Catechol-O-methyltransferase (COMT): Biochemistry, Molecular Biology, Pharmacology, and Clinical Efficacy of the New Selective COMT Inhibitors

1. Brain COMT.

The cellular localization of COMT has been studied in several ways. A number of lesion studies (Rivett et al., 1983; Kaakkola et al., 1987) and immunohistochemical studies (Karhunen et al., 1995a; Lundström et al., 1995) have demonstrated that there is no significant COMT activity in presynaptic dopaminergic neurons, but some activity is present in postsynaptic neurons and substantial activity is located in glial cells.

Immunoelectron microscopy studies suggest that COMT resides in astrocytic processes around synapses and capillary walls and in postsynaptic dendritic spines. Some primary neurons also show immunoreactivity to COMT (Karhunen et al., 1995a). Because there are no S-COMT- and MB-COMT-specific antisera available, it has not been possible to separately analyze the tissue distribution of the two enzymes forms. Immunoblotting analysis of the primary cell cultures of the rat brain cells shows that both isoforms of COMT occur in astrocytes, oligodendrocytes, and neurons (Karhunen et al., 1995b).

Some indication of subcellular distribution can be obtained through differential centrifugation. The mammalian S-COMT activity resides in the nonsedimenting, cytoplasmic fractions, and MB-COMT activity resides in the sedimenting fractions, the equivalent of the microsomal fraction (Jeffery and Roth, 1984; Roth, 1992).

With immunoblotting and COMT activity determinations for the analysis of the COMT isoforms, it was shown that both in rat brain and in baculovirus-infected insect cells, the MB-COMT polypeptide resides in the subcellular fractions containing endoplasmic reticulum and cell membranes. These two components could not be separated in this study (Tilgmann et al., 1992). In mammalian cell preparations overexpressing either S-COMT or MB-COMT, the subcellular localization has been studied in detail (Ulmanen et al., 1997). Overexpressed S-COMT was localized in cytosol and in the nucleus, whereas MB-COMT was a microsomal protein. This was determined through double immunostaining against an established marker protein of the rough endoplasmic reticulum. MB-COMT was located in the rough endoplasmic reticulum, facing the cytoplasm. It is worth noting that no MB-COMT has been found in the cell membrane (Ulmanen et al., 1997).

When the cellular distribution of COMT in the rat striatum was studied in an attempt to selectively destroy glial cells with fluorocitrate, we found that initially there was a decrease in COMT activity at 1 and 2 days after lesioning, followed by a marked increase at 3 days. At the same time, a microglial marker (alkaline phosphodiesterase) was increased. Immunohistochemical analysis also revealed a major increase in microglia (OX-42-immunoreactive cells) but not of astroglia (glial fibrillary acidic protein immunoreactive cells). No neuronal COMT immunoreactivity was detectable in these studies (Reenilä et al., 1997).

2. COMT in Other Tissues.

COMT has been found in practically all mammalian tissues investigated. The highest COMT activity in both rat and humans is in the liver, followed by the kidneys and gastrointestinal tract (both stomach and intestine; Nissinen et al., 1988b; Schultz and Nissinen, 1989; Männistö et al., 1992b). These findings have been confirmed and expanded to spleen and submaxillary glands through the use of immunohistochemistry (Karhunen et al., 1994; Lundström et al., 1995). In pancreas, COMT immunoreactivity was found in β and δ cells but not in α cells (Karhunen et al., 1994). The importance of pancreatic COMT activity has also been demonstrated through other means. After the pretreatment of conscious rats with OR-486 (dinitrocatechol), a potent nitrocatechol-type COMT inhibitor, the uptake of radioactivity from [¹³C]l-dopa into the pancreas was increased by 4-fold. Most of the radioactivity was derived from [¹³C]dihydroxyphenylalanine ([¹³C]DOPAC; Bergström et al., 1997). Positron emission tomography (PET) studies have demonstrated high COMT activity in kidney, liver, intestine, stomach, spleen, lungs, and heart of mice (Ding et al., 1996); they also confirmed high COMT activity in baboon liver and kidney.

In kidney, COMT activity is found in proximal tubular epithelial cells, in fact, in the same cells in which dopamine is synthesized. Dopamine acts as a local hormone, exerting diuretic and natriuretic effects (Lee, 1993). COMT mRNA (mostly that of S-COMT) has also been visualized in epithelial cells of proximal tubules, the thick ascending limb of loop of Henle, and the ureter (Meister et al., 1993), where the enzyme is thought to regulate the metabolism of dopamine and other catecholamines. COMT mRNA has been detected in the prenatal kidney at gestational day 18 (Meister et al., 1993). After birth, theK _m values of rat kidney COMT activity appear to increase as a function of age from 3.3 μM at age 3 days to 16.9 μM at age 30 days (see Other Enzymological Aspects).V _max values (which are dependent on the total amount of enzyme in the tissue, which was not measured) were not much altered (Vieira-Coelho and Soares-da-Silva, 1996). The same authors also claim that the inhibitory activity of tolcapone on kidney COMT would be radically altered as a function of the age. However, because the changes in the COMT protein content were not measured, this conclusion remains uncertain. The same is true for the comparison between kidney and liver COMT inhibition with tolcapone.

Human kidney COMT activity was also assayed by De Santi et al. (1998b), who found it to be 159 pmol/min × mg protein when DBA was used as the substrate. COMT activity in the liver was three times higher than kidney activity, and in the duodenum, activity about 30% less than that in kidney was noted. In that study, entacapone was found to be severalfold more active than tolcapone as a COMT inhibitor in all tissues studied. This is an unexpected result that has not been confirmed.

Although it has been demonstrated that the metabolism of the newly formed dopamine in the rat kidney is primarily mediated through MAO, with only a minor involvement of COMT (Fernandes and Soares-da-Silva, 1994), both nitecapone (Eklöf et al., 1997) and entacapone (Hansell et al., 1998) induced a copious diuresis and natriuresis, which in both cases was inhibited by a dopamine D₁ antagonist. However, the amounts of dopamine excreted were only marginally increased by entacapone but much more byN-(2-pyridone-6-yl)-N′N′-di-n-propylformamidine (CGP 28014), which is not at all a COMT inhibitor (see below; Hansell et al., 1998). Nitecapone was shown to enhance Na⁺,K⁺-ATPase inhibition in the proximal tubular cells (Eklöf et al., 1997). Finally, there seems to be a weak but significant correlation of histamine-N-methyltransferase and COMT activities in the human renal cortex (De Santi et al., 1998a).

Opossum kidney cells in culture can synthesize and metabolize dopamine (by MAO and COMT) in a manner similar to rat renal tubular cells. Therefore, these cultured cells can be used as an easily available and standardized model system to explore the role of dopamine in kidney function (Guimarães et al., 1997).

In hamster kidney, a 2- or 4-week estrogen treatment induces COMT immunoreactivity, as well as in the nucleus, where only S-COMT was seen (Weisz et al., 1998b), supporting the nuclear localization of S-COMT as discussed. This probably is a compensatory mechanism inducing a metabolizing enzyme to oppose the formation of electrophilic quinones and semiquinones from catecholestrogens, particularly in this animal species (see Estrogen Metabolism and Role of COMT and COMT Inhibitors).

