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Oulu, Finland

Korjamo T.,Novamass Ltd | Korjamo T.,Orion Corporation | Tolonen A.,Admescope Ltd. | Ranta V.-P.,University of Eastern Finland | And 2 more authors.
Frontiers in Pharmacology | Year: 2012

Oxycodone is commonly used to treat severe pain in adults and children. It is extensively metabolized in the liver in adults, but the maturation of metabolism is not well understood. Our aim was to study the metabolism of oxycodone in cryopreserved human hepatocytes from different age groups (3 days, 2 and 5 months, 4 years, adult pool) and predict hepatic plasma clearance of oxycodone using these data. Oxycodone (0.1, 1, and 10 μM) was incubated with hepatocytes for 4 h, and 1 μM oxycodone also with CYP3A inhibitor ketoconazole (1 μM). Oxycodone and noroxycodone concentrations were determined at several time points with liquid chromatography-mass spectrometry. In vitro clearance of oxycodone was used to predict hepatic plasma clearance, using the well-stirred model and published physiological parameters. Noroxycodone was the major metabolite in all batches and ketoconazole inhibited the metabolism markedly in most cases. A clear correlation between in vitro oxycodone clearance and CYP3A4 activity was observed. The predicted hepatic plasma clearances were typically much lower than the published median total plasma clearance from pharmacokinetic studies. The data suggests that there are no children-specific metabolites of oxycodone. Moreover, CYP3A activity seems to be the major determinant in metabolic clearance of oxycodone regardless of age group or individual variability in hepatocyte batches. © 2012 Korjamo, Tolonen, Ranta, Turpeinen and Kokki.

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.

Murtomaa M.,University of Oulu | Viitala P.,University of Oulu | Hokkanen J.,Novamass Ltd | Pelkonen O.,University of Oulu | Rautio A.,University of Oulu
Environmental Toxicology and Pharmacology | Year: 2010

Previous studies in bank vole (Myodes glareolus) and field vole (Microtus agrestis) living at the old sawmill area contaminated by chlorinated dibenzo-p-dioxins and -furans (PCDD/Fs) showed that these two relatively close species have a significant difference in their body burden of PCDD/Fs, bank voles having significantly higher concentrations. The aim of this study was to clarify more comprehensively the basic xenobiotic metabolism in wild bank voles and examine whether PCDD exposure would affect xenobiotic metabolism in bank voles more generally. The activity of cytochrome P450 enzymes was studied by fluorometric analyzes as well as by the aid of an earlier developed N-in-one CYP-selective activity cocktail, and immunoblotting assay. Several CYP-associated activities were considerably and statistically significantly elevated in the liver of animals living in the contaminated area. Increases in several CYP1A- and CYP2A/B-associated activities are probably due to the exposure to variable dioxin- and PCB-class inducers. The results of this comprehensive screening seemed to be in agreement with the existing knowledge of CYP enzyme induction by dioxin-like substances. © 2009 Elsevier B.V. All rights reserved.

Pellinen P.,Santen Oy | Huhtala A.,University of Tampere | Tolonen A.,Novamass Ltd | Lokkila J.,Santen Oy | And 2 more authors.
Current Eye Research | Year: 2012

Purpose: To investigate the cytotoxicity of benzalkonium chloride (BAC)-containing ophthalmic solutions of prostaglandin analogs (latanoprost, travoprost, bimatoprost, and preservative-free (PF) tafluprost), BAC mixture (BACmix) and BAC homologs with different alkyl chain lengths using human corneal epithelial (HCE) and conjunctival epithelial (IOBA-NHC) cell cultures. The distribution of BAC homologs in rabbit ocular surface tissues in vivo was examined. Methods: The cells were exposed for one hour to prostaglandin analogs, BACmix and three homologs. Cytotoxicity was assessed with the WST-1 and lactate dehydrogenase (LDH) assays for cellular viability and cell membrane integrity. BAC 0.02% solution was instilled on the rabbit eye daily for 14 days and the concentrations of BAC homologs in external ocular tissues were determined. Results: The order of decreasing cytotoxicity in the WST-1 test was latanoprost ≥ travoprost > bimatoprost ≥ PF tafluprost. IOBA-NHC cells were more sensitive than HCE cells. In HCE, only latanoprost diluted to 10% increased LDH leakage. In IOBA-NHC, LDH leakage was statistically significant with 310% travoprost and 10% latanoprost. The order of decreasing cytotoxicity of preservatives was C14 > C12 > BACmix > C16 in HCE and C12 > C14 > BACmix > C16 in IOBA-NHC. Following treatment with BAC 0.02% solution, the amounts of BAC-C12, -C14 and -C16 in rabbit cornea and conjunctiva, respectively were: 0.37±0.08 and 2.64±0.27ng/mg; 0.42±0.07 and 4.77±0.43ng/mg; 0.04±0.01 and 0.54±0.05ng/mg. Conclusions: The cytotoxic effects of latanoprost, travoprost, and bimatoprost were dependent on the BAC concentration in their formulations. BACmix was cytotoxic at the concentrations above those corresponding to 0.001% BAC in ophthalmic medications. PF tafluprost was the least toxic of the drugs tested. Within studied BAC homologs, those with longer alkyl chain and higher lipophility penetrated effectively into rabbit external ocular tissues. © 2012 Informa Healthcare USA, Inc.

Liquid chromatography in combination with mass spectrometry (LC/MS) is a superior analytical technique for metabolite profiling and identification studies performed in drug discovery and development laboratories. In the early phase of drug discovery the analytical approach should be both time- and cost-effective, thus providing as much data as possible with only one visit to the laboratory, without the need for further experiments. Recent developments in mass spectrometers have created a situation where many different mass spectrometers are available for the task, each with their specific strengths and drawbacks. We compared the metabolite screening properties of four main types of mass spectrometers used in analytical laboratories, considering both the ability to detect the metabolites and provide structural information, as well as the issues related to time consumption in laboratory and thereafter in data processing. Human liver microsomal incubations with amitriptyline and verapamil were used as test samples, and early-phase 'one lab visit only' approaches were used with all instruments. In total, 28 amitriptyline and 69 verapamil metabolites were found and tentatively identified. Time-of-flight mass spectrometry (TOFMS) was the only approach detecting all of them, shown to be the most suitable instrument for elucidating as comprehensive metabolite profile as possible leading also to lowest overall time consumption together with the LTQ-Orbitrap approach. The latter however suffered from lower detection sensitivity and false negatives, and due to slow data acquisition rate required slower chromatography. Approaches with triple quadrupole mass spectrometry (QqQ) and hybrid linear ion trap triple quadrupole mass spectrometry (Q-Trap) provided the highest amount of fragment ion data for structural elucidation, but, in addition to being unable to produce very high-important accurate mass data, they suffered from many false negatives, and especially with the QqQ, from very high overall time consumption. © 2010 John Wiley & Sons, Ltd.

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