Eur J Clin Pharmacol (2000) 56: 145±151
Ó Springer-Verlag 2000
PHARMACOKINETICS AND DISPOSITION
C. MartõÂ nez á G. Gervasini á J. A. G. AguÂndez J. A. Carrillo á S. I. Ramos á F. J. GarcõÂ a-Gamito L. Gallardo á J. BenõÂ tez
Modulation of midazolam 1-hydroxylation activity in vitro by neurotransmitters and precursors Received: 28 October 1999 / Accepted in revised form: 10 February 2000
Abstract Objective: The aim of this study was to ®nd whether endogenous substances could modulate CYP3A activity. There is evidence that CYP3A, a major phase-I xenobiotic metabolizing enzyme, is present in human brain but, at the present time, endogenous substrates for such an enzyme remain to be identi®ed. A possible linkage between the CYP2D6 enzyme and serotonergic transmission has been recently reported by our group. In the same manner, structurally related enzymes such as CYP3A could also be related to endogenous compounds. Methods: CYP3A activity was measured using the enzyme-speci®c substrate midazolam in human liver microsomes. Several neurotransmitters, precursors, and their metabolites, corresponding to three dierent metabolic routes, were assayed as putative modulators of CYP3A enzyme activity. These comprised serotonergic, catecolaminergic, and GABAergic transmitters and precursors. The inhibitory capacity of ketoconazole, a competitive inhibitor of CYP3A, was also analyzed for comparison. Results: The kinetic analysis of the midazolam 1-hydroxylase activity measured in microsomes from ®ve human liver samples indicated Km values (mean SD) of 5.8 4.9 lM, and Vmax values of 1.7 1.4 nmol min)1 per mg microsomal protein in all the samples used in the study. Of the 14 substances analyzed, adrenaline, serotonin, and 5-hydroxytriptofol were full inhibitors of CYP3A enzyme activity (Ki values of 42.3, 26.4, and
C. MartõÂ nez á G. Gervasini á J. A. G. AguÂndez á J. A. Carrillo S. I. Ramos á L. Gallardo á J. BenõÂ tez (&) Department of Pharmacology, Medical School, University of Extremadura, Avda. de Elvas s/n, E-06071 Badajoz, Spain e-mail:
[email protected] Tel.: +34-924-289458; Fax +34-924-271100 F. J. GarcõÂ a-Gamito Service of General Surgery, INSALUD Hospital Merida, Badajoz, Spain
43 lM, respectively). The remaining substances were weak inhibitors or had no inhibitory eect. Conclusion: Brain CYP3A activity could be modulated by some neurotransmitters and precursors. Key words CYP3A á Metabolism á Indoleamines
Introduction The human CYP3A subfamily of the cytochrome P450 is involved in the metabolism of a large number of drugs, xenobiotics, and endogenous substrates (Wrighton and Stevens 1992). Known drug substrates of CYP3A include nifedipine (Guengerich et al. 1986), midazolam (Kronbach et al. 1989), etoposide (Relling et al. 1994), lidocaine (Bargetzi et al. 1989), terfenadine (Yun et al. 1993), cocaine (LeDue et al. 1993), and cyclosporin (Combalbert et al. 1989). The human CYP3A subfamily is composed of at least three dierent genes: CYP3A4, CYP3A5, and CYP3A7 (Wrighton and Stevens 1992). The existence of CYP3A3, formerly believed to be an allelic variant almost identical to CYP3A4, has been questioned (Gorski et al. 1994; Kolars et al. 1994; Nelson et al. 1996; Janardan et al. 1996). CYP3A4 is predominantly located in liver and intestine, being the major CYP3A isoform in both tissues (De Waziers et al. 1990). This enzyme accounts for about 30% on average of the total cytochrome P450 present in adult human liver (Shimada et al. 1994). The CYP3A5 enzyme exhibits similar catalytic activity to CYP3A4. Recent studies have found CYP3A5 mRNA and low levels of protein in as many as 74% of the liver samples tested (Kolars et al. 1994; JounaõÈ di et al. 1996). This isozyme is also commonly expressed in small intestinal tissue, albeit at much lower levels that CYP3A4, and represents the major isoform in several extrahepatic tissues. CYP3A7 is the predominant human fetal liver CYP3A isoform (Wrighton and Stevens 1992; Nelson et al. 1993). Recent evidence indicates that some indoleamines could modulate CYP2D6 and CYP1A2 activities
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(MartõÂ nez et al. 1997; AguÂndez et al. 1998). This raised the possibility that other cytochrome P450 enzymes could also be modulated by endogenous compounds in a similar manner. CYP3A enzyme activity has been shown to be present in brain tissue. Farin and Omiecinsky (1993) detected CYP3A mRNA expressed in brain, with the highest levels present in the pons region. In the same way, CYP3A4 mRNA was found in both basal ganglia and frontal cortex, while CYP3A5 mRNA was identi®ed mainly in the midbrain (McFayden et al. 1998). Furthermore, several authors have suggested that CYP3A protein could be present in brain (Jayyosi et al. 1992; Bhamre et al. 1993; Kirches et al. 1999). The presence of the CYP3A enzyme in brain, along with the relevance of CYP3A in the metabolism of psychoactive drugs, raised the possibility that the role of endogenous modulatory substances of CYP3A enzyme activity in neural tissue could in¯uence drug response, as they could be responsible for drug metabolism modulation at the site of action of psychoactive drugs. In an attempt to elucidate the occurrence of such endogenous modulators, we have investigated whether several neurotransmitters, precursors, and metabolites could in¯uence CYP3A activity.
