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Department of Pharmaceutics (J.Z.P, R.J.S.) and Department of Medicinal Chemistry (R.P.R.), College of Pharmacy, University of Minnesota, Minneapolis, Minnesota
(Received January 7, 2004; accepted April 23, 2004)
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; Van Vleet et al., 2002). CYP1A1 and CYP1A2 are highly homologous proteins with more than 80% amino acid similarity (Yabusaki et al., 1984). The substrate selectivity of these two enzymes often overlaps. CYP1A1 is expressed constitutively in extrahepatic tissues, such as small intestine and lungs (Traber et al., 1992). However, neither mRNA nor protein expression for CYP1A2 has been found in either small intestine or lungs. In liver, CYP1A2 is constitutively expressed and comprises approximately 5% of the total P450, whereas CYP1A1 expression is very low in the uninduced state.
Induction of enzyme expression is commonly used to study regulation and substrate selectivity. CYP1A1 is highly induced by polycyclic aromatic hydrocarbons such as 3-methylcholanthrene (3-MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin, as well as by the prototypical inducers ß-naphthoflavone and omeprazole. These compounds also induce CYP1A2 (Burke et al., 1977). Without induction, the amount of CYP1A1 protein in liver is very low; however, after pretreatment of rats with ß-naphthoflavone or 3-MC, the amount of CYP1A1 is actually higher than that of CYP1A2 in liver due to the greater inducibility of CYP1A1 (Burke et al., 1994).
Mechanism-based inactivation is a unique type of enzyme inhibition. By definition, a mechanism-based inactivator itself does not inactivate the enzyme, but it is metabolized by the enzyme to a reactive electrophilic intermediate which, without prior release from the active site, binds (most often covalently) to nucleophilic activesite amino acids resulting in a complete or partial loss of activity (Silverman, 1988).
There are several important clinical implications when a drug is a mechanism-based inactivator (suicide substrate) of P450. First of all, the drug concentration after multiple dosing will be much higher than what is expected from linear kinetics. Second, elevated drug concentrations may more likely lead to side effects and toxicity. Finally, if a patient is taking another drug metabolized by the same P450 enzyme, then a drug-drug interaction may be anticipated (Okuda et al., 1997).
Tacrine (1,2,3,4-tetrahydro-9-acridinamine, THA; Cognex) is a cholinesterase inhibitor that has been used in the treatment of patients with Alzheimer's disease and dementia of the Alzheimer type. It is eliminated from the body mainly by metabolism. Its metabolites, 1-hydroxytacrine (1HT), 2-hydroxytacrine (2HT), 4-hydroxytacrine (4HT), and 7-hydroxytacrine (7HT), are formed in mouse, rat, dog, and human microsomes, with only quantitative differences existing across species (Madden et al., 1995). In rats, 1HT and 2HT are the major metabolites and are further metabolized to dihydroxy metabolites or conjugated to form glucuronides. CYP1A1 and CYP1A2 were reported to be the isozymes primarily responsible for tacrine metabolism (Woolf et al., 1993; Spaldin et al., 1994).
In two previous studies conducted in our laboratory, THA showed nonlinear kinetics after consecutive doses measured by both blood microdialysis (Brundage, 1996) and direct blood sampling (Peng, 2002). In the latter study, THA (5 mg/kg) was given to rats intraduodenally, and 8 h later a second identical dose was given. Although THA, 1HT, and 2HT concentrations were close to or below the detection limit when the second dose was given, the THA second dose AUC was doubled compared with the first dose AUC, whereas the AUC 2nd/1st ratio for 2HT was 1.44, and for 1HT, it was 0.69. The pharmacokinetic results suggest that pre-exposure of THA may be inactivating CYP1A enzymes involved in first-pass metabolism. It has also been found in human subjects taking multiple doses of tacrine that steady-state tacrine concentrations were much higher than would be predicted from normal accumulation (Forsyth et al., 1989; Cutler et al., 1990; Johansson et al., 1996). The goal of the present study was to investigate the underlying mechanism of this phenomenon; particularly, to carry out in vitro enzyme inhibition studies and explore whether tacrine or its metabolite may be a mechanism-based inhibitor of CYP1A enzymes.
