalities related to venlafaxine, but the presence of interacting drugs was more common in cases showing high venlafaxine concentrations. Nicotine findings in deceased young adults were roughly three times more prevalent than the smoking frequency estimation of living population. Contrary to previous studies, no difference in the proportion of suicides was observed between nicotine users and non-nicotine users. However, findings of abused substances, including abused prescription drugs, were more common in the nicotine users group than in the non-nicotine users group. The results of the thesis are important for forensic and clinical medicine, as well as for public health. The possibility of drug interactions and pharmacogenetic issues should be taken into account in cause-of-death investigations, especially in unclear cases, medical malpractice suspicions and cases where toxicological findings are scarce. Post-mortem toxicological epidemiology is a new field of research that can help to reveal problems in drug use and prescription practises.

Iäkkäiden lääkehoitoihin liittyvien riskien kartoittaminen – Arviointityökalun kehittäminen Delfoi-menetelmällä

Tutkimuksen taustalla on väestön ikääntyminen ja iäkkäiden lisääntynyt lääkkeiden käyttö. Ikääntyminen aiheuttaa elimistössä useita muutoksia, jotka voivat muuttaa lääkevastetta ja altistaa potilaan haittavaikutuksille. Iäkkäillä riski joutua sairaalaan lääkkeen haittavaikutuksen vuoksi on arvioitu olevan neljä kertaa suurempi kuin nuoremmilla. Monet lääkkeiden yhteis- ja haittavaikutukset olisivat ehkäistävissä välttämällä iäkkäillä tiettyjä lääkeaineita. Iäkkäiden lääkehoitojen seurantaa ja arviointia varten on kehitetty erilaisia suosituksia sekä Suomessa että kansainvälisesti. Tutkimuksen tavoitteena oli luoda Suomen oloihin soveltuva, hoitajien koulutettuina käytettävissä oleva, avohoidon yli 65-vuotiaiden iäkkäiden lääkitykseen liittyviä riskejä arvioiva työkalu. Työkalun avulla voitaisiin löytää ne potilaat, joiden lääkitykseen liittyy paljon riskejä. Tutkimuksen aineistona olivat laaja kansainvälinen kirjallisuuskatsaus iäkkäiden lääkehoitoihin liittyvistä tekijöistä sekä asiantuntijakommentoinnit, joiden perusteella luotiin alustava arviointityökalu. Alustava arviointityökalu validoitiin kolmikierroksisella Delfoi-menetelmällä. Delfoi-menetelmä on laadullinen konsensusmenetelmä, joka perustuu asiantuntijoiden arvioihin tutkittavasta asiasta. Kaksi ensimmäistä Delfoi-kierrosta mittasivat alustavan arviointityökalun kohtien soveltuvuutta ja kolmas kierros kohtien tärkeyttä arvioitaessa iäkkään lääkityksiin liittyviä riskejä. Tutkimuksen Delfoi-kierroksien vastaajiksi pyydettiin 33 geriatrian asiantuntijaa, joista vastaajiksi lupautui 11 lääkäriä, 3 proviisoria ja 4 sairaanhoitajaa. Delfoi-kierrosten tuloksia analysoitiin sekä kvantitatiivisesti että kvalitatiivisesti. Tutkimuksen tuloksena saatu arviointityökalu käsittää 19 iäkkäiden lääkehoitoa arvioivaa kohtaa. Asiantuntijapaneelin mukaan kaikki arviointityökalun lääkitykseen liittyviä riskejä mittaavat kohdat ovat tärkeitä tai jokseenkin tärkeitä, joten työkalun voidaan olettaa olevan validi mitattaessa lääkitykseen liittyviä riskejä. Jatkotutkimuksissa työkalun käytettävyyttä sekä riskien mittaamiskykyä tulee testata sekä arviointityökalun käyttäjien että potilaiden keskuudessa. Kehitettyä työkalua voidaan jatkossa hyödyntää esimerkiksi koulutustarkoituksissa sen varsinaisen käyttötarkoituksen lisäksi. Työkalun avulla iäkkäiden lääkehoitoa voidaan tulevaisuudessa toteuttaa entistä turvallisemmin ja tarkoituksenmukaisemmin.

Anti-TNF treatment in juvenile idiopathic arthritis and associated uveitis : Clinical perspectives on growth, uveitis, and drug survival