Materials and methods Chemicals Quinidine sulfate, furafylline, tryptophan, 5-hydroxytryptophan, serotonin, 5-hydroxyindoleacetic acid, 5-hydroxytryptophol, tryptamine, tyrosine, dopamine, noradrenaline, adrenaline, homovanillic acid, vanillylmandelic acid, glutamic acid, c-aminobutyric acid (GABA), and cytochrome c were from Sigma Chem. Co. (Madrid, Spain). Midazolam, 1¢-hydroxymidazolam, 4-hydroxymidazolam, and the internal standard diazepam were kindly supplied by Homan-La Roche (Basel, Switzerland). Ketoconazole was from ICN Biomedical Inc. (Ohio, USA). Reduced nicotinamide adenine dinucleotide phosphate (NADPH), glucose-6-phosphate, and glucose-6-phosphate dehydrogenase were purchased from Boehringer Mannheim (Barcelona, Spain). These and all other chemicals used in this study were of analytical grade. Water was ®ltered through a Milli Q water system (Millipore Corp., Bedford, Mass). Preparation of human liver microsomes Human liver samples were obtained from unrelated white Spanish patients who underwent laparotomy for pathologies not related to liver disease (e.g., cholelitiasis without cholestasis, nonhepatic abdominal tumors). The samples were ¯ash-frozen and stored at )80 °C until analysis. Informed consent was obtained from all patients. The protocol of the study was approved by the ethics committee of the Infanta Cristina University Hospital (Badajoz, Spain). The preparation of microsomal fractions and the measurement of protein concentration were performed as described elsewhere (GarcõÂ a-AguÂndez et al. 1990). Assay for CYP3A enzyme activity The CYP3A activity was measured using the enzyme-speci®c substrate midazolam (Gorski et al. 1994). Midazolam 1¢-hydroxylation was measured using the method described by Kronbach et al. (1989), with minor modi®cations. The incubations were carried out
in a ®nal volume of 200 ll which contained 5 mM MgCl2, 1 mM NADPH, and 100 mM sodium phosphate buer, pH 7.4. A NADPH-regenerating system was included in the reaction mix in order to maintain a constant NADPH concentration during all incubation time. For this, 1 mM glucose-6-phosphate and 0.4 U of glucose-6-phosphate dehydrogenase from yeast were added to the reaction mixture. The reaction was started by the addition of 1.5 mg microsomal protein and stopped after 30 min incubation at 37 °C by the addition of 200 ll ice-cold methanol-acetonitrile (62.5:37.5; v:v). The samples were then frozen for 10 min and centrifuged for 10 min in a microfuge at maximum speed. The supernatant (50 ll) was analyzed using high-pressure liquid chromatography (HPLC). The analysis of midazolam and its main metabolites, 1¢-hydroxy midazolam and 4-hydroxy midazolam, using HPLC was carried out as described by Carrillo et al. (1998). Unless speci®ed, all the measurements were done using low midazolam concentrations (0.5±20 lM). These concentrations were in the same range as those described in previous reports to test CYP3A activity in human liver microsomes (Gascon and Dayer 1991). All the measurements were done within incubation time and under linear conditions for microsomal protein concentration. All the assays included samples that were stopped at time 0, and blanks without microsomes and/or without the corresponding neurotransmitters or other inhibitors were assayed. Assay for adrenaline and serotonin metabolism The metabolism of adrenaline, serotonin, and 5-hydroxytryptophol (a serotonin metabolite) was studied according to Mills et al. (1991). The composition of the reaction mixtures and details of the incubation conditions are described elsewhere (MartõÂ nez et al. 1997). After completion of the incubation time, the reaction was stopped by addition of HClO4 up to a ®nal concentration of 5%, and the mixture was frozen for 10 min. After centrifugation, the samples were analyzed for parent drugs (adrenaline and serotonin) and corresponding metabolites (5-hydroxytryptophol, 5-hydroxyindoleacetic acid, 3,4-dihydroxyphenylethylene glycol, and 3methoxy-4-hydroxyphenyl glycol) by reverse-phase HPLC with ¯uorescence detection (Mills et al. 1991). Statistical analysis All the experiments were done at least in triplicate in dierent (three or more) human liver samples. All the results given are mean SD of three or more measurements done under identical conditions.
