Comparison of the Hepatic Safety of Catechol-O-Methyltransferase Inhibitors Entacapone and Tolcapone with Special Reference to Uncoupling of Oxidative Phosphorylation

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Comparison of the hepatic safety of catechol-O-methyltransferase inhibitors entacapone and tolcapone with special reference to uncoupling of oxidative phosphorylation

Kristiina Haasio

ACADEMIC DISSERTATION

To be presented with the permission of the Faculty of Veterinary Medicine for public examination in auditorium Maximum, Hämeentie 57, 00580 Helsinki, on May 28th, 2003, at 12 noon Supervised by Professor Antti Sukura, DVM, PhD and Docent Erkki Nissinen, PhD and Docent Esa Heinonen, MD, PhD

Reviewed by Professor Anthony Schapira, DSc, MD, FRCP, FMedSci and Professor Raimo Tuominen, MD, PhD

Opponent Professor Hannu Raunio, MD, PhD Department of Pharmacology and Toxicology Faculty of Pharmacy University of Kuopio

Cover: A rat liver hepatocyte with nucleus and mitochondria, transmission electron micrograph

ISBN 952-91-5877-7 (paperback) ISBN 952-10-1178-5 (PDF)

A5 Repropalvelu Tampere 2003 http://ethesis.helsinki.fi/ To Jussi, Armi and Lassi

Anything is possible if you wish hard enough (web pages 21.4.2003, Orion Pharma) Contents

Contents

1. Abstract ...... 6 2. Original publications ...... 7 3. Abbreviations ...... 8 4. Introduction ...... 9 5. Review of the literature ...... 11 5.1. COMT and catecholamine metabolism ...... 11 5.2. Other catecholamine-metabolizing enzymes ...... 13 5.3. COMT inhibitors in the treatment of Parkinson’s disease ...... 14 5.3.1. Rationale for the use of COMT inhibitors...... 14 5.3.2. First-generation COMT inhibitors ...... 16 5.3.3. Second-generation COMT inhibitors entacapone and tolcapone ...... 17 5.4. Drug induced hepatotoxicity ...... 21 5.4.1. Mechanisms, clinical symptoms and signs of hepatotoxicity ...... 21 5.4.2. Hepatotoxicity induced by COMT inhibitors ...... 23 5.5. Uncoupling as a phenomenon ...... 25 5.5.1. Uncoupling at cellular level ...... 25 5.5.2. Signs of uncoupling in vivo ...... 27 5.5.3. Uncoupling induced by COMT inhibitors...... 28 5.5.4. COMT inhibition and uncoupling in relation to protein binding...... 29 5.6. Pathological findings in tissues in connection with uncoupling of oxidative phosphorylation ...... 30 5.6.1. Histological findings ...... 30 5.6.2. Electron microscopy findings ...... 31 6. Aims ...... 33 7. Materials and methods ...... 34 7.1. Animals and treatment ...... 34 7.2. Test substances ...... 34 7.3. Methods in in vivo studies ...... 35 7.3.1. Clinical signs, mortality, body weight and organ weights (I, II, V) ...... 35 7.3.2. Behavioural studies (V) ...... 35 7.3.3. Body temperature measurements (I, II, IV) ...... 35 7.3.4. Haematology and clinical chemistry (I, II, V) ...... 35 7.3.5. Determination of catecholamines from plasma (V) ...... 35 7.3.6. Determination of MAO-A and MAO-B in liver homogenates (V) ...... 36 7.3.7. Analysis of drug concentrations in plasma and liver (I, II) ...... 36 7.3.8. Histopathology (I, II, III, V)...... 36 7.3.9. Electron microscopy (III) ...... 36 4 Contents

7.4. Material and methods in studies at cellular level ...... 37 7.4.1. Adenosine nucleotides from liver mitochondria and liver tissue (II) ...... 37 7.4.2. Mitochondrial oxygen consumption (II) ...... 37 7.4.3. Mitochondrial membrane potential studies (IV)...... 37 7.4.4. Measurement of COMT activity in protein-binding studies (IV) ...... 37 7.5. Statistics...... 38 8. Results ...... 39 8.1. Comparative toxicity of entacapone and tolcapone in vivo ...... 39 8.1.1. Clinical signs, mortality, body weight and organ weights (I, II, V) ...... 39 8.1.2. Haematology and clinical chemistry (I, II, V) ...... 39 8.1.3. Catecholamine plasma levels (V) ...... 40 8.1.4. Activity of MAO-A and MAO-B in the liver (V) ...... 40 8.1.5. Plasma and liver tissue concentrations (I, II) ...... 40 8.1.6. Pathology induced by treatment with entacapone and tolcapone ...... 41 8.1.6.1. Histopathological findings (I, II, III, V)...... 41 8.1.6.2. Electron microscopy findings (III) ...... 41 8.2. Uncoupling effects related to entacapone and tolcapone ...... 42 8.2.1. Signs of uncoupling in vivo ...... 42 8.2.1.1. Body temperature (I, II, IV) ...... 42 8.2.2. Signs of uncoupling at cellular level ...... 43 8.2.2.1. Adenosine nucleotide concentrations in liver mitochondria and liver tissue (II)...... 43 8.2.2.2. Mitochondrial oxygen consumption (II)...... 44 8.2.2.3. Mitochondrial membrane potential (IV) ...... 44 8.2.3. COMT activity in relation to protein binding (IV) ...... 44 8.3. Summary of the results in vivo and in vitro ...... 47 9. Discussion ...... 48 9.1. Comparative toxicology of entacapone and tolcapone in vivo ...... 48 9.1.1. Clinical signs and mortality...... 48 9.1.2. Hepatotoxic properties of entacapone and tolcapone ...... 49 9.1.3. Exposure of animals to entacapone and tolcapone ...... 52 9.2. Uncoupling effects related to entacapone and tolcapone ...... 52 9.2.1. Signs of uncoupling in vivo ...... 52 9.2.2. Signs of uncoupling at cellular level...... 53 10. Conclusions ...... 56 11. Acknowledgements ...... 57 12. References ...... 59 Original publications (I-V) ...... 73 5 Abstract

1. Abstract

Entacapone and tolcapone are novel COMT activity in otherwise healthy catechol-O-methyltransferase (COMT) animals. inhibitors developed for adjunctive Entacapone was well tolerated at high treatment with levodopa in Parkinson’s oral repeated doses by rats. Tolcapone disease (PD). In clinical use, tolcapone has induced clinical signs and increased induced severe hepatic dysfunction, mortality at liver concentrations that caused including three fatal cases. Tolcapone, but no adverse effects with entacapone. Rectal not entacapone, has also been shown to be body temperature increased with tolcapone an uncoupler of oxidative phosphorylation and DNP treatments. Rigor mortis in vitro at low micromolar concentrations. occurred instantly after death in tolcapone- The toxicity of entacapone and tolcapone treated rats. The mitochondrial ATP/ADP was compared in animal studies in vivo and ratio decreased after tolcapone and DNP in vitro. 2,4-dinitrophenol (DNP), a known treatments. Hepatotoxicity was expressed as uncoupling agent, was used as a reference an increase in liver enzymes as well as substance. In the in vivo studies, rats were histopathological changes in tolcapone- treated with high repeated oral doses. treated and to a lesser degree in DNP-treated Clinical symptoms, mortality and rectal animals. No pathological findings in tissues body temperature were recorded. Further- were observed in mice lacking COMT more, clinical chemistry and haematological activity. parameters were measured, and at necropsy, The study showed that tolcapone has tissue samples for histological examination potential hepatotoxic properties at high and biochemical determinations were doses in animals. The findings in vivo, at extracted. At cellular level, mitochondrial the cellular level and in vitro are consistent energy status was determined after oral with those observed after treatment with treatment in rats. In vitro, mitochondrial DNP, suggesting that hepatotoxic properties membrane potential in isolated rat liver of tolcapone may be due to uncoupling of mitochondria was determined to obtain oxidative phosphorylation. Entacapone was data on the direct effects of these agents on well tolerated and did not express any signs uncoupling of oxidative phosphorylation. related to uncoupling. Total depletion of To evaluate the role of total COMT COMT activity did not induce any liver inhibition in the toxicity of COMT problems in the knock-out mice, indicating inhibitors, mice lacking the Comt -gene were that hepatotoxicity is not a class effect of used to assess COMT inhibition-induced these COMT inhibitors. pathology in organs expressing high

6 Original publications

2. Original publications

This thesis is based on the following original papers referred to in the text by their Roman numerals:

I Haasio, K., Sopanen, L., Vaalavirta, L., Lindén, I.-B., Heinonen, E.H., 2001. Comparative toxicological study on the hepatic safety of entacapone and tolcapone in the rat. J Neural Transm 108:79-91.

II Haasio, K., Nissinen, E., Sopanen, L., Heinonen, E.H., 2002. Different toxicological profile of two COMT inhibitors in vivo: the role of uncoupling effects. J Neural Transm 109:1391-1401.

III Haasio, K., Lounatmaa, K., Sukura, A., 2002. Entacapone does not induce conformational changes in liver mitohondria or skeletal muscle in vivo. Exp Toxic Pathol 54: 9-14.

IV Haasio, K., Koponen, A., Penttilä, K.E., Nissinen, E., 2002. Effects of entacapone and tolcapone on mitochondrial membrane potential. Eur J Pharm 453: 21-26.

V Haasio, K., Huotari, M., Nissinen, E., Männistö, P.T. Tissue histopathology, clinical chemistry and behaviour of adult Comt-gene disrupted mice. J Appl Toxicol, in press.

7 Abbreviations

3. Abbreviations

A adrenaline levodopa L-dihydroxyphenylalanine, ADP adenosine diphosphate L-dopa ALAT alanine aminotransferase MAO monoamino oxidase AMP adenosine monophosphate MB-COMT membrane-bound COMT APHOS alkaline phosphatase N number of observations/ ASAT aspartate aminotransferase animals ATP adenosine triphosphate NA noradrenaline AUC area under curve NAD no abnormalities detected BBB blood-brain barrier OFF time sudden loss of effectiveness

Cmax peak plasma concentration with abrupt onset of akinesia

Ctlast last measurable plasma (Fahn 1974) concentration 3-OMD 3-O-methyldopa CNS central nervous system ON time sudden return of COMT catechol-O-methyl trans- effectiveness, may be ferase accompanied by hyperkinesia DA dopamine (Fahn 1974) DDC dopa decarboxylase PD Parkinson’s disease DHPG 3,4-dihydroxyphenylglycol PST phenolsulphotransferase DNP 2,4-dinitrophenol RCR respiratory control ratio DOPA 3,4-dihydroxyphenylalanine = (oxygen consumption rate DOPAC 3,4-dihydroxyphenyl acetic with ADP)/(oxygen con- acid sumption rate without ADP)

EC50 median effective dose S-COMT soluble COMT HPLC high-performance liquid SD standard deviation chromatography SDH sorbitol dehydrogenase HPLC-EC HPLC with colourimetric TEM transmission electron electrochemical detection microscopy HVA homovanillic acid ULN upper limit of normal

IC50 median inhibitory concentration

8 Introduction

4. Introduction

The catechol-O-methyltransferase (COMT; first-generation inhibitors were based on EC 2.1.1.6) enzyme was characterized by catechol structure (Ross and Haljasmaa Axelrod and Tomchick in 1958 (Axelrod 1964a; 1964b; Baldessarini and Greiner and Tomchick 1958; Axelrod and LaRoche 1973; Gugler and Dengler 1973). Gallates, 1959; Guldberg and Marsden 1975; catechols, tropolone and its derivatives and Männistö and Kaakkola 1999). COMT is pyrogallol were all tested in vitro and in vivo present in most mammalian tissues, with and appeared to be effective but were usually the highest activities occurring in the liver extremely short-acting (Belleau and Burba and kidneys (Guldberg and Marsden 1975; 1961; Ross and Haljasmaa 1964a; Dorris Nissinen et al. 1988; Karhunen et al. 1994). and Dill 1977). They were also toxic, COMT catalyses the inactivation of inducing convulsions in experimental catecholamines by methylation of the 3- animals. In clinical trials with e.g. N- hydroxyl group of the catechol ring both in butylgallate and U-0521, they were the periphery and the central nervous system established to be non-effective in the (CNS) (Figure 1, page 14). Besides treatment of PD, offering no advantage catecholamines, levodopa, the cornerstone when compared with dopa decarboxylase in treatment of Parkinson’s disease (PD), is (DDC) inhibitors administered together one of the substrates of COMT. The gradual with levodopa (Ericsson 1971). None of destruction of dopaminergic neurons in the the first-generation COMT inhibitors was brain leads to a dopamine (DA) deficiency taken into routine clinical use. and the onset of symptoms of PD (Schapira Second-generation COMT inhibitors 2001; Orth and Schapira 2002). Levodopa were developed simultaneously by three is administered together with dopa laboratories during the late 1980s decarboxylase (DDC) inhibitors, which (Bäckström et al. 1989; Borgylua et al. diminish the peripheral metabolism of 1989; Waldmeier et al. 1990). Approxi- levodopa and increase its bioavailability mately ten years later, a long-acting inhibitor, (Teräväinen et al. 2001). To induce further BIA 3-202, with limited access to the brain, increases of DA in the brain, COMT was reported to have beneficial effects on inhibitors were designed to be used in the brain DA metabolism in the rat (Parada et treatment of PD as adjuncts to levodopa al. 2001). All of the second-generation and DDC inhibitors. COMT inhibitors are selective and orally The first generation of COMT inhibitors active, and all except one are structurally was developed during the 1960s. Pyrogallol nitrocatechols. Two of them were taken was reported to be a potent COMT into clinical use in the late 1990s. inhibitor already in 1959 by Axelrod and Entacapone mainly acts peripherally, while LaRoche, and it was later used as a standard tolcapone crosses the blood-brain barrier inhibitor in the development of new agents (BBB) and acts in both the periphery and (Ross and Haljasmaa 1964a). Several of the CNS (Männistö and Kaakkola 1999). Both

9 Introduction are designed to be administered cases were reported within a short time span concomitantly with levodopa, tolcapone (Assal et al. 1998; Mayoral et al. 1999; three times a day, and entacapone up to ten Olanow 2000; Spahr et al. 2000; Watkins times a day with each levodopa dose 2000). In 1998, the European Medicines (Dingemanse 1997; Männistö and Evaluation Agency recommended Kaakkola 1999). Although they are relatively suspension of the marketing authorization similar structurally, their kinetics and for tolcapone in the European Union metabolic pathways differ. In non-clinical (European Medicine Evaluation Agency studies, both inhibitors proved to be safe 1998). At the same time, the European after undergoing extensive toxicity testing Medicines Evaluation Agency evaluated the in several animal species (Schläppi et al. clinical data for entacapone and found no 1996a; 1996b; 1996c; Tasmar Product reason to restrict its use. Monograph 1997; Entacapone Product Since entacapone and tolcapone are Monograph 1999). In clinical studies, they structurally related nitrocatechols, concern were well tolerated, with the main side has arisen regarding hepatotoxicity of this effects being gastro-intestinal and class of COMT inhibitors. As no signs of dopaminergic (Männistö and Kaakkola liver toxicity in non-clinical studies or in 1999). clinical trials of these inhibitors have been After being on the market for about one observed, comparison of these agents at high year and when 60 000 patients had been doses is the next logical step in evaluating exposed to tolcapone, the compound was possible hepatotoxic properties and found to induce liver toxicity and three fatal mechanisms.

