Korvikealkoholien ja lääkkeiden aiheuttamien myrkytyskuolemien analysointi : painopiste aineenvaihduntatuotteissa

In Finland, 800-900 deaths are caused by fatal poisonings due to drugs or alcohols annually. Of these, around ten are caused by toxic alcohols, mainly methanol and ethylene glycol, and 400-500 are caused by drugs, both medicinal and illicit. Despite the fact that the majority of fatal poisonings are caused by relatively few individual compounds, toxicological laboratories must be capable of identifying and quantifying a wide range of possible toxicants. Small sample volumes and post-mortem changes impose special demands on both the laboratory investigation and the interpretation of results. The two main objectives of the present thesis are, first, to develop new analytical laboratory methods for toxic alcohols and drugs and, second, to apply these methods to post-mortem toxicology cases in order to generate reference data on lethal concentrations of especially metabolites for interpretation purposes. Metabolites of toxic substances constitute an interpretive resource that is far from fully exploited in post-mortem toxicology. The therapeutic, toxic, and lethal concentrations of alcohols and drugs are usually expressed as the concentration of the parent compound in blood. Yet the toxicity of methanol and ethylene glycol, for example, is mainly caused by their toxic metabolites and many therapeutic drugs are transformed to metabolites that possess pharmacological activity similar to or greater than that of the parent drug. A method for the quantitative analysis of ethylene glycol, glycolic acid, and formic acid and for the screening of volatile and hydroxylic organic compounds was developed, using headspace in-tube extraction gas chromatography-mass spectrometry. In fatal poisonings by methanol or ethylene glycol, the concentration of the parent alcohol varied significantly, whereas the concentrations of formic acid and glycolic acid in blood were found to be more uniform. Fatal metabolite threshold concentrations were established for those poisoning cases that had not received hospital treatment. In cases involving putrefaction, significant post-mortem production of formic acid was detected, and consequently the concentrations of metabolites should always be interpreted together with those of the parent alcohols. A comprehensive method for the quantitative monitoring of over 150 basic drugs and metabolites in blood samples was developed utilizing ultra-high performance liquid chromatography coupled with two consecutive detectors: diode array detector and corona charged aerosol detector (UHPLC-DAD-CAD). Based on the universal response of CAD, the method was evaluated for the quantification of drug metabolites using a secondary calibration method with the corresponding parent drug as a calibration standard. This approach offers a straightforward way of quantifying especially N-demethylated metabolites. It was found that the metabolite to parent ratios of certain toxicologically relevant drugs in post-mortem blood samples were generally comparable to the standard ratios defined in clinical therapeutic drug monitoring. With the highest post-mortem parent drug concentrations, the ratios were below the clinical normal ranges, suggesting acute ingestion or poisoning. These findings encourage forensic toxicologists to actively use the metabolite to parent ratio in the interpretation of post-mortem toxicology results.Alkoholien ja lääkeaineiden aiheuttamiin myrkytyksiin kuolee Suomessa vuosittain noin 800 900 henkeä. Näistä niin kutsuttujen korvikealkoholien, pääasiassa metanolin ja etyleeniglykolin, aiheuttamia on noin 10 ja lääkeaineiden ja huumeiden aiheuttamia 400 500. Vaikka valtaosa myrkytyksistä on muutaman aineen aiheuttamia, täytyy laboratorioilla olla valmius tunnistaa ja määrittää suuri määrä mahdollisesti myrkyllisiä aineita pienestä näytemäärästä. Tämän väitöstutkimuksen ensimmäinen tavoite oli kehittää uusia analyysimenetelmiä haihtuvien aineiden, korvikealkoholien ja emäksisten lääkeaineiden sekä niiden aineenvaihduntatuotteiden eli metaboliittien määrittämiseksi biologista näytteistä. Toisena tavoitteena oli kerätä tietoa erityisesti metaboliittien pitoisuuksista ruumiinavauksissa otetuissa näytteissä ja tutkia niiden merkitystä tulosten tulkinnassa. On yleisesti tiedossa, että korvikealkoholien myrkyllisyys johtuu pääasiassa niiden happamista metaboliiteista, muurahaishaposta ja glykolihaposta. Monien lääkeaineiden metaboliittien vaikutus on lähes yhtä suurta tai suurempaa kuin lääkeaineella itsellään. Tiedetään myös, että joidenkin lääkeaineiden pitoisuudet voivat muuttua merkittävästi kuoleman jälkeen. Tästä huolimatta alkoholien ja lääkeaineiden terapeuttisten ja myrkyllisten pitoisuuksien viitearvoina käytetään aineen itsensä pitoisuutta veri- tai seeruminäytteessä. Tässä tutkimuksessa kehitetyllä kaasukromatografisella menetelmällä pystytään määrittämään etyleeniglykoli, glykolihappo ja muurahaishappo sekä tunnistamaan haihtuvia aineita. Nestekromatografisella menetelmällä voidaan määrittää yli 150 emäksistä lääkeainetta ja metaboliittia verestä samanaikaisesti. Lisäksi menetelmällä voidaan määrittää joitain metaboliitteja ilman vertailuaineita hyödyntäen vastaavaa lääkeainetta. Korvikealkoholien aiheuttamissa myrkytyskuolemissa metanolin ja etyleeniglykolin pitoisuuksien havaittiin vaihtelevan merkittävästi. Sen sijaan metaboliittien pitoisuudet veressä olivat yhtenäisempiä ja saavuttivat raja-arvon jota voidaan pitää tappavana. Lisäksi havaittiin, että kuolemanjälkeiset muutokset nostavat muurahaishapon pitoisuutta verinäytteissä. Näiden tulosten perusteella kuolinsyynselvityksessä olisi parasta käyttää korvikealkoholin ja sen metaboliitin pitoisuuksia yhdessä. Lääkeaineiden metaboliittien ja lääkeaineiden pitoisuuksien suhteiden huomattiin vastaavan kliinisesti määritettyjä suhteita myös silloin kun lääkeaineen pitoisuus ylitti terapeuttisen alueen. Vain kaikista isoimmilla lääkeainepitoisuuksilla suhde alitti kliiniset raja-arvot, viitaten akuuttiin käyttöön tai yliannostukseen. Tämä tulos kannustaa hyödyntämään metaboliitin ja lääkeaineen pitoisuuksien suhdetta toksikologisten tulosten tulkinnassa