Results Characterization of the CYP3A enzyme activity in the samples used in the study The kinetic analysis of the midazolam 1¢-hydroxylase activity measured in microsomes from ®ve human liver samples indicated Km values (mean SD) of 5.8 4.9 lM (range 1.4±10 lM) and Vmax values of 1.7 1.4 nmol min)1 per mg microsomal protein (range 0.6±2.8 nmol min)1 per mg). Figure 1 shows a typical experiment. It should be pointed out that midazolam concentrations over 15 lM resulted in a decrease of the Vmax, as is shown in Fig. 1. This is a highly reproducible phenomenon that was observed in all the samples analyzed (n 5). All further analyses were performed under conditions that assured that such inhibition was not present (i.e., with low concentrations of midazolam).
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Fig. 1 Kinetic parameters of midazolam in human liver microsomes. Inset: double reciprocal plot of the data corresponding to midazolam concentrations less than 15 lM
tion of midazolam was ®xed at 10 lM. Table 1 shows the Imax, Ki, and Hill's coecient values for all substances analyzed. Three of the 14 substances analyzed were full inhibitors of midazolam 1¢-hydroxylase activity, two of them neurotransmitters, namely serotonin and adrenaline. Figure 2 and Figure 3 show typical inhibition experiments as obtained with serotonin and adrenaline, respectively. The serotonin metabolite 5-hydroxytryptophol also showed an inhibitory eect on CYP3A activity (Fig. 4). The rest of the substances analyzed were poor inhibitors of activity or did not have an inhibitory eect (Table 1). Incubation of the microsomes in the presence of increasing concentrations of serotonin resulted in a concentration-dependent inhibition of midazolam 1¢-hydroxylase activity characterized by an increase in the Km values, whereas the Vmax values had no major changes (Km values of 1.4, 6.2, and 12.9 lM for a serotonin concentration of 0, 30, and 140 lM, respec-
The inhibitory capacity of ketoconazole, a competitive inhibitor of CYP3A (Gascon and Dayer 1991), was also analyzed in all the samples used in the study. In all cases, the Imax value was 100% of the activity. The Ki ranged from 11 lM to 19 lM (mean SD 15 4.6 lM), and the Hill's coecient was close to the unit (1.1 0.4, range 0.8±1.4). All these results are consistent with those reported previously (Gascon and Dayer 1991; Back et al. 1989), thus indicating the adequacy and reproducibility of the analytical methods used. Eect of the neurotransmitters studied on CYP3A activity Three metabolic routes of neurotransmitters were analyzed as putative modulators of CYP3A activity. These were that of serotonergic, catecolaminergic, and GABAergic transmitters. The concentration of substances tested ranged from 0.1 lM to 500 lM. The concentraTable 1 Inhibitory eect of neurotransmitters, precursors and metabolites on human liver microsomal CYP3A activity, measured as midazolam 1¢-hydroxylation
Substance Full inhibitors Serotonin Adrenaline 5-Hydroxytryptophol Poor inhibitors Dopamine Tyrosine Tryptamine L-Tryptophan Noradrenaline Vanillylmandelic acid 5-Hydroxytryptophan 5-Hydroxyindoleacetic acid Homovanillic acid GABA Glutamic acid
Fig. 2 Inhibition of midazolam 1¢-hydroxylase by serotonin. The percentage of activity is in the absence of inhibitor. The inhibition parameters for the experiment shown are: Imax 100%; Ki 51 lM; Hill's coecient 1.4
Imax (% activity)
K0.5 (lM) (range; n = 5)
Hill's coecient (range; n = 5)
100 90 100
26.4 22.7 (2.9±52) 42.3 6.6 (28.3±50.1) 43 21 (19.7±67)
1.6 0.2 (1.1±1.8) 1.1 0.2 (0.8±1.3) 1.7 0.4 (1.4±1.9)
420 310 >500 >500 330 >500 380 >500 >500 >500 >500
± ± ± ± ± ± ± ± ± ± ±
55 73 0 0 77 0 83 0 0 0 0