10 Review of the literature

5. Review of the literature

5.1. COMT and catecholamine of two distinct forms of COMT in rat metabolism erythrocytes was demonstrated (Assicot and Bohuon 1971): one fraction was in soluble Catechol-O-methyltransferase (COMT) is form (S-COMT), and the other was bound an intracellular enzyme that catalyses the O- to membranes (MB-COMT) (Assicot and methylation of catechol-structured Bohuon 1971; Borchardt et al. 1974). compounds, such as catecholamine neurotransmitters, catechol hormones and Cellular and tissue distribution of COMT xenobiotic catechols, in a variety of tissues The distribution and cellular localization of in mammals (Figure 1, page 14) (Axelrod COMT have been evaluated in man and in and Tomchick 1958; Guldberg and several animal species (Guldberg and Marsden 1975; Bonifati and Meco 1999). Marsden 1975; Nissinen et al. 1992; The COMT enzyme was initially Karhunen et al. 1994). The highest activity characterized in the 1950s, when both of COMT is found in the liver and kidney, adrenaline (A) and noradrenaline (NA) were but activity has also been detected in several found to undergo an O-methylation other organs, including the brain, lung, reaction (Axelrod 1957). Adrenaline was spleen, mammary gland, uterus, stomach, metabolized in the rat liver fraction in the intestines and adrenals (Guldberg and presence of S-adenosylmethionine and Marsden 1975; Nissinen et al. 1988; magnesium ions to metanephrine. This led Karhunen et al. 1994; De Santi et al. 1998). to the finding that endogenous amines, Axelrod and Tomchick compared liver including NA, A and dopamine (DA), are COMT activities in several mammalian O-methylated to normetanephrine, species and found the highest activity in the metanephrine and 3-methoxytyramine, rat, followed by the mouse and guinea pig, respectively, when S-adenosylmethionine while the activity of COMT in human liver donates its methyl group to one of the was about one-tenth of that in the rat liver hydroxy groups of catecholamines (Axelrod (Axelrod and Tomchick 1958; Guldberg and Tomchick 1958). Later it has been and Marsden 1975). Although MB- found that besides A, NA and DA, the COMT represents only 2-10% of total physiological substrates of COMT also COMT activity in the rat liver and brain, it include 3,4-dihydroxyphenylalanine has a 3- to 100-fold higher affinity for (DOPA) and catecholestrogens (Guldberg catechol substrates than S-COMT, thus and Marsden 1975; Männistö and Kaakkola being responsible for O-methylation at 1999). These amines are inactivated by physiologically relevant concentrations of methylation of the 3-hydroxyl group of the neurotransmitters (Assicot and Bohuon catechol ring, and COMT catalyses this 1971; Aprille and Malamud 1975; Rivett reaction using S-adenosyl-L-methionine as et al. 1983; Jeffery and Roth 1984; Roth the methyl donor. In the 1970s, the presence 1992). The activity of S-COMT pre-

11 Review of the literature dominates when MB-COMT is saturated neuronal localization (Karhunen et al. 1994). (Roth 1992; Vieira-Coelho and Soares-da- The most intensely stained areas were lateral, Silva 1999). The subcellular localization of third and fourth ventricles, followed by cells MB-COMT using rat liver preparations has of the choroid plexus (Karhunen et al. been postulated to be on mitochondrial or 1994). plasma membranes (Grossman et al. 1985; In the human liver, the activity is Tilgmann et al. 1992). Ulmanen et al. significantly higher in men than in women, (1997) have shown by immuno- and it increases with age (Agathopoulos et cytochemistry in cell culture studies using al. 1971; De Santi et al. 1998). MB- both mammalian cell lines and rat primary COMT is distributed among all tissues, neurons that MB-COMT is located on with the highest levels being found in the intracellular membranes like the rough liver (Roth 1992). In the human brain, both endoplasmic reticulum rather than on isoforms of COMT are present, but MB- plasma membranes, while S-COMT is COMT is the functionally significant form expressed both in the nuclei and cytoplasm. (Roth 1992; Karhunen et al. 1994; Hong In the mouse liver, most of the COMT et al. 1998). Since the highest ratio of MB- activity is associated with membranes COMT to S-COMT is found in brain (Aprille and Malamud 1975). The liver tissue, this suggests that MB-COMT may membranes contain 70-90% of the total be localized in neuronal cells of the CNS in COMT activity in homogenate. The man (Roth 1992; Tenhunen et al. 1994). specific activity in mouse membranes is 8 In human breast tissue, COMT, but no to 9-fold higher than that in rat membranes, other methyltransferase enzyme, has been whereas the activity of S-COMT is reported to be present (Assicot et al. 1977). comparable in the mouse and rat (Aprille An increase in COMT activity has been and Malamud 1975). observed in the majority of primary S-COMT is the predominant form of mammary carcinoma samples of high the enzyme expressed in the periphery of malignancy (Assicot et al. 1977; Hoffman most rat tissues (Karhunen et al. 1994; Lotta et al. 1979). Using immunocytochemical et al. 1995). Rat liver shows highest activity staining methods, S-COMT has been for S-COMT, while the lowest activity of localized in the cytoplasm of epithelial cells both S-COMT and MB-COMT is found of secretory tubules in both human and in the heart (Ellingson et al. 1999). MB- rodent mammary glands; there is a strong COMT is detected mainly in the liver and reaction in malignant cells, particularly in kidneys and in smaller amounts in the brain tumours (Amin et al. 1983; Tenhunen et and intestines (Karhunen et al. 1994). The al. 1999). In human mammary epithelial cellular localization of S-COMT in rat liver cells, nuclear COMT has been observed in is limited to hepatocytes, and Kuppfer cells normal cells as well as cancer cells (Weisz et show no activity (Karhunen et al. 1994). al. 2000). Using immunohistochemical staining methods, S-COMT has been found to be the major form in the rat brain with non-

12 Review of the literature

5.2. Other catecholamine- mitochodrial membrane. metabolizing enzymes Role of PST Other metabolic pathways in inactivating Sulphation of catecholamines is performed catecholamines include oxidative removal by several types of PST (Kopin 1985). The of their amino group by monoamine oxidase highest PST concentrations are observed in (MAO) and sulphoconjugation by the rat brain and liver, but enzyme activity phenolsulphotransferase (PST) (Kopin is also found in the adrenals, lung, kidneys 1994) (Figure 1). MAO metabolizes and testes (Foldes and Meek 1974). In the catecholamines to 3-methoxy-4- rat, sulphoconjugation of 3,4- hydroxyphenylglycol. There are two dihydroxyphenylacetic acid (DOPAC) and isozymes of MAO, A and B, which have homovanillic acid (HVA) plays a more slightly different substrates; e.g. NA is important role than in e.g. dogs and preferentially metabolized by MAO-A, primates (Kopin 1985). whereas DA is a substrate of both types in Gene-manipulated mice deficient in most species (O’Carroll et al. 1983; Kopin catecholamine-metabolizing enzymes 1994). In the human brain, approximately 70% of total MAO activity is of type B, The effects of total COMT or MAO which oxidizes most of the DA in man inhibition in the brain has been evaluated (Riederer et al. 1986). However, in rodents, in COMT- or MAO-deficient mice (Gogos DA is oxidized mainly by MAO-A et al. 1998; Shih et al. 1999a; Huotari et al. (Johnston 1968). 2002). In COMT knock-out mice, striatal, hypothalamic and cortical MAO-dependent Cellular and tissue localization of MAO metabolite DOPAC and 3,4-dihydroxy- In the periphery, MAO is expressed in phenylglycol (DHPG) levels were increased, several mammalian tissues, the highest and COMT-dependent metabolites HVA concentrations being in the liver and kidney and 3-methoxy-4-hydroxyphenylglycol (Kalaria and Harik 1987; Kopin 1994). were absent in mice lacking the Comt -gene. Some tissues, e.g. bovine liver and kidney, A trend for DA and NA to increase in striatal contain mainly MAO-B, whereas rat and hypothalamic brain homogenates of skeletal muscle contains only minor COMT knock-out mice has also been found amounts of both forms (Shih et al. 1999b). (Huotari et al. 2002). Gogos et al. (1998) In the brain, the distribution of MAO- reported slightly aggressive behaviour of A and -B shows little species variation. male heterozygous mice. Mice lacking MAO-A is predominantly found in MAO-A or MAO-B have been observed catecholaminergic neurons, MAO-B is express different changes in behaviour (Shih most abundant in serotonergic and et al. 1999b). Since MAO-A is reported to histaminergic neurons and glial cells, being be responsible for 5-hydroxytryptamine, the dominant isozyme in the human brain NA and DA metabolism, mice deficient in (Kopin 1985; Riederer et al. 1986; Kalaria MAO-A have elevated brain levels of 5- and Harik 1987; Shih et al. 1999b). Both hydroxytryptamine and thus show more MAO isozymes are located on the outer aggressive behaviour. MAO-B is responsible 13 Review of the literature for phenylethylamine metabolism and 5.3. COMT inhibitors in the knock-out animals do not exhibit aggres- treatment of Parkinson’s disease sion (Shih et al. 1999b).

In humans, due to the role of MAO in the 5.3.1. Rationale for the use of COMT metabolism of catecholamines in the brain, inhibitors both MAO-A and MAO-B inhibitors have In Parkinson’s disease, dopaminergic been taken into clinical use. Selective neurons in the brain are gradually destroyed, inhibition of MAO-B by e.g. selegiline is leading to a DA deficiency. When clinical used in the treatment of PD to increase the symptoms appear, ca. 70-80 % of the concentration of DA in the human brain dopaminergic neurons have already been lost independently of levodopa (Deleu et al. (Schapira 2001; Teräväinen et al. 2001; Orth 2002). and Schapira 2002). The decrease in DA

COMT DOPA DHPG MHPG COMT MAO dopadecarboxylase aldehydereductase 3-OMD dopamine-β- phenylethanolamine-N- hydroxylase methyltransaminase DA NA A COMT

MAO COMT MAO COMT MAO MN

3-MT DOPAC NM DOMA MAO COMT MAO COMT MAO VMA HVA

Figure 1. Biosynthesis and catabolism of catecholamines. DOPA, 3,4-dihydroxyphenylalanine; DA, dopamine; NA, noradrenaline; A, adrenaline; NM, normetanephrine; DOMA, 3,4- dihydroxymandelic acid; MN, metanephrine; VMA, vanillylmandelic acid; 3-MT, 3- methoxytyramine; DOPAC, 3,4-dihydroxyphenylacetic acid; DHPG, 3,4-dihydroxyphenylglycol; HVA, homovanillic acid; MHPG, 3-methoxy-4-hydroxyphenylglycol; 3-OMD, 3-O- methyldopa.

14 Review of the literature levels in the striatum provokes the typical (DDC). As a COMT substrate, a smaller symptoms of PD (Schapira 2001). The amount of levodopa is metabolized by aetiology of neuronal destruction is COMT through O-methylation to form unknown. More than one aetiological factor 3-O-methyldopa (3-OMD) (Figure 2). has been postulated to cause PD, including Peripherally acting selective DDC inhibitors, genetic and environmental factors (Orth and such as carbidopa and benserazide, have been Schapira 2002). Levodopa, the most used as adjuncts to levodopa treatment in effective symptomatic treatment available PD patients (Bartholini and Pletscher 1975; for PD, has been in clinical use since the Rivest et al. 1999). DDC inhibitors increase 1960s (Birkmayer and Hornykiewicz the bioavailability of levodopa in the brain, 1961). Levodopa is a DA precursor, that but due to the increased metabolism by the can cross the BBB and is further COMT pathway, less than 10% of the decarboxylated to form DA. In the levodopa dose reaches the brain (Nutt and periphery, after oral administration, Fellman 1984; Männistö and Kaakkola approximately 70% of levodopa is 1990). The plasma concentration of 3- metabolized to DA by dopa decarboxylase OMD increases due to the long half-life of

3-OMD 3-OMD

COMT COMT inhibitor BBB inhibitor levodopa levodopa

DDC inhibitor

DA DA

Periphery Brain

Figure 2. Effect of COMT inhibitors on levodopa metabolism. DA, dopamine; 3-OMD, 3-O- methyldopa; DDC, dopa decarboxylase; BBB, blood-brain barrier.

15 Review of the literature this metabolite and remains high during inhibitors include e.g. pyrogallol and its chronic therapy (Kuruma et al. 1971). The derivatives, tropolones, 3,4-dihydroxy-2- methylated product may further reduce the methylpropiophenone (U-0521) and utilization of levodopa by competing with catechol. The COMT inhibition activity of it on the BBB level (Nutt and Fellman all of these compounds was weak or non- 1984; Reches and Fahn 1984; Männistö selective, they had poor bioavailability and and Kaakkola 1990). By blocking the O- they tended to be short-acting. None of the methylation pathway of levodopa, COMT first-generation COMT inhibitors was inhibitors increase its bioavailability and therefore taken into routine clinical use reduce the formation of 3-OMD in PD (Ericsson 1971; Reches and Fahn 1984). patients (Männistö 1994). With Pyrogallol (1,2,3-trihydroxybenzene) concomitant use of COMT inhibitors and (Figure 3) was shown to be a potent COMT levodopa, daily doses of levodopa can be inhibitor in vitro and in vivo by Axelrod and reduced while retaining the clinical benefit LaRoche in 1959. It was a good substrate (Nutt et al. 1994; Kurth et al. 1997). The of COMT, acting as a competitive inhibitor, administration of COMT inhibitors to PD and had a short duration in vivo (Belleau patients slows the elimination of levodopa and Burba 1961), but it induced toxic effects from the plasma, which increases the ON due to properties other than COMT time in these patients (ON time, sudden inhibition (Wylie et al. 1960; Angel and return of effectiveness that may be Rodgers 1968). Pyrogallol acts both in the accompanied by hyperkinesia) (Fahn 1974; CNS and periphery (Angel and Rodgers Nutt et al. 1994; Dingemanse 1997). 1968), but with regular use for two weeks, In the brain, reuptake of catecholamines COMT activity in the rat heart and brain into presynaptic neurons and metabolism showed no major changes after a sub- by MAO are the most important routes in cutaneous daily dose of 50 mg/kg (Maitre eliminating released catecholamines; 1966). While pyrogallol is not suitable for COMT plays only a minor role. COMT clinical use because of these properties, it inhibition is reported not to alter has been used as a tool in studies on COMT catecholamine levels in plasma noticeably inhibition. (Illi et al. 1994; Li et al. 1998; Männistö Gallates are effective COMT inhibitors and Kaakkola 1999; Rojo et al. 2001). in vitro and in vivo but have an extremely short duration of action in the rat, only 15- 5.3.2. First-generation COMT 30 minutes after an intraperitoneal injection inhibitors (Dorris and Dill 1977). A few clinical trials Following description and partial performed with N-butyl gallate (Figure 3) characterization of the COMT enzyme in failed to reveal any advantage over DDC the late 1950s and the reported occurrence inhibitors administered together with of 3-O-methylated metabolites of levodopa (Ericsson 1971). When ten catecholamines in mammals, the develop- parkinsonian patients in whom the ment of first-generation COMT inhibitors maximum dosage of levodopa had been began (Axelrod and Tomchick 1958). These reached were treated with an initial dose of