Juvenile idiopathic arthritis (JIA) is a heterogeneous group of childhood chronic arthritides, associated with chronic uveitis in 20% of cases. For JIA patients responding inadequately to conventional disease-modifying anti-rheumatic drugs (DMARDs), biologic therapies, anti-tumor necrosis factor (anti-TNF) agents are available. In this retrospective multicenter study, 258 JIA-patients refractory to DMARDs and receiving biologic agents during 1999-2007 were included. Prior to initiation of anti-TNFs, growth velocity of 71 patients was delayed in 75% and normal in 25%. Those with delayed growth demonstrated a significant increase in growth velocity after initiation of anti-TNFs. Increase in growth rate was unrelated to pubertal growth spurt. No change was observed in skeletal maturation before and after anti-TNFs. The strongest predictor of change in growth velocity was growth rate prior to anti-TNFs. Change in inflammatory activity remained a significant predictor even after decrease in glucocorticoids was taken into account. In JIA-associated uveitis, impact of two first-line biologic agents, etanercept and infliximab, and second-line or third-line anti-TNF agent, adalimumab, was evaluated. In 108 refractory JIA patients receiving etanercept or infliximab, uveitis occurred in 45 (42%). Uveitis improved in 14 (31%), no change was observed in 14 (31%), and in 17 (38%) uveitis worsened. Uveitis improved more frequently (p=0.047) and frequency of annual uveitis flares was lower (p=0.015) in those on infliximab than in those on etanercept. In 20 patients taking adalimumab, 19 (95%) had previously failed etanercept and/or infliximab. In 7 patients (35%) uveitis improved, in one (5%) worsened, and in 12 (60%) no change occurred. Those with improved uveitis were younger and had shorter disease duration. Serious adverse events (AEs) or side-effects were not observed. Adalimumab was effective also in arthritis. Long-term drug survival (i.e. continuation rate on drug) with etanercept (n=105) vs. infliximab (n=104) was at 24 months 68% vs. 68%, and at 48 months 61% vs. 48% (p=0.194 in log-rank analysis). First-line anti-TNF agent was discontinued either due to inefficacy (etanercept 28% vs. infliximab 20%, p=0.445), AEs (7% vs. 22%, p=0.002), or inactive disease (10% vs. 16%, p=0.068). Females, patients with systemic JIA (sJIA), and those taking infliximab as the first therapy were at higher risk for treatment discontinuation. One-third switched to the second anti-TNF agent, which was discontinued less often than the first. In conclusion, in refractory JIA anti-TNFs induced enhanced growth velocity. Four-year treatment survival was comparable between etanercept and infliximab, and switching from first-line to second-line agent a reasonable therapeutic option. During anti-TNF treatment, one-third with JIA-associated anterior uveitis improved.

External signal-activated liposomal drug delivery systems

Modern drug discovery gives rise to a great number of potential new therapeutic agents, but in some cases the efficient treatment of patient may not be achieved because the delivery of active compounds to the target site is insufficient. Thus, drug delivery is one of the major challenges in current pharmaceutical research. Numerous nanoparticle-based drug carriers, e.g. liposomes, have been developed for enhanced drug delivery and targeting. Drug targeting may enhance the efficiency of the treatment and, importantly, reduce unwanted side effects by decreasing drug distribution to non-target tissues. Liposomes are biocompatible lipid-based carriers that have been studied for drug delivery during the last 40 years. They can be functionalized with targeting ligands and sensing materials for triggered activation. In this study, various external signal-assisted liposomal delivery systems were developed. Signals can be used to modulate drug permeation or release from the liposome formulation, and they provide accurate control of time, place and rate of activation. The study involved three types of signals that were used to trigger drug permeation and release: electricity, heat and light. Electrical stimulus was utilized to enhance the permeation of liposomal DNA across the skin. Liposome/DNA complex-mediated transfections were performed in tight rat epidermal cell model. Various transfection media and current intensities were tested, and transfection efficiency was evaluated non-invasively by monitoring the concentration of secreted reporter protein in cell culture medium. Liposome/DNA complexes produced gene expression, but electrical stimulus did not enhance the transfection efficiency significantly. Heat-sensitive liposomal drug delivery system was developed by coating liposomes with biodegradable and thermosensitive poly(N-(2-hydroxypropyl) methacrylamide-mono/dilactate polymer. Temperature-triggered liposome aggregation and contents release from liposomes were evaluated. The cloud point temperature (CP) of the polymer was set to 42 °C. Polymer-coated liposome aggregation and contents release were observed above CP of the polymer, while non-coated liposomes remained intact. Polymer precipitates above its CP and interacts with liposomal bilayers. It is likely that this induces permeabilization of the liposomal membrane and contents release. Light-sensitivity was introduced to liposomes by incorporation of small (< 5 nm) gold nanoparticles. Hydrophobic and hydrophilic gold nanoparticles were embedded in thermosensitive liposomes, and contents release was investigated upon UV light exposure. UV light-induced lipid phase transitions were examined with small angle X-ray scattering, and light-triggered contents release was shown also in human retinal pigment epithelial cell line. Gold nanoparticles absorb light energy and transfer it into heat, which induces phase transitions in liposomes and triggers the contents release. In conclusion, external signal-activated liposomes offer an advanced platform for numerous applications in drug delivery, particularly in the localized drug delivery. Drug release may be localized to the target site with triggering stimulus that results in better therapeutic response and less adverse effects. Triggering signal and mechanism of activation can be selected according to a specific application.