Rather high COMT activities have been described in human lung by De Santi et al. (1998b) and in rat lung by Bryan-Lluka (1995). There also is a substantial amount of COMT in the eye, in both the ciliary body and the retinal ganglion cell layer (Karhunen et al., 1994). COMT activity has also been demonstrated in spinal membranes of monkeys and pigs (Kern et al., 1995).

COMT activity is detectable in the skin, with the activity being higher in epidermis than in dermis (Bamsdah, 1969). COMT is found in keratocytes, where it may metabolize epinephrine, but also in melanocytes, which make up only 3 to 5% of the epidermal cell population (Smit and Pavel, 1995). The epidermis of patients with vitiligo with contains more COMT than does the epidermis from healthy controls (Lepoole et al., 1994). Intermediate indolic compounds, like 5,6-dihydroxyindole-2-carboxylic acid, are formed in the synthesis of eumelanin from tyrosine. These intermediates can be readily oxidized to toxic quinone derivatives. The polymerization of these potentially toxic intermediates to melanin can be considered a detoxification process. COMT activity may further reduce the amount of dihydroxyindoles and therefore protect normal melanocytes against their own reactive compounds generated during melanogenesis. If the metabolism of dihydroxyindoles by COMT is inhibited, these metabolites can accumulate and become harmful to normal skin (Smit and Pavel, 1995). However, there is a possibility for a novel therapeutic role for COMT inhibition in melanoma treatment (Shibata et al., 1993; Smit et al., 1994). It has been shown that malignant melanocytes contain both S-COMT and MB-COMT. At physiological concentrations of dihydroxyindoles, MB-COMT may be more relevant and functionally more significant than S-COMT (Shibata et al., 1993; Smit and Pavel, 1995). In melanoma cells, the accumulation of reactive dihydroxyindoles after effective COMT inhibition could selectively damage melanoma cells (Smit et al., 1994; Smit and Pavel, 1995).

Some tumors, notably pheochromocytomas, contain high amounts of COMT, particularly MB-COMT. High metanephrine levels in patients with pheochromocytoma are derived from catecholamines produced and metabolized within the tumors. Hence, measurement of plasma-free metanephrine concentrations may be important for the rapid diagnosis of these patients (Eisenhofer et al., 1998).

Human (Keränen et al., 1994), hamster, guinea pig, rabbit, hen, dog and monkey (Zürcher et al., 1996), and rat (Nissinen et al., 1992) erythrocytes contain some COMT activity. The activity varies from species to species, but it is high in rat and quite low in humans. Erythrocyte COMT offers a convenient way of monitoring the COMT inhibition in the body during COMT inhibitor therapy. Several human studies have shown an excellent correlation between the inhibition of erythrocyte COMT activity and the concentration of a COMT inhibitor in plasma or the decrease of 3-O-methyl-dopa (3-OMD) levels in plasma (Keränen et al., 1994; Dingemanse et al., 1995b). Even human lymphocytes contain a significant amount of COMT, almost half of which appears to be MB-COMT (Sladek-Chelgren and Weinshilboum, 1981).

1. Substrates of COMT.

The physiological substrates of COMT include l-dopa, catecholamines (dopamine, norepinephrine, epinephrine), their hydroxylated metabolites, catecholestrogens (Ball and Knuppen, 1980), ascorbic acid, and dihydroxyindolic intermediates of melanin. Several dietary and medicinal products are also COMT substrates, such as triphenols and substituted catechols, dobutamine, isoprenaline, rimiterol, α-methyldopa, benserazide, carbidopa, dihydroxyphenyl serine (Maruyama et al., 1996), flavonoids, and dihydroxy derivatives of tetrahydroxyisoquinolones. The general function of COMT is the elimination of biologically active or toxic catechols and some other hydroxylated metabolites. During the first trimester of pregnancy, COMT protects the placenta and the developing embryo from activated hydroxylated compounds formed from aryl hydrocarbons by hydroxylases (Barnea and Avigdor, 1990). COMT also acts as an enzymatic detoxicating barrier between the blood and other tissues shielding against the detrimental effects of xenobiotics (e.g., in the intestinal mucosa and the brain). COMT may also serve some unique or indirect functions in the kidney and intestine tract by modulating the dopaminergic tone; the same may be true in the brain: COMT activity may regulate the amounts of active dopamine and norepinephrine in various parts of the brain and therefore be associated with mood and other mental processes.

2. Quantitative Role of COMT in Metabolism of Catecholamines.

The relative importance of enzymes in metabolizing catecholamines and the uptake processes of catecholamines have been clarified. Without exogenous levodopa loading, the high-affinity neuronal reuptake (uptake₁) is an efficient elimination system for the released catecholamines, being responsible for most of their elimination both in peripheral tissues and the brain (Kopin, 1985;Männistö et al., 1992b; Cass et al., 1993). The role of the extraneuronal transport is less clear (Friedgen et al., 1996b). The contribution of metabolism, including COMT, is unimportant, and therefore COMT inhibition does not affect dopamine levels to a detectable degree. It is equally clear that inhibition of MAO (by pargyline) and COMT (by tolcapone), each separately, has little, if any, effect on the removal of norepinephrine, epinephrine, and dopamine on passage through the systemic and pulmonary circulation. The pulmonary, but not the systemic, clearance of catecholamine can be reduced by a combined blockade of MAO and COMT (Friedgen et al., 1996a). Thus, inhibition of COMT does not greatly alter the elimination of infused catecholamines or exercise-induced elevation of catecholamines, which is an important safety-increasing factor in the use of potent COMT inhibitors (see later). However, the elimination pathways are adaptable. When MAO is blocked, both COMT and phenolsulfotransferase activities are increased, but these two pathways do not compete with each other (Buu, 1985).

The situation is dramatically altered when exogenous levodopa is administered. When levodopa is administered alone, it is predominantly decarboxylated to dopamine in the peripheral tissues, and only a small fraction of the dose ever reaches the brain to be used for dopamine synthesis. If dopa decarboxylase (DDC) is inhibited, the majority of surplus l-dopa is metabolized, now preferably by peripheral COMT (Kuruma et al., 1972; Messiha et al., 1972; Fahn, 1974; Da Prada et al., 1984; Männistö et al., 1992b). During the combination therapy, 3-OMD is the major metabolite (Fahn, 1974;Rivera-Calimlim et al., 1977; Reilly et al., 1980; Da Prada et al., 1984; Hardie et al., 1986), and the role of COMT inhibitors becomes extremely meaningful.

The interplay of catecholamine uptake (uptake₁) and COMT has been explored in several cell lines with COMT activity and artificially expressing the recombinant amine transporters (Eshleman et al., 1997). COMT inhibition by tropolone or a nitrocatechol-type inhibitor, Ro 41-0960, augmented by 4-fold the transport of ligands that were COMT substrates but did not affect substrates that were not metabolized by COMT. The uptake of serotonin was not altered by COMT inhibitors; however, no such potentiation was seen with dopamine uptake into mouse neostriatal synaptosomes. That may be due to the fact that dopamine is protected by intracellular sequestration into synaptic vesicles, organelles that were lacking from the cell lines used in cell culture.