16 Review of the literature

N-butyl gallate 250 mg, followed by 1964b). Tropolone 100 mg/kg increasing doses of up to 750 mg daily, the administered intraperitoneally caused an symptoms were alleviated. However, the increase in DA and DOPAC concentrations clinical improvement was not as effective in rat striatum, while simultaneously as that obtained with DDC inhibitors reducing the concentration of HVA (Broch (Ericsson 1971). 1972). However, the central COMT Catechol (1,2-dihydroxybenzene) and inhibition was quite modest and was not several of its derivatives are COMT correlated with the concentration of inhibitors in vitro and in vivo (Ross and tropolone. Moreover, tropolones have been Haljasmaa 1964a; Baldessarini and Greiner reported to be toxic in mice, rats and rabbits 1973), but they are toxic in vivo because of at doses inducing convulsions (Ri 1951). properties unrelated to COMT inhibition Since the majority of first-generation (Angel and Rodgers 1968; Bakke 1970). COMT inhibitors had metal-chelating Some of them, e.g. catechol-configurated properties, a chelation mechanism was flavonoids, are more potent inhibitors of proposed for complex formation between COMT in vitro than pyrogallol (Gugler and catecholamines and the COMT enzyme Dengler 1973). Catechol itself is highly (Senoh et al. 1959). Metal chelators of toxic, inducing convulsions in the rat after various structures were tested in vitro in an oral dose of 100 mg/kg (Angel and mouse brain homogenates for their capacity Rodgers 1968; Bakke 1970). U-0521 (3, to inhibit the COMT enzyme. Some of 4-dihydroxy-2-methylpropriophenone) these compounds, e.g. 8-hydroxyquinoline, (Figure 3) was reported to effectively were more potent COMT inhibitors than diminish plasma and brain 3-O-methyldopa pyrogallol in vitro, but the majority failed (3-OMD), enhancing the accumulation of to express any COMT inhibitory effects in both DOPA and DA in rat striatum after mouse brain extract in vitro, indicating that an intraperitoneal injection of 100 mg/kg the metal-chelating effect alone was or more (Fahn et al. 1979; Reches and Fahn insufficient to produce COMT inhibition 1984). However, it was not effective when (Ross and Haljasmaa 1964a). administered orally to rats or in clinical use with parkinsonian patients (Fahn et al. 5.3.3. Second-generation COMT 1979; Reches and Fahn 1984). inhibitors entacapone and tolcapone Tropolone (2-hydroxycyclohepta- A new generation of COMT inhibitors was trienone) (Figure 3) and its derivatives discovered in the 1980s,and these inhibit both peripheral and central COMT compounds were shown to inhibit COMT in vitro and in vivo but are short-acting and to alter levodopa metabolism in vivo. (Belleau and Burba 1961; 1963; Ross and Entacapone, tolcapone, nitecapone and the Haljasmaa 1964a). The inhibitory effects recently described BIA 3-202 and 3-335 are are comparable with those of pyrogallol in all structurally nitrocatechols, while CGP the periphery; however, in the brain, 28014 is a pyridine derivative (Figure 3) tropolone shows much less activity than (Nissinen et al. 1988; Borgylua et al. 1989; pyrogallol (Ross and Haljasmaa 1964a; Waldmeier et al. 1990; Parada et al. 2001;

17 Review of the literature

HO N HO O HO CN OH HO

NO2

Pyrogallol Entacapone

O O OH HO

NO2

Tropolone Tolcapone

O HO HO O O

HO HO O

OH NO2

n-Butyl gallate Nitecapone

O O HO HO HO HO NO2

U-0521 BIA 3-202

HO NO2

HO N N N O NO2

2,4 -Dinitrophenol CGP 28 014 Figure 3. Structure of first-generation COMT inhibitors (pyrogallol, tropolone, n-Butyl gallate, U-0521), second-generation inhibitors (entacapone, tolcapone, nitecapone, BIA 3-202, CGP 28014) and the uncoupling agent 2,4-dinitrophenol.

18 Review of the literature

Bonifacio et al. 2002). The nitrocatechol- with levodopa and carbidopa to rats or structured COMT inhibitors entacapone cynomolgus monkeys reduced the and tolcapone are known as tight-binding formation of 3-OMD and elevated serum inhibitors, but the binding to COMT is levodopa levels (Cederbaum et al. 1991; reversible (Nissinen et al. 1992; Lotta et al. Nissinen et al. 1992). The increases in 1995). They are highly selective inhibitors levodopa, DA and DOPAC concentrations with virtually no action on tyrosine were dose-dependent, and the lowest hydroxylase, dopamine ß-hydroxylase, effective dose in the rat was 3 mg/kg, which DDC or MAO-A and MAO-B (Bäckström also reduced 3-OMD concentrations et al. 1989; Zürcher et al. 1990b). (Nissinen et al. 1992). However, even at a Entacapone acts mainly peripherally, dose of 30 mg/kg, the HVA concentration whereas tolcapone crosses the BBB (Zürcher in the rat was not decreased, indicating that et al. 1990a; Nissinen et al. 1992; central COMT activity is not inhibited by Dingemanse 1997). entacapone (Nissinen et al. 1992). Tolcapone, in contrast, induced about 90% Pharmacology in vitro and in vivo decrease in the levels of HVA and 3-MT in Entacapone selectively inhibits rat S- the rat brain at the dose of 30 mg/kg p.o. COMT from the duodenum, liver, brain (Zürcher et al. 1990b). After oral and erythrocytes at low nanomolar administration to rats, tolcapone 0.2 mmol concentrations in vitro (Nissinen et al. (54.6 mg/kg) markedly reduced the 1992). After an oral dose of 10 mg/kg peripheral metabolism of exogenous DOPA entacapone S-COMT acitivity is greatly (100 mg/kg p.o.) to 3-OMD when given reduced in rat duodenum, liver and together with benserazide (50 mg/kg p.o.), erythrocytes. The longest duration of thus increasing the bioavailability of DOPA inhibition is in the duodenum, but striatal in the brain (Zürcher et al. 1990b). COMT activity is suppressed only Tolcapone inhibits brain COMT activity transiently (Nissinen et al. 1992). in the rat at single doses of 3-10 mg/kg, Tolcapone, by contrast, markedly inhibits while 30 mg/kg or more of entacapone is both peripheral and central COMT needed for temporary suppression of (Zürcher et al. 1990b). In several human COMT activity (Zürcher et al. 1990a; peripheral organs (liver, kidneys, Männistö et al. 1992a; 1992b). Extracellular duodenum, lungs), both entacapone and DOPAC tends to increase and HVA to tolcapone have been shown to be potent decrease in the rat striatum after a dose of COMT inhibitors ex vivo (De Santi et al. 10 mg/kg tolcapone intraperitoneally, 1998). Hepatic COMT activity of human whereas the respective dose of entacapone liver ex vivo was inhibited by entacapone at has no effect (Kaakkola and Wurtman an IC50 151 nmol/l, while that for 1993). At an intraperitoneal dose of 30 mg/ tolcapone was 773 nmol/l; the results for kg, neither entacapone nor tolcapone has other organs were similar (De Santi et al. any effect on extracellular levels of free 1998). catecholamines in the brain of anaesthetized Oral entacapone administered together rats, although tolcapone does reduce

19 Review of the literature

COMT-dependent metabolites (Li et al. Acute toxicity of both entacapone and

1998). In rabbits, 30 mg/kg of tolcapone tolcapone is low; oral LD50 values in the rat intravenously increases catecholamine are higher than 2 g/kg, and toxicity is not baseline levels in plasma ca. 300% (Garrido modified by the combined administration et al. 1994). of levodopa and carbidopa (Borgulya et al. 1991; Törnwall and Männistö 1991; In vivo Parkinson animal models Kaakkola et al. 1994). The metabolic routes Proof of principle has been shown in animal of tolcapone are more complicated than models of PD for both entacapone and those of entacapone. The main metabolic tolcapone. When neurological deficits pathway for entacapone and tolcapone is resembling those seen in PD were induced glucuronidation, but tolcapone is also in monkeys by administration of neurotoxin metabolized through methylation, 1-methyl-4-phenyl-1,2,3,6-tetrahydro- reduction and hydroxylation (Wikberg et pyridine, co-administration of entacapone al. 1993; Dingemanse et al. 1995). 12.5 mg/kg p.o. or tolcapone 15 or 30 mg/ kg p.o. prolonged the antiparkinsonian Clinical use activity of levodopa/carbidopa (Smith et al. The symptoms of PD are alleviated by 1997; Tasmar Product Monograph 1997). administration of levodopa. It improves the Unilateral destruction of dopaminergic disability of patents with PD more than any neurons in the substantia nigra of rats by other drug. Since levodopa is actively neurotoxin 6-hydroxydopamine led to metabolized, it is given concomitantly with contralateral circling behaviour when rats DDC inhibitors, which inhibit the were treated with levodopa/carbidopa. conversion of levodopa to DA, thus Entacapone 3 or 10 mg/kg and tolcapone increasing its bioavailability. However, most 30 mg/kg prolonged the circling response of the exogenous levodopa is methylated to levodopa/carbidopa in these rats (Da to 3-OMD by COMT. The administration Prada et al. 1993; Törnwall and Männistö of entacapone or tolcapone together with 1993). levodopa and a DDC inhibitor increases Entacapone and tolcapone inhibit both plasma concentrations of levodopa dose- S- and MB-COMT, but tolcapone inhibits dependently without significantly affecting MB-COMT 10 times more effectively than the peak plasma concentration of levodopa entacapone (Lotta et al. 1995). Tolcapone (Nutt et al. 1994; Fahn 1998; Teräväinen is very potent in inhibiting MB-COMT in et al. 2001). The concentrations of DOPAC rat liver and brain tissue in vivo after oral and HVA, the main metabolites of DA, are administration (Vieira-Coelho and Soares- increased after treatment with entacapone, da-Silva 1999). However, the expression of while after tolcapone DOPAC is increased MB-COMT in tissues is far less than that and HVA decreased. Although the central of S-COMT, although the former possesses actions of entacapone and tolcapone differ, higher affinity for catecholamines than the systemic administration leads to an increased S-COMT (Guldberg and Marsden 1975; level of DA in the brain. Both inhibitors Lotta et al. 1995). are eliminated rapidly after an oral dose. The

20 Review of the literature plasma elimination half-life after entacapone 5.4. Drug induced hepatotoxicity is about 1-2 hours, whereas the half-life of tolcapone is approximately 2-3 hours. The Although most hepatotoxic compounds are bioavailability of entacapone (35%) is less detected during non-clinical testing and are than that of tolcapone (65%). Entacapone not developed further, hepatotoxicity is 98% and tolcapone 99.9% bound to remains the leading cause of withdrawal of plasma proteins. Since entacapone is drugs from the market (Lasser et al. 2002; metabolized more rapidly than tolcapone, Thomas 2002). This is because drug- it is administered in clinical use up to 10 induced hepatotoxicity is generally only seen times a day with each dose of levodopa, after a drug has been marketed and used by while tolcapone is administered three times tens of thousands of patients, as opposed a day. Both inhibitors provide benefits to phase II clinical trials with limited numbers parkinsonian patients in concomitant of participants (Batt and Ferari 1995; Lasser dosing with levodopa (Keränen et al. 1994; et al. 2002). Dingemanse 1997; Männistö and Kaakkola 1999). One to two months’ treatment with 5.4.1. Mechanisms, clinical symptoms entacapone or tolcapone at a clinical dose and signs of hepatotoxicity of 200 mg (entacapone with each levodopa Drug-induced hepatotoxicity is typically dose, tolcapone three times daily) inhibits observed as a hepatocellular or cholestatic the formation of 3-OMD by 50% and injury (Watkins 2000). Cholestatic injury 80%, respectively (Kaakkola 2000). Both is reflected as a selective interference of the entacapone and tolcapone significantly drug with the liver’s ability to make and increase the ON time and decrease the OFF secrete bile (Watkins 2000). In humans, it time in patients with advanced PD is characterized by an elevation in serum (Kaakkola 2000). The increase in daily ON alkaline phosphatase (APHOS) and time varies from 1 to 2 hours with bilirubin. In liver tests, cholestatic injury is entacapone and from 0 to 2.5 hours with suspected when the value of APHOS is two tolcapone (Kaakkola 2000). times the upper limit of normal (ULN) or The plasma levels of DA, NA, A and total the ratio of alanine transaminase (ALAT) catecholamines were reported to increase in to APHOS is less than two (Benichou seven out of eight PD patients at a mean 1990; Kaplowitz 2001). However, in the tolcapone dose of ca. 270 mg/day (Rojo et rat, APHOS is predominantly of intestinal al. 2001). One patient simultaneously had origin, and thus, it cannot be used as a elevated liver enzymes and a 513% increase biomarker of cholestatic injury as such but in catecholamine levels. Entacapone has not should be used together with γ-glutamyl been reported to change plasma cate- transferase and bilirubin (Loeb and Quimby cholamine levels in healthy volunteers (Illi 1999). In hepatocellular injury, liver cells et al. 1994; Scheinin et al. 1998). are damaged, and the toxic signs manifest as a rise in clinical serum chemistry parameters (Benichou 1990; Kaplowitz 2001). In man, alanine aminotransferase (ALAT),

21 Review of the literature as a relatively liver-specific enzyme, is the and dogs (Balazs et al. 1961; Loeb and most useful marker of hepatocellular injury Quimby 1999). Balazs et al. (1961) have in patients. In animals, especially in mice, reported a distinct correlation between rats and dogs, hepatic ALAT activity is 3- ALAT activity and the degree of necrosis, 10 times higher than in any other tissue although in other studies the correlation is (Loeb and Quimby 1999). ALAT is less clear (VanVleet and Alberts 1968). It localized both the cytoplasma and has been postulated that due to changed mitochondria of cells, but the activity is permeability of the cell membranes ALAT several times higher in the former. It is and SDH can leak into serum before cell released to serum when liver cells are dying, death and that histological necrosis is seen thus elevating the normal levels of serum variably (Loeb and Quimby 1999). ALAT. In patients, a serum ALAT level two- The lack of specific clinical or histological or threefold the ULN or an ALAT/APHOS features of drug-induced liver lesions is one ratio of five or more is considered clinically of the reasons behind the sporadic incidence significant and a sign of hepatocellular liver of liver injury since injuries produced by injury (Benichou 1990; Dossing and Sonne drugs may be indistinguishable from those 1993; Watkins 2000; Kaplowitz 2001). In due to other reasons (Dossing and Sonne non-clinical toxicity studies, ALAT is used 1993; Watkins 2000). Some drugs cause as a marker of hepatocellular injury, with liver toxicity in a dose-related manner with activity values of treated and untreated a reaction arising only after a sufficient control animals being compared. In amount of the drug is taken. Dose- addition to ALAT activity, increased sorbitol independent reactions occur as a rare dehydrogenase (SDH) activity appears early complication of therapeutic doses of the in the course of liver injury in animals. SDH drug (Dossing and Sonne 1993). is a cytosolic enzyme with the highest Hepatocellular necrosis can occur within activity in the liver and kidneys. A positive minutes after a toxic insult and is usually correlation between increased ALAT and associated with severe metabolic SDH activities and hepatocellular changes disturbances. Cell necrosis is a typical in the rat has been reported (Travlos et al. consequence of acute hepatic injury. The 1996). ALAT activity is cleared in about onset of cell death is characterized by four days, and SDH activity returns to breakdown of the plasma membrane normal even faster (Mizrahi et al. 1987; permeability barrier, which leads to loss of Loeb and Quimby 1999). Although both metabolic intermediates, leakage of enzymes these enzymes are located periportally in the and collapse of all electrical and ionic liver, the main target for the injury is the gradients across the plasma membrane centrilobular area due to diminished oxygen (Mehendale et al. 1994). Fountoulakis et during the passage through periportal areas al. (2002) reported a threefold elevation of to the centrilobular area (Haschek et al. ALAT six hours after carbon tetrachloride 2002). A few reports are available on dosing of rats, and at 24 hours, the increase histological hepatic necrosis in connection in ALAT values was extremely pronounced. with the elevation of serum ALAT in rats Typical centrilobular hepatocellular