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Animals and Pretreatment. Male Sprague-Dawley rats (Harlan, Indianapolis, IN) weighing approximately 320 g were used for the studies. There were three rats each in control and induced groups. For the induced group, 80 mg/kg 3-MC in corn oil was given intraperitoneally daily for 3 days. For the control group, only corn oil was given. After the final injection, the animals were fasted for approximately 24 h until they were sacrificed. On the day of microsomal preparation, animals were sedated in a CO2 chamber and decapitated, prior to removal of the liver and intestine.
Preparation of Liver and Intestinal Microsomes. Liver microsomes were prepared as previously described (Nelson, 1998). Intestinal microsomes were prepared by a modification of the method of Bonkovsky et al. (1985) and Weiser (1973). Mesentery was separated from freshly harvested small intestine. Proximal and middle sections of the small intestine (approximately the first two-thirds of the small intestine) were excised and rinsed twice with ice-cold 0.01 M potassium phosphate buffer with 1.15% KCl (pH 7.4) to remove the contents in the lumen. Then, the intestine was filled with solution A (1.5 mM KCl + 96 mM NaCl + 27 mM sodium citrate + 8 mM KH2PO4 + 5.6 mM Na2HPO4 + 40 µg/ml PMSF) and incubated in a 37°C water bath for 15 min. After the incubation, the solution was discarded and filled with ice-cold solution B (phosphate-buffered saline + 1.5 mM EDTA + 0.5 mM dithiothreitol + 40 µg/ml PMSF). The lumen was wound around a middle finger and tapped gently against the finger. The upper villus cells were released into solution B during this process. The tapping and harvesting of cells in solution B were carried out three times for each small intestine, and the collected cells were pooled. The pooled solution was centrifuged at low speed (200 rpm, 
10g) for 5 min at 4°C on a Beckman GS-15R centrifuge (Beckman Coulter, Fullerton, CA). The supernatant was discarded, and approximately 20 ml of ice-cold solution C (5 mM histidine + 0.25 M sucrose + 0.5 mM EDTA + 40 µg/ml PMSF) was added into each centrifuge tube; then the tube was inverted twice. Following a second centrifugation, the supernatant was discarded, and the cells were resuspended in fresh ice-cold solution C (repeated twice). Then the cells in solution C were homogenized with a Pyrex glass Potter-Elvehjem homogenizer. The homogenate was centrifuged at 10,000g for 20 min at 4°C. The supernatant was then centrifuged at 100,000g for 65 min at 4°C. The microsomal pellet was resuspended in 0.2 mM EDTA/20% glycerol/80% 0.1 M phosphate buffer (pH 7.4), homogenized, and stored in cryovials at -80°C. The protein content of rat microsomes was measured using a BCA Protein Assay Kit (Pierce). Fraction V bovine serum albumin was used as the protein standard.
Inhibition Assays. All reactions were carried out in 96-well plates (Sarstedt, Inc., Newton, NC). The biological buffer system used was 0.1 M Tris buffer (pH 7.4 at 37°C). THA, ER, and NADPH solutions were freshly prepared. Microsomes (RIM-cont, RLM-MC, recombinant CYP1A1 or CYP1A2) were thawed on ice and diluted to desired concentrations. In primary mixture, 20 µl of THA of different concentrations and 30 µl of NADPH were preincubated at 37°C for 3 min. Then, 50 µl of microsomes were added and the mixture was incubated at 37°C for different durations. The final concentrations of THA, NADPH, and microsomes in 100 µl were different for each pool of microsomes (see the legends and captions for Figs. 1, 2, and 4 under Results). At the end of the incubation, 100 µl of Tris buffer were added to dilute the tacrine, and then 20 µl of diluted primary mixture were transferred into a separate well containing 180 µl of a prewarmed secondary mixture that consisted of 50 µl of ER (0.5 µM final concentration), 70 µl of 0.1 M Tris buffer, and 60 µl of NADPH (0.25 mM final concentration). Resorufin formation was detected at 37°C for 5 min in a Bio-Tek FL600 Microplate Fluorescence Reader (Bio-Tek Instruments, Winooski, VT) with an excitation wavelength of 530 nm and emission of 585 nm. ER at 0.5 µM was chosen because preliminary studies indicated that this was near the Km and that the production of resorufin was readily detectable (Peng, 2002). The control enzyme activity (without THA) was taken as 100% and microsomal incubations pretreated with THA were compared with the control. Six determinations were carried out for each THA concentration and incubation time combination. The rate of EROD activity was calculated based on a standard curve of fluorescence versus resorufin concentration. The mean and standard deviation (S.D.) of the percentage of remaining activity were reported.