3. COMT Knockout Mice.

The ultimate importance of COMT will probably be clarified in a new strain of animals lacking the COMT gene. Such mice were recently produced by Gogos et al. (1998). The mice appeared to be normal, they had only minor changes in their behavior, and the brain neurochemistry of catecholamines was virtually unaltered. The mice were also able to breed normally. Surprisingly, all of the changes were sex and region specific. Mutant male mice, totally lacking COMT activity, had an almost 3-fold increase of dopamine levels in the frontal cortex but not in the striatum or hypothalamus, but females showed no changes. It is noteworthy that despite the complete lack of COMT gene and protein, residual homovanillic acid (HVA) levels were detectable in several brain areas. This points to the possibility that there exists a still-unidentified methylation pathway in the brain. Female knockout mice had impaired emotional reactivity in the black-white box. On the other hand, heterozygous males (but not homozygous males or any females) were more aggressive than their wild-type counterparts. These findings suggest that 1) the importance of COMT in the behavior has probably been underestimated and 2) a complete lack of COMT can be effectively compensated, at least in mice.

1. COMT Inhibition.

Nitrocatechols are so-called tight-binding inhibitors of COMT, although their binding to COMT is fully reversible (Schultz and Nissinen, 1989; Lotta et al., 1995; Borges et al., 1997). Prolongation of the preincubation time with an inhibitor markedly reduces the IC₅₀ values of nitrocatechol-type inhibitors (Schultz and Nissinen, 1989). This particular property has been the source of many problems when the kinetic parameters have been characterized. It is necessary to perform inhibition kinetic studies in a special way to avoid invalid conclusions. In the presence of varying inhibitor concentrations, the reaction velocity of COMT increases progressively with increasing enzyme concentrations and parallels the velocity curve without an inhibitor if sufficiently high amounts of enzyme are used. Also, the effect of the nitrocatechol-type inhibitors can be gradually abolished by dialysis (about 50% in 5 h, fully reversed within 24 h; Schultz and Nissinen, 1989).

Entacapone [OR-611; (E)-2-cyano-N,N-diethyl-3-(3,4-dihydroxy-5- nitrocinnamamide] and nitecapone [OR-462; 3-(3,4-dihydroxy-5-nitro-benzylidene)-2,4-pentanedione; Fig. 2] are highly effective inhibitors of rat S-COMT with IC₅₀ values of about 150 to 300 nM in the liver and 10 to 20 nM in the brain tissues.K _i values in the rat liver have been 145 and 23 nM for entacapone and nitecapone, respectively (Schultz and Nissinen, 1989; Nissinen et al., 1992). When analyzed with pure recombinant COMT enzyme forms, K _ivalues of nitecapone and entacapone were around 1 nM or even lower (Lotta et al., 1995). They are also selective because their in vitro IC₅₀ values for tyrosine hydroxylase, dopamine-β-hydroxylase, DDC, and MAO-A and -B are in the micromolar range (Männistö et al., 1988; Nissinen et al., 1988a,1992). They strongly inhibit the COMT activity in a variety of tissues. An oral dose of 10 mg/kg nitecapone or entacapone causes nearly complete inhibition of duodenal COMT activity for 1 to 3 h and highly significant inhibition in erythrocyte and liver COMT activity for several hours. Full recovery of duodenal COMT activity is achieved at 8 to 12 h after drug administration. The striatal COMT activity is slightly and transiently suppressed by entacapone administration but not at all by nitecapone administration; full recovery is attained at 3 h. Oral ID₅₀ values of nitecapone and entacapone have been in a range of about 1 to 5 mg/kg in the liver and duodenum but at least 25 mg/kg or higher in the brain (Schultz and Nissinen, 1989; Zürcher et al., 1990a; Nissinen et al., 1992).

Tolcapone [Ro 40-7592; 4′-methyl-3,4-dihydroxy-5-nitro-benzophenone] resembles chemically entacapone and nitecapone (Fig. 2; Zürcher et al., 1990b). It seems to be slightly more potent at inhibition of liver COMT activity than entacapone and nitecapone, with IC₅₀ and K _ivalues of 36 nM in the rat liver (Zürcher et al., 1990a, 1991;Borgulya et al., 1991). In both recombinant COMT forms, theK _i value of tolcapone was about 0.3 nM (Lotta et al., 1995). Tolcapone can penetrate into the brain and inhibits the brain COMT activity in vivo with an ID₅₀ value of 26 to 28 mg/kg (Da Prada et al., 1991; Zürcher et al., 1991). A high oral dose of tolcapone (100 mg/kg) causes virtually complete inhibition of rat heart and kidney COMT activity at 15 to 30 min. Liver and brain COMT activity is less effectively suppressed (maximally by 70% at 30 min). About 50% inhibition is measured at 11 h (heart and kidney), at 8 h (liver), and at 6 h (brain) after dosing. Full recovery of COMT activity occurs by 16 h. The inhibition of COMT activity by tolcapone is fully reversible, as it is with entacapone and nitecapone. Tolcapone does not affect MAO, hydroxyindole-O-methyltransferase, histamine-N-methyltransferase, or phenyl-ethanolamine-N-methyltransferase activities or adrenergic (α, β), serotonergic, or cholinergic receptors (Zürcher et al., 1990a,b).

Nitrocatechols with NO₂ group in positions other than that in the “classic” nitrocatechols have also been described (Fig. 2; Perez et al., 1992, 1993, 1994). These dihydroxyvinyl-type compounds are able to bind to the active site of COMT (Vidgren and Ovaska, 1997), but their efficacy against both human and pig enzymes is generally lower than that of compounds with the NO₂ in the classic site of the catechol ring. The situation is complicated by the fact that the in vitro comparisons have usually been made using pig liver COMT, which differs from rat and human enzymes by having a polar arginine in position 38 instead of hydrophobic Trp38. This difference leads to reduced affinity (higherK _m value for catechol substrates) and higher K _i values for inhibitors. However, even in human COMT, 2-(3,4-dihydroxy-2-nitrophenyl)vinyl phenylketone (vinylphenylketone, or QO IIR) has 4- to 13-fold higher K _i values than nitecapone, entacapone, and tolcapone (Vidgren and Ovaska, 1997). Vinylphenylketone is a tight-binding, reversible COMT inhibitor that resembles the classic nitrocatechols (Perez et al., 1993, 1994).

Also an endogenous COMT inhibitor, 6-nitronorepinephrine (Fig. 2) has been described and quantified in pig and rat brain, where its concentration is around 75 pg/g (Shintani et al., 1996). This compound is generated from the reaction between nitric oxide and norepinephrine. If the synthesis of nitric oxide is inhibited, the amount of 6-nitronorepinephrine is decreased. Perfusion of 6-nitronorepinephrine into the paraventricular nucleus elevated norepinephrine levels and decreased 3-methoxy-4-hydroxyphenyl glycol (MHPG) levels. In vitro, COMT inhibition is modest because the IC₅₀ value is as high as 7.5 μM. However, 6-nitronorepinephrine inhibits also the reuptake of norepinephrine into the synaptosomes, with an IC₅₀ value of 31 μM. In summary, 6-nitronorepinephrine is a potential, although fairly weak, endogenous signal molecule that may link the actions of catecholamines and nitric oxide (Shintani et al., 1996). However, 6-nitronorepinephrine cannot be used as a drug.

CGP 28014, a hydroxypyridine compound (Fig. 2), was first described byWaldmeier et al. (1990a,b). CGP 28014 and its major metabolite, 2-amino-6-hydroxypyridine, are not COMT inhibitors in vitro until millimolar concentrations are reached. CGP 28014 does not affect receptors of the various neurotransmitters and other endogenous substances (Waldmeier et al., 1990a).

An active iron chelator, 1,2-dimethyl-3-hydroxypyridine-4-one (L1, CP20), is a further example of another type of COMT inhibitor that, however, also inhibits tyrosine and tryptophan hydroxylases (Waldmeier et al., 1993).

