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Valitalo P.,University of Eastern Finland | Kuusisto M.,University of Eastern Finland | Ranta V.P.,University of Eastern Finland | Raatikainen K.,Kuopio University Hospital | Hautajarvi H.,Admescope Ltd
British Journal of Anaesthesia | Year: 2014

BackgroundDespite being increasingly used for pain management, only two studies, with controversial results, have evaluated the epidural use of oxycodone.MethodsTwenty-four women, aged 26-64 yr, undergoing elective gynaecological surgery were enrolled in this randomized, double-blinded, parallel group study. The subjects were administered either i.v. oxycodone and epidural placebo (IV group; n=12) or epidural oxycodone and i.v. placebo (EPI group; n=12) after operation. Oxycodone was administered as a single dose of 0.1 mg kg-1. An epidural catheter for drug administration was placed at T12/L1 and a spinal catheter for cerebrospinal fluid (CSF) sampling at L3/4. Plasma and CSF were frequently collected for the analysis of oxycodone and its major metabolites. The primary outcomes were the peak concentration (C max), time to peak concentration (Tmax), and the exposure (AUClast) of oxycodone in CSF and plasma. The secondary outcome was the analgesic efficacy, measured as the total dose of rescue fentanyl during the first four postoperative hours.ResultsIn the EPI group, the median oxycodone Cmax and AUClast in the CSF were 320-and 120-fold higher, respectively, compared with the IV group. The total dose of rescue fentanyl was significantly lower in the EPI group (seven subjects needed 16 doses) than in the IV group [12 subjects needed 71 doses (P=0.001)]. No serious or unexpected adverse events were reported.ConclusionsEpidural oxycodone provides much higher CSF concentrations and possibly better analgesic efficacy than does i.v. oxycodone.Clinical trial registrationEudraCT reference number: 2011-000125-76. © 2014 The Author .

Heikkinen A.T.,University of Eastern Finland | Heikkinen A.T.,Admescope Ltd | Lignet F.,Roche Holding AG | Cutler P.,Roche Holding AG | Parrott N.,Roche Holding AG
Proteomics - Clinical Applications | Year: 2015

Pharmacokinetics (PK) refers to the time course of drug concentrations in the body and since knowledge of PK aids understanding of drug efficacy and safety, numerous PK studies are performed in animals and humans during the drug development process. In vitro to in vivo extrapolation and physiologically based pharmacokinetic (PBPK) modeling are tools that integrate data from various in silico, in vitro, and in vivo sources to deliver mechanistic quantitative simulations of in vivo PK. PBPK models are used to predict human PK and to evaluate the effects of intrinsic factors such as organ dysfunction, age, and genetics as well as extrinsic factors such as co-administered drugs. In recent years, the use of PBPK within the industry has greatly increased. However, insufficient data on how the abundance of metabolic enzymes and membrane transporters vary in different human patient populations and in different species has been a limitation. A major advance is therefore expected through reliable quantification of the abundance of these proteins in tissues. This review describes the role of PBPK modeling in drug discovery and development, outlines the assumptions involved in integrating protein abundance data, and describes the advances made and expected in determining abundance of relevant proteins through mass spectrometric techniques. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

PubMed | University of Trieste, University of Oulu, Novozymes AS, University of Bonn and Admescope Ltd
Type: Journal Article | Journal: Applied microbiology and biotechnology | Year: 2016

Endophytes, microorganisms living inside plant tissues, are promising producers of lead compounds for the pharmaceutical industry. However, the majority of endophytes are unculturable and therefore inaccessible for functional studies. To evaluate genetic resources of endophytes, we analyzed the biodiversity of fungal microbiome of black crowberry (Empetrum nigrum L.) by next-generation sequencing and found that it consists mainly of unknown taxa. We then separated the host and the endophyte genomes and constructed a fosmid expression library from the endophytic DNA. This library was screened for antibacterial activity against Staphylococcus aureus. A unique antibacterial clone was selected for further analysis, and a gene En-AP1 was identified with no similarity to known sequences. The expressed, folded protein En-AP1 was not active against S. aureus, while tryptic digests exhibited antimicrobial activity. Seven out of twelve synthesized peptides, predicted antibacterial in silico, exhibited in vitro activity towards both S. aureus and Escherichia coli. We propose that the En-AP1 protein is degraded in the library host E. coli and antimicrobial fragments are released from the cell, explaining the in vitro antibacterial activity of the clone. This is the first report of a novel gene expressed in vitro derived from an endophytic microbiome, demonstrating the potential of finding novel genes and compounds from unculturable endophytes.

