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Inhibitory and Stimulatory Effects of Selective Serotonin Reuptake Inhibitors on Cytochrome P450 2D6-mediated Dopamine Formation from p-Tyramine

- PURPOSE: The effects of selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine and paroxetine on dopamine formation from p -tyramine, mediated by cytochrome P450 (CYP) 2D6.2 (Arg296Cys, Ser486Thr) and CYP2D6.10 (Pro34Ser, Ser486Thr), were compared with their effects on CYP2D6.1 (wild type)-mediated dopamine formation, to investigate the influence of a CYP2D6 polymorphism on neuroactive amine metabolism in the brain. METHODS: The Michaelis constants ( K m ) and maximal velocity ( V max ) values of dopamine formation mediated by CYP2D6.1, CYP2D6.2, and CYP2D6.10 (expressed in recombinant Escherichia coli ), and inhibition constants ( K i ) of the SSRIs toward dopamine formation catalyzed by the CYP2D6 variants were estimated. RESULTS: The K m values for CYP2D6.2 and CYP2D6.10 decreased at lower fluoxetine concentrations, while the V max values for all CYP2D6 variants increased, indicating that fluoxetine stimulated dopamine formation. Conversely, paroxetine competitively inhibited dopamine formation mediated by CYP2D6.1, CYP2D6.2, and CYP2D6.10 with K i values of 0.47, 1.33, and 31.3 µM, respectively. CONCLUSIONS: These results suggest that the inhibition/stimulation of CYP2D6-mediated dopamine formation by these SSRIs would be affected by CYP2D6 polymorphisms in the brain.


INTRODUCTION
Cytochrome P450s (P450s or CYPs) catalyze the oxidation of endogenous compounds, including steroid hormones and neuroactive amines, as well as a wide variety of exogenous drugs (1,2). CYP2D6 accounts for only 2-9% of all P450s present in human livers (3,4), but metabolizes approximately 20% of all therapeutic agents (5,6). It is also expressed in the brain (midbrain) (7), howbeit with unknown physiological and pharmacological functions, specifically in the human brain.
Tyramine is exogenously present in fermented foods such as cheese and wine, and endogenously present in the brain, especially in the basal ganglia and limbic system (18).
p-Tyramine, dopamine, fluoxetine hydrochloride, and paroxetine maleate were obtained from Tokyo Chemical Industry (Tokyo, Japan). All other reagents and organic solvents used were of the highest purity commercially available.

Determination of dopamine formation
Dopamine formation from p-tyramine in the presence or absence of SSRIs was determined as previously described (19,21,31). Briefly, the incubation mixture consisted of 10 nM CYP2D6 variants, 0.05-10 mM p-tyramine, 1 mM NADPH, 50 µl of water or 0.5-1000 µM SSRIs dissolved in water, and 100 mM potassium phosphate buffer (pH 7.4) in a final volume of 500 µl. After pre-incubation at 37°C for 3 min, the reaction was activated by adding NADPH, and the mixture was incubated at 37°C for 10 min. Dopamine concentrations in the mixtures were measured using high performance liquid chromatography (HPLC). Tosoh (Tokyo, Japan) TSK-gel ODS-120T (5 µm, 4.6×250 mm) analytical columns were used, and the fluorescence intensities were determined at an excitation wavelength of 280 nm and an emission wavelength of 340 nm (19,21,31). The linearity of the reaction with P450 concentrations and incubation times were confirmed for CYP2D6.1 and its variants in preliminary experiments. Unless stated otherwise, the inhibitory/stimulatory effects of SSRIs on CYP2D6.1-and CYP2D6.2-mediated dopamine formation were investigated at 0.1 mM substrate concentration, and 1 mM substrate concentration was used for CYP2D6.10-mediated reactions. These substrate concentrations were close to or <Km values previously reported (21,31).

DATA ANALYSES
All data were analyzed using the means of duplicate or triplicate reactions, and Km, Vmax, and Ki values and the standard deviations (S.D.) as indices of the precision of the calculated parameters were calculated from Michaelis-Menten kinetics using nonlinear least squares regression by means of MULTI (32). Statistical significance was determined by Student's t-test, and the significance level was set at p<0.05. JMP 5 software (SAS Institute Inc, Cary, NC, USA) was used for the statistical analysis.