2. Effects on l-Dopa and Catecholamine Metabolism.

In rats, oral administration of entacapone and nitecapone (3–30 mg/kg) in combination with levodopa and carbidopa effectively reduce 3-OMD formation and elevate serum and brain l-dopa, dopamine, DOPAC, and HVA levels (Männistö et al., 1988, 1992a;Nissinen et al., 1988a, 1992). Similar findings have been reported inCynomolgus monkeys in whom the i.v. efficacy of entacapone and nitecapone was equipotent (Cedarbaum et al., 1991). Part of the levodopa saved from COMT is metabolized through alternative pathways because the increase of serum l-dopa is smaller than the decrease in 3-OMD would lead one to expect.

In microdialysis studies, the pharmacokinetics of l-dopa in plasma and gluteal muscle was studied in pentobarbital anesthetized dogs treated with levodopa alone (20 mg/kg i.v.), with levodopa and carbidopa (repeated doses of 100 mg p.o. and 100 mg i.v. at the beginning of anesthesia) and levodopa, carbidopa, and entacapone [15 mg/kg i.v., 1 h before levodopa (Deleu et al., 1995)]. It was found that carbidopa had an l-dopa-sparing effect in both plasma and muscle, and this effect was further enhanced with entacapone. The T _½β ofl-dopa was prolonged from 0.76 to 2.66 h by entacapone, whereas it was only 0.4 h without carbidopa and entacapone. A similar shift occurred in the muscle although to a lesser extent. The T _½α ofl-dopa in plasma was not altered by either treatment. Formation of 3-OMD was greatly reduced in both plasma (by 98%) and muscle (by 85%) with entacapone. Skeletal muscle may be an important l-dopa storage site (Deleu et al., 1995) whose capacity can be significantly increased with entacapone (Ordonez et al., 1974).

The effective decrease in 3-OMD, HVA, and 3-MT levels, as well as in COMT activity in the brain, can be used as criteria for the classification of tolcapone as a brain-penetrating COMT inhibitor (Zürcher et al., 1990a,b, 1991; Männistö et al., 1992a). In the time course studies, 13 mg/kg tolcapone p.o. decreased several 3-O-methylated metabolites of catecholamines (3-MT, HVA, and MHPG). The formation of 3-MT was suppressed by 90% as early as 1 h after dosing, and that of HVA and MHPG was suppressed more slowly and modestly (80 and 60%, respectively) at about 4 h after tolcapone. Whole brain dopamine was not changed, whereas DOPAC levels were doubled for 6 to 8 h (Zürcher et al., 1991).

After the oral administration of 30 mg/kg tolcapone with 10 mg/kg levodopa and 15 mg/kg benserazide, plasma and whole brainl-dopa levels increased by 4-fold and 3-OMD decreased to very low levels in rats. Dopamine formation in the whole brain increased for at least 6 h (Zürcher et al., 1990a,b).

In a further study on rats, 20 mg/kg levodopa (combined with 15 mg/kg benserazide) has been compared with 10 mg/kg levodopa (and 15 mg/kg benserazide) plus 30 mg/kg tolcapone. The triple treatment approximately doubled the area under the curve (AUC) of plasmal-dopa compared with the conventional double treatment with twice as high a dose of levodopa. Thus, the bioavailability of levodopa was increased 3.5-fold by the addition of tolcapone (Zürcher et al., 1991). However, it is worth noting that theT _1/2 of levodopa was not markedly prolonged. The plasma levels of 3-OMD, which normally exceed the plasmal-dopa levels by a factor of 2, remained low and near the detection limit for at least 8 h (Zürcher et al., 1991).

In rats, tolcapone (30 mg/kg i.p.) was also able to prolong the elimination half-life (+116%) and area under the plasma apomorphine concentration-time curve (+31%), and apomorphine was present in striatum for a 85% longer time period (Coudoré et al., 1997). Even 2-(3,4-dihydroxy-2-nitrophenyl)vinyl phenylketone (QO IIR, or vinylphenylketone) has recently been proved to act as a peripherally acting COMT inhibitor in in vivo studies in rats (Rivas et al., 1999).

The in vivo effects of CGP 28014 in rats mimicked those of the other COMT inhibitors. The ED₅₀ values are 2 to 8 mg/kg p.o. when the endpoints were the decrease in striatal HVA, the decline of striatal 3-MT after clorgyline treatment (both without exogenous levodopa), or the formation of 3-OMD from the exogenous levodopa (without a DDC inhibitor). It was long acting when administered at high doses of 100 mg/kg (>12 h) or 300 mg/kg (>24 but <36 h). CGP 28014 increased striatal AdoMet levels. However, it unexpectedly increased striatal 5-hydroxyindoleacetic acid and tryptophan levels (Waldmeier et al., 1990a,b).

In our studies in rats, CGP 28014 proved to be a poor COMT inhibitor in the periphery, or at least its inhibitory effect on 3-OMD formation was slow, becoming significant only by 3 h. However, it behaved as an efficient COMT inhibitor-like compound in the brain, preventing both HVA and 3-MT formation (Männistö et al., 1992a). This kind of brain priority has not been described previously for the COMT inhibitors. In fact, in earlier studies, CGP 28014 inhibited both 3-OMD and HVA formation to an equal degree after both p.o. and i.p. administration (Waldmeier et al., 1990a,b). However, in these studies, either CGP 28014 was administered without levodopa or levodopa was administered without the inhibition of the peripheral DDC. Therefore, only moderate amounts of 3-OMD were produced in the periphery because most of the l-dopa was decarboxylated to dopamine. This unusual behavior of CGP 28014 was also seen in rat kidney studies, where it enhanced dopamine and DOPAC secretion but did not affect sodium secretion. This is in contrast to the effect of entacapone, whose action on dopamine secretion was much less but whose action on sodium secretion was much stronger (Hansell et al., 1998).

3. Microdialysis Studies.

In vivo microdialysis technique is a widely used method for the estimation of extracellular levels of various substances, such as neurotransmitters and their metabolites. The effects of entacapone, tolcapone, and CGP 28014 alone or in combination with levodopa and DDC inhibitors on brainl-dopa and dopamine metabolism have been investigated in rats. The aims have been, on one hand, to clarify the mechanisms of brain dopamine metabolism and, on the other hand, to confirm the beneficial effect of combination of a COMT inhibitor with levodopa on brain dopamine formation. Only a few studies have dealt with norepinephrine levels and metabolism (Li et al., 1998).

4. Voltammetric Studies.

Garris and Wightman (1995) studied the effect of various drugs, including tolcapone (40 mg/kg), on the dopamine efflux from the rat caudate-putamen and basolateral amygdaloid nucleus, elicited by electrical stimulation of ascending dopamine fibers at various frequencies. Dopamine efflux was monitored by fast-scan cyclic voltammetry. Tolcapone caused negligible effects in both regions at frequencies of 30 Hz or higher. However, at 20 Hz, these workers did detect a nearly 50% increase in dopamine release. In contrast, the effects of two uptake blockers, nomifensine and cocaine, to stimulate dopamine efflux were robust in both brain regions. Similar results were recently obtained by Budygin et al. (1999) with 30 mg/kg tolcapone and 10 mg/kg GBR 12909, a new dopamine uptake blocker. These results suggest that under normal conditions, uptake₁, rather than transmitter metabolism, regulates extracellular levels of dopamine.