Febuxostat, But Not Allopurinol, Markedly Raises the Plasma Concentrations of the Breast Cancer Resistance Protein Substrate Rosuvastatin

Xanthine oxidase inhibitors febuxostat and allopurinol are commonly used in the treatment of gout. Febuxostat inhibits the breast cancer resistance protein (BCRP) in vitro. Rosuvastatin is a BCRP substrate and genetic variability in BCRP markedly affects rosuvastatin pharmacokinetics. In this study, we investigated possible effects of febuxostat and allopurinol on rosuvastatin pharmacokinetics. In a randomized crossover study with 3 phases, 10 healthy volunteers ingested once daily placebo for 7 days, 300 mg allopurinol for 7 days, or placebo for 3 days, followed by 120 mg febuxostat for 4 days, and a single 10 mg dose of rosuvastatin on day 6. Febuxostat increased the peak plasma concentration and area under the plasma concentration-time curve of rosuvastatin 2.1-fold (90% confidence interval 1.8-2.6; P = 5 x 10(-5)) and 1.9-fold (1.5-2.5; P = 0.001), but had no effect on rosuvastatin half-life or renal clearance. Allopurinol, on the other hand, did not affect rosuvastatin pharmacokinetics. In vitro, febuxostat inhibited the ATP-dependent uptake of rosuvastatin into BCRP-overexpressing membrane vesicles with a half-maximal inhibitory concentration of 0.35 mu M, whereas allopurinol showed no inhibition with concentrations up to 200 mu M. Taken together, the results suggest that febuxostat increases rosuvastatin exposure by inhibiting its BCRP-mediated efflux in the small intestine. Febuxostat may, therefore, serve as a useful index inhibitor of BCRP in drug-drug interaction studies in humans. Moreover, concomitant use of febuxostat may increase the exposure to BCRP substrate drugs and, thus, the risk of dose-dependent adverse effects.Peer reviewe

Comparative Hepatic and Intestinal Metabolism and Pharmacodynamics of Statins

The study aimed to comprehensively investigate the in vitro metabolism of statins. The metabolism of clinically relevant concentrations of atorvastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin, and their metabolites were investigated using human liver microsomes (HLMs), human intestine microsomes (HIMs), liver cytosol, and recombinant cytochrome P450 enzymes. We also determined the inhibitory effects of statin acids on their pharmacological target, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. In HLMs, statin lactones were metabolized to a much higher extent than their acid forms. Atorvastatin lactone and simvastatin (lactone) showed extensive metabolism [intrinsic clearance (CLint) values of 3700 and 7400 mu l/min per milligram], whereas the metabolism of the lactones of 2-hydroxyatorvastatin, 4-hydroxyatorvastatin, and pitavastatin was slower (CLint 20-840 mu l/min per milligram). The acids had CLint values in the range SIGNIFICANCE STATEMENT The present comparison of the in vitro metabolic and pharmacodynamic properties of atorvastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin and their metabolites using unified methodology provides a strong basis for further application. Together with in vitro drug transporter and clinical data, the present findings are applicable for use in comparative systems pharmacology modeling to predict the pharmacokinetics and pharmacological effects of statins at different dosages.Peer reviewe

