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.
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.,Novamass Ltd. |
Rousu T.,University of Oulu |
Herttuainen J.,A.P.Pharma |
Tolonen A.,Novamass Ltd.
Rapid Communications in Mass Spectrometry | Year: 2010
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.
Kublbeck J.,University of Eastern Finland |
Laitinen T.,University of Eastern Finland |
Jyrkkarinne J.,University of Eastern Finland |
Rousu T.,Novamass Ltd |
And 10 more authors.
Biochemical Pharmacology | Year: 2011
The so-called human xenosensors, constitutive androstane receptor (hCAR), pregnane X receptor (hPXR) and aryl hydrocarbon receptor (hAhR), participate in drug metabolism and transport as well as in several endogenous processes by regulating the expression of their target genes. While the ligand specificities for hPXR and hAhR are relatively well described, this property of hCAR still remains fairly unclear. Identifying hCAR agonists for drug development and for studying hCAR biology are hindered mainly by the unique properties of the receptor, such as the high constitutive activity and complex signaling network but also by the lack of robust and reliable assays and cellular models. Here, validated reporter assays for these three xenosensors are presented and thereafter used to screen a large set of chemicals in order to find novel selective hCAR ligands. We introduce a novel selective hCAR agonist, FL81, which can be used as a stable positive control in hCAR activity assays. Our established receptor-selective ligand identification methods consisting of supporting biological assays and molecular modeling techniques are then used to study FL81 as well as other discovered ligands, such as diethylstilbestrol, o,p′-DDT, methoxychlor and permethrin, for their ability to specifically activate hCAR and to regulate the CYP enzyme expression and function. © 2011 Elsevier Inc.
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.
Kajula M.,University of Oulu |
Tejesvi M.V.,University of Oulu |
Kolehmainen S.,University of Oulu |
Makinen A.,University of Oulu |
And 4 more authors.
Fungal Biology | Year: 2010
Production of extracellular siderophores is typical for many plant-associated microbes, both mutualistic and antagonistic. Various strains of mycorrhizal fungi produce siderophores, and siderophore production by pathogenic fungi is typically associated with virulence. We analyzed extracellular siderophore production along with production of antibacterial and antioxidant compounds in foliar endophytic fungi of Scots pine (Pinus sylvestris L.) and Labrador tea (Rhododendron tomentosum Harmaja). The siderophore produced in vitro was ferricrocin, quantities ranging between 7.9 and 17.6 μg/l. Only the fungi with antibacterial activity produced ferricrocin and any well-known siderophores were not detected in the broths of antioxidant-producing fungi. Therefore, production of ferricrocin is typical for some, but not all foliar endophytic fungi. Ferricrocin was detected in the leaves of Labrador tea, which suggests that ferricrocin may play a role in vivo in the interaction between the endophyte and plant host. © 2010 The British Mycological Society.
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.
PubMed | Admescope Ltd., University of Eastern Finland and Novamass Ltd.
Type: Journal Article | Journal: Journal of pharmaceutical sciences | Year: 2016
The human intestinal Caco-2 cell line has been extensively used as a model of small intestinal absorption but it lacks expression and function of cytochrome P450 enzymes, particularly CYP3A4 and CYP2C9, which are normally expressed in the intestinal epithelium. In order to increase the expression and activity of CYP isozymes in these cells, we created 2 novel Caco-2 sublines expressing chimeric constitutive androstane or pregnane X receptors and characterized these cells for their metabolic and absorption properties. In spite of elevated mRNA expression of transporters and differentiation markers, the permeation properties of the modified cell lines did not significantly differ from those of the wild-type cells. In contrast, the metabolic activity was increased beyond the currently used models. Specifically, CYP3A4 activity was increased up to 20-fold as compared to vitamin D treated wild-type Caco-2 cells.
Heikkinen A.T.,University of Eastern Finland |
Heikkinen A.T.,Biocenter Kuopio |
Korjamo T.,Novamass Ltd |
Korjamo T.,Orion Corporation |
And 4 more authors.
Molecular Pharmaceutics | Year: 2010
P-Glycoprotein mediated efflux is one of the barriers limiting drug absorption from the intestine. Predictions of the intestinal P-glycoprotein function need to take into account the concentration dependency because high intestinal drug concentrations may saturate P-glycoprotein. However, the substrate binding site of P-glycoprotein lies inside the cells and the drug concentration at the binding site cannot be measured directly. Therefore, rigorous determination of concentration dependent P-glycoprotein kinetics is challenging. In this study, the effects of the aqueous boundary layers, extracellular pH and cellular retention on the apparent saturation kinetics of P-glycoprotein mediated transport of quinidine in an in vitro cell permeation setting were explored. The changes in the experimental conditions caused 1 order of magnitude variation in the apparent affinity to P-glycoprotein (K m,app) and a 5-fold difference in the maximum effective P-glycoprotein mediated transport rate of quinidine (Vmax,app). However, fitting the concentration data into a compartmental model which accounted for the aqueous boundary layers, cell membranes and cellular retention suggested that the P-glycoprotein function per se was not altered, it was the differences in the passive transfer of quinidine which changed the apparent transport kinetics. These results provide further insight into the dynamics of the P-glycoprotein mediated transport and on the roles of several confounding factors involved in in vitro experimental setting. Further, the results confirm the applicability of compartmental model based data analysis approach in the determination of active transporter kinetics. © 2010 American Chemical Society.