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Effect of NADPH and Trapping Agent. GSH was used as a trapping agent. For sample 0 (control), the primary mixture consisted of 50 µl of Tris buffer; for sample 1, 10 µl of THA + 40 µl of Tris; for sample 2, 10 µl of THA + 10 µl of Tris + 30 µl of NADPH; for sample 3, 10 µl of THA + 10 µl of GSH + 30 µl of NADPH. All four samples were preincubated at 37°C for 3 min. Then, 50 µl of CYP1A2 were added. The final concentrations of THA, GSH, NADPH, and CYP1A2 were 5 µM, 2 mM, 250 µM, and 100 µg of protein/ml, respectively. After incubation at 37°C for 20 min, 100 µl of Tris buffer were added. Then 20 µl of primary mixture were transferred to pre-warmed 180 µl of secondary mixture with an experimental protocol identical to that described above. Six determinations for each sample were carried out.
Metabolism of THA by Recombinant Enzymes. All reactions were carried out in 1.5-ml polypropylene microcentrifuge tubes (Fisher Scientific Co., Pittsburgh, PA). To each tube, 50 µl of CYP1A1 (final concentration 80 µg of protein/ml in the 100-µl reaction mixture) and 20 µl of THA solution of a specific concentration (THA final concentration 5, 10, 25, 50, 100, 250, or 500 µM) were added and preincubated in a 37°C water bath for 2 min. NADPH (30 µl, final concentration 1 mM) was added to start the reaction. The reaction was carried out at 37°C for 40 min and terminated by adding 100 µl of ice-cold acetonitrile. Each tube was vortexed for 30 s and kept on ice for at least 15 min followed by centrifugation at 1500g for 10 min with an IEC Centra-4B centrifuge (International Equipment Co., USA). Then, 100 µl of supernatant was transferred to a clean microcentrifuge tube, diluted with 100 µl of a 10 mM sodium acetate buffer (pH 4), and finally transferred into injection vials. No internal standard was added into these samples. All determinations were in duplicate.
Metabolism of THA by CYP1A2 was carried out in the same way except that 50 µl of CYP1A2 (final concentration 100 µg of protein/ml) were added instead of CYP1A1, with an incubation time of 30 min.
HPLC Assay for THA Metabolites. The mobile phase consisted of 25% v/v acetonitrile and 75% v/v 10 mM sodium acetate buffer. The aqueous buffer was adjusted to pH 4.0 with glacial acetic acid and filtered before being mixed with acetonitrile. A Shimadzu LC-10AD HPLC pump (Shimadzu, Kyoto, Japan) was used to deliver the mobile phase. A Jasco FP-821 fluorescence detector Jasco (Tokyo, Japan) was used to detect peaks of interest with the excitation and emission wavelengths of 240 and 355 nm, respectively, at an attenuation of 1, gain x100, and slow response. HPLC samples were injected onto a reverse-phase IB-SIL 5CN, 150 x 4.6 mm column. For the detection of 2HT peaks, the flow rate was 0.78 ml/min and injection volume was 50 µl; for the detection of 1HT and 4HT, a flow rate of 0.5 ml/min and 20-µl injection volume were used for better separation of these two peaks.