Pulmonary administration of a dry powder formulation of the antifibrotic drug tilorone reduces silica-induced lung fibrosis in mice

The aim of this work was to study the antifibrotic effect of pulmonary administration of tilorone to lung fibrosis. L-leucine coated tilorone particles were prepared and their aerosolization properties were analyzed using two dry powder inhalers (Easyhaler and Twister). In addition, the biological activity and cell monolayer permeation was tested. The antifibrotic effect of tilorone delivered by oropharyngeal aspiration was studied in vivo using a silica-induced model of pulmonary fibrosis in mice in a preventive setting. When delivered from the Easyhaler in an inhalation simulator, the emitted dose and fine particle fraction were independent from the pressure applied and showed dose repeatability. However, with Twister the aerosolization was pressure-dependent indicating poor compatibility between the device and the formulation. The formulation showed more consistent permeation through a differentiated Calu-3 cell monolayer compared to pristine tilorone. Tilorone decreased the histological fibrosis score in vivo in systemic and local administration, but only systemic administration decreased the mRNA expression of type I collagen. The difference was hypothesized to result from 40-fold higher drug concentration in tissue samples in the systemic administration group. These results show that tilorone can be formulated as inhalable dry powder and has potential as an oral and inhalable antifibrotic drug.Peer reviewe

A comprehensive pharmacogenomic study indicates roles for SLCO1B1, ABCG2 and SLCO2B1 in rosuvastatin pharmacokinetics

AimsThe aim was to comprehensively investigate the effects of genetic variability on the pharmacokinetics of rosuvastatin.MethodsWe conducted a genome-wide association study and candidate gene analyses of single dose rosuvastatin pharmacokinetics in a prospective study (n = 159) and a cohort of previously published studies (n = 88).ResultsIn a genome-wide association meta-analysis of the prospective study and the cohort of previously published studies, the SLCO1B1 c.521 T > C (rs4149056) single nucleotide variation (SNV) associated with increased area under the plasma concentration–time curve (AUC) and peak plasma concentration of rosuvastatin (P = 1.8 × 10−12 and P = 3.2 × 10−15). The candidate gene analysis suggested that the ABCG2 c.421C > A (rs2231142) SNV associates with increased rosuvastatin AUC (P = .0079), while the SLCO1B1 c.388A > G (rs2306283) and SLCO2B1 c.1457C > T (rs2306168) SNVs associate with decreased rosuvastatin AUC (P = .0041 and P = .0076). Based on SLCO1B1 genotypes, we stratified the participants into poor, decreased, normal, increased and highly increased organic anion transporting polypeptide (OATP) 1B1 function groups. The OATP1B1 poor function phenotype associated with 2.1-fold (90% confidence interval 1.6–2.8, P = 4.69 × 10−5) increased AUC of rosuvastatin, whereas the OATP1B1 highly increased function phenotype associated with a 44% (16–62%; P = .019) decreased rosuvastatin AUC. The ABCG2 c.421A/A genotype associated with 2.2-fold (1.5–3.0; P = 2.6 × 10−4) increased AUC of rosuvastatin. The SLCO2B1 c.1457C/T genotype associated with 28% decreased rosuvastatin AUC (11–42%; P = .01).ConclusionThese data suggest roles for SLCO1B1, ABCG2 and SLCO2B1 in rosuvastatin pharmacokinetics. Poor SLCO1B1 or ABCG2 function genotypes may increase the risk of rosuvastatin-induced myotoxicity. Reduced doses of rosuvastatin are advisable for patients with these genotypes.</p

"Se rauhoittaa, sinne saa purettua ne tunteet" Leijonaemot: Blogikirjoittamisen merkitys kirjoittajalle

