Drug Metabolism and Disposition Fast Forward
First published on June 2, 2005; DOI: 10.1124/dmd.105.003772
0090-9556/05/3311-1717-1722$20.00
DMD 33:1717-1722, 2005
ROLE OF CYP2E1 IN DERAMCICLANE METABOLISM
Katalin Monostory,
Krisztina Köhalmy,
Eszter Hazai,
László Vereczkey, and
László Kóbori
Chemical Research Center, Hungarian Academy of Sciences (K.M., K.K., E.H., L.V.), Budapest, Hungary; and Transplantation and Surgery Clinic, Semmelweis University (L.K.), Budapest, Hungary
(Received January 19, 2005;
accepted May 27, 2005)
 |
Abstract
|
|---|
The aim of our study was to identify the form(s) of cytochrome P450 responsible for the metabolism of deramciclane, a new anxiolytic drug candidate. The main routes of biotransformation in hepatic microsomes were side chain modification (N-demethylation or total side chain cleavage) and hydroxylation at several points of the molecule. Although several cytochrome P450 forms were involved in the metabolism, the role of CYP2E1 should be emphasized, since it catalyzed almost all steps. Production of deramciclane metabolites was significantly inhibited by diethyl-dithiocarbamate and was elevated in liver microsomes of isoniazid-treated rats. Furthermore, cDNA-expressed rat CYP2E1 generated the metabolites formed by side chain modification and hydroxylation. Neither deramciclane nor its primary metabolite, N-desmethyl deramciclane were able to influence directly the activity of CYP2E1. However, during the biotransformation, one or more metabolites must have been formed which were potent inhibitors of CYP2E1.
Variability in drug response is often due to interindividual differences in drug metabolism. A patient's individual response to a drug depends on the activities of drug-metabolizing enzymes that determine the fate and elimination of the drug from the body. One of the reasons for variable drug metabolism is the polymorphic expression of these enzymes, mainly cytochromes P450 (P450s). Although poor metabolizers often have a low frequency in the population, being a poor metabolizer can cause severe side effects if a drug is metabolized through a polymorphic enzyme, particularly if it is the only pathway of the elimination (Tamási et al., 2003
). Another aspect of interindividual variation involves enzyme induction and inhibition (Rendic and Di Carlo, 1997; Maurel, 1998). Not only drugs, but smoking, alcohol consumption, or foodstuffs can act as inducers or inhibitors of drug-metabolizing enzymes. Environmental influences (including drug interactions) and clinical consequences can be predicted from the identification of the enzyme(s) responsible for the metabolic steps. The procedures to determine the P450 form(s) involved in drug metabolism can use P450 form-selective inhibitors as well as microsomes containing cDNA-expressed P450 proteins (Ono et al., 1996).
Deramciclane, developed by EGIS Pharmaceuticals Ltd. (Budapest, Hungary), is a new non-benzodiazepine-type anxiolytic agent that is more effective than diazepam or chlordiazepoxide (Gacsályi et al., 1988; Berényi et al., 1990). Its anticonvulsant activity is exerted via inhibition of synaptosomal 
-aminobutyric acid uptake (Kovács et al., 1989). Our previous work (Monostory et al., 2005) identified the structures of the metabolites formed during the biotransformation of deramciclane in rat, mouse, rabbit, dog, and human primary hepatocytes. Deramciclane underwent side chain modification and oxidation at several positions of the molecule. The side chain modification led to the formation of N-desmethyl deramciclane and phenylborneol. The oxidation of deramciclane resulted in several hydroxy-, carboxy-, and N-oxide derivatives. The hydroxylation took place at primary or secondary carbon atoms of the camphor ring as well as at the side chain. Furthermore, the side chain-modified metabolites were also oxidized to hydroxy- or carboxy-derivatives. Our present work has made an attempt to identify the P450 form(s) participating in deramciclane metabolism.
|
Materials and Methods
|
|---|
Chemicals. Deramciclane and the presumable metabolites N-desmethyl deramciclane, 9-hydroxy-deramciclane, N-desmethyl 9-hydroxy-deramciclane, and phenylborneol were provided by EGIS Pharmaceuticals Ltd. (Budapest, Hungary). [14C]Deramciclane labeled on the phenyl-ring (1.3394 GBq/mmol) was synthesized at the Chemical Research Center of the Hungarian Academy of Sciences (Budapest, Hungary). D-Glucose 6-phosphate, glucose-6-phosphate dehydrogenase, diethyl-dithiocarbamate, dichloromethane, acetonitrile, perchloric acid, and ethyl acetate were purchased from Merck (Darmstadt, Germany). Triethylamine was obtained from Loba Feinchemie (Fischamend, Austria). 