For the standard curves, appropriate volumes of aqueous stock solutions of 1HT, 2HT, and 4HT were added to obtain the desired concentrations, and a volume of 10 mM sodium acetate buffer (pH 4) was added to make the total aqueous volume 750 µl. Then, 250 µl of acetonitrile were added. The standard curve was run twice, with one at flow rate of 0.78 ml/min and injection volume of 50 µl and the other at 0.5 ml/min and injection volume of 20 µl. Equally weighted linear least-squares regression of peak height as a function of the nominal standard concentration was performed with KaleidaGraph (Abelbeck/Synergy, Reading, PA).
Effect of THA Metabolites on CYP1A1 or CYP1A2 Activities. 1HT and 2HT were tested for their ability to inactivate CYP1A1/2 activity, with an experimental protocol similar to that described under Inhibition Assays. In the primary mixture, 20 µl of 1HT or 2HT in 0.1 M Tris buffer were added to obtain a final concentration of 100 µM in the primary mixture incubation. Incubation times varied from 0 to 10 min. The control enzyme activity (without 1HT/2HT) was taken as 100% and those with 1HT/2HT were compared with the control. Six determinations were carried out for each 1HT/2HT concentration and incubation time combination.
Data Analysis. In the in vitro inhibition studies, THA was observed to inhibit CYP1A2 activity in a time- and concentration-dependent manner, suggesting that tacrine may be a mechanism-based inhibitor of CYP1A2. Therefore, it would be inappropriate to use a simple Michaelis-Menten equation to analyze the data for THA metabolism studies. A modified Michaelis-Menten equation was derived and used to describe the amount of 1HT formed from tacrine catalyzed by recombinant CYP1A2 in the in vitro metabolism studies. Here we assumed that the 1-hydroxylation pathway is affected by enzyme inactivation as suggested by the data from the in vivo pharmacokinetic studies (Brundage, 1996; Peng, 2002).
Under the assumption that the loss of the amount of active enzyme (ENZact) follows first-order kinetics (mechanism-based inhibition) and kobs is the observed inactivation rate constant, then the rate of change of ENZact can be expressed as eq. 1,
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If ENZact versus t is plotted, a monoexponential curve will be observed. The area under the amount of active enzyme curve from time 0 to t is
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 | (3) |
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A time-averaged amount of active enzyme (ENZact,ave) from time 0 to t can be calculated from eq. 5,
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Under the assumption that enzyme inactivation affects Vmax but not Km, and Vmax is proportional to ENZact,ave, then the Michaelis-Menten equation can be modified and a time-averaged formation rate (Vave) can be written as follows,
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Vmax(0) is the maximal formation rate at time 0. The amount of metabolite formed from time 0 to t (Amt 0->t) is shown in eq. 7,
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Equation 7 was used to fit the amount of 1HT formed from tacrine catalyzed by recombinant CYP1A2 for 30 min (i.e., t = 30 min). JMP (Version 4.0.4, SAS Institute) was used for data fitting, with weight = 1.
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Recombinant enzymes CYP1A1 and CYP1A2 from BD Gentest were also used in the inhibition studies for confirmatory purposes and better estimation of inactivation parameters. Figure 2 shows the time- and concentration-dependent loss of recombinant CYP1A2 activity by THA. THA concentrations used in this study were much lower than those in the rat microsomal inhibition study. When there was no preincubation, enzyme activities were approximately 90% of the control. However, after 20 min of incubation, only 20% of CYP1A2 activity remained at the highest concentration of tacrine (25 µM). A monoexponential equation was used to analyze the data, and the observed inactivation rate constant, kobs, was estimated from the slopes of the lines in Fig. 2. Then, kobs versus tacrine concentration was plotted (Fig. 3) and the equation kobs = kinact x s/(Ki + s) was used in analyzing the data with KaleidaGraph (Version 3.09, Abelbeck/Synergy), with weight = 1. The mean (standard error) Ki was found to be 1.94 (0.33) µM and the maximal inactivation rate constant, kinact, was 0.091 (0.0041) min-1.