Tolonen A.,Novamass Ltd. | Tolonen A.,Admescope Ltd. | Koskimies P.,Hormos Medical Ltd. | Turpeinen M.,University of Oulu | And 3 more authors.
Drug Metabolism and Drug Interactions | Year: 2013

Background: The metabolism of ospemifene, a novel nonsteroidal selective estrogen receptor modulator, was investigated as part of its development. Methods: Metabolite identification, tentative quantitation, and CYP assignment of ospemifene were performed in human liver microsomes or homogenate incubations and in plasma samples from volunteer humans. The potential contributions of CYP enzymes were determined by recombinant human CYPs. Metabolite identification and tentative quantification were performed by liquid chromatography-mass spectrometry. Results: The relative abundances of metabolites produced were dependent on ospemifene concentration and liver preparation, but the largest quantities of 4- and 4'-hydroxy-ospemifene (and their glucuronides in smaller quantities) were produced in human liver microsomes at low ospemifene concentrations. Other metabolites were detected in in vitro incubation with human liver including a direct glucuronide of ospemifene and some metabolites with only minor abundance. In human plasma samples, 4-hydroxy-ospemifene was the most abundant metabolite, representing about 25% of the abundance of the parent compound. All the other metabolites detected in plasma, including 4'-hydroxyospemifene, represented <7% of the abundance of ospemifene. Several CYP enzymes participated in 4-hydroxylation, including CYP2C9, CYP2C19, CYP2B6, and CYP3A4, whereas CYP3A enzymes were the only ones to catalyze 4'-hydroxylation. Conclusions: In vitro incubations with liver preparations provided a rather reliable starting point in the search for potential metabolites in clinical settings. The in vitro metabolite profile is informative for the in vivo metabolite profile, especially regarding the major hydroxylated metabolites. However, it is anticipated that extended in vivo exposures may result in an increased production of more distal metabolites from major metabolites.

Background: The use of high-resolution MS systems for quantitative bioanalysis is a growing field, even though a clear majority of bioanalytical methods are still based on MS/MS with triple quadrupole (QqQ) instrumentation. The recent advances in TOF-MS technology have provided increased linear range and a high selectivity of detection by increased mass resolution and mass accuracy, making these instruments attractive for quantitative analysis due to lack of a need for compound-specific detection reaction optimization and their capability to collect data for a high number of compounds by sensitive wide mass range data acquisition. Materials & Methods: Here, 11 steroids spiked to human plasma were analyzed by LC-MS using both a QqQ MS system and a TOF instrument operating at 12,000 mass resolution. Sample preparation was performed by hybrid SPE technology. Results: The LOD were 0.5-5 and 0.5-20 ng/ml in plasma for all analytes with QqQ and TOF-MS detection, respectively. Conclusion: Although the results show wider linear range and slightly better sensitivity for most of the compounds with QqQ in comparison to TOF, acceptable performance was obtained for most of the compounds within the range of LOD to 2000 ng/ml (in plasma), this was also the case with LC-TOF-MS analysis. The main problem in TOF-MS analysis at 12,000 mass resolution from plasma was selectivity rather than sensitivity or linear range. © 2012 Future Science Ltd.