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Fig. 3 Inhibition of midazolam 1¢-hydroxylase by adrenaline. The percentage of activity is in the absence of inhibitor. The inhibition parameters for the experiment shown are: Imax 100%; Ki 57 lM; Hill's coecient 1.3
tively, and Vmax values of 0.82, 2.1, and 0.2 nmol min)1 mg)1 for the same concentrations of transmitter). This is consistent with a competitive-type inhibition of the CYP3A activity. These ®ndings are consistent with those reported by our group on the competitive inhibition of serotonin on other cytochrome P450 enzymes, such as CYP2D6 and CYP1A2, with Ki values similar to those found for CYP3A in this study (MartõÂ nez et al. 1997; AguÂndez et al. 1998). Metabolism of serotonin and adrenaline in human liver microsomes The fact that serotonin competitively inhibits CYP3A activity, displaying Hill's values close to unit, is
Fig. 4 Inhibition of midazolam 1¢-hydroxylase by 5-hydroxytryptophol. The percentage of activity is in the absence of inhibitor. The inhibition parameters for the experiment shown are: Imax 100%, Ki 53 lM, Hill's coecient 2.1
compatible with serotonin being partly metabolized by CYP3A. We have analyzed the metabolism of serotonin in human liver microsomes after inhibition of monoamine oxidase (MAO) activity by the use of 2.5 mM pargyline. Such a concentration of pargyline was chosen after several experiments that proved that 2.5 mM pargyline inhibited MAO activity present in human liver microsomes, whereas it did not inhibit CYP3A or NADPH reductase activities (results not shown). Under these conditions, serotonin is metabolized in human liver microsomes by a low-anity activity, with a Km value of 480 56 lM, to an unidenti®ed product that co-eluted with 5-hydroxyindole acetic acid (AguÂndez et al. 1998). Such enzyme activity was not inhibited in the presence of the CYP3A-speci®c inhibitor ketoconazole at a concentration of 500 lM. In the same way, serotonin-metabolizing enzyme activity was not inhibited in the presence of the CYP2D6speci®c inhibitor quinidine 500 lM (not shown) or the CYP1A2-speci®c inhibitor furafylline at a concentration of 500 lM (AguÂndez et al. 1998). Under our experimental conditions, we could not evidence adrenaline metabolism in human liver microsomes.
Discussion The enzymes of the CYP3A subfamily are among the most abundant cytochrome P450 enzymes in man and are present at hepatic and extrahepatic sites. A large number of drugs and other xenobiotics and endogenous substrates are metabolized by these enzymes, and several drug interactions are a direct result of interactions with CYP3A enzymes (Spina et al. 1992; Kerr et al. 1994). We have used midazolam as a probe for CYP3A activity in human liver microsomes, as midazolam 1¢hydroxylation seems to be mediated almost exclusively by CYP3A isoforms (Kronbach et al. 1989). This benzodiazepine has been previously characterized as a speci®c substrate probe of CYP3A metabolism both in vitro and in vivo (Watkins et al. 1994; Wrighton and Ring 1994). Our results showed a decrease in Vmax with midazolam concentrations over 15 lM. This is consistent with previous studies in which diminishing reaction velocities at high substrate concentrations were observed (Gascon and Dayer 1991; von Moltke et al. 1996; Greenblatt et al. 1996). This pattern was previously described for in vitro formation of a-hydroxy-midazolam from midazolam (Kronbach et al. 1989) and was reported as consistent with Michaelis-Menten kinetics and uncompetitive substrate inhibition (Segel 1975). Studies in rat have shown that one full-length cDNA clone (Wang et al. 1996), designated as 3aH15, was isolated from male rat brain cDNA library. Clone 3aH15 was thus named CYP3A9. In human brain, mRNA corresponding to CYP1A1, CYP1A2, CYP2C, CYP2D6, CYP2E1, and CYP3A has been isolated (Farin and Omiecinski 1993; McFayden et al. 1998). Furthermore, a study by Jayyosi et al.