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When clone-expressed CYP1A1 was incubated with tacrine, concentration-dependent but time-independent inhibition of CYP1A1 activity was observed (Fig. 4), indicating that this process was reversible. The results from inhibition studies with the clone-expressed enzymes were consistent with those in rat liver/intestinal microsomes.
Effect of NADPH and Trapping Agent. To determine whether catalytic processing of the inhibitor was required for inactivation, the effect of NADPH was examined. In this study, a concentration of 5 µM tacrine and an incubation time of 20 min were chosen so that approximately 20% of the activity remained when all the components were included. The control (only CYP1A2 in the primary mixture) activity was taken as 100%. When tacrine was incubated with CYP1A2 but in the absence of NADPH, the enzyme activity remained almost completely intact (mean ± S.D., 91.4 ± 10.0% of the control). However, when 5 µM tacrine, CYP1A2, and NADPH were incubated for 20 min, only 21.9 ± 3.2% of enzyme activity remained, suggesting an absolute requirement for NADPH for inactivation. The presence of glutathione (2 mM), an exogenous nucleophile, did not have any effect on the inactivation (23.7 ± 3.9% activity remained), suggesting that the reactive intermediate was not released from the active site before the inactivation occurred.
Metabolism of THA by Recombinant Enzymes. The upper panel of Fig. 5 shows the formation of three metabolites at different concentrations of THA incubated with CYP1A1. The y-axis, formation rate (pmol/min/pmol P450), was plotted on a log scale so that all three metabolites could be included on the same plot. Under the in vitro condition, the 1HT formation rate was the highest, and it was approximately 10-fold higher than the 4HT formation rate and 100-fold higher than the 2HT formation rate. When THA was incubated with CYP1A2, again 1HT was the major metabolite, followed by 4HT, and the amount of 2HT formed was very low at all concentration levels of THA (Fig. 5 lower panel).
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Equation 7 was used to fit the amount of 1HT formed from tacrine catalyzed by recombinant CYP1A2, and convergence was reached in the default gradient. Figure 6 (upper panel) shows the predicted line and observed data points. The point estimate and standard error for each parameter are listed in the upper panel of Table 1.
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For comparison purposes, the same data set was also fit to Michaelis-Menten eq. 8. The point estimate and standard error for Vmax and Km are listed in the lower panel of Table 1.
 | (8) |
As shown in Fig. 6 (upper panel), eq. 7 describes the data well. However, when the unmodified Michaelis-Menten equation was used, although convergence was achieved, the model appears to poorly predict the data (Fig. 6, lower panel). The predicted line tends to overestimate the amount of 1HT formed in the middle THA concentration range and to underestimate it in the high THA concentration range.
Partition Ratio Estimation. Incubation of 25 µM tacrine with recombinant CYP1A2 for 20 min caused a loss of 78.4 ± 6.6% of enzyme activity (from inhibition assay). The initial enzyme content was 3.7 pmol (provided by BD Gentest); therefore, the loss of the amount of active enzyme during this 20-min incubation was 2.9 ± 0.2 pmol. From THA metabolism data, the amount of THA consumed during this 20 min was estimated to be 63.5 pmol (by summation of the amounts of 1HT, 2HT, and 4HT formed). Therefore, the partition ratio was estimated to be approximately 22. In other words, in every 23 turnovers, 1 leads to inactivation and 22 result in stable metabolite formation.
Effect of THA Metabolites on CYP1A1 or CYP1A2 Activities. To examine whether THA metabolites have any effect on the enzyme activity, 100 µM 1HT or 2HT was incubated with CYP1A1 or CYP1A2 in the presence of NADPH, and then the EROD activity was measured. With or without preincubation, 1HT or 2HT had little effect on either enzyme (Table 2).