Tolonen A.,Admescope Ltd | Pelkonen O.,University of Oulu
Toxicology | Year: 2015

For quantitative in vitro-. in vivo extrapolation (QIVIVE) of metabolism for the purposes of toxicokinetics prediction, a precise and robust analytical technique for identifying and measuring a chemical and its metabolites is an absolute prerequisite. Currently, high-resolution mass spectrometry (HR-MS) is a tool of choice for a majority of organic relatively lipophilic molecules, linked with a LC separation tool and simultaneous UV-detection. However, additional techniques such as gas chromatography, radiometric measurements and NMR, are required to cover the whole spectrum of chemical structures. To accumulate enough reliable and robust data for the validation of QIVIVE, there are some partially opposing needs: Detailed delineation of the in vitro test system to produce a reliable toxicokinetic measure for a studied chemical, and a throughput capacity of the in vitro set-up and the analytical tool as high as possible. We discuss current analytical challenges for the identification and quantification of chemicals and their metabolites, both stable and reactive, focusing especially on LC-MS techniques, but simultaneously attempting to pinpoint factors associated with sample preparation, testing conditions and strengths and weaknesses of a particular technique available for a particular task. © 2013 Elsevier Ireland Ltd.

Rousu T.,University of Oulu | Rousu T.,Novamass Ltd. | Tolonen A.,University of Oulu | Tolonen A.,Admescope Ltd.
Rapid Communications in Mass Spectrometry | Year: 2011

Reactive metabolites are estimated to be one of the main reasons behind unexpected drug-induced toxicity, by binding covalently to cell proteins or DNA. Due to their high reactivity and short lifespan, reactive metabolites are analyzed after chemical trapping with nucleophilic agents such as glutathione or cyanide. Recently, unexplained and uncharacterized methylated reaction products were reported in a human liver microsome based reactive metabolite trapping assay utilizing potassium cyanide as a trapping agent. Here, a similar assay was utilized to produce mono- or dimethylated and further cyanide-trapped reaction products from propranolol, amlodipine and ciprofloxacin, followed by ultra-performance liquid chromatography/time-of-flight mass spectrometry (UPLC/TOF-MS) and ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) experiments for their more detailed structural elucidation. Formation of all observed cyanide-trapped products was clearly NADPH-dependent and thus metabolism-mediated. The suggested reaction pathways included N-methylation leading to iminium formation in primary and/or secondary amines preceded by cytochrome P450 (CYP)-mediated reactions. As the methylation reaction was suggested to be involved in formation of the actual reactive iminium ion, the observed cyanide-trapped products were experimental artifacts rather than trapped reactive metabolites. The results stress that to avoid overestimating the formation of reactive metabolites in vitro, this methylation phenomenon should be taken into account when interpreting the results of cyanide-utilizing reactive metabolite trapping assays. This in turn emphasizes the importance of identification of the observed cyano conjugates during such studies. Yet, metabolite identification has a high importance to avoid overestimation of in vitro metabolic clearance in the cases where this kind of metabonate formation has a high impact in the disappearance rate of the compound. Copyright © 2011 John Wiley & Sons, Ltd.

PubMed | University of Eastern Finland, Kuopio University Hospital and Admescope Ltd
Type: Journal Article | Journal: Clinical drug investigation | Year: 2016

Based on earlier literature, etoricoxib may have a delayed analgesic effect in postoperative setting when analgesic efficacy of nonselective nonsteroidal anti-inflammatory drug dexketoprofen is rapid. This may be caused by slow penetration of etoricoxib into the central nervous system (CNS). Therefore we decided to determine the plasma and cerebrospinal fluid (CSF) pharmacokinetics and pharmacodynamics of dexketoprofen and etoricoxib in patients with hip arthroplasty.A total of 24 patients, scheduled for an elective primary hip arthroplasty were enrolled. After surgery, 12 subjects were randomized to received a single intravenous dose of dexketoprofen, and 12 subjects were given oral etoricoxib. Paired blood and CSF samples were taken up to 24h for measurement of drug concentrations, interleukin (IL)-6, IL-1ra and blood for interleukin 10.In CSF the highest measured concentration (C max) of dexketoprofen was 4.0 (median) ng/mL (minimum-maximum 1.9-13.9) and time to the highest concentration (t max) 3h (2-5), and for etoricoxib C max 73ng/mL (36-127) and t max 5h (1-24), respectively. Opioid consumption during the first 24 postoperative hours was similar in the two groups. Dexketoprofen and etoricoxib had a similar effect on the postoperative inflammatory response. No significant differences considering pain relief or adverse events were found between the two groups.Dexketoprofen and etoricoxib entered the CNS readily, already at 30 min after administration dexketoprofen was detected in the CSF in most subjects and etoricoxib after 60 min. A single dose of dexketoprofen and etoricoxib provided a similar anti-inflammatory and analgesic response after major orthopaedic surgery.