5. Estrogen Metabolism and Role of COMT and COMT Inhibitors.

Estrogen metabolism, including the crucial role of COMT in the metabolism of 2- and 4-hydroxylated estrogens (catecholestrogens), was reviewed by Ball and Knuppen (1980) and recently by Zhu and Conney (1998). Some of the highlights and safety aspects are described here. A schematic summary of the main pathways, and of the role of COMT in particular, is given in Figs.3 and4.

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Figure 3

Main estrogen metabolites and their proposed estrogenic (or antiestrogenic) activity according to Zhu and Conney (1998). The theoretical effects of COMT inhibition on the relative amounts of the metabolites, and the subsequent changes in hormonal activities, are also indicated. 2-OH-E, 2-OH-estrogen; 4-OH-E, 4-OH-estrogen; 2-CH₃O-E, 2-methoxyestrogen; 4-CH₃O-E, 4-methoxyestrogen; 16α-OH-E, 16α-hydroxy estrogen.

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Figure 4

Main metabolic routes of catecholestrogens modified according to Cavalieri et al. (1997) and Cavalieri and Rogan (1998). The formation of catecholestrogen semiquinones and quinones and further formation of stable (with 2,3-semiquinones and 2,3-quinones) and depurinating DNA adducts (with 3,4-semiquinones and 3,4-quinones) are shown. CE, catecholestrogen; P-450; cytochrome P-450; GSHS-transferase; glutathioneS-transferase.

Estradiol (17β-estradiol), which is reversibly oxidized to estrone, can undergo numerous metabolic routes. Among these, the NADPH-dependent hydroxylation reactions, catalyzed mainly by multiple forms of cytochrome P-450 enzymes, are relevant to this review. There are many hydroxylation products that are formed, but only the catecholestrogens are substrates of COMT, and at high concentrations they may act as competitive COMT inhibitors. Catecholestrogens and theirO-methylated products, 2- and 4-methoxy estradiols or 2- and 4-methoxy estrones, are not inert metabolites but may possess unique activities that are not necessarily directly associated with the actions of their parent hormones (Zhu and Conney, 1998).

It is difficult to estimate the relative amounts of various hydroxylated estrogens, but in women, 2-OH-estradiol, which is formed mainly in the liver, is the most abundant catecholestrogen detected in urine (50% or more; Dannan et al., 1986; Kerlan et al., 1992; Zhu et al., 1993; Suchar et al., 1995). The other hydroxylated estrogens are much less abundant; 4-OH-estradiol represents about 5% (<15% of that of 2-OH-estradiol) and the 16 α-hydroxylated product represents more than 10% of the total. It is noteworthy that in extrahepatic “target” tissues, 4-hydroxylation is much more common (see below).

There are very low levels of 2-OH-estrogens in the systemic circulation, mainly because they are rapidly further metabolized by COMT and conjugation enzymes (sulfation, glucuronidation; Ball et al., 1978; Fishman and Martucci, 1979; Emons et al., 1983, 1987; Zhu and Liehr, 1996). In addition to liver, 2-OH-estrone and 2-OH-estradiol are also formed locally in target tissues like uterus and breast. 2-OH-estrogens have multiple effects in the body, only a few of which are relevant here. First, they bind to estrogen receptor but only with low affinity, and their hormonal potency is much less than that of their parent compounds. This means that their net effect is antiestrogenic (MacLusky et al., 1983; van Aswegen et al., 1989;Feigelson and Henderson, 1996). Second, they may generate estrogen semiquinones and quinones and highly toxic oxygen radicals (Liehr et al., 1986b; Liehr, 1990; Liehr and Roy, 1990). The latter possibility seems rather theoretical because instead of having tumorigenic activity in routine tests, they tend to inhibit spontaneous tumorigenesis in estrogen-sensitive tissues (Liehr et al., 1986a; Li and Li, 1987; see below). Even a subsequent O-methylation product of 2-OH-estradiol, 2-methoxy estradiol, inhibits angiogenesis, and is a strong inhibitor of tumor cell proliferation (Seegers et al., 1989;Lottering et al., 1992; D‘Amato et al., 1994; Fotsis et al., 1994;Hamel et al., 1996; Klauber et al., 1997). Interestingly, in patients with depression, the formation of 2-methoxyestrogens is increased, whereas that of 4-hydroxylation is reduced (Banger et al., 1990). In summary, 2-OH-estrogens and their O-methylation products are evidently beneficial and anticarcinogenic (Fig. 3).

4-Hydroxylation of estrogens is not extensive in the liver, but this reaction is much more common in the extrahepatic target tissues of estrogens, such as pituitary (Bui and Weisz, 1988), myometrium and myomal tissues (Lier et al., 1995), and breast (Lier et al., 1995;Hayes et al., 1996). This point has been thoroughly discussed by Weisz (1991, 1994) and recently by Weisz et al. (1998a,b) and Cavalieri’s group (Cavalieri et al., 1997; Cavalieri and Rogan, 1998; Stack et al., 1998). The actions of 4-OH-estrogens are completely different from those of 2-OH-derivatives. First, 4-OH-estradiol binds to estrogen receptors as effectively as estradiol itself, and its binding is long lasting (Martucci and Fishman, 1976; MacLusky et al., 1983; van Aswegen et al., 1989). Consequently, 4-OH-estrogens are hormonally very active (Martucci and Fishman, 1976; Franks et al., 1982). Second, 4-OH-estradiol undergoes a metabolic redox cycling to generate free radicals and chemically very reactive semiquinone and quinone intermediates, which can damage DNA and other cellular components, induce cell transformation, and initiate tumorigenesis (Liehr et al., 1986b; Liehr, 1990; Liehr and Roy, 1990; Fig. 4). Third, 4-OH-estradiol is a strong carcinogen in the hamster kidney. Injections of either 4-OH-estradiol or estrone-3,4-quinone cause kidney and liver tumors but not mammary tumors (Liehr et al., 1986a; Li and Li, 1987).

The above quinone hypothesis, bypassing the importance of pure estrogen receptor stimulation as a key factor in estrogen-dependent cancer, has obtained strong support from recent studies on the abundant appearance of cytochrome P-4501B1, converting estrogens to 4-OH-estrogens in the breast tissue, and apparently also in uterus and ovaries, more effectively than in tissues not prone to estrogen-linked cancer (Hayes et al., 1996; Spink et al., 1998). Catecholestrogens can be metabolized further by COMT, conjugated to glucuronide or sulfate but also oxidated to semiquinones and quinones. Particularly reactive is estrogen-3,4-quinone, which has been shown to bind to DNA, creating so-called depurinating adducts both in in vitro studies and in animals. These adducts are quickly falling off, taking with them two bases of DNA: adenine and guanine. The gaps formed in the DNA have a strong potential to create gene mutations and eventually cause cancer (Cavalieri et al., 1997). These events are schematically summarized in Fig. 4. Because the formation of 4-OH-estrogens is very low, at least in humans, it is quite unclear how large a risk this metabolic route may have. However, any procedure inhibiting the normal metabolism of estrogens may increase formation of 4-OH-estrogens and can be regarded as potentially dangerous.