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). In a study carried out by Woolf et al. (1993), it was found that the recovery of radioactivity in the supernatant fractions from postmicrosomal incubations containing radiolabeled THA and NADPH was lower than anticipated, suggesting that THA-derived radioactivity was associated with the protein precipitate. In the same study, the precipitated microsomal protein was resuspended and subjected to exhaustive extraction. The amount of THA-derived radioactivity bound to the final protein pellet was much higher than that in control incubation mixtures, and this phenomenon was also NADPH-dependent. Their results suggested that the binding was irreversible and the species that bound to microsomal protein was catalyzed from THA. These results are in good agreement with what was found in the current studies. However, it was not clear from Woolf's study (Woolf et al., 1993) the specific protein(s) that was covalently modified by tacrine. Woolf et al. (1993) also examined the effect of glutathione, and they found that GSH (5 mM) coincubation inhibited approximately half the irreversible binding of THA-derived radioactivity in 3-MC-induced rat liver preparations. Interestingly, we found that the trapping agent GSH did not have any effect on the inactivation, suggesting that the reactive intermediate was not released from the active site before the inactivation occurred. There may be some release of a reactive intermediate into the medium surrounding the enzyme in microsomes, but the covalent binding to the external periphery of the enzyme does not result in inactivation of CYP1A2. Several different enzyme sources were used in the current studies. Rat intestinal microsomes, either control or induced, contain CYP1A1. As shown in Fig. 1 (lower panel), the inhibitory effect of THA on this enzyme was concentration-dependent but not time-dependent, indicating that this process was reversible with a rapid equilibrium. In 3-MC-induced rat liver microsomes, both CYP1A1 and 1A2 are present, and thus a more complicated kinetic profile of inhibition was observed, consisting of both competitive inhibition of CYP1A1 and mechanism-based inactivation of CYP1A2. The time-dependent inactivation component was confirmed in incubations with recombinant CYP1A2 (Fig. 2).
Although time-dependent inactivation is a key characteristic of mechanism-based inhibition, it is not the sole criterion. In some cases, time-dependent inhibition could be caused by slow- and/or tight-binding or slow release of the product, such that the observed enzyme kinetic profile is reflective of covalent inactivation (Imperiali and Abeles, 1986; Stein et al., 1987). If indeed tacrine is a slow- and tight-binding inhibitor rather than a mechanism-based inactivator, the timing of the second dose would be important since there would be some amount of tacrine remaining from the first dose still bound to the enzyme active site.
The loss of CYP1A2 activity by metabolic inactivation would have greater impact on the hepatic extraction during first pass after oral administration compared with intravenous administration, since the liver is exposed to high initial concentrations via the portal vein. After intravenous administration, rapid distribution of tacrine will significantly reduce tacrine concentrations in the liver, and the large amount of liver CYP1A2 would be capable of eliminating tacrine efficiently from the body. The resulting effect on overall exposure (AUC) between initial and subsequent doses is therefore magnified with oral administration.
The exact mechanism by which tacrine is catalyzed into a reactive intermediate leading to CYP1A inactivation is not yet known. Since the metabolites (1HT and 2HT) are not mechanism-based inhibitors, CYP1A2 inactivation may occur during their formation. Assuming the inactivation is a result of C-hydroxylation, one possible mechanism may involve hydrogen atom abstraction by an iron-oxo species to produce a carbon-centered radical intermediate at the benzylic position (Fig. 7, Species A). Radical abstraction followed by oxygen rebound is thought to be a very fast reaction (<70 ps), but rearrangement of radical to produce other products or inactivation complexes has been demonstrated for several substrates (Newcomb et al., 2003). Shaik and coworkers have suggested that some of the data with radical clock substrates such as trans-2-methylphenyl-cyclopropane support the existence of two spin states of the iron-oxo species with the high spin state resulting in rearrangement (Kumar et al., 2004). Alternatively, Newcomb et al. (2003) have proposed the existence of two oxidants (iron-oxo and hydroperoxy-iron species). Insertion of OH+ into a C-H bond by a hydroperoxy-iron species with subsequent loss of water could produce a carbocation (Chandrasena et al., 2004) (Fig. 7, Species B). Cation-derived rearrangement products have been observed in P450 reaction with substrates such as methylcubane and norcarane (Newcomb et al., 2003). Rapid rearrangement of the carbocation or radical and subsequent covalent binding of the tacrine moiety would result in enzyme inactivation. An alternative inactivation mechanism may also occur that involves oxidation of the primary amine (hydroxylamine formation). One-electron transfer to the nitrogen to form a radical cation followed by rearrangement to an aminium cation and subsequent proton donation to form an aminyl radical has also been postulated as reactive intermediates capable of P450 inactivation (Sasaki et al., 2002). However, one might expect to observe inactivation with 1HT and 2HT as well as tacrine if P450-catalyzed attack at nitrogen predominates. The observed inactivation results (with only tacrine) could be explained if 1HT and 2HT were unable to return to the active site for N-oxygenation.