Opinnäytetyön tavoitteena oli selvittää blogikirjoittamisen merkitystä kirjoittajalle. Opinnäytetyön työelämäkumppanina oli Leijonaemot ry ja tutkimuksen kohteena oli Leijonaemojen blogi. Leijonaemot ry on vertaistukea tarjoava yhdistys erityistä tukea tarvitsevan lapsen vanhemmille. Tutkimukseen osallistui kuusi blogikirjoittajaa. Teoriaosuudessa tarkasteltiin Leijonaemot-yhdistystä, blogikirjoittamista, erityistä tukea tarvitsevaa lasta sekä vertaistukea sen eri muodoissa. Lisäksi teoriassa esiteltiin myös lainsäädäntöä liittyen blogin kirjoittamiseen ja erityislapsiperheisiin. Aineisto tutkimusta varten kerättiin haastattelemalla sähköpostin välityksellä kuutta blogikirjoittajaa. Haastateltaville annettiin kirjoituspyyntö ja viisi haastattelukysymystä, jotka rajasivat kirjoittamista. Tutkimus oli kvalitatiivinen ja se analysoitiin teemoittelua käyttämällä. Tutkimuksessa todettiin, että Leijonaemojen blogin kirjoittamisella haluttiin ottaa kantaa yhteiskunnallisesti sekä vaikuttaa ja tuoda esiin asioita, jotka koskevat erityislapsiperheitä. Kirjoittamista pidettiin myös tärkeänä itselle. Kirjoittamisen kuvattiin toimivan terapiamuotona, jonka avulla omia ajatuksia voi jäsentää. Kirjoittaminen selkiyttää myös omia tunteita.The aim for this thesis was to find out the meaning of blog writing for the writer. This thesis collaborated with Leijonaemot association (Lion mothers) and the target of this survey was Leijonaemot blog. Leijonaemot association offers peer support for parents of children with special needs. Six different blog writers participated in this survey. The theory part examined the Leijonaemot association, blog writing, children with special needs and peer support in different forms. The theoretical background also introduced legislation concerning blog writing and families who have children with special needs. Data for the survey were collected by interviewing six blog writers via email. The blog writers were asked to answer five questions, which delimited the writing process. The survey was qualitative and the results were analyzed by means of a thematic analysis. Based on the survey, the Leijonaemo blog writers wanted to address social issues, influence and adduce things that pertain to families who have children with special needs. Writing a blog is important for the writers themselves. Writing is like therapy, which helps to structure one’s thoughts. It also clarifies your feelings

An automated cocktail method for in vitro assessment of direct and time-dependent inhibition of nine major cytochrome P450 enzymes-application to establishing CYP2C8 inhibitor selectivity

We developed an in vitro high-throughput cocktail assay with nine major drug-metabolizing CYP enzymes, optimized for screening of time-dependent inhibition. The method was applied to determine the selectivity of the time-dependent CYP2C8 inhibitors gemfibrozil 1-O-beta-glucuronide and clopidogrel acyl-beta-D-glucuronide. In vitro incubations with CYP selective probe substrates and pooled human liver microsomes were conducted in 96-well plates with automated liquid handler techniques and metabolite concentrations were measured with quantitative UHPLC-MS/MS analysis. After determination of inter-substrate interactions and Km values for each reaction, probe substrates were divided into cocktails I (tacrine/CYP1A2, bupropion/CYP2B6, amodiaquine/CYP2C8, tolbutamide/CYP2C9 and midazolam/CYP3A4/5) and II (coumarin/CYP2A6, S-mephenytoin/CYP2C19, dextromethorphan/CYP2D6 and astemizole/CYP2J2). Time-dependent inhibitors (furafylline/CYP1A2, selegiline/ CYP2A6, clopidogrel/CYP2B6, gemfibrozil 1-O-beta-glucuronide/CYP2C8, tienilic acid/CYP2C9, ticlopidine/ CYP2C19, paroxetine/CYP2D6 and ritonavir/CYP3A) and direct inhibitor (terfenadine/CYP2J2) showed similar inhibition with single substrate and cocktail methods. Established time-dependent inhibitors caused IC50 fold shifts ranging from 2.2 to 30 with the cocktail method. Under time-dependent inhibition conditions, gemfibrozil 1-O-beta-glucuronide was a strong (90% inhibition) and selective (<< 20% inhibition of other CYPs) inhibitor of CYP2C8 at concentrations ranging from 60 to 300 mu M, while the selectivity of clopidogrel acyl-beta-D-glucuronide was limited at concentrations above its IC80 for CYP2C8. The time-dependent IC50 values of these glucuronides for CYP2C8 were 8.1 and 38 mu M, respectively. In conclusion, a reliable cocktail method including the nine most important drug-metabolizing CYP enzymes was developed, optimized and validated for detecting timedependent inhibition. Moreover, gemfibrozil 1-O-beta-glucuronide was established as a selective inhibitor of CYP2C8 for use as a diagnostic inhibitor in in vitro studies.Peer reviewe

A comprehensive pharmacogenomic study indicates roles for SLCO1B1, ABCG2 and SLCO2B1 in rosuvastatin pharmacokinetics