DISCUSSION
Human CYP2D6 is expressed in the liver and brain, especially the midbrain (7). Previously we observed that dopamine formation from p-tyramine as well as progesterone hydroxylation were affected by CYP2D6 polymorphism (21,31). In addition, many of the reported kinetic parameters such as Km, Vmax, and Vmax/Km, of CYP2D6.2 and CYP2D6.10 were different from those of CYP2D6.1 (wild type) (9). Unlike reports on metabolic activities, only a few reports exist on the inhibitory effects of CYP2D6 polymorphism on the inhibition of CYP2D6mediated reactions induced by exogenous compounds such as antidepressants. We recently observed that the Ki values of quinidine (a typical strong inhibitor of CYP2D6 in vitro, as recommended in the guidance for drug interaction studies by US FDA (33), EMA (34), and Japanese PMDA (35)) and quinine (a potent inhibitor of the rat CYP2D subfamily, including rat brain CYP2D4 (36-39)) against CYP2D6.1 were lower than those against CYP2D6. 10. Furthermore, we previously demonstrated that psychotropic drugs such as imipramine, desipramine, fluoxetine, and mazindol, inhibited 21-hydroxylation of progesterone and/or allopregnanolone (a neuroactive steroid), mediated by CYP2D6 and CYP2D4. However, fluoxetine increased the Km and Vmax values of CYP2D6mediated progesterone 21-hydroxylation (40). Imipramine and desipramine inhibited dopamine formation from p-tyramine and the Ki values for CYP2D6.10 were higher than those for CYP2D6.1 (25). In this study, paroxetine inhibited dopamine formation with a higher Ki value for CYP2D6.10 compared with that for CYP2D6.1. These results suggested that the inhibition of CYP2D6 by various psychotropic drugs, including these antidepressants, would be affected by CYP2D6 polymorphism in the brain.
We have reported that steroid hormones such as progesterone and testosterone, stimulate the metabolism of CYP3A4 substrates; however, selection of the enzyme source (recombinant P450s or liver microsomes) can affect enzyme activation (41). More so, some researchers have demonstrated the activation of CYP3A4-mediated metabolic reactions, with no information on the detailed mechanism (40)(41)(42). This activation has similarly been demonstrated for other P450s such as CYP1A2, CYP2C8, CYP2C9, CYP2D6, and CYP3A7 (42). We previously observed that fluoxetine increased the Km and Vmax values for CYP2D6-mediated progesterone 21-hydroxylation (40), and that fluvoxamine and other SSRIs increased the Km and Vmax values for dopamine formation mediated by CYP2D6.1, CYP2D6.2, and CYP2D6.10, but decreased the Km value for CYP2D6.10 to the level for CYP2D6.1 and CYP2D6.2 (25). Fluoxetineinduced activation of CYP2D6-mediated progesterone 21-hydroxylation was the first finding in relation to CYP2D6 (40,42). In this study, fluoxetine also increased Km and Vmax values for dopamine formation mediated by CYP2D6.1 and its variants and decreased the Km value for CYP2D6.10 to similar levels as those of CYP2D6.1 and CYP2D6.2. These results suggest that the CYP2D6 activation pattern would be affected by CYP2D6 polymorphism. Apart from selective inhibition of serotonin reuptake, some SSRIs such as fluoxetine and fluvoxamine that are metabolized by CYP2D6, exhibit novel physiological actions via the activation of dopamine formation and 21-hydroxylation of neurosteroids, including progesterone and allopregnanolone, mediated by brain CYP2D6 (43). Interestingly, fluoxetine and fluvoxamine but not paroxetine have a trifluoromethylphenoxy group in their chemical structure (Fig. 1), suggesting that this functional moiety may be essential for activation. On the other hand, not only paroxetine but also fluoxetine were demonstrated to inhibit CYP2D6catalyzed sparteine oxidation in human liver microsomes (44,45), indicating that the activation/inhibition depends on the substrates. Further investigations like using three-dimensional structural analyses such as molecular docking simulation (46,47), should be conducted to determine the underlying reason.
In conclusion, this study suggests that unlike paroxetine, fluoxetine stimulate CYP2D6-mediated dopamine formation from p-tyramine, and CYP2D6 polymorphism affect the inhibitory/stimulatory potency of SSRIs. However, further clinical studies are required to confirm the therapeutic relevance of our findings.