-Naphthoflavone, quinidine, troleandomycin, sulfaphenazole, isoniazid, and bovine serum albumin were the products of Sigma Chemie GmbH (Deisenhofen, Germany). Chlorzoxazone was purchased from Aldrich Chemical Co. (Steinheim, Germany). 6-Hydroxy-chlorzoxazone was obtained from Ultrafine Chemicals (Manchester, UK). Methanol and butanol were obtained from ChemoLab (Budapest, Hungary) as chromatography-grade products. All other chemicals were obtained from Reanal (Budapest, Hungary).
Microsomes. Male Wistar rats (Charles River, Budapest, Hungary), weighing 150 to 200 g, were pretreated with isoniazid (50 mg/kg i.p.) for 3 consecutive days. Controls were administered the same volume of saline. After an overnight fast, the rats were killed by decapitation, and the livers were excised. Human liver tissues from kidney transplant donors were obtained from Transplantation and Surgery Clinic, Semmelweis University (Budapest, Hungary). The permission of the Local Research Ethics Committee was obtained to use human tissues. Liver microsomes were prepared as described by van der Hoeven and Coon (1974). Protein concentrations were determined by the method of Lowry et al. (1951) using bovine serum albumin as the standard. Microsomes from human lymphoblastoid cells expressing rat CYP2E1 and NADPH-P450 reductase were purchased from BD Gentest (Woburn, MA).
Inhibition of Deramciclane Biotransformation. The incubation mixture (in a final volume of 1 ml) consisted of 0.1 M Tris-HCl buffer (pH 7.4), NADPH-generating system (0.4 mM NADP, 4 mM glucose 6-phosphate, 2 mM MgCl2, 1 unit/ml glucose-6-phosphate dehydrogenase), 1.5 mg/ml microsomal protein, and 50 µM[14C]deramciclane (266.4 MBq/mmol). In inhibition studies, selective P450 inhibitors (10 µM -naphthoflavone, 10 µM sulfaphenazole, 2 µM quinidine, 100 µM diethyl-dithiocarbamate, or 50 µM troleandomycin) were also added to the incubation mixtures (Miners et al., 1988; Kobayashi et al., 1989; Guengerich et al., 1991; Roos et al., 1993; Siess et al., 1995; Maurel, 1996). Diethyl-dithiocarbamate and troleandomycin were preincubated with microsomes and NADPH-generating system for 15 min. Deramciclane biotransformation was carried out aerobically at 37°C for 30 min. Deramciclane and its metabolites were extracted from the aqueous phase three times in 5-ml dichloromethane, the organic phase was evaporated to dryness, and the residue was dissolved in 0.1 ml of methanol. After extraction, the total radioactivity of organic and aqueous phases was determined by liquid scintillation techniques (LKB 1217 Rackbeta type; PerkinElmer Wallac, Turku, Finland). Samples (10 µl) of the organic phases were analyzed by thin-layer chromatography on 0.2-mm-thick DC-Alufolien Kieselgel 60 F254 plates (Merck) using ethyl acetate/triethylamine (25:1, v/v) solvent delivery system. The amounts of deramciclane metabolites were calculated on the basis of digital autoradiography by a Berthold digital autoradiograph type LB-287 (EG&G; Berthold Technologies, Bad Wildbad, Germany). The metabolites were identified on the basis of their Rf values on thin layer compared with authentic standards or on the basis of mass spectrometric analysis as previously described (Monostory et al., 2005). The rates of metabolite formation in the presence or absence of inhibitors were determined.
Assays with Liver Microsomes and cDNA-Expressed CYP2E1. Microsomes from the liver of control and isoniazid-treated rats and from a human lymphoblastoid cell line expressing rat CYP2E1 were used in the study to analyze the biotransformation rates of [14C]deramciclane and 14C-N-desmethyl deramciclane. The parameters of the assays were the same as those in the inhibition studies, except for HPLC analyses. The separation was performed on a Hypersil 150 x 4 mm C18, 3-µm column (BST, Budapest, Hungary) with gradient elution. The mobile phases consisted of acetonitrile/water/perchloric acid, 35:65:0.35 (v/v/v) at 0 to 4 min and acetonitrile/water/perchloric acid, 50:50:0.35 (v/v/v) at 4.5 to 55 min. The pH of the mobile phases was adjusted to 3.0 by the addition of triethylamine. The column was eluted at a rate of 0.8 ml/min at room temperature. The effluent was analyzed on the basis of radioactivity using a Packard Radiomatic 150TR flow scintillation analyzer (Canberra Industries, Meriden, CT).
Monooxygenase Assays. Chlorzoxazone 6-hydroxylase activity of CYP2E1 was determined by the method of Peter et al. (1990). Hydroxylation of p-nitrophenol was measured according to the method of Reinke and Moyer (1985). In experiments performed to determine the inhibitory effect of deramciclane or N-desmethyl deramciclane on CYP2E1, the compounds were preincubated with microsomal fractions and NADPH-regenerating system for 0, 15, 30, and 60 min before fresh NADPH-regenerating system and chlorzoxazone (50 µM) or p-nitrophenol (50 µM) were added. The inhibition constant (Ki) for chlorzoxazone 6-hydroxylation was determined after preincubation with different concentrations of deramciclane (0.1–200 µM) for 30 min. Ki was calculated from Dixon plots of velocity–1 versus deramciclane concentration at various chlorzoxazone concentrations (2–200 µM). The apparent Ki was estimated from the intercept of the lines of the Dixon plots and expressed as mean ± standard deviation of the intercepts.