Aims The aim was to comprehensively investigate the effects of genetic variability on the pharmacokinetics of rosuvastatin. Methods We conducted a genome-wide association study and candidate gene analyses of single dose rosuvastatin pharmacokinetics in a prospective study (n = 159) and a cohort of previously published studies (n = 88). Results In a genome-wide association meta-analysis of the prospective study and the cohort of previously published studies, the SLCO1B1 c.521 T > C (rs4149056) single nucleotide variation (SNV) associated with increased area under the plasma concentration-time curve (AUC) and peak plasma concentration of rosuvastatin (P = 1.8 x 10(-12) and P = 3.2 x 10(-15)). The candidate gene analysis suggested that the ABCG2 c.421C > A (rs2231142) SNV associates with increased rosuvastatin AUC (P = .0079), while the SLCO1B1 c.388A > G (rs2306283) and SLCO2B1 c.1457C > T (rs2306168) SNVs associate with decreased rosuvastatin AUC (P = .0041 and P = .0076). Based on SLCO1B1 genotypes, we stratified the participants into poor, decreased, normal, increased and highly increased organic anion transporting polypeptide (OATP) 1B1 function groups. The OATP1B1 poor function phenotype associated with 2.1-fold (90% confidence interval 1.6-2.8, P = 4.69 x 10(-5)) increased AUC of rosuvastatin, whereas the OATP1B1 highly increased function phenotype associated with a 44% (16-62%; P = .019) decreased rosuvastatin AUC. The ABCG2 c.421A/A genotype associated with 2.2-fold (1.5-3.0; P = 2.6 x 10(-4)) increased AUC of rosuvastatin. The SLCO2B1 c.1457C/T genotype associated with 28% decreased rosuvastatin AUC (11-42%; P = .01). Conclusion These data suggest roles for SLCO1B1, ABCG2 and SLCO2B1 in rosuvastatin pharmacokinetics. Poor SLCO1B1 or ABCG2 function genotypes may increase the risk of rosuvastatin-induced myotoxicity. Reduced doses of rosuvastatin are advisable for patients with these genotypes.Peer reviewe

An automated cocktail method for in vitro assessment of direct and time-dependent inhibition of nine major cytochrome P450 enzymes-application to establishing CYP2C8 inhibitor selectivity

We developed an in vitro high-throughput cocktail assay with nine major drug-metabolizing CYP enzymes, optimized for screening of time-dependent inhibition. The method was applied to determine the selectivity of the time-dependent CYP2C8 inhibitors gemfibrozil 1-O-beta-glucuronide and clopidogrel acyl-beta-D-glucuronide. In vitro incubations with CYP selective probe substrates and pooled human liver microsomes were conducted in 96-well plates with automated liquid handler techniques and metabolite concentrations were measured with quantitative UHPLC-MS/MS analysis. After determination of inter-substrate interactions and Km values for each reaction, probe substrates were divided into cocktails I (tacrine/CYP1A2, bupropion/CYP2B6, amodiaquine/CYP2C8, tolbutamide/CYP2C9 and midazolam/CYP3A4/5) and II (coumarin/CYP2A6, S-mephenytoin/CYP2C19, dextromethorphan/CYP2D6 and astemizole/CYP2J2). Time-dependent inhibitors (furafylline/CYP1A2, selegiline/ CYP2A6, clopidogrel/CYP2B6, gemfibrozil 1-O-beta-glucuronide/CYP2C8, tienilic acid/CYP2C9, ticlopidine/ CYP2C19, paroxetine/CYP2D6 and ritonavir/CYP3A) and direct inhibitor (terfenadine/CYP2J2) showed similar inhibition with single substrate and cocktail methods. Established time-dependent inhibitors caused IC50 fold shifts ranging from 2.2 to 30 with the cocktail method. Under time-dependent inhibition conditions, gemfibrozil 1-O-beta-glucuronide was a strong (90% inhibition) and selective (<< 20% inhibition of other CYPs) inhibitor of CYP2C8 at concentrations ranging from 60 to 300 mu M, while the selectivity of clopidogrel acyl-beta-D-glucuronide was limited at concentrations above its IC80 for CYP2C8. The time-dependent IC50 values of these glucuronides for CYP2C8 were 8.1 and 38 mu M, respectively. In conclusion, a reliable cocktail method including the nine most important drug-metabolizing CYP enzymes was developed, optimized and validated for detecting timedependent inhibition. Moreover, gemfibrozil 1-O-beta-glucuronide was established as a selective inhibitor of CYP2C8 for use as a diagnostic inhibitor in in vitro studies