PubMed | Leiden University, University of Eastern Finland, Kuopio University Hospital and Admescope Ltd
Type: | Journal: British journal of clinical pharmacology | Year: 2016

This study aimed to characterize the pharmacokinetics of oxycodone and its major metabolites in infants and covered the age range between extremely preterm neonates and 2-year-old infants.Seventy-nine infants (gestational age 23-42 weeks; postnatal age 0-650 days) received intravenous oxycodone hydrochloride trihydrate at a dose of 0.1mgkgOxycodone pharmacokinetics changed markedly with patient age. Preterm neonates were found to have the highest pharmacokinetic variability out of the study population. In extremely preterm neonates (n=6) median of elimination half-life was 8.8h (range 6.8-12.5), in preterm (n=11) 7.4h (4.2-11.6), and in older neonates (n=22) 4.1h (2.4-5.8), all of which were significantly longer than that in infants aged 6-24 months (n=12) 2.0h (1.7-2.6). Median renal clearance was fairly constant in all age groups, whereas non-renal clearance markedly increased with age. Noroxycodone was the major metabolite in plasma and urine.Oxycodone elimination is slower and pharmacokinetic variability more pronounced in neonates when compared to older infants. These findings highlight the importance of careful dose titration for neonates.

PubMed | University of Oulu and Admescope Ltd
Type: Journal Article | Journal: Rapid communications in mass spectrometry : RCM | Year: 2016

Menthofuran is a hepatotoxin and a major metabolite of pulegone, a monoterpene found in the essential oils of many mint species. It is bioactivated by cytochrome P450 (CYP) enzymes to reactive metabolites, which may further react with glutathione to form S-linked and N-linked conjugates. The tandem mass spectrometric (MS/MS) fragmentation pathways of rarely observed N-linked conjugates, and the differences to fragmentation of S-linked conjugates, have not been reported in the literature previously, although this information is essential to enable comprehensive MS/MS-based screening methods covering the both types of conjugates.(R)-(+)-Pulegone, (S)-(-)-pulegone, and menthofuran were incubated with a human liver S9 fraction with glutathione (GSH) as the trapping agent. Conjugates were searched with ultra-performance liquid chromatography (UPLC)/orbitrap MS and their MS/MS spectra were measured both in the negative and positive ionization polarities. Menthofuran was also incubated with recombinant human CYP enzymes and GSH to elucidate the CYPs responsible for the formation of the reactive metabolites.Four GSH conjugates of menthofuran were detected and identified as S- and N-linked conjugates based on MS/MS spectra. N-linked conjugates lacked the characteristic fragments of S-linked conjugates and commonly produced fragments that retained parts of glutamic acid. CYP1A2, 2B6 and 3A4 were observed to produce more GSH conjugates than other CYP isoforms.Furans can form reactive aldehydes that react in Schiff-base fashion with the free glutamyl-amine of GSH to form N-linked conjugates that have distinct MS/MS spectra from S-linked adducts. This should be taken into account when setting up LC/MS/MS-based detection of glutathione conjugates to screen for reactive metabolites, at least for compounds with a furan moiety. Neutral loss scanning of 178.0412 Da and 290.0573 Da in the positive ionization mode, or neutral loss scanning of 256.0695 Da and 290.0573 Da and precursor ion scanning of m/z 143.0462 in the negative ionization mode, is recommended. Copyright 2016 John Wiley & Sons, Ltd.

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