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A modified Michaelis-Menten equation with mechanism-based inhibition was derived. The data of 1HT formation in the in vitro metabolism studies were analyzed with this modified equation. Reasonable fit was obtained based on visual inspection of the fit (Fig. 6, upper panel) and a plot of residuals (data not shown), indicating that this equation is suitable to describe metabolism data when the substrate is a mechanism-based inhibitor of the enzyme. The Vmax(0) obtained from the fit to eq. 7 was 6.5 nmol/min/mg protein, a value that was much larger than the observed maximal formation rate. A time-averaged Vmax value of 0.37 nmol/min/mg protein was obtained when the data were subjected to nonlinear regression of the Michaelis-Menten equation. The values of Ki (32.1 µM) and kinact (0.52 min-1) obtained from THA metabolism data in recombinant CYP1A2 fit to the modified Michaelis-Menten equation were significantly different from those obtained in the inactivation studies with EROD as a probe (1.94 µM and 0.091 min-1, respectively). It seems that THA inactivates its own metabolism in a low-affinity and high-capacity manner, while inactivating EROD with a high affinity and low capacity. There are two possible explanations for these observations. First, the Ki value in the inactivation studies is reflective of mechanism-based inactivation as well as competitive inhibition of CYP1A2, since there is a residual amount of THA transferred from the primary mixture into the secondary mixture. In addition, the 1-hydroxylation pathway may reflect only a partial picture of enzyme inactivation. The exact reason for this discrepancy is not known.
In summary, tacrine was a rapid inactivator of CYP1A2 (0.091 min-1) with a partition ratio of 22. The absolute requirement for NADPH for inactivation confirms that catalytic processing of the inhibitor is required before inactivation can occur. The trapping agent GSH did not have any effect on CYP1A2 inactivation. Tacrine inhibited CYP1A1 in a concentration-dependent but time-independent manner, suggesting that this process was reversible. Tacrine metabolites 1HT and 2HT did not show any effect on CYP1A activity, eliminating the possibility of product inhibition. Microsomal metabolism studies with tacrine were also carried out, and the data were examined by nonlinear regression, using a modified Michaelis-Menten equation which considers mechanism-based inhibition. Although a reasonable fit was obtained, differences in individual parameter values were large between experiments.
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ABBREVIATIONS: P450, cytochrome P450; AUC, area under the curve; EROD, 7-ethoxyresorufin O-deethylation; 3-MC, 3-methylcholanthrene; THA, 1,2,3,4-tetrahydro-9-acridinamine (tacrine); HT, hydroxytacrine; ER, ethoxyresorufin; HPLC, high-performance liquid chromatography; PMSF, phenylmethylsulfonyl fluoride; RIM-cont, uninduced rat intestinal microsomes; RLM-MC, methylcholanthrene-induced rat liver microsomes; GSH, glutathione.
1 Current address: Abbott Laboratories, Clinical Pharmacokinetics (Dept. R4PK, Bldg. AP13A), 100 Abbott Park Road, Abbott Park, IL 60064-6104. 
Address correspondence to: Dr. Ronald J. Sawchuk, Department of Pharmaceutics, College of Pharmacy, University of Minnesota, Weaver-Densford Hall, 308 Harvard Street S.E., Minneapolis, MN 55455. E-mail: sawch001{at}umn.edu
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