22 Review of the literature necrosis induced by carbon tetrachloride is in state 4 oxygen consumption in isolated reported to be associated with catecholamine rat liver mitochondria, the cytotoxicity is release (Schwetz and Plaa 1969; Roberts et linked to uncoupling of oxidative al. 1997). A and NA can potentiate the phosphorylation (Nakagawa and Tayama sensitivity of the liver to carbon 1995). tetrachloride, but administered alone they The non-clinical toxicity studies fail to induce any toxicity. The toxicity is performed for the marketing applications characterized by increased serum of second-generation COMT inhibitors aminotransferase activities and hepato- entacapone and tolcapone revealed no signs cellular necrosis (Schwetz and Plaa 1969). of hepatotoxicity (Eckhardt et al. 1996; Endogenous A and NA are also capable of Schläppi et al. 1996a; 1996b; 1996c; Tasmar potentiating the toxicity induced by carbon Product Monograph 1997; Entacapone tetrachloride, as well as several other Product Monograph 1999). In non-clinical adrenergic drugs, in the rat (Roberts et al. studies with tolcapone in mice, only slight 1997). The mechanism behind the to moderate hepatocellular hypertrophy was adrenergic agents’ increased toxicity is observed after 18 months’ treatment (dose suggested to be related to induced hypoxia not given) (Olanow 2000). within the liver cells (Zempel et al. 1983). In vivo, COMT inhibition alone has not been reported to induce hepatotoxicity. 5.4.2. Hepatotoxicity induced by COMT knock-out mice, developed by COMT inhibitors means of homologous recombination in The first-generation COMT inhibitors have embryonic stem cells leading to a mouse been studied in relation to hepatotoxic strain in which the gene encoding the properties. Tropolone has been reported to COMT enzyme is disrupted, have shown induce non-specific hepatitis in mice in vivo no signs of liver injury (Gogos et al. 1998; after a single dose of 400 mg/kg; this is also Huotari et al. 2002). The mice have been viable and fertile, with only minor changes the oral LD50 for mice (Table 1) (Ri 1951; Nakagawa and Tayama 1998). In isolated in catecholamine metabolism (Huotari et rat hepatocytes, tropolones induced a dose- al. 2002). and time-dependent loss of cell viability, The first-generation COMT inhibitors which is associated with a decrease of were not taken into clinical use because of intracellular adenosine triphosphate (ATP) properties connected to short duration of (Ri 1951; Nakagawa and Tayama 1998). action, toxicity or inefficiency in clinical Tropolone is also reported to suppress the studies (Ericsson 1971; Reches and Fahn 1984). Hepatotoxic signs were observed in growth of murine hepatocytes, with the IC50 being ca. 1.23 µg/ml (Inamori et al. 1993). clinical use up to 12 weeks with N-butyl Gallates induced a concentration-depen- gallate, when markedly abnormal ALAT dent cell death in rat hepatocytes in vitro, activities (values not given) were reported which was preceded by the depletion of ATP in four out of 11 patients receiving dosages (Table 1) (Nakagawa and Tayama 1995). above 2250 mg daily (Simpson and Varga Since propyl gallate also induced an increase 1972).

23 Review of the literature

The use of entacapone has been reported November 1998, marketing authorization to be associated with hepatotoxicity in one for tolcapone was suspended in the patient after three weeks’ treatment (Fisher European Union because of increasing et al. 2002). Two other cases were earlier concern about reports of severe reported to the Australian Adverse Drug hepatotoxicity (European Medicines Reaction Advisory Committee (ADRAC). Evaluation Agency 1998). Findings of severe However, in two of these three cases, the centrilobular hepatic necrosis with patients had concomitant medications with inflammatory infiltrates (plasma cells, hepatotoxic potential, and the third case had eosinophils) due to tolcapone treatment a history of long-standing alcohol abuse and were reported in three parkinsonian patients alcohol-induced liver cirrhosis (Beck et al. (Olanow 2000). In one patient who died 2002). In none of these cases was after tolcapone treatment, fulminant hepatotoxicity hepatocellular, instead being hepatitis was verified histologically (McCaul cholestatic injury in terms of ALAT activity et al. 1986; Spahr et al. 2000). In all three or the ALAT/APHOS ratio (Watkins 2000; tolcapone-treated patients, ALAT was Kaplowitz 2001). increased to at least three times the ULN In clinical use of tolcapone, severe (Olanow 2000). The incidence of hepatic hepatotoxicity has been observed in several failure was 10-100 times higher than in the PD patients (Table 1). A total of nine cases general population (Mayoral et al. 1999). of abnormal hepatic reactions have been In clinical trials, 3.5% of tolcapone-treated reported, three with a fatal outcome (Assal patients had elevated liver enzymes three et al. 1998; Bonifati and Meco 1999; times higher than the ULN compared with Colossimo 1999; Olanow 2000). In only 1% of placebo-treated patients

Table 1. Hepatotoxic properties of COMT inhibitors.

COMT Toxicity to hepatocytes In clinical use Reference inhibitor In vitro In vivo Serum Liver aminases toxicity

Tropolone yes hepatitis not in not in Ri 1951; Inamori et al. 1993; clinical use clinical use Nakagava and Tayama1998 Gallates yes no data ↑ NAD Simpson and Varga 1972; Nakagava and Tayama 1995 U-0521 no data no data NAD NAD Reches and Fahn 1984 Entacapone no data NAD NAD NAD Keränen et al. 1994; Vaalavirta et al. 1998 Tolcapone no data necrosis ↑ hepatitis Assal 1998; Vaalavirta et al. 1998; Olanow 2000

NAD = no abnormalities detected

24 Review of the literature

(Mayoral et al. 1999; Watkins 2000). In leading to mitochondrial depolarization, addition, a clear dose-response effect was uncoupling of oxidative phosphorylation, evident. ATP depletion and cell death (Mehendale et al. 1994).

5.5. Uncoupling as a 5.5.1. Uncoupling at cellular level phenomenon Oxidative phosphorylation is the major In healthy cells, ATP-driven Ca2+ pumps mechanism by which aerobic cells produce maintain a Ca2+ gradient across the plasma ATP using a respiratory assembly located membrane. Since calcium precipitates are in the inner mitochondrial membrane (Berg prominent in necrotic tissue, an increase in et al. 2002). In oxidative phosphorylation, free Ca2+ may be one of the first responses ATP synthesis is coupled to the flow of to cell injury. However, in at least some electrons towards oxygen by a proton models of drug-induced liver injury caused gradient across the inner mitochondrial by hypoxia, Ca2+ does not rise before the membrane (Figure 4). Electron flow results toxic lesion (Harman et al. 1992; in pumping of protons out of the Mehendale et al. 1994). Uncouplers of mitochondrial matrix and generation of oxidative phosphorylation, such as Br- membrane potential. The membrane A23187, can cause bioenergetic cell death potential increases as the electrons flow rather than Ca2+-mediated cell killing down the respiratory chain to oxygen. The (Mehendale et al. 1994). Thus, cell killing mitochondrial membrane potential across in models of hypoxic injury to hepatocytes the inner membrane is the driving force for is not dependent on Ca2+, but mitochondria phosphorylation since ATP is synthesized are important targets of hepatocellular injury when protons flow back to the matrix by toxicants that causes oxidative stress, (Fromenty et al. 1990; Brown 1992). The

H+ + H+ H+ H+ + + + H O O 2 - - - DNP 2

- + + e H H + + + H H ADP + Pi H H2O ADP + Pi H2O ATP ATP Matrix Matrix Membrane Membrane

A. B.

Figure 4. In mitochondria, oxidation and ATP synthesis are coupled by transmembrane proton fluxes (A). When an uncoupling agent is present (B), a protonated form of e.g. DNP transports protons across the inner mitochondrial membrane, neutralizing the proton gradient. 25 Review of the literature tight coupling of electron transport and results in a rise in body temperature (Tainter phosphorylation in mitochondria can be and Wood 1934; Kaiser 1964; Terada 1990; disrupted by uncoupling agents, which act Brown 1992). as proton carriers that allow protons to flow The five different metabolic states of into the mitochondrial matrix without mitochondria have been determined passing through the transmembrane protein experimentally by Chance and Williams complex that synthesizes ATP from (1955) (Table 2) to clarify steady-state levels adenosine diphosphate (ADP) (Terada during passage of electrons from the 1990; Krähenbuhl 2001). In the presence substrate to oxygen. In state 1, mito- of an uncoupling agent, electron transport chondria display a slow respiration rate proceeds in the normal fashion, but ATP is (Chance and Williams 1955; Hackenbrock not formed since the proton-motive force 1966). State 2 is characterized by high across the inner membrane is dissipated. The concentrations of ADP and respiration is rate of oxygen consumption in the absence close to zero. State 3 is the active state of of ADP in respiratory substrate- respiration and phosphorylation (Chance supplemented mitochondria is low. With and Williams 1955). In aerobic state 4, ADP the addition of an uncoupler, the respiratory is totally phosphorylated and the respiration rate increases suddenly in a dose-dependent rate is again low. In state 5, oxygen is lacking manner, and at the same time, the and the ADP level is high. The adding of mitochondrial membrane potential uncoupling agents converts this state to state decreases (Terada 1990). The loss of 2 (Chance and Williams 1956). In all states, respiratory control leads to increased oxygen mitochondria show the typical consumption and to decreased ATP morphological features determined by production as a compensatory function of Hackenbrock (1966). The level of the cell. The energy is liberated as heat, which respiration in vitro is expressed as a respiratory

Table 2. Metabolic states of mitochondria according to Chance and Williams (1955).

Characteristics Ultrastructural findings State ADP level Respiration Rate-limiting Mitochondrial morphology rate substance (Hackenbrock 1966)

1 low slow ADP condensed formation, intramitochondrial granules 2 high slow substrate highly condensed matrix 3 high fast respiratory chain matrix ↓, intracristal space ↑, small granules in matrix, inner membrane foldings 4 low slow ADP condensed formation, intramitochondrial granules 5 high 0 oxygen

26 Review of the literature control ratio (RCR), which is the gastro-intestinal symptoms, such as nausea, relationship between the respiration rate in vomiting and loss of appetite, were state 3 and the respiration rate in state 4. If common. With long-term use, cutaneous the respiration rate in state 4 increases, the lesions were frequent and were complicated mitochondrial membrane potential by polyneuritis, otitis media and cataracts. decreases, no ATP is formed and electron As several patients developed cataracts and transport and phosphorylation is uncoupled some died due to overdose, the treatment (Berg et al. 2002). of obesity with DNP was discontinued in Various compounds are known to be the 1940s (Horner 1942; Simon 1953). uncouplers, but weakly acidic uncoupling Most fatal cases have, however, been among agents are the most potent uncouplers, workers in the agricultural industry since speculated to produce uncoupling by their DNP was used as a pesticide and herbicide protonophoric action on the mitochondrial (Bidstrup and Payne 1951). membrane. Typical examples of uncoupling The symptoms of intoxication in man agents include salicylic acids, non-steroidal consist of body temperature exceeding inflammatory drugs and some local 40ºC, extreme fatigue, profuse sweating and anaesthetics (Krähenbuhl 2001). As a weak dehydration, laboured respiration and rapid acid, the uncoupling effect of DNP is onset of rigor mortis after death (Figure 5) attributable to its protonophoric nature (Perkins 1919; Cutting and Tainter 1933; (Terada 1990). Entacapone and tolcapone Poole and Haining 1934; Horner 1942). are both structurally related to the known Fatal doses of DNP in the treatment of uncoupling agent DNP. Nissinen et al. obesity have been between 2.66 mg/kg/day (1997) have shown that tolcapone interacts for 14 days and 46 mg twice 7 days apart with mitochondrial oxygen consumption, (Masserman and Goldsmith 1934; Poole being a more potent uncoupler than DNP and Haining 1934). In rats and dogs, oral in vitro. treatment with DNP has induced increased heart rate and abnormal electrocardiographic 5.5.2. Signs of uncoupling in vivo readings, elevation of body temperature, Uncoupling agents inducing disruption increased oxygen consumption and between electron transport and phos- increased metabolic rate (Tainter and phorylation are generally weakly acidic, Cutting 1933b; Kaiser 1964; Bakke and lipid-soluble substances, which enables them Laurence 1965). The cause of death in acute to carry protons across the inner mito- animal studies is generally considered to be chondrial membrane (Terada 1990). DNP, a result of the pyretic effect of DNP, a known uncoupling agent of oxidative produced by an increase in metabolic rate. phosphorylation, was used in the 1930s for Spencer et al. (1948) indicated that rats weight reduction in humans, until it was treated once by gavage either died within 1- found to be a toxic compound (Figure 3). 2 hours or recovered completely. Since Horner et al. (1942) reported that more than uncoupling of oxidative phosphorylation 4500 patients used “one capsule three times induces a reduction in mitochondrial energy a day after meals”. Among these patients, production, leading to usage of glycogen in

27 Review of the literature the liver and muscles, instantly occurring related to uncoupling in parkinsonian rigor mortis expresses very rapid patients. consumption of ATP and glycogen from Pyrogallol, which structurally resembles the skeletal muscle after death (Gracey DNP, is reported to be a 4-5 times weaker 1981). In acute DNP poisoning, the uncoupling agent than DNP in vitro (Ross glycogen content in muscles and liver is and Haljasmaa 1964a; Conyers et al. 1968). diminished (Stoner et al. 1952). Rigor It uncouples oxidative phosphorylation in mortis has been reported to either occur vitro in isolated mitochondria of both rat within 10 minutes of death due to DNP kidney and beef heart (Table 3) (Conyers et treatment (Figure 5) (Tainter and Wood al. 1968). Complete uncoupling was 1934) or already be complete at the time of achieved at a concentration of 1 µM death (Barnes 1969; Parker 1973). expressed as the ratio of phosphate esterified to oxygen consumed. In the same 5.5.3. Uncoupling induced by COMT experiment, complete uncoupling by DNP inhibitors was achieved at a concentration of 0.2 µM Several of the first-generation COMT (Conyers et al. 1968). inhibitors have been shown to possess Tropolone and its derivatives reduced uncoupling properties in vitro. None of intracellular ATP levels in isolated rat them has been taken into long-term clinical hepatocytes, and this reduction is associated use. The second-generation COMT with the loss of cell viability (Nakagawa and inhibitors have also been reported to express Tayama 1998). In isolated rat liver uncoupling properties in vitro, but in clinical mitochondria, tropolones also caused a use, they have not induced any symptoms concentration-dependent increase in the rate

Hypoxia Respiration Ï

ATP/ADP Ð Energy Body temperature Ï metabolism Ï

RCR Ð Glycogen Ð Rapid rigor mortis

Mitochondrial Liver damage dysfunction

Figure 5. Biological effects of uncoupling of oxidative phosphorylation in mitochondria. RCR, respiratory control ratio.

28 Review of the literature of state 4 oxygen consumption, thus having µM), while entacapone is a weak uncoupler uncoupling properties of oxidative at high concentrations only (Table 3) phosphorylation (Table 3) (Nakagawa and (Nissinen et al. 1997). Borroni et al. (2001) Tayama 1998). This indicates that the reported uncoupling properties of tolcapone cytotoxicity induced by tropolones is at a similar concentration range (3 µM), and associated with ATP depletion via tolcapone also shows cytotoxic properties mitochondrial dysfunction related to at the same concentration in rat hepatocytes. oxidative phosphorylation (Nakagawa and Tayama 1998). 5.5.4. COMT inhibition and Propyl gallate and related gallates have uncoupling in relation to protein been reported to express uncoupling effects binding on mitochondria isolated from rat liver Entacapone is 98% and tolcapone 99.9% (Table 3) (Nakagawa and Tayama 1995). bound to plasma proteins (Keränen et al. As a sign of uncoupling of oxidative 1994; Dingemanse et al. 1995). The strong phosphorylation, propyl gallate induced an protein binding is postulated to diminish increase in the state 4 oxygen consumption the pharmacologically active concentration, in rat liver mitochondria as well as a thus also affecting the uncoupling properties depletion of ATP in rat hepatocytes of the drug (Borroni et al. 2001). In (Nakagawa and Tayama 1995). practice, the plasma concentration needed Tolcapone has proven to be a potent for clinical response to most of the drugs is uncoupler of oxidative phosphorylation in less than the saturated concentration, and vitro at low micromolar concentrations (2.6 therefore, an equilibrium exists between the

Table 3. Uncoupling and hepatotoxic properties of COMT inhibitors.