Levosimendan in patients with ischaemic heart disease

Levosimendan is a drug developed for the treatment of heart failure. Its mechanism of action includes calcium sensitization of contractile proteins and the opening of ATP-sensitive potassium channels. The combination of positive inotropy with possible anti-ischaemic effects via potassium channel opening may offer benefits in comparison with currently available intravenous inotropes, which are contraindicated in patients with ongoing myocardial ischaemia. The active levosimendan metabolite OR-1896 significantly prolongs the duration of the haemodynamic effects of levosimendan. The aims of the present study were to investigate: 1) the clinical effects and safety of intravenous and oral levosimendan and 2) the pharmacodynamics and pharmacokinetics of intravenous and oral levosimendan and its metabolites in patients with ischaemic heart disease. Levosimendan was administered intravenously or orally in four studies to 557 patients with ischaemic heart disease with or without concomitant heart failure. One study included patients with acute myocardial infarction, while the other three studies included stable ischaemic patients. Non-invasive haemodynamic measurements were used in all studies, and blood samples for pharmacokinetics were drawn in three studies. Safety was followed by ECG recordings, adverse event inquiries and laboratory assessments. Intravenous levosimendan, administered as a 6-hour infusion did not cause clinically significant hypotension or ischaemia in comparison with placebo and reduced worsening heart failure and short- and long-term mortality. Increase in incidence of hypotension and ischaemia was seen only with the highest dose (0.4 µg/kg/min). Both intravenous and oral levosimendan possessed a moderate positive inotropic effect. Vasodilatory effect was more pronounced with intravenous levosimendan. A chronotropic effect was seen in all studies; however, it was not accompanied by any increase in arrhythmic events. The formation of levosimendan metabolites after oral dosing increased linearly with the daily dose of the parent drug, leading to increased inotropic and chronotropic response. Levosimendan was well tolerated in all studies. In conclusion, levosimendan was safe and effective in the treatment of patients with acute or chronic ischaemia. The risk-benefit ratio of intravenous levosimendan is favourable up to the dose of 0.2 µg/kg/min. The daily dose of oral levosimendan in patients with ischaemic heart failure should not exceed 4 mg due to an increase in chronotropic response.

Inhibition of CYP1A2-mediated drug metabolism in vitro and in humans : With special emphasis on rofecoxib and other NSAIDs

The cytochrome P450 1A2 (CYP1A2) is one of the major metabolizing enzymes. The muscle relaxant tizanidine is a selective substrate of CYP1A2, and the non-steroidal anti-inflammatory drug (NSAID) rofecoxib was thought to modestly in-hibit it. Cases suggesting an interaction between tizanidine and rofecoxib had been reported, but the mechanism was unknown. Also other NSAIDs are often used in combination with muscle relaxants. The aims of this study were to investigate the effect of rofecoxib, several other NSAIDs and female sex steroids on CYP1A2 ac-tivity in vitro and in vivo, and to evaluate the predictability of in vivo inhibition based on in vitro data. In vitro, the effect of several NSAIDs, female sex steroids and model inhibitors on CYP1A2 activity was studied in human liver microsomes, without and with preincubation. In placebo controlled, cross-over studies healthy volunteers ingested a single dose of tizanidine after a pretreament with the inhibitor (rofecoxib, tolfenamic acid or celecoxib) or placebo. Plasma (and urine) concentrations of tizanidine and its metabolites were measured, and the pharmacodynamic effects were recorded. A caffeine test was also performed. In vitro, fluvoxamine, tolfenamic acid, mefenamic acid and rofecoxib potently in-hibited CYP1A2. Ethinylestradiol, celecoxib, desogestrel and zolmitriptan were moderate, and etodolac, ciprofloxacin, etoricoxib and gestodene were weak inhibi-tors of CYP1A2. At 100 µM, other tested NSAIDs and steroids inhibited CYP1A2 less than 35%. Rofecoxib was found to be a mechanism-based inhibitor of CYP1A2. In vivo, rofecoxib greatly increased the plasma concentrations (over ten-fold) and the pharmacodynamic effects of tizanidine. Also the metabolism of caf-feine was impaired by rofecoxib. Despite the relatively strong in vitro CYP1A2 inhibitory effects, tolfenamic acid and celecoxib did not have a significant effect on tizanidine and caffeine concentrations in humans. Competitive inhibition model and the free plasma concentration of the inhibitor predicted well the effect of fluvoxam-ine and the lack of effect of tolfenamic acid and celecoxib on tizanidine concentra-tions in humans, and mechanism-based inhibition model explained the effects of rofecoxib. However, the effects of ciprofloxacin and oral contraceptives were un-derestimated from the in vitro data. Rofecoxib is a potent mechanism-based inhibitor of CYP1A2 in vitro and in vivo. This mechanism may be involved in the adverse cardiovascular effects of rofecoxib. Tolfenamic acid and celecoxib seem to be safe in combination with tizanidine, but mefenamic acid might have some effect on tizanidine concentrations in vivo. Con-sidering the mechanism of inhibition, and using the free plasma concentration of the inhibitor, many but not all CYP1A2 interactions can be predicted from in vitro data.