Interestingly, after 2 or 4 weeks of estrogen treatment, COMT immunoreactivity was increased in hamster kidneys, and it was seen even in the nucleus (Weisz et al., 1998b). This is unexpected because in contrast to human COMT, hamster kidney COMT lacks nuclear localization signal sequence. The nuclear entry of COMT was also selective because another second phase II enzyme, CuZn-superoxide dismutase, remained extranuclear. The authors suggest that the nuclear translocation of COMT after estrogen treatment is a protective response to the threat of the genome by electrophilic products of catechols, quinones and semiquinones (Weisz et al., 1998b). It can be concluded that 4-OH-estradiol is a potentially harmful compound, a strong tumorigenic substance that may be associated with the genesis of estrogen-dependent cancers (Figs. 3 and 4).

The role of COMT in the O-methylation of estrogens and how this is altered by potent COMT inhibitors are areas of concern. Catecholestrogens are generally good substrates of COMT. 2-OH-estrone in particular is rapidly O-methylated, and 2-methoxy estrone is one of the most abundant estrogen metabolites in human plasma and urine (Kraychy and Gallagher, 1957; Ball et al., 1979). Both 2-methoxy estradiol and 2-methoxy estrone have very high affinity for the sex hormone-binding protein (Dunn, 1983). Little is known about the role of 4-methoxy derivatives, but their activity is inferior to that of the corresponding 2-methoxy derivatives.

Mono-O-methylated estrogens have little or no affinity for estrogen receptors, and they do not exert any estrogenic effect on target tissues (Merriam et al., 1980). It seems, therefore, thatO-methylation of catecholestrogens is primarily a detoxification pathway. However, 2-methoxy estradiol has a number of effects on its own, not related to estradiol, 2-OH-estradiol or other methoxy derivatives. It inhibits the proliferation of several cancer cell lines in vitro (Seegers et al., 1989; Lottering et al., 1992;Fotsis et al., 1994; Cushman et al., 1995; Klauber et al., 1997). For example, human breast cancer cell lines were particularly sensitive to 2-OH-estradiol, and it depressed the growth of some implanted cancers (Fotsis et al., 1994; Klauber et al., 1997). 2-Methoxy estradiol also disturbs the function of microtubules (D'Amato et al., 1994; Hamel et al., 1996), and it is one of the most potent endogenous inhibitors of angiogenesis known (Fotsis et al., 1994; Klauber et al., 1997). Even the inhibitory effects of some enzyme inducers, like phenobarbital and indole-3-carbinol, on spontaneous breast carcinogenesis in mice may be mediated by enhanced formation of 2-OH-estradiol and its rapid metabolism to 2-methoxy estradiol (Osborne et al., 1990; Bradlow et al., 1991; Jellinck et al., 1991, 1993).

The 16 α-hydroxylation of estrogens does not have any direct connection to COMT. However, this route may be activated if the COMT pathway is inhibited, and therefore the properties of 16 α-OH-estrogens are important (Fig. 3). Both 16 α-OH-estrone and 16 α-OH-estradiol retain most of the hormonal activity of their parent compounds (Fishman and Martucci, 1980). 16 α-OH-estrone seems to bind covalently to the estrogen receptor and may activate the classic estrogen-mediated oncogene expression and evoke long-term growth stimulation (Hsu et al., 1991). It is fairly well established, based on a number of different experiments, that increased formation of 16 α-OH-estrogens is associated with an increased risk of mammary cancer in mice and humans (Bradlow et al., 1986, 1995; Fishman et al., 1995).

We want to stress that the above summations have not been confirmed in other types of experiments and that they are not universally accepted. Also, other groups have demonstrated that increased urinary excretion of catecholestrogens, but not 16 α-OH-estrogens, can be correlated with an increased risk for nonfamilial breast cancer (Lemon et al., 1992). The same outcome was reached in a Finnish study where catecholestrogens dominated compared with 16 α-OH-estrogens in the population at high risk to develop breast cancer (Adlercreutz et al., 1994a,b). Finally, 16 α-OH-estrone and 16 α-OH-estradiol are quite weak carcinogens in the hamster kidney tumor model (Li and Li, 1987; Seegers et al., 1989; Li et al., 1995). Furthermore, during pregnancy, women excrete large amounts of 16 α-OH-estrogens, but full-term pregnancy does not increase the risk of breast cancer (Merrill, 1958; MacMahon et al., 1970; Fotsis, 1987; Yuan et al., 1988;Henderson et al., 1996). Our conclusion is that there is not sufficiently strong evidence to conclude that 16 α-OH-estrogens are carcinogenic but also that their harmful effects cannot be excluded. It seems that they do not possess any beneficial effects on estrogen-dependent cancers.

The intention of this discussion is to demonstrate that there is a possibility that inhibition of COMT by new drugs may seriously interfere with the metabolism of catecholestrogens. However, to the best of our knowledge, no studies have been conducted on this matter.

6. Behavior.

Generally, when administered alone, COMT inhibitors have virtually no effect on the motor behavior of rodents (Maj et al., 1990; Männistö et al., 1992b;Männistö, 1998). However, a compound that interferes with adrenergic transmission would be predicted to have important cognition-enhancing effects via improved attention and motivation or secondarily via enhanced cholinergic functions (Levin et al., 1990;Kelland et al., 1993). In fact, we have found that COMT inhibitors affect several phases of learning in a simple passive avoidance paradigm (Khromova et al., 1997). In more sophisticated studies, spatial working memory (radial-arm maze) of intact rats was facilitated after the pretraining i.p. administration of tolcapone (10 mg/kg). Similarly, tolcapone improved the performance of senescent poor performers in a spatial memory task (linear arm maze). However, tolcapone was not able to counteract the performance deficits in rats whose memory had been impaired by scopolamine or bilateral lesions in the nucleus basalis magnocellularis (Liljequist et al., 1997).

In an electric brain self-stimulation model, tolcapone administered alone has shown antidepressant activity (Moreau et al., 1994). In our studies, however, levodopa also was needed to reveal the antidepressant-like effect of tolcapone in two other rat models of depression (Männistö et al., 1995). In addition, it is quite interesting that entacapone (30 mg/kg i.p., a dose that apparently penetrates the blood-brain barrier to some extent), administered with levodopa (100 mg/kg i.p.) induced a place preference in rats (Katajamäki et al., 1998).

Maj et al. (1990) described behavioral effects of tolcapone. It increased exploratory activity of rats at doses of 10 and 30 mg/kg p.o., and the hyperactivity and stereotypy induced by amphetamine and nomifensine were potentiated by tolcapone. Tolcapone slightly antagonized fluphenazine-induced catalepsy but did not protect against pimozide- or haloperidol-induced catalepsy. Also, Himori and Mishima (1994) found that pretreatment with tolcapone (30 mg/kg) slightly potentiated the antagonist effect of levodopa and benserazide on haloperidol-induced catalepsy in the mice administered 1-methyl-4-phenylpyridium intracerebroventricularly. Very high doses of 3-OMD (800 mg/kg) attenuated this effect of tolcapone.

The antiakinetic action of a subthreshold dose of levodopa (5 mg/kg) in the MPTP-treated mice was potentiated by small doses of tolcapone (1 and 3 mg/kg). The same was true with two MAO inhibitors: pargyline (5 mg/kg) and selegiline (l-deprenyl; 1, 5 and 10 mg/kg). When given together, tolcapone and pargyline increased both the peak effect and the duration of the antiakinetic action. In the case of tolcapone plus l-deprenyl, only the duration of action was prolonged (Fredriksson and Archer, 1995).