COMT Uncoupling effects Liver Central References inhibitor toxicity effects µ Entacapone EC50* 58.0 M no no Nissinen et al., 1992, 1997; Vaalavirta et al., 1998 Tropolone intracellular ATP ↓, yes ±0 Ri 1951; Nakagava and Tayama 1995, 1998 state 4 respiration ↑ Pyrogallol 1 mM (complete) no data yes Wylie et al. 1960; Ross and Haljasmaa 1964a; Conyers et al. 1968 Gallates intracellular ATP ↓, yes yes Wylie et al. 1960; Ross and Haljasmaa state 4 respiration ↑ 1964a; Simpson and Varga 1972; Nakagava and Tayama 1995 µ Tolcapone EC50* 2.6 M yes yes Nissinen et al. 1997; Vaalavirta et al. 1998; Männistö and Kaakkola, 1999

*EC50 is the concentration for half of the maximal stimulation of succinate-supported mitochondrial respiration.

29 Review of the literature free and bound drug in plasma. The protein- used as an experimental example of the bound fraction is stored in the albumin and lesions induced by uncoupling. Most of the the free fraction penetrates such tissues as experimental studies have been performed the liver, where the drug accumulates if it is in isolated hepatocytes in vitro or on rat not efficiently metabolized by, e.g. skeletal muscle in vivo (Hackenbrock 1966; glucuronidation or sulphation. Despite Buffa et al. 1970; Melmed et al. 1975; extensive protein binding, both inhibitors Sahgal et al. 1979; Shah et al. 1982; 1985; have proved to be potent COMT inhibitors Kawahara et al. 1991; Nakagawa and in clinical use (Kaakkola 2000). The Tayama 1995). Only in a few in vivo studies concentration that produces COMT with DNP have possible histological lesions inhibition is about equal for both inhibitors been sought; liver tissue in particular has been (Table 4), but tolcapone expresses studied. After an oral single-dose treatment uncoupling properties at a much lower of DNP to rats, lesions in the kidney, liver concentration than entacapone (Nissinen et and spleen have been observed (Spencer et al. 1997; Borroni et al. 1999; 2001). al. 1948; Arnold et al. 1976). In the kidneys, tubular necrosis or degeneration is typically present, in the spleen congestion and 5.6. Pathological findings in haemosiderosis, and in the liver cloudy tissues in connection with swelling and slight congestion. However, the uncoupling of oxidative liver of dogs treated with single doses of phosphorylation DNP revealed no histological findings (doses not given) (Tainter and Cutting 1933b). In skeletal muscle, the only light 5.6.1. Histological findings microscopy findings, reported in Histological findings due to uncoupling of experimental rat studies with DNP, consist oxidative phosphorylation in the tissues are of subsarcolemmal accumulations of rare. Since the uncoupling of oxidative mitochondria seen as ragged red fibres after phosphorylation is behind the toxic modified Gomori trichrome staining mechanism of DNP, it has been extensively (Melmed et al. 1975; Sahgal et al. 1979). The red fibres were confined to oxidative type I muscle fibres. Large subsarcolemmal Table 4. Concentrations of entacapone and aggregates were verified to be mitochondria tolcapone inducing a 50% COMT inhibition in transmission electron microscopy (TEM)

(IC50) and the effective concentration inducing studies (Sahgal et al. 1979). half of the maximal uncoupling (EC50) In humans, most of the histological (Nissinen et al. 1997). lesions concentrate in the heart and lungs, and a few liver findings have been reported COMT inhibitor IC (µM) EC (µM) 50 50 due to DNP poisoning. Poole and Haining entacapone 0.25 58.0 (1934) found degeneration of liver cells and tolcapone 0.25 2.6 pyknotic nuclei after five days of DNP treatment at 7 mg/kg. Necrosis of hepato-

30 Review of the literature cytes was also identified in a woman who 5.6.2. Electron microscopy findings received DNP for one week (dose not given) The ultrastructure of mitochondria in (Lattimore 1934), and “an acute different experimental metabolic states was degenerative hepatitis” with early necrosis determined in mouse liver by Hackenbrock and fatty change was reported in a case of a in 1966 (Table 5). In state 1, at a slow man dying in a chemical plant where DNP respiration rate, the ultrastructure is was being manufactured (Warthin 1918). characterized by condensed formation with However, the latest histological reports on large intramitochondrial granules (Chance humans date back to the 1940s, and in most and Williams 1955; Hackenbrock 1966). cases, no autopsy or histological evaluation The mitochondria in this state are was performed. The liver does not appear indistinguishable from those in state 4. In to be a sensitive organ for DNP toxicity in state 2, the mitochondria show highly humans or animals exposed orally, and no condensed formation. The volume of their consistent light microscopy liver findings outer compartment seems to have more have been associated with uncoupling of volume than the inner compartment oxidative phosphorylation induced by DNP (Hackenbrock 1966). When oxidative (Research Triangle Institute 1995).

Table 5. Histological findings in liver and skeletal muscle and TEM findings in mitochondria after treatment with DNP as an uncoupling agent.

Liver findings References

Histology necrosis of hepatocytes Lattimore 1934; Poole and Haining cloudy swelling, congestion 1934; Spencer et al. 1948 degeneration of liver cells TEM, isolated contraction of the inner membrane; Buffa et al. 1970 mitochondria in vitro reduction of the total volume

Skeletal muscle findings

Histology ragged red fibres Melmed et al. 1975 TEM accumulations of mitochondria; Kawahara et al. 1991 swelling; abnormal cristae; rough endoplasmic reticulum disappeared accumulations of mitochondria; Sahgal et al. 1979 inclusions; concentric laminar bodies; vacuoles, dilated sarcotubules increase in number; swelling; Shah et al. 1982, 1985 inclusions; concentric laminar bodies; unfolded cristae; vacuoles, dilated sarcotubules unfolded cristae Melmed et al. 1975

31 Review of the literature phosphorylation is activated (state 3), the 1985; Kawahara et al. 1991). In several cases, volume of mitochondrial matrix decreases, the number of mitochondria was increased, simultaneously with an increase in the especially in skeletal muscle under the volumes of the outer compartment and sarcolemma, whereas in the perinuclear or intracristal space. The matrix is associated intermyofibrillar region the number of with small granules and the inner membrane mitochondria was unaffected (Melmed et forms irregular foldings (Hackenbrock al. 1975; Sahgal et al. 1979; DiMauro et al. 1966). When uncoupling agents are added, 1985; Shah et al. 1985; Kawahara et al. the morphology shows some similarities 1991). Large aggregates of mitochondria with that of state 2 (Chance and Williams were also observed in light microscopy 1956). Although histological findings may (Melmed et al. 1975; Sahgal et al. 1979). be lacking, typical mitochondrial changes Isolated rat liver mitochondria after in TEM studies of isolated rat liver incubation with pentachlorophenol, mitochondria or skeletal muscle cells have another uncoupling agent, showed an been observed after the treatment with DNP increased inner mitochondrial space and a (Table 5). Buffa et al. (1970) reported matrix containing constricted or indistinct contracted conformation of the inner cristae (Weinbach et al. 1967). Mito- mitochondrial sac formed by the inner chondria with a ruptured outer membrane membrane and matrix in isolated rat liver were also seen. However, when rats were mitochondria. Mitochondrial swelling with treated intraperitoneally with 60 mg/kg of reduced density in the matrix and loss of pentachlorophenol, the mitochondria in cristae have also been observed (Melmed et liver sections did not differ from those of al. 1975; Sahgal et al. 1979; Shah et al. control animals (Weinbach et al. 1967).

32 Aims

6. Aims

Entacapone and tolcapone, two phosphorylation, a possible connection nitrocatechol-structured COMT inhibitors, between hepatotoxicity and uncoupling by have been used as adjuncts to levodopa in COMT inhibition was sought. the treatment of Parkinson’s disease for several years. Tolcapone has been shown to These studies were performed to examine: induce liver toxicity in clinical use, and three 1. Comparative toxicity profiles of fatal cases have been reported. Since these entacapone and tolcapone in vivo (I, II). two COMT inhibitors are structurally 2. Role of uncoupling of oxidative related to each other, exploring differences phosphorylation in these toxicity profiles in their toxicity profiles was warranted. in vivo and in vitro (II, III, IV). Furthermore, as some data existed on 3. Effects of total COMT inhibition in tolcapone being an uncoupler of oxidative mice in vivo (V).

33 Materials and methods

7. Materials and methods

7.1. Animals and treatment transferred for breeding purposes to the University of Kuopio, Finland. The In comparative toxicity studies in vivo, (I, genotype of mice was determined as ® II, III), outbred Crl:CD BR rats (Sprague- described in Huotari et al. (2002). The Dawley origin, supplied by Charles River commercially available Standard Rodent Wiga, Sulzfeld, Germany) were used. The Diet R36 (Ewos AB, Sweden) and tap water dosing for these studies was conducted in a from the public supply were available ad semi-barriered, limited-access animal room. libitum. Animal care was performed The test suspensions were administered according to the regulations of the Council orally for eight days (I) or two weeks (I, II, of Europe (1990) and the National Research III) to each rat at 24-hour intervals by Council, USA (1996). The study protocols gavage using a flexible plastic catheter and a were approved by the Provincial State Office syringe. In the first study, the doses of of Southern Finland or by the Animal entacapone and tolcapone were 200 and 400 Ethics Committee of Orion Corporation, mg/kg/day, and in the second study, 300 Finland. and 500 mg/kg/day, with DNP 20 mg/kg/ day serving as a positive reference substance. Entacapone doses were based on previous 7.2. Test substances toxicological studies (Entacapone Product Entacapone and tolcapone were synthesized Monograph 1999). Tolcapone doses were at Orion Pharma, Espoo, Finland selected according to non-clinical and clinical (Bäckström et al. 1989; Borgylua et al. exposure data (Tasmar Product Monograph 1989). The drug substances were identified 1997). The dose of DNP was selected to by comparing the substances to standards be the highest dose not inducing mortality using infra-red spectra. The impurities in (Spencer et al. 1948). The control animals both entacapone and tolcapone, determined received the vehicle 0.5% methyl cellulose. by high-performance liquid chromato- The commercially available rodent SDS diet graphy (HPLC), were under 0.1%. In RM1 (E) SQC (Special Diet Services Ltd, comparative toxicity studies, DNP served Witham, Essex, England) and tap water as a positive reference compound. All of from the public supply, filtered twice, were these test substances were suspended in available ad libitum. In in vitro studies on autoclaved 0.5% methyl cellulose, and the uncoupling properties (II, IV), the same dosing suspensions were prepared daily strain of rat served as a liver donor. immediately before the oral dosing. To evaluate the complete lack of the COMT enzyme in vivo (V), COMT- deficient mice were used. This knock-out strain was developed by Gogos et al. (1998) at Rockefeller University, USA, and

34 Materials and methods

7.3. Methods in in vivo studies floating times were measured.

7.3.3. Body temperature measurements 7.3.1. Clinical signs, mortality, body (I, II, IV) weight and organ weights (I, II, V) Rectal body temperature was measured The animals were inspected at least once a twice prior to the dosing period to adapt day to monitor clinical signs or reactions to the animals to this measurement. During the treatment and mortality. Individual body the study the rectal temperature was weight and body weight gain as well as measured on several days, once in the body-related organ weights (II, liver; V, liver morning immediately before dosing and and kidneys) were recorded. one or two hours after the dosing of test substances or vehicle. A digital thermometer 7.3.2. Behavioural studies (V) with a plastic-covered stainless probe was The activity of all three genotypes was used. determined using the ten-channel IRS Actometer System (custom-designed by L. 7.3.4. Haematology and clinical Yaviz and E. Koivisto at the University of chemistry (I, II, V) Kuopio) for determining the effects of Blood samples were taken from fasted COMT deficiency on the circadian rhythm animals on the day of terminal necropsy for of locomotor activity and on diurnal haematological (I, V) or clinical chemistry rhythm. The measurements were taken at (I, II, V) analyses. The haematological 30-minute consecutive intervals for a total analyses were performed using a Sysmex F- of 72 hours. An elevated plus-maze test was 800 Microcellcounter (Toa Medical performed in mice as described by Handley Electronics Co. Ltd., Kobe, Japan). In and Mithani (1994) with some modifi- clinical chemistry determinations, the main cations (Vasar et al. 1993). During a three- target was the effect of the treatment on minute observation session the following liver function. The analyses were carried out measurements were taken: latency period of using a BM/Hitachi 911 E automatic first open part entry, number of attempts analyzer (Boehringer Mannheim, to enter the central square, number of line Mannheim, Germany). crossings in open arms and in the central square, total time spent in the open part 7.3.5. Determination of catecholamines and open arms, and number of visits to open from plasma (V) and closed arms of plus-maze. Immediately Blood samples were collected into tubes with after the elevated plus-maze test, general ethylenediamintetraacetic acid as an anti- motor activity of each mouse was tested for coagulant. The concentrations of NA, A and two minutes in a dimly illuminated round DHPG in plasma were determined at the box by counting the number of line Clinical Research Services, Turku University, crossings and rearings. A forced swimming Turku, Finland, using high-performance test was performed as described by Porsolt liquid chromatography with colorimetric et al. (1977), and active swimming and electrochemical detection (HPLC-EC), as 35 Materials and methods described previously (Scheinin et al. 1991). mean plasma concentration (AUC) of entacapone and tolcapone was calculated 7.3.6. Determination of MAO-A and compartment-model independently using MAO-B in liver homogenates (V) a linear trapezoidal approximation of up to

The enzyme activity of MAO-A and MAO- 12 hours (Ctlast). The area prior to the first B was determined from mouse liver sampling point (10 minutes) was estimated homogenates (V) using an enzymatic with no lag time in absorption. The area radiochemical method. The MAO-A assay beyond the time Ctlast was approximated was performed according to Young et al. using a triangular area up to the next (1986), using 14C-hydroxytryptamine sampling point. The proportion of the creatinine sulphate as the substrate. MAO- approximated area of the area for the 24- 14 B activity was determined using C- hour dosing interval (AUC0-24h) was a phenylethylamine hydrochloride as the maximum of 8%. substrate, with some modifications to the For liver tissue sampling, the animals were two methods described by Keller & al sacrificed by inhaled carbon dioxide two (1987) and Koulu & al (1989). The results hours after the dosing (II). The medial lobe were expressed as pmol of deaminated of the liver was removed, and liver metabolite, 5-hydroxyindoleacetic acid (for concentrations of entacapone and tolcapone MAO-A) or phenylacetic acid (for MAO- were determined by validated HPLC B), formed per min mg protein. In both methods (Timm and Erdin 1992; Wikberg determinations, the protein content was et al. 1993). The limit of quantification was measured according to Peterson (1977), with 50 ng/g of fresh liver tissue. bovine serum as the standard. 7.3.8. Histopathology (I, II, III, V) 7.3.7. Analysis of drug concentrations Tissue samples from the liver were preserved in plasma and liver (I, II) in 4% buffered formaldehyde. The tissues Exposure of the animals to entacapone and were embedded in paraffin wax, sections cut tolcapone was monitored by determining into 4 µm and stained with haematoxylin the plasma and liver concentrations of and eosin (H & E). Tissues were examined entacapone and tolcapone during the dosing under a light microscope. period. For plasma concentration analysis (I, II), 7.3.9. Electron microscopy (III) samples were drawn at several time points Samples for transmission electron after the dosing. Blood was collected into microscopy were taken from the liver and lithium-heparinized tubes and kept in ice skeletal muscle (M. gastrocnemius). Tissue until centrifuged. The plasma was separated samples of a maximum size of 1 mm3 were and the concentrations of entacapone and prefixed in phosphate-buffered (pH 7.2) tolcapone were determined using validated 2.5% glutaraldehyde. Postfixation was HPLC methods. The limit of detection was performed with phosphate-buffered 1% 25 ng/ml for both analytes. osmium tetroxide. The thin sections were For systemic exposure, the area under the studied after dehydration with ethanol,

36 Materials and methods embedding in epoxy resin, thin-sectioning carbonyl cyanide chlorophenylhydrozone. with an ultramicrotome and post-staining The respiratory control ratio [= (oxygen with uranyl acetate and lead citrate. consumption rate with ADP, state 3)/ (oxygen consumption rate without ADP, state 4)] (RCR) was calculated. The protein 7.4. Material and methods in content of the samples was estimated by the studies at cellular level method of Lowry (1951).