149
(1992) identi®ed an immunoreactive protein related to cytochrome P450p (CYP3A) in rat brain. In the same manner, Bhamre et al. (1993) puri®ed from the cerebral cortex of a single human brain a P450 protein that was reactive weakly with an antibody to human hepatic CYP3A4, although the speci®c isolated form was not clearly identi®ed. Moreover, in a study carried out by Kirches et al. (1999), antibodies to human liver CYP3A4 stained a subfraction of tumor cell cultures (gliobastomas and astrocytomas) and, to some extent, neurons in normal brain areas. All this evidence taken together raises the thought that CYP3A protein, and not only mRNA, could be present in brain. The regional expression levels of these CYPs suggests that some psychoactive drugs' metabolism could take place directly in brain tissue. The physiological role of CYP3A in human brain remains to be elucidated, and no endogenous substrates for such an enzyme have been identi®ed so far. However, a possible endogenous substrate for CYP2D6 has been recently identi®ed (MartõÂ nez et al. 1997). Due to the partial overlapping of substrate speci®cities that characterize the cytochrome P450 superfamily of enzymes, it is likely that other cytochrome P450 enzymes could metabolize neurotransmitters. In this study, we have shown that CYP3A does not play an important role in serotonin metabolism, but serotonin could in¯uence CYP3A activity. So far, a modulation of microsomal CYP2D6 (MartõÂ nez et al. 1997), CYP1A2 (AguÂndez et al. 1998), and CYP3A (this paper) enzyme activities by endogenous compounds has been shown. These endogenous substances show an isozyme-speci®c eect. Table 2 shows the eect of some neurotransmitters and related compounds in cytochrome P450 activities studied so far. In this regard, it is noteworthy that neurotransmitters, and not the precursors or metabolites, are the substances that most potently inhibit cytochrome P450 enzyme activities (Table 2). The occurrence of a similar inhibitory eect of serotonin in three cytochrome P450 enzymes with dierent substrate speci®cities raised the possibility that the inhibition could be partly due to interaction with the NADPH reductase activity coupled to CYP enzyme Table 2 Eect of neurotransmitters, and related compounds on cytochrome P450 enzyme activities. +++-Ki < 50 lM; ++-Ki 50±300 lM; +-Ki 300±500 lM; )Ki > 500 lM or no inhibitory eect Substance
CYP3Aa
CYP2D6b
CYP1A2c
Serotonin Tryptamine Adrenaline Noradrenaline Dopamine
+++ ) +++ ) )
+++ +++ +++ ++ +
+++ +++ ++ + )
a
This paper b MartõÂ nez et al.1997 c AguÂndez et al. 1998
activities. However, we have previously shown that the substances studied in this paper as possible inhibitors of CYP3A activity do not signi®cantly inhibit NADPH reductase activity at concentrations as high as 500 lM, with a single exception ± adrenaline at a concentration of 500 lM causes a partial inhibition of NADPH. However, at a concentration of 100 lM, adrenaline, which causes a strong inhibition on CYP3A activity (Table 1), does not inhibit NADPH reductase activity (AguÂndez et al. 1998). It is a relevant point whether the real concentrations of the substances shown as inhibitors of CYP3A activity in this study are high enough to cause a signi®cant inhibition of this enzyme in brain tissue. Values for serotonin plasma concentration ranging from 0.03 lM to 1.1 lM have been reported (Dalsgaard-Nielsen et al. 1982; Huang et al. 1996) and a serotonin concentration as high as 6 lM in rat brain homogenates has been published (Mosseau 1993). Values ranging from 0.17 nM to 0.3 nM for adrenaline plasma concentration have also been reported (Ensinger et al. 1992; Zadik et al. 1980). With regard to 5-hydroxytryptophol, values from human cerebrospinal ¯uid (CSF) (4.22 nM) (Beck et al. 1984a), plasma (0.9 nM) (Beck et al. 1984b), and human post-mortem brain (range 10.9±387 pmol/g) (Beck et al. 1984b) can be found in the literature. Taking into consideration the K0.5 values shown in this study, these concentrations, with the single exception of serotonin, would not be enough to cause a signi®cant inhibition of CYP3A activity. However, there are several limitations to establish the level of these substances in brain, as they are not homogeneously distributed in this tissue and wide inter-individual dierences exist (PerezCruet et al. 1974; Sedvall et al. 1980). Therefore, higher local brain concentrations of these substances should be expected. Further studies should focus on the role of endogenous compounds on the modulation of drug-metabolizing enzymes. This could be a major cause of inter-individual variability for local brain drug metabolism, and hence this mechanism could underlie variability in drug response. As CYP3A is involved in the metabolic pathways of many neuro- and psychoactive drugs, a local modulation of brain CYP3A activity could be very relevant for pharmacological response to these drugs. Acknowledgements This work has been partly supported by grants CICYT-SAF96-0006 from Comision Interministerial de Ciencia y TecnologõÂ a, UE97-0001 from SecretarõÂ a de Estado de Universidades, InvestigacioÂn y Desarrollo (Madrid, SPAIN), BMH4-CT960291 from European Union, PRI96060023 and PRI97C120 from Junta de Extremadura (Merida, Spain).
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