Entacapone potentiated the turning behavior induced by levodopa and carbidopa in rats with unilateral nigral lesions (Etemadzadeh et al., 1989). Nitecapone resembled entacapone in this respect (Männistö et al., 1988). Comparative studies have confirmed that inhibition of COMT outside the brain (e.g., after entacapone and nitecapone) contributed nearly equally to the levodopa-induced turning behavior as that occurring also within the brain (i.e., after tolcapone; Törnwall and Männistö, 1993). We were not able to show any positive interaction between entacapone (3 mg/kg) and clorgyline (3 mg/kg) on the levodopa/carbidopa-induced turning behavior (Törnwall and Männistö, 1993). Entacapone potentiated the behavioral effects of levodopa also in common marmosets (Smith et al., 1997).

When given with levodopa and benserazide, tolcapone potentiated the increase in motor activity plus the antihypothermic effect and anticataleptic activity of levodopa or levodopa plus pargyline (Maj et al., 1990). Tolcapone potentiated levodopa/carbidopa-induced turning behavior at doses of 3 to 30 mg/kg (Törnwall and Männistö, 1993).

The effects of tolcapone and MAO inhibitors, separately and together, on levodopa-induced turning were thoroughly studied by Heeringa et al. (1997). There was a significant potentiation of levodopa-induced (10 mg/kg levodopa, 15 mg/kg benserazide) contralateral turning by tolcapone (30 mg/kg) or an MAO-A inhibitor, Ro 41-1049, but not with an MAO-B inhibitor, Ro 19-6327. Pretreatment with tolcapone and either of the MAO inhibitors further potentiated the levodopa-induced turning. The blockade of three dopamine-metabolizing enzymes (COMT, MAO-A, MAO-B) at the same time, leaving only conjugation reactions intact, most effectively prolonged the turnings.

In addition, a vinylphenylketone-type COMT inhibitor (QO IIR) was able to potentiate the reversal of reserpine-induced akinesia by levodopa/carbidopa in rats at a dose range of 7.5 to 30 mg/kg i.p. and catalepsy and hypothermia in mice at 30 mg/kg i.p., as did the reference compounds nitecapone and Ro 41-0960 at 30 mg/kg (Rivas et al., 1999). CGP 28104 was as effective as entacapone and tolcapone in potentiating the turning behavior induced by levodopa and carbidopa (Törnwall and Männistö, 1993).

7. S-Adenosyl-l-Methionine-Saving Effect of COMT Inhibitors.

It is well known that the levodopa therapy in PD patients depletes their body levels of AdoMet levels, and this depletion also occurs in the brain (Benson et al., 1993). This can be explained by enhanced O-methylation of the large doses ofl-dopa present, which consumes the methyl groups of AdoMet. Long-term levodopa treatment leads to compensatory enhancement of the synthesis of AdoMet by increasing the activity of methionine adenosyl transferase (Benson et al., 1993). In turn, when high doses of exogenous AdoMet were administered, they depleted nigrostriatal and forebrain tyrosine hydroxylase and brain dopamine (Charlton, 1997). When the COMT-induced O-methylation was effectively inhibited by first-generation COMT inhibitors (like tropolone but not pyrogallol; Waldmeier and Feldtrauer, 1987) and second-generation compounds (like tolcapone; Da Prada et al., 1994;Miller et al., 1997; Yassin et al., 1998), AdoMet levels in the brain were increased. Homocysteine levels were restored by tolcapone (Da Prada et al., 1994; Miller et al., 1997). Tolcapone enhanced the activity of methionine adenosyl transferase (Yassin et al., 1998). Even CGP 28014, an atypical inhibitor of O-methylation that does not inhibit the COMT enzyme itself, elevated AdoMet levels in the brain (Waldmeier et al., 1990a). Interestingly, MAO inhibition appeared to have just the opposite effect as that observed after COMT inhibition (Yassin et al., 1998).

Enhanced AdoMet levels in the brain during tolcapone therapy may explain the antidepressive (Moreau et al., 1994; Männistöet al., 1995) and cognition-improving (Khromova et al., 1995, 1997) effects of COMT inhibitors. Some studies have shown that the quality of life is increased in PD patients receiving COMT inhibitor therapy (Welsh et al., 1995; Baas et al., 1997; Waters et al., 1997). One report has also described improved cognition during tolcapone therapy (Gasparini et al., 1997). In fact, these findings fit well to the animal data demonstrating both antidepressive and cognition-improving properties of COMT inhibitors (Moreau et al., 1994;Männistö et al., 1995; Khromova et al., 1997; Liljequist et al., 1997).

8. Other Effects of COMT Inhibitors.

The nitrocatechols, of which nitecapone and entacapone have been studied in detail, are quite effective antioxidants (Suzuki et al., 1992), nitric oxide radical scavengers (Marcocci et al., 1994), and iron chelators (Haramaki et al., 1995; Orama et al., 1997), and they can protect cells from lipid peroxidation (Haramaki et al., 1995). Nitecapone has also been shown to prevent ischemia-reperfusion injury in experimental heart surgery in rats (Haramaki et al., 1995). In all these studies, the concentrations used (high micromolar or even millimolar) were very much higher than those needed to inhibit COMT. These actions do not appear to be related to COMT inhibition.

We have found that the repeated administration of 3 mg/kg tolcapone, when started before toxin administration, partially restored the memory deficit induced by bilateral infusions of a cholinotoxin, acetylcholine mustard, to the basal magnocellular nuclei of Meynert (Khromova et al., 1995). It is interesting that tolcapone (at 1 or 100 nM), like selegiline (at 1 nM to 10 μM), can protect aggregation cultures of rat brain exposed to a combination of anoxia and hypoglycemia for 30 min. Tolcapone (10 μM) was itself toxic. The beneficial effect was seen after only 1 day in culture, not at 2- or 4-day after ischemia (Ekblom et al., 1998).

Nitecapone increased bicarbonate secretion from rat and human duodenum after both i.v. and intraluminal administration (Flemström et al., 1993; Knutson et al., 1993; Flemström and Säfsten, 1994). High concentrations of nitecapone (maximum effect at 225 μM) increased synthesis and secretion of gastric sulfomucin (Slomiany et al., 1993). Although these findings point to some gastroprotective properties, nitecapone has not been found to be of any use as an antiulcer therapy in humans.

The mechanism of tolcapone-induced dose-dependent diarrhea has been studied in conscious dogs bearing 40-cm jejunal segment loops. Buffered physiological solution was perfused, and the secretions were analyzed at 15-min intervals. Acute oral administration of tolcapone (10 or 30 mg/kg) was well tolerated but induced a dose-dependent efflux of intestinal fluid, sodium, potassium, chloride, and bicarbonate (Larsen et al., 1998). A similar enhancement of bicarbonate excretion has been seen in gastric mucosa studies (Flemström et al., 1993; Knutson et al., 1993; Flemström and Säfsten, 1994). The coadministration of levodopa and benserazide accelerated the onset of tolcapone-induced secretion. No diarrhea developed in animals in these studies, nor did the treatments cause any signs of mucosal damage. The administration of tolcapone or the drug combinations for 7 days reduced secretory responses, demonstrating that tolerance did develop (Larsen et al., 1998). The importance of these findings to the pathogenesis of diarrhea remained open, but it cannot be excluded that the increased secretion may induce diarrhea in some susceptible patients. It is somewhat puzzling that dopamine itself has been reported to have an antisecretory effect in the rat jejunum preparation (stripped epithelial sheets). This action was mediated through α₂-adrenergic receptors (Vieira-Coelho and Soares-da-Silva, 1998).