7.4.3. Mitochondrial membrane 7.4.1. Adenosine nucleotides from liver potential studies (IV) mitochondria and liver tissue (II) Mitochondria from rat liver (IV) were Mitochondria were isolated immediately isolated with differential centrifugation and from fresh liver samples of rats by prepared as reported earlier (Nissinen et al. homogenization and differential centrifu- 1997). Mitochondrial membrane potential gation and used for determination of was measured using a fluorometric method adenosine nucleotides. The liver tissue originally described by Åkerman and samples were prepared according to Faupel Wikström (1976), and slightly modified by et al. (1972). Kauppinen and Hassinen (1984) and The adenosine nucleotide [adenosine Fromenty et al. (1990). monophosphate (AMP), ADP, ATP] Mitochondrial membrane potential was concentrations in mitochondria and liver presented as percentage of membrane homogenates were determined by HPLC potential in fully energized mitochondria using ultraviolet detection as described by using the following formula: Carter and Muller (1990). The levels of Membrane potential (% of maximum) adenosine nucleotides were expressed as an = (F – F )/(F – F ) · 100, where ATP/ADP ratio. The energy status of the max n max min F = Fluorescence maximum (zero cell was also expressed as an energy charge max membrane potential). The reading was [= (½ ADP + ATP) / (AMP + ADP +ATP)]. taken at the start of the measurement before addition of succinate. F = Fluorescence 7.4.2. Mitochondrial oxygen min minimum (maximal membrane potential). consumption (II) The reading was taken after addition of The oxygen consumption of isolated succinate. Fn = Fluorescence after the mitochondria was assayed with an oxygen addition of the test compound. electrode connected to an amplifier and a chart recorder (Trounce et al. 1996; Nissinen 7.4.4. Measurement of COMT activity et al. 1997) using 5 mM succinate as the in protein-binding studies (IV) respiration substrate. The rate of oxygen COMT activity in vitro was determined consumption before (state 4) and after (state with and without serum proteins using a 3) adding 2 µl of ADP was recorded and soluble COMT preparation from rat liver finally the mitochondria were maximally as described earlier (Nissinen et al. 1992) uncoupled by adding a known uncoupler, and 3,4-dihydroxybenzoic acid as the

37 Materials and methods

substrate. The results were expressed as IC50 points: 0, 2, 26, 50, 74, 98, 122 hours). values (µM) of the test substances to induce The values at the commencement of half of the maximal inhibition of COMT treatment (0 hours) were used as a covariate. (%). In Study V, body weight, organ weights, body weight related organ weights, behavioural data, biochemical deter- 7.5. Statistics minations and clinical chemistry values of Biochemical parameters, clinical chemistry the different COMT knock-out genotypes and body weight related organ weights were were evaluated using two-way analysis of analysed using one-way analysis of variance variance with two between-factors (genotype in Studies I and II. Log transformation was and sex). Tukey’s test was used for further applied to assure the assumptions of the individual comparison in behavioural model (normality of residuals). For the studies. Before statistical analysis, all clinical overall tests, a p-value (two-sided) of less chemistry parameters were log-transformed than 0.05 was considered statistically to assure the assumption of normality. For significant. If statistically significant results the overall tests, a p-value of less than 0.05 were found, Bonferroni-corrected contrasts was considered statistically significant. If the were applied to characterize these results in main effects of genotype or the interactions more detail. Statistical evaluation of the between genotype and sex were statistically body temperature was performed using the significant, pair-wise comparisons were used analysis of covariance for unbalanced to characterize these results in more detail. repeated measures with one between-factor Statistical analyses were performed with ® (dose groups) and one within-factor (time SAS statistical software (version 6.12).

38 Results

8. Results

8.1. Comparative toxicity of Entacapone did not cause changes in entacapone and tolcapone in vivo body or liver weight of treated animals. Tolcapone (300 and 500 mg/kg/day) or DNP treatment (20 mg/kg/day) resulted in 8.1.1. Clinical signs, mortality, body an increase of about 15% in body weight weight and organ weights (I, II, V) related liver weight (p<0.01). The treatment Entacapone was well tolerated in rats; even had no effect on body weight gain of the at a dose of 600 mg/kg/day up to 15 days, animals, except in the high-dose tolcapone no clinical signs of toxicity or deaths group, where body weight gain was slightly occurred. Tolcapone, in turn, induced decreased. At the age of one year, the clinical symptoms, including tachypnea, homozygous animals of the COMT knock- laboured breathing, decreased spontaneous out strain had lower body weight than wild- motor activity and drowsiness, on the fourth type animals (p<0.01). Relative kidney day after treatment of 400 mg/kg/day. weight was higher in homozygous than in Mortality due to tolcapone treatment was wild-type animals (p<0.01). high, and the tolcapone-treated animals were terminated because of poor condition at the 8.1.2. Haematology and clinical latest on the eighth day of treatment. At chemistry (I, II, V) necropsy, all of the deaths were considered Treatment with entacapone 600 mg/kg/day to be treatment-related. The reference or tolcapone 200 mg/kg for 14 days in the substance, DNP, induced no clinical signs rat induced no changes in haematological at the dose of 20 mg/kg/day during the 15 parameters. In COMT knock-out mice, the days of treatment. Sudden onset of rigor parameters did not vary between mortis was observed after tolcapone homozygous, heterozygous and wild-type treatment already at a dose of 300 mg/kg/ animals. day and DNP of 20 mg/kg/day. Clinical chemistry parameters were No mortality or clinical signs due to total unaffected by entacapone treatment (Table COMT inhibition were present in COMT- 6). However, tolcapone caused a significant deficient mice aged up to one year. The decrease (p<0.001) in protein and globulin motor activity of homozygous females was concentrations in serum even at the lower significantly higher than that of wild-type dose of 300 mg/kg/day. In addition, serum mice (p<0.05). In Porsolt’s forced albumin was decreased (p<0.001) in swimming test, male homozygous mice had animals treated with tolcapone 500 mg/kg/ a shorter floating time (p<0.05) (less day. ALAT was significantly higher in this depression) than their heterozygous dose group than in the control group counterparts, while both heterozygous and (p<0.001). There was also an increase in homozygous females had longer floating SDH for DNP 20 mg/kg/day (p<0.01), and times than wild-type females (p<0.05). a marginal increase for tolcapone 300 mg/ 39 Results kg/day (p=0.09). The serum glucose 8.1.5. Plasma and liver tissue concentration was elevated in the tolcapone concentrations (I, II) 500 mg/kg/day group and in the DNP The exposure factors (exposure in animal/ group (p<0.001). maximum clinical exposure in man) for In the 12-month-old homozygous entacapone and tolcapone and AUC0-24h COMT knock-out mice, clinical chemistry values from mean plasma concentrations values for APHOS, ALAT, calcium, total were calculated in relation to the AUC0-24h proteins (Prot), albumin (Alb), urea and reached in humans after maximum clinical glucose (Gluc) were higher than in the wild- doses of entacapone (10 x 200 mg daily) or type mouse (V). tolcapone (3 x 200 mg daily) (Tasmar Product Monograph 1997; Entacapone 8.1.3. Catecholamine plasma levels (V) Product Monograph 1999). The exposure The plasma levels of A, NA or DHPG did factor achieved at the highest entacapone not vary between homozygous and wild- dose level (600 mg/kg/day) was 21. With type mice of the COMT knock-out strain. tolcapone, an exposure factor of 14 was achieved at a dose of 400 mg/kg/day. 8.1.4. Activity of MAO-A and MAO-B The concentrations of entacapone and in the liver (V) tolcapone found in liver tissue two hours No statistically significant differences in the after dosing of 300 and 500 mg/kg/day enzyme activities of MAO-A or MAO-B were comparable (Figure 6). The mean in the liver tissue were present in different plasma concentrations of tolcapone were genotypes of the COMT knock-out mice. higher than those achieved after entacapone treatment in the respective dose groups (300 and 500 mg/kg/day).

Table 6. Clinical chemistry values (mean ± SD) of the rats treated with entacapone (Enta), tolcapone (Tolca) or 2,4-dinitrophenol (DNP). N=6, except in Enta groups N=12 and in Tolca 300 group N=11 (II).

Treatment S-Prot S-Alb S-Glob S-ALAT SDH S-Gluc mg/kg/day (g/l) (g/l) (g/l) (U/l) (U/l) (mmol/l) Control 0 66 ± 1.9 33 ± 1.4 34 ± 0.8 57 ± 12.9 15.9 ± 1.5 6.7 ± 1.3 Enta 300 65 ± 3.2 33 ± 1.3 32 ± 2.5 53 ± 10.0 15.5 ± 5.4 6.2 ± 1.0 Enta 500 64 ± 3.7 33 ± 1.3 31 ± 2.9 56 ± 8.1 18.2 ± 5.5 6.9 ± 0.9 Tolca 300 59 ± 2.2*** 31 ± 0.9 28 ± 1.9*** 56 ± 6.8 21.8 ± 5.3 7.1 ± 0.7 Tolca 500 54 ± 3.4*** 28 ± 1.5*** 26 ± 1.9*** 109 ± 9.1*** 16.1 ± 9.4 11.2 ± 2.3*** DNP 20 63 ± 4.2 32 ± 2.1 32 ± 2.3 60 ± 16.2 27.4 ± 10.4** 9.6 ± 1.0***

** p<0.01, when compared with the control group, *** p<0.001 S-Prot, serum proteins; S-Alb, serum albumin; S-Glob, serum globulin; S-ALAT, serum ALAT; S-Gluc, serum glucose

40 Results

8.1.6. Pathology induced by treatment were considered to be normal for this strain with entacapone and tolcapone of rats and thus were of no toxicological significance. 8.1.6.1. Histopathological findings (I, II, In the COMT knock out-mice, special III, V) emphasis was given to liver, kidneys and Histological examination of liver tissue mammary glands in the homozygous and from entacapone-treated rats at any dose wild-type animals. The only finding was level did not reveal treatment-related changes decreased midzonal or centrilobular (Figure 7). As a sign of hepatotoxicity, vacuolization of the liver in homozygous diffuse centrilobular necrosis at tolcapone male mice as compared with the wild-type 400 mg/kg/day was observed in one out of animals. Anisocaryosis was observed in both five animals (Figure 7). In the rats treated genotypes. All other findings in different with the high dose of tolcapone (600 mg/ organs were distributed evenly amongst the kg/day), necrotic foci or single cell necrosis different genotype groups, thus being was seen. Centrilobular hypertrophy was normal background data. observed in the groups treated with tolcapone 500 mg/kg/day or DNP 20 mg/ 8.1.6.2. Electron microscopy findings (III) kg/day. Minor necrotic foci or single cell Changes occurred in the shape and internal necrosis was also found in the liver tissue of structure of mitochondria of liver cells in rats treated with DNP. All other findings DNP- and tolcapone-treated rats compared

120 25

100

10 40 Concentration ng/g wet tissue wet ng/g Concentration Concentration µg/ml plasma µg/ml Concentration

5 20

0 0 Enta Enta Tolca Tolca Enta Enta Tolca Tolca 300 500 300 500 300 500 300 500 Figure 6. Concentrations of entacapone (Enta) and tolcapone (Tolca) in rat liver tissue (ng/g) and in plasma (µg/ml) two hours after dosings at different levels (300 or 500 mg/kg/day) (mean, SD). 41 Results with entacapone-treated animals (Figure 8). 8.2. Uncoupling effects related to The DNP- induced changes consisted of entacapone and tolcapone swollen mitochondria, deformed or broken cristae and reduced matrix density. With tolcapone, the matrix density was also 8.2.1. Signs of uncoupling in vivo reduced and mitochondria were swollen. In addition, intracellular oedema was observed 8.2.1.1. Body temperature (I, II, IV) in the liver cells of DNP- and tolcapone- Entacapone had no effect on body treated animals. The mitochondria were temperature even at a dose of 600 mg/kg/ similar to each other in the entacapone and day. By contrast, after each dose of tolcapone control groups. (300 mg/kg/day) body temperature In the skeletal muscle, intermyofibrillar increased within one or two hours from oedema and swelling of transverse (T)- dosing about one centigrade, the highest tubules were seen in both the tolcapone and increase being 1.1°C (p<0.01) (Figure 9). DNP groups but more prominently in the DNP 20 mg/kg/day also induced a tolcapone group. Contracted sarcomeres, significant increase in rectal body visualized as shortened I-bands, were also temperature compared with controls seen. Mitochondrial swelling and decreased (1.3°C, p<0.001). The increased tem- matrix density were less prominent in peratures decreased to basal level within 24 skeletal muscle than in liver tissue, but the hours. A more marked rise in body changes were still identifiable in the DNP- temperature up to 41°C preceded the deaths and tolcapone-treated animals. In the of six rats treated with tolcapone 600 mg/ entacapone and control groups, no apparent kg/day (Figure 10). The combination of findings were made. carbidopa and levodopa did not induce any

Figure 7. Liver of entacapone (left) and tolcapone (right) treated rat. Centrilobular necrosis is seen in the tolcapone-treated rat liver. Magnification 100x, H&E staining (I).

42 Results changes in body temperature. When with oxidative mitochondrial energy entacapone (400 mg/kg) was added to the production (Table 7). Nor did entacapone treatment, the temperature decreased induce changes in whole liver ATP synthesis. slightly (p<0.05). Tolcapone increased body Tolcapone treatment of 500 mg/kg/day temperature at a dose of 50 mg/kg even in reduced both mitochondrial ATP synthesis the rats treated with carbidopa/levodopa and whole liver ATP concentration (p<0.01). (p<0.001). Mitochondrial ATP production was diminished, as seen in the ATP/ADP 8.2.2. Signs of uncoupling at cellular ratio (p<0.01). The energy charge in the level mitochondria and liver tissue (p<0.01) was also significantly reduced. DNP 20 mg/kg/ 8.2.2.1. Adenosine nucleotide day decreased the mitochondrial ATP concentrations in liver mitochondria and concentration, which was reflected as a liver tissue (II) reduction in the whole liver ATP Treatment with entacapone did not interfere production (p<0.01), and thus, the ATP/

Figure 8. Mitochondrial changes in rat liver treated with DNP 20 mg/kg/day (a) or tolcapone 500 mg/kg/day (b). Mitochondrial swelling and reduced matrix density are seen. Liver from entacapone-treated (500 mg/kg/day) (c) and control rat (d). M, mitochondria; N, nucleus; Bar, 500 nm.

43 Results

ADP ratio was decreased in the mitochondria 8.2.2.3. Mitochondrial membrane as well as in the whole liver of DNP-treated potential (IV) rats. There was also a significant reduction Entacapone had no effect on mitochondrial of energy charge in DNP-treated membrane potential when the cumulative mitochondria (p<0.001). final concentration of entacapone remained µ 8.2.2.2. Mitochondrial oxygen consumption under 100 M (Figure 11). Tolcapone (II) added in increasing concentrations gradually disrupted the membrane potential. The Treatment with tolcapone produced a mild concentration required to decrease the effect on mitochondrial energy production, membrane potential by 50% was 3.6 µM. reflected in the marginal decrease in the DNP caused a concentration-dependent respiratory control rate (RCR) (p = 0.08). decrease of mitochondrial membrane The RCR ratio of DNP-treated rats was potential at slightly lower concentrations lowered by only a few percentage points than tolcapone. The concentration required (Table 7). to decrease the membrane potential by 50% was 1.7 µM. At concentrations of 5 µM or above, tolcapone and DNP further suppressed the membrane potential (Figure 11).

3 8.2.3. COMT activity in relation to protein binding (IV) Entacapone and tolcapone inhibited 2 COMT at equal concentrations (IC50 values of 0.25 µM and 0.24 µM, respectively; Figure 12), as measured by COMT activity ***

** without preincubation in rat serum. 1 ** However, when the compounds were

Increasebody in temperature (ºC) preincubated in serum for 30 minutes, the COMT inhibitory activity of entacapone

was somewhat reduced (IC50 value 0.54 0 µ Enta Enta Enta Tolca Tolca Tolca Tolca DNP M), while that of tolcapone was shifted 300 Enta 500 600 300 400 600 600 400 µ to micromolar level (IC50 value 2.62 M).

Figure 9. Mean change in rectal body temperature of rats measured one or two hours after entacapone (Enta), tolcapone (Tolca) and DNP treatment at doses of 300–600 mg/ kg of entacapone and tolcapone and 20 mg/ kg of DNP. Bars represent SD, ** p<0.01, *** p<0.001. 44 Results

† † 41 †

40 † †

39 †

Body temperature (°C) 37

36 Dosing 2 h 35 Day1 Day 2 Day 3 Day 4 Day 5 Day 6

Figure 10. Rectal body temperature after tolcapone treatment of individual rats that died at various time points during the study. The temperature was measured just before death. Doses were tolcapone 400 or 600 mg/kg/day tolcapone. † = time of death (I).

Table 7. Adenosine nucleotide ratios; mitochondrial and liver tissue values (mean ± SD) of the rats treated with entacapone (Enta), tolcapone (Tolca) or 2,4-dinitrophenol (DNP). N=6 (II).

Treatment Mitochondria Liver tissue mg/kg/day ATP/ADP EC1 ATP/ADP EC1 RCR2 Control 0 3.12 ± 0.36 0.83 ± 0.02 3.07 ± 0.54 0.83 ± 0.04 5.01 ± 0.49 Enta 500 2.84 ± 0.44 0.81 ± 0.04 2.78 ± 0.43 0.81 ± 0.03 5.45 ± 0.27 DNP 20 1.46 ± 0.35*** 0.63 ± 0.07*** 2.12 ± 0.54 0.74 ± 0.08** 4.87 ± 0.37 Control3 0 3.55 ± 0.73 0.85 ± 0.04 3.79 ± 0.46 0.86 ± 0.02 5.37 ± 0.61 Tolca 500 1.88 ± 0.69** 0.69 ± 0.10** 2.44 ± 0.57*** 0.76 ± 0.07** 4.85 ± 0.32§

1 EC = energy charge 2 RCR = Respiratory Control Ratio 3 Control for the group treated with tolcapone 500 mg/kg/day ** p<0.01, when compared with the control group); *** p<0.001 § p = 0.08

45 Results

100

60 EC50 40 tolcapone 2,4-dinitrophenol

20 entacapone Membrane potential (%) 0

1 1,7 3,6 10 100 Concentration (µM)

Figure 11. Concentration dependency for the effect of entacapone, tolcapone or 2,4- dinitrophenol on mitochondrial membrane potential (% of maximum). Mitochondria were isolated from rat liver. N=1-5. EC50 is the concentration which induces a 50% inhibition of membrane potential (IV).

100

60 IC50 40 entacapone, no serum tolcapone, no serum entacapone, with serum 20

COMT inhibition (%) tolcapone, with serum

0 0,1 0,24 0,54 1 2,62 10 0.25 Concentration (µM)

Figure 12. Effect of addition of rat serum on COMT inhibition activity (%) of entacapone and tolcapone using soluble COMT from rat liver. N=3. IC50 indicates the concentration at which the COMT inhibition is 50% of the maximum (IV).

46 Results

8.3. Summary of the results in vivo and in vitro Table 8 summarizes the results of in vivo and in vitro studies.

Table 8. Summary of the signs in relation to hepatotoxicity and oxidative phosphorylation in vivo and in vitro in rats after treatment with entacapone, tolcapone or 2,4-dinitrophenol (DNP).

Finding Entacapone Tolcapone DNP

In vivo Mortality — ↑ — Relative liver weight — ↑↑ Liver enzymes — ↑↑ Serum proteins — ↓ — Body temperature — ↑↑ Onset of rigor mortis — < 10 min < 10 min Liver histology — necrosis necrotic foci Liver mitochondria — swelling; deformed swelling;deformed cristae; matrix density ↓ cristae; matrix density ↓ Skeletal muscle — intermyofibrillar oedema intermyofibrillar oedema ultrastructure contracted sarcomeres contracted sarcomeres Skeletal muscle — swelling; matrix density ↓ swelling; matrix density ↓ mitochondria

At cellular level ATP/ADP mitochondria — ↓↓ EC1 liver tissue — ↓↓ RCR2 mitochondria —(↓)— Mitochondrial membrane — ↓↓ potential

1 EC = energy charge 2 RCR = respiratory control ratio (↑/ ↓) marginal increase/decrease — not affected

47 Discussion

9. Discussion

9.1. Comparative toxicology of suddenly. The symptoms after tolcapone entacapone and tolcapone in vivo treatment are consistent with those in Spencer et al. (1948), who reported that rats after oral dosing of 30 mg/kg or more of 9.1.1. Clinical signs and mortality DNP either died within one to two hours COMT inhibition has been postulated to or recovered completely. This is also in be toxic, inducing hepatotoxicity and accordance with a mouse study on DNP, leading to further complications in clinical where peak concentrations of DNP were use (Rivest et al. 1999; Olanow 2000). Since reported to be reached within one hour in entacapone and tolcapone are structurally plasma and within two hours in liver tissue related COMT inhibitors, they have been after oral dosing (Robert 1986). In Study speculated to have similar toxic effects. Both II, the selected dose of DNP (20 mg/kg/ have undergone extensive non-clinical day) was approximated to be the highest toxicity testing in several animal species, and dose not to induce any mortality or clinical no toxicity has been reported, even with symptoms (Spencer et al. 1948). The doses high-dose treatment (Tasmar Product of entacapone were selected according to Monograph 1997; Entacapone Product previous toxicity studies (Entacapone Monograph 1999). Despite the safety Product Monograph 1999), while studies and clinical trials, several cases of tolcapone doses were based on non-clinical hepatotoxicity due to clinical treatment with and clinical exposure data (Tasmar Product tolcapone have emerged (Assal et al. 1998; Monograph 1997). In these earlier Mayoral et al. 1999; Olanow 2000). toxicological profile studies, tolcapone had The toxicity of entacapone and tolcapone proved to be well tolerated (Eckhardt et al. proved to be different in rats receiving high 1996; Schläppi et al. 1996a; 1996b; Tasmar oral doses (I, II). Entacapone was well Product Monograph 1997), with only a tolerated up to 15 days at high oral doses few clinical symptoms, consisting of with no signs of toxicity. However, the first respiratory difficulties in connection with signs of toxicity after tolcapone treatment premature deaths or some female rats lying were expressed on the third day, and on the side for several minutes after the mortality increased during the next eight dosing (300 mg/kg/day), reported (Tasmar days of treatment such that the remaining Product Monograph 1997). In the four- animals in the tolcapone-treated groups week oral toxicity study in rats, three animals were euthanized. The clinical symptoms in died within 15 to 60 minutes after dosing rats consisted of laboured breathing, (Schläppi et al. 1996a). In one case, the decreased spontaneous motor activity and cause of death was speculated to be due to drowsiness. The symptoms usually faded exaggerated pharmacological activity or away after two to three hours from dosing, hypoxia (Schläppi et al. 1996a). However, although some of the rats died quite the conditions in non-clinical long-term 48 Discussion toxicity studies were different from those decreased significantly in tolcapone-treated in Studies I and II since in the former studies rats, signalling the diminished capacity of tolcapone had been administered to rats as the liver to synthesize proteins. In DNP a feed mixture, and in dietary studies, the treatment, SDH was significantly increased. exposure remains lower and no high peak Robert (1986) has reported treatment of plasma concentrations occur. Thus, the high mice with a single dose of DNP 22.5 mg/ mortality following tolcapone treatment in kg, where plasma ALAT activity was not Studies I and II is considered to be related significantly different from the control to the treatment. levels; SDH was not measured. A positive In the behavioural studies with COMT correlation between hepatocellular damage knock-out mice, the differences between the and an increase of serum ALAT and SDH genotypes were marginal and always less activities in the rat has been observed earlier than differences between the sexes. Thus, (Travlos et al. 1996), with ALAT values also reduced or total lack of COMT enzyme reflecting the severity of hepatocellular activity does not appear to be associated with necrosis (Balazs et al. 1961). Liver cell any clinical symptoms or changed necrosis and hypertrophy in animal studies behaviour. are reported to be associated with increased serum concentrations of either SDH or 9.1.2. Hepatotoxic properties of ALAT in about 40-58% of the cases entacapone and tolcapone (Travlos et al. 1996). The elevations in Drug-induced hepatotoxicity is the leading ALAT and SDH activities were reflected in reason for withdrawal of a drug from the liver histology as necrotic foci and single cell market (Lee 1995; Lasser et al. 2002; necrosis in both tolcapone- and DNP- Thomas 2002). Hepatotoxicity is also the treated rats (I, II). Hepatocellular hyper- most serous problem in tolcapone-induced trophy was also observed, with increased toxicity in clinical use (Assal et al. 1998; liver weight of about 15% (II). Electron Mayoral et al. 1999; Olanow 2000; microscopy examination of liver cells of Watkins 2000). Three fatal cases due to tolcapone- and DNP-treated rats revealed tolcapone treatment have been reported, and swelling of mitochondria, deformation of two of them were characterized by hepatic cristae and reduced matrix density (III). necrosis (Assal et al. 1998; Olanow 2000). These findings are consistent with those in The first indication of drug-induced a tolcapone-treated patient dying from hepatotoxicity is the rise in clinical chemistry fulminant drug-induced hepatitis (Assal et parameters e.g. ALAT (Watkins 2000). In al. 1998; Spahr et al. 2000). Electron micro- laboratory animals, SDH is the second scopy findings showed mitochondrial choice marker of hepatotoxicity (Loeb and swelling with reduced density in the matrix Quimby 1999). Tolcapone induced a and loss of cristae (McCaul et al. 1986; twofold elevation in rat ALAT values as Spahr et al. 2000). compared with the control group, indicating Tolcapone is reported to induce slight to a mild hepatocellular disturbance (II). In moderate hepatocellular hypertrophy in mice addition, the serum protein values were after 18 months of treatment (Olanow

49 Discussion

2000), but in a four-week study in the rat, histological findings in the liver supported while liver weight increased 10%, no treatment-related hepatotoxicity. hypertrophy was observed (Schläppi et al. Centrilobular necrosis follows circulatory 1996a). Hypertrophy is usually regarded as shock, as oxygen is depleted by the passage an adaptive response to drug-induced of blood through the periportal areas. Since microsomal enzyme induction in the liver, centrilobular hepatocytes have a much although in some cases, this enhanced higher content of drug-metabolizing metabolism may give rise to long-term enzymes than periportal hepatocytes, the toxicity (Greaves 1990). toxic response takes place in the centrilobular Centrilobular necrosis was observed in area. Necrosis can occur within minutes of one rat treated with tolcapone for three days. a toxic insult and is usually associated with Since the peak serum enzyme rise usually severe metabolic disturbances. The onset of occurs at about 24 hours after the dosing, cell death is characterized by breakdown of and induced liver damage, depending on the the plasma membrane permeability barrier, hepatotoxic agent, the enzyme value and the which leads to loss of metabolic inter- degree of histological necrosis do not mediates and leakage of liver enzymes into correlate with each other to the same extent the circulation (Mehendale et al. 1994). later (Dixon et al. 1975). By two to three When the metabolic system is disrupted, weeks from the beginning of treatment, the concentration of toxic substances serum SDH is more predictive in treatment- increases in centrilobular areas, leading to related rat liver histopathological lesions degeneration and further necrosis of than serum ALAT (Travlos et al. 1996). In hepatocytes. Even if destruction of tissue is non-clinical toxicity testing of tolcapone, extensive, inflammatory cell infiltration may no elevations of serum enzyme levels in rats still be limited. The necrosis begins to or dogs have been reported (Schläppi et al. regenerate within 24 hours, and within one 1996a; 1996b; 1996c; Tasmar Product week can be totally healed (Haschek et al. Monograph 1997). However, total protein 2002). values in female rats were significantly The specific mechanism of drug-induced decreased after four weeks’ oral treatment hepatotoxicity remains unknown. It might (400/300 mg/kg/day) (Schläppi et al. be an idiosyncratic reaction of partly genetic 1996a), which is consistent with the background or may be associated with findings in Study II. By contrast, entacapone mitochondrial toxicity (Dossing and Sonne did not induce elevation in the liver enzymes 1993; Acuna et al. 2001). With few or protein values in rats, even at the high exceptions, hepatotoxic reactions induced doses used. by drugs are not a dose-related phenomenon Centrilobular necrosis in liver tissue is the (Lee 1995; Watkins 2000). However, in most frequent form of drug-induced clinical use, tolcapone did induce a clear hepatocytic necrosis (Haschek et al. 2002). dose-response effect with respect to liver This was also observed in a liver biopsy of a enzyme elevation (Mayoral et al. 1999; tolcapone-treated patient (Assal et al. 1998). Olanow 2000; Watkins 2000). Gasser and The patient died of hepatic failure, and Smit (2001) have also reported that 5.7%