Nitrocatechols may inhibit oxidative phosphorylation and uncouple mitochondrial energy production. One study compared entacapone and tolcapone; it was reported that tolcapone was severalfold more effective as an uncoupler than entacapone. The effective concentrations of tolcapone were in the same range as those achieved in plasma during tolcapone therapy (Nissinen et al., 1997). The relevance of this finding to the adverse effects of tolcapone (e.g., to the hepatic failure, rhabdomyolysis, and diarrhea) remains to be clarified.

9. Physicochemical Properties and Animal Pharmacokinetics.

Although the COMT-inhibitingK _i and IC₅₀values of nitrocatechols are in the low nanomolar range in vitro, doses as high as 10,000 nM/kg are needed to achieve adequate COMT inhibition in vivo. Evidently, these compounds have poor general intracellular availability. There are very little published data on the physicochemical properties of the new COMT inhibitors. Nitrocatechols are weak acids, and the pK _a values of both entacapone and tolcapone are 4.5. The water solubility of both compounds is low, less than 0.005% (<0.05 μg/ml); it is particularly poor in acidic milieu and improved considerably at pH 7.4 (entacapone, 7 mg/ml). The solubility in organic solutions and alcohols is substantially better. The log P (octanol/0.1 N HCl) of entacapone is 2.01; that of tolcapone is 2.6, indicating the better lipid solubility of the latter compound (Roche, 1997; P. T. Männistö and S. Kaakkola, unpublished data).

There is only restricted information about the actual brain and tissue penetration of the nitrocatechols. For instance, Dingemanse (1997)mentions unpublished observations that the brain extraction ratio of entacapone is 3% and is not dependent on the concentration in plasma. In contrast, he claims that tolcapone has a concentration-dependent (5–100 μg/l) brain penetration of 24 to 46% but does not provide any experimental details to amplify these statements. These values are extremely high compared with our unpublished data generated after the i.v. injection of varying doses of entacapone and tolcapone. The brains were made blood free by saline perfusion before collection. Our brain/plasma ratios were from less than 0.1 to 0.27% for entacapone and from 0.6 to 1% for tolcapone during the first 60 min after i.v. injection of the drugs (P. T. Männistö and S. Kaakkola, unpublished data, 1995). Both experiments agree on one point: the brain penetration of tolcapone is severalfold higher than that of entacapone.

Studies into the pharmacokinetics of tolcapone in rats have been published (Funaki et al., 1994, 1995). After 10 mg/kg i.v., theT _1/2 is less than 60 min, the AUC is 21.8 μg × h/ml, the total clearance is 7.84 ml/min × kg, and the V _D is 0.291 liters/kg. Oral doses of 20 and 40 mg/kg led to a slightly longerT _1/2. The maximum drug concentration in plasma (C _max) values were 15.9 and 20.2 μg/ml and the AUC values were 21.1 and 49.0 μg × h/ml after 20 and 40 mg/kg, respectively. It can be calculated that the oral bioavailability of tolcapone in the rat is 48 to 56%.

Data on entacapone rat kinetics in rats have not been published, but in general, the kinetic values after i.v. administration are close to those of tolcapone. However, after oral administration, the plasma entacapone levels are lower than those obtained with tolcapone. The oral bioavailability appears to be less than 20% in the rat (P. T. Männistö and S. Kaakkola, unpublished data, 1995).

Nitrocatechols are extensively metabolized, and only a small fraction is recovered intact in the urine. The metabolic profile is described in more detail in relation to the human pharmacokinetics.

The reason for the low oral bioavailability of nitrocatechols is still unknown. Their poor water solubility could indicate poor absorption, but also their abundant metabolism may start during the first pass. It seems likely that both factors contribute to their incomplete oral bioavailability.

10. Toxicity.

A full preclinical toxicology program has been conducted with entacapone and tolcapone; both drugs have recently been marketed throughout the world. A discussion on human toxicology appears in the clinical section of this review (see Safety). The acute toxicity of nitrocatechols is generally low (Borgulya et al., 1989). The acute i.p. LD₅₀ value of Ro 41-0960, a structural analog of tolcapone, in mice is about 100 mg/kg, whereas that of nitecapone and entacapone is about 500 mg/kg (Törnwall and Männistö, 1991). Levodopa and carbidopa treatments do not significantly enhance the acute toxicity of COMT inhibitors. Oral LD₅₀ values for tolcapone are 1600 to 1800 mg/kg in mice and greater than 2 g/kg in rats (Borgulya et al., 1991). The same is true for entacapone and nitecapone. Because their effective doses are in the range of 3 to 30 mg/kg, the therapeutic index for oral administration in rodents is more than 50. The lethal threshold for tolcapone in rats and dogs was more than 100 μg/ml (Roche, 1997). These values can be compared with the approximate therapeutic plasma levels of 3 to 6 μg/ml attained after 100 and 200 mg of tolcapone delivered in three doses each day.

Some toxicity emerged in long-term toxicity and carcinogenicity studies after fairly high doses of tolcapone (Roche, 1997). Moderate epithelial hyperplasia of the nonglandular part of the rodent stomach (not present in humans) was seen in rats and mice. In the same studies, 35% of rats developed renal epithelial tumors, depending on the dose. The safety margin of this phenomenon was about 10-fold above the therapeutic plasma concentrations of tolcapone. Finally, an increased incidence of uterine adenocarcinomas was seen at the 250 mg/kg/day dosage level. This finding may be related to the fact that dopamine agonists produced uterine tumors that are specific to rats. In addition, the dopamine-prolactin-estrogen axis may be disturbed. Catecholestrogens are also very good substrates of COMT, and a blockade of their metabolism will lead to accumulation (Ball et al., 1972; Ball and Knuppen, 1980; see Estrogen Metabolism and Role of COMT and COMT Inhibitors). It is worth noting that the liver was not a target of tolcapone toxicity in rodents.

We are not aware of the detailed results of the long-term toxicity studies of entacapone. However, genotoxicity and carcinogenicity studies have not revealed any serious toxicity. Some instances of anemia has been noted during repeated administration, probably due to iron chelation by entacapone. In reproduction toxicity studies in rabbits, there was some retardation of fetal growth and bone development (Orion, 1998).

11. Conclusions from Animal Studies.

The nitrocatechol-type COMT inhibitors alter l-dopa metabolism and potentiate the action of levodopa plus DDC inhibitors much more effectively than the first-generation COMT inhibitors. Some of the new compounds (entacapone, nitecapone) hardly penetrate the blood-brain barrier but still significantly increase striatal dopamine levels and potentiate the behavioral effects of levodopa in the same way as the brain-penetrating compounds (tolcapone, Ro 41-0960).

The mechanism of action of CGP 28014 is not known. Because it or its known metabolite do not directly inhibit COMT, it might inhibit the transfer of the COMT substrates to the enzyme (i.e., uptake₂). However, in glioma cells, no such inhibition could be seen (M. Törnwall and P. T. M., unpublished results, 1993). A further possibility would be that CGP 28014 forms an unknown metabolite, and this is not formed in vitro, only in vivo. Nevertheless, CGP 28014 mimics the other COMT inhibitors in being an inhibitor of O-methylation, preferably that acting in the brain. It is also positive in alleviating the symptoms in animal model of PD.