50 Discussion of patients (N=3848) receiving 200 mg showed slightly higher levels of certain three times daily of tolcapone experienced clinical chemistry values as well as liver function abnormalities. Moreover, in hyperglycemia associated with lower body all three fatal cases due to hepatotoxicity after weight than wild-type mice (V). It could tolcapone treatment clinically, serum ALAT be assumed that in these homozygous mice was raised to more than three times the ULN the levels of catecholamines are higher than (Assal et al. 1998; Mayoral et al. 1999; in wild-type mice, leading to a mild Olanow 2000). Entacapone, by contrast, has catabolic condition reflected as not induced severe hepatotoxicity in clinical hypermetabolism, hyperglycemia and a use (Watkins 2000). The mechanism behind lower body weight. Since endogenous A and tolcapone-induced hepatotoxicity may be NA are capable of potentiating the associated with the lipid solubility and the hepatotoxicity induced by several drugs in metabolism of tolcapone, which differ from the rat (Roberts et al. 1997), the total those of entacapone (Nissinen et al. 1992; COMT inhibition with elevated Wikberg et al. 1993; Dingemanse et al. catecholamines in plasma could be expressed 1995; Dingemanse 1997). If a drug is not as liver toxicity in the COMT knock-out efficiently metabolized, e.g. by glucu- mouse. However, a complete lack of ronidation or sulphation after dissociating COMT activity in mice did not cause from serum proteins, it can accumulate in significant liver problems (V). Experi- such tissues as the liver. Thus, intracellular mentally, in rabbits as well as in clinical use, concentrations of tolcapone might be tolcapone has been shown to increase increased in the liver tissue of patients whose catecholamine plasma levels, and one patient glucuronidation rate is poor, which is was reported to simultaneously have had reflected in liver function abnormalities. elevated liver enzymes (Garrido et al. 1994; Some parkinsonian patients, for instance, Rojo et al. 2001). However, the levels of have a deficiency in mitochondrial catecholamines in plasma of knock-out respiratory chain function that affects the mice were not significantly increased despite first enzyme complex involved in oxidative high DHPG levels, nor did the activities of phosphorylation (Schapira 1994; Schapira the two MAO isoenzymes in the liver (V) 2001). This genetically determined complex differ between the genotypes. There were I defect may be exacerbated by DA, which no increases in general motor activity or in increases free radical formation and may the levels of anxiety and depression in the damage the mitochondrial respiratory chain mice lacking COMT, supporting the view function. The dysfunction can reduce ATP that there was no enhanced catechol- synthesis, leaving the cell with inadequate aminergic tone in these animals. To be a class energy (Schapira 2001). If treatment with a effect of COMT inhibitors or COMT COMT inhibitor results in uncoupling of inhibition, liver toxicity should be induced oxidative phosphorylation, these patients are by both entacapone and tolcapone in then more vulnerable to toxic effects clinical use due to the close structural (Schapira 2001; Orth and Schapira 2002). relationship of these agents. Liver toxicity Homozygous COMT knock-out mice should also be expressed as histopathology

51 Discussion in COMT-deficient mice (Watkins 2000). exposure factor in entacapone- and Since no toxicity as histopathology or tolcapone-treated animals was 21 and 14, elevated liver enzyme values were noted after respectively. At the same time point, the entacapone treatment or in homozygous concentrations of entacapone and tolcapone COMT knock-out mice, COMT were equally high in liver tissue, indicating inhibition as such does not appear to cause that both compounds can penetrate the liver significant liver toxicity. similarly although highly bound to plasma proteins (Dingemanse 1997). At com- 9.1.3. Exposure of animals to parable liver concentrations, entacapone entacapone and tolcapone caused no toxic effects in rats, while The plasma concentration of tolcapone two tolcapone did induce toxic signs in vivo. hours after the dosing was clearly higher than that of entacapone (I, II). Moreover, 9.2. Uncoupling effects related to in clinical use, a peak concentration of approximately 6 µg/ml is reached after an entacapone and tolcapone oral tolcapone dose of 200 mg, while after an equal dose of entacapone the peak 9.2.1. Signs of uncoupling in vivo concentration is 1.8 µg/ml (Table 9) The effect of entacapone and tolcapone on (Keränen et al. 1994; Dingemanse et al. the body temperature of rats measured one 1995). Thus, the AUC of tolcapone at the or two hours after the dosing was clearly maximal recommended dosage in man is different (I, II). Entacapone caused no much higher than that of entacapone (for elevation, while both tolcapone and DNP tolcapone 75 h•µg/ml, dosage 3 x 200 mg/ treatments induced a significant increase in day; for entacapone 20 h•µg/ml, dosage 10 body temperature of rats. Furthermore, x 200 mg/day). However, the calculated

Table 9. In vitro concentrations of entacapone and tolcapone inducing 50% COMT inhibition

(IC50) and in vivo (clinical) data on peak plasma concentrations (Cmax) after a single dose of 200 mg of entacapone or tolcapone, and the concentrations in the liver tissue in rats in vivo (dose 500 mg/kg/day).

Compound COMT inhibition in vitro Clinical data Liver tissue concentration µ µ µ µ IC50 ( M) IC50 ( M) Cmax Cmax ( M) Cmax ( M)* (ng/g without serum with serum (µg/ml) total unbound wet tissue) proteins proteins

Entacapone 0.24 0.54 1.8 5.9 0.118 22.9 ± 2.4 Tolcapone 0.25 2.62 6.3 23.0 0.023 19.1 ± 3.1

*calculated from the clinical data as unbound to serum proteins (Keränen et al. 1994; Dingemanse et al. 1995)

52 Discussion some of the premature deaths of animals administering uncoupling agents, the after tolcapone treatment were preceded by amount of ATP is already low before death, body temperature increasing up to 41ºC. and therefore, rigor mortis should occur at In several cases of DNP poisoning in clinical the time of death or directly after it. In use, body temperature has been shown to skeletal muscles, intermyofibrillar oedema be markedly increased (Horner 1942). In together with prominent contraction of the presence of levodopa and carbidopa (IV), sarcomeres in the tolcapone groups tolcapone induced an elevation of body resembles findings during the early changes temperature after a single oral dose, while of rigor mortis (Collan and Salmenperä entacapone had a slight decreasing effect on 1976; Kobayashi et al. 1999). Melmed et temperature. Dopamine agonists are known al. (1975) have postulated that gross to induce a decrease in body temperature morphological changes in mitochondria through stimulation of dopamine D2 only occur when the concentration of an receptors (Cox and Tha 1975; Faunt and uncoupler in the cell is very high. The Crocker 1987). Since concomitant dosing situation in vivo differs from that in vitro of COMT inhibitors with DDC inhibitors since in in vivo studies the concentration of increases the bioavailability of levodopa, the an uncoupling agent in the liver or skeletal decrease in body temperature after muscle cell after one dose may not reach entacapone treatment may be due to a the same concentrations as in isolated higher DA concentration in the brain. As mitochondria in an incubation medium in levodopa treatment has been reported not vitro. To induce alterations in the mito- to interfere with mitochondrial respiration chondrial morphology in vivo, the test in rat skeletal muscle (Dagani et al. 1991), substance should be administered repeatedly the mechanism by which tolcapone induces and over a longer period of time to allow it a rise in body temperature is apparently to accumulate in the tissue and cell. During unrelated to DA concentration but may be the several days of dosing in Study III the a result of uncoupling of oxidative mitochondrial changes due to uncoupling phosphorylation. should also have become apparent. In the Rigor mortis was not observed in liver cells, swelling of mitochondria, entacapone-treated animals during the 20- deformation of cristae and reduced matrix to 30 minute necropsy; however, it was seen density were observed in DNP- and immediately after death in tolcapone- and tolcapone-treated rats, and these findings are DNP-treated rats (I, II). The rapid onset of comparable with those reported by Spahr rigor mortis has also been reported in et al. (2000) in a clinical case report after previous non-clinical testing of tolcapone tolcapone treatment. and toxicity studies with DNP in the rat (Spencer et al. 1948; Eckhardt et al. 1996; 9.2.2. Signs of uncoupling at cellular Schläppi et al. 1996b). Instantly occurring level rigor mortis indicates very rapid usage of The liver mitochondrial ATP/ADP ratio ATP and glycogen from the skeletal muscle was unaffected by treatment with high oral cells upon the death (Gracey 1981). After doses of entacapone (II). The ratio decreased

53 Discussion significantly after treatment with tolcapone reported that the lowest effective dose of or DNP, indicating uncoupling of oxidative tolcapone to induce a concentration- phosphorylation at the cellular level dependent increase in respiration rate is (Cutting and Tainter 1933; Kaiser 1964; 1 µM. At a concentration range of 1 to 300 Bakke and Laurence 1965). Both tolcapone µM, tolcapone further reduced the and DNP have earlier been shown to be mitochondrial membrane potential in a potent uncouplers in vitro in isolated rat liver dose-dependent fashion. When uncoupling mitochondria (Loomis and Lipmann 1948; of oxidative phosphorylation decreases the Terada 1990; Nissinen et al. 1997). Study amount of ATP, the synthesis of ATP and II demonstrated that tolcapone has the ADP from glucose in the liver is diminished potential to interfere with oxidative energy and energy is generated mainly from metabolism in the liver in vivo. The glycolysis. Increased liver pyruvate induces hampered oxidative ATP production was gluconeogenesis, which is reflected as an also seen in the lowered energy charge in elevation of short duration in serum glucose the tolcapone-treated rat liver mitochondria concentration (Berg et al. 2002), as was also and at the level of the whole liver tissue. shown in the DNP- and tolcapone-treated Decreased ATP production leads to increased rats (II). The reason for the greater potential oxygen consumption as a compensatory of tolcapone than entacapone to cause function of the cell and cell death may occur. uncoupling might relate to tolcapone’s lipid In isolated rat liver mitochondria, both solubility (Dingemanse 1997). Since tolcapone and DNP had also a direct tolcapone is a more lipophilic compound, disruptive effect on mitochondrial it more easily penetrates the cell membrane membrane potential at the same con- and crosses the mitochondrial bilayer than centration range, EC50 being lower than 5 the less lipid-soluble entacapone, inducing µM (IV). Entacapone did not have any a disruption in membrane potential. effect on membrane potential at Borroni et al. (2001) have speculated concentrations lower than 100 µM. Thus, about the discrepancy between in vitro and entacapone has no influence on cellular in vivo findings regarding uncoupling. They respiration or mitochondrial oxygen assumed that because the free fraction of a consumption until concentrations are drug is diminished by extensive protein beyond the physiological, and does not binding in vivo, the uncoupling effect of uncouple oxidative phosphorylation by both tolcapone and DNP is counteracted disrupting the mitochondrial membrane (Borroni et al. 1999; 2001). As entacapone potential. EC50 for stimulation of succinate- is 98% and tolcapone 99.9% bound to supported mitochondrial respiration of plasma proteins in vitro, the effective free tolcapone has earlier been reported to be 2.6 concentrations would be 2% and 0.1%, µM, which is in the same concentration respectively (Dingemanse 1997). However, range as the concentration for disruption of entacapone and tolcapone are equally potent membrane potential in Study IV (Nissinen COMT inhibitors in vitro (IV), with an µ et al. 1997). These results are also consistent IC50 value of ca. 0.25 M, when a rat liver with those of Borroni et al. (2001), who soluble COMT preparation without added

54 Discussion rat serum is used. After adding the serum uncoupler if it is not effectively further to the incubation mixture, a tenfold metabolized. However, the liver concentration of tolcapone is needed to concentrations of both tolcapone and achieve a similar IC50 value of COMT entacapone are equally high after oral dosing inhibition, whereas only twice as much to rats (II), indicating that both compounds entacapone is needed for a 50% COMT can penetrate the liver despite being highly inhibition in the presence of serum (Table bound to plasma proteins. Thus, the 9). However, in clinical use, both inhibitors extensive binding of a drug to serum have proved to be potent COMT inhibitors proteins does not directly affect tissue despite extensive protein binding (Kaakkola concentrations or prevent uncoupling of 2000). If protein binding noticeably oxidative phosphorylation in vivo. decreases the pharmacological activity of the The dissimilarities in the hepatic safety compounds in vivo, the unbound concen- of entacapone and tolcapone may be due to tration of entacapone and tolcapone in different metabolic routes as well as the clinical use should be twice and ten times as lipophilicity of the two compounds. As a high, respectively, to achieve effective more lipophilic compound, tolcapone COMT inhibition (Table 8), which readily crosses mitochondrial membranes. contradicts the results of Borroni et al. The only significant metabolic route for (2001). entacapone is glucuronidation, whereas Findings in vitro and in vivo are not tolcapone also undergoes oxidation and directly comparable since in vitro the methylation. Uncoupling of oxidative reactions are carried out over a few minutes, phosphorylation may have a role in the whereas in vivo the drug is present for hours. hepatotoxicity caused by tolcapone Due to the equilibrium between the protein- treatment, as uncoupling properties bound and free fractions of a drug in comparable with those of DNP are plasma, the free fraction penetrates tissues expressed by tolcapone both in vivo and in and can act as a potential protonophoric vitro.

55 Conclusions

10. Conclusions

The non-clinical safety of entacapone and liver and the ultrastructure of liver and tolcapone, two nitrocatechol-structured skeletal muscle mitochondria. Moreover, COMT inhibitors designed as adjuncts to tolcapone was shown to cause uncoupling levodopa treatment of Parkinson’s disease, of respiration on mitochondrial membranes was compared in vivo and in vitro. in vitro. Taken together, uncoupling of After repeated high oral doses to rats, oxidative phophorylation may have a role entacapone did not cause toxicity, while in the hepatotoxicity caused by tolcapone. equal doses of tolcapone induced severe COMT inhibition as such did not induce toxic signs. These signs were comparable toxic signs in mice lacking COMT activity, with those induced by DNP, an uncoupler nor did it cause any liver problems. The of oxidative phophorylation. Histo- results indicate that toxicity induced by one pathological findings after tolcapone COMT inhibitor cannot be generalized to treatement were of the same type as in all agents in the same class. DNP-treated rats, including changes to the

56 Acknowledgements

11. Acknowledgements

The present study was carried out at the knowledge about kinetics. Department of Basic Veterinary Sciences, Professor Pekka T. Männistö, MD, PhD, Section of Veterinary Pathology of the for initiating the COMT project and for Faculty of Veterinary Medicine, University pushing me forward every now and then. of Helsinki, and at Orion Pharma, Research My colleagues Docent Inge-Britt Lindén, Centre, Espoo. PhD, and Malla Toivonen, MSc, for valuable comments on the manuscript. I wish to express my deepest gratitude to: Co-workers Marko Huotari, PhD, Anita Koponen, MSc, Docent Kari Lounatmaa, Professor Antti Sukura, DVM, PhD, for PhD, Kai Penttilä, PhD and Leila Vaalavirta, his support, generous advice and discussions MD, PhD for contribution during the over these trying years. His never-failing different phases of this thesis. enthusiasm, humour and good mood Jarmo Pystynen, MSc, for providing me encouraged me to keep going. with valuable information on nitrocatechols Docent Erkki Nissinen, PhD, for his and molecular structures. continuous support and encouragement as Pasi Hakulinen, BSc, for helping me well as his sharp but valuable criticism with statistics and for advice on how to throughout this work. His door was always apply the methods to non-clinical work. open. Pia Friman for helping me with figures, Docent Esa Heinonen, MD, PhD, for and Helena Helander and Olli Louhimo for providing the idea of this thesis and for efficiently providing me with articles. guidance, especially during the first years. Ulla Hirvensalo, MSc, Aino Pippuri, He also taught me how to write scientific MSc, and Heikki Brandt, MD, for our publications, even though it sometimes took meetings on Wednesday mornings and for numerous versions over a period of months. discussions that went beyond science and Professor Anthony Schapira, DSc, MD, work. Without Aino, this thesis would not FRCP, FMedSci, and Professor Raimo have come reality, as she is the person who Tuominen, MD, PhD, for constructive synthesized entacapone. criticism which is largely responsible for the The whole staff at the Department of text’s final form. Toxicology, Orion Pharma, as well as my My colleague Leena Sopanen, MSc, for colleagues on the second floor of the ideas and new ways of thinking about Research Centre. It has been a pleasure to problems and for cheering me up on rainy work with you. days. Carol Ann Pelli, HonBSc, for editing the My colleague Arja Vuorela, MSc, for English of the manuscript and for her teaching me everything I know about kindness in conforming to my time pharmacokinetics. I hope that I’ve gained schedules. at least a small part of her enthusiasm and My dear husband Jussi, MD, PhD, for

57 Acknowledgements preparing the layout of this thesis, and Armi would never have been completed. and Lassi for just being there. Thank you The financial support of Orion Pharma all three so much. You brought cheer to hard and the Finnish Academy is greatly days with music played on the piano or appreciated. accordion. Without your support, this work

Espoo, April 2003

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