Estimation of Ka (Association Constant). Difference spectra were produced upon titration with chlorzoxazone (10–100 µM) or deramciclane (10–100 µM) of liver microsomes (1 mg/ml). A double reciprocal plot of the titration data (spectral changes–1 versus substrate concentration–1) yielded a Lineweaver-Burk plot with an intercept of substrate concentration–1 axis estimating Ka values of the compounds. Ka values were considered to be the reciprocal values of Ks (the equilibrium dissociation constant) and expressed as µM–1.
|
Results
|
|---|
Inhibition of Deramciclane Biotransformation. Thin-layer chromatography was first used to ascertain the full spectrum of metabolites formed during microsomal metabolism of deramciclane. Six metabolites generated by rat liver microsomes were separated on thin-layer plates (Fig. 1). Deramciclane underwent side chain modification, which led to the production of N-desmethyl deramciclane (M4, the main metabolite) and phenylborneol (M8). Hydroxylation of deramciclane resulted in the formation of 9-hydroxy-deramciclane (M5) and hydroxy-deramciclane II (M6). N-Desmethyl 9-hydroxyderamciclane (M2) and N-desmethyl hydroxy-deramciclane II (M3), the corresponding hydroxy-derivatives of M4, were also detected. The rate of total deramciclane metabolism by rat liver microsomes was 0.453 ± 0.136 nmol/mg protein/min.
Microsomal fractions from human liver tissues were also incubated with deramciclane. The total metabolism by human microsomes was lower (0.139 ± 0.013 nmol/mg protein/min) than that by rat microsomes. Human microsomal incubations produced primarily side chain-modified metabolites: N-desmethyl deramciclane (M4) as a major metabolite, and phenylborneol (M8).
Various form-selective inhibitors for P450s were used to identify the individual isozymes participating in deramciclane metabolism (Table 1). Diethyl-dithiocarbamate was the strongest inhibitor of deramciclane biotransformation. In the presence of this CYP2E1 inhibitor, total deramciclane metabolism was inhibited by 62.3 ± 10.66% and 60.25 ± 6.65% in rat and human microsomal fractions, respectively. The formation of N-desmethyl deramciclane (M4) by rat and human microsomes was reduced by about 50% and 35%, respectively, whereas phenylborneol (M8) could not be detected at all. Addition of diethyl-dithiocarbamate to rat liver microsomal fractions decreased the production of 9-hydroxy-deramciclane (M5) by about 40% and totally abolished the formation of N-desmethyl 9-hydroxyderamciclane (M2), N-desmethyl hydroxy-deramciclane II (M3), and hydroxy-deramciclane II (M6). Other inhibitors used in the metabolic assays (-naphthoflavone, sulfaphenazole, quinidine and troleandomycin) exhibited modest or no inhibitory effect on deramciclane metabolism. The production of phenylborneol (M8) decreased in the presence of -naphthoflavone, sulfaphenazole, or troleandomycin. -Naphthoflavone was also able to inhibit the formation of N-desmethyl 9-hydroxy-deramciclane (M2) in rat liver microsomes. Interestingly, the amount of N-desmethyl 9-hydroxy-deramciclane (M2) increased significantly in the presence of sulfaphenazole (375 ± 108.6% in rat microsomes).
View this table:
[in this window]
[in a new window]
|
TABLE 1 Inhibition of microsomal biotransformation of deramciclane by P450-specific inhibitors
Liver microsomes (1.5 mg/ml) were incubated with 50 µM deramciclane in the absence or presence of P450 form-specific inhibitors (NF, -naphthoflavone; SF, sulfaphenazole; QUIN, quinidine; DDC, diethyl-dithiocarbamate; TAO, troleandomycin). The metabolites were separated on a thin-layer plate using ethyl acetate/triethylamine (25:1, v/v). The amounts of metabolites are presented as the percentage of uninhibited activity.
|
|
Biotransformation of Deramciclane by Microsomes of Isoniazid-Pretreated Rat Liver and cDNA-Expressed Rat CYP2E1. HPLC analysis of the extracts of rat microsomal incubations detected all deramciclane metabolites that were separated by thin-layer chromatography, but an additional derivative (M9) was also observed (Fig. 2A). Pretreatment of rats with isoniazid (the specific CYP2E1 inducer; Ryan et al., 1985) significantly stimulated the hydroxylation of deramciclane (Fig. 3A). The formation of 9-hydroxy-deramciclane (M5), hydroxy-deramciclane II (M6), N-desmethyl 9-hydroxy-deramciclane (M2), and N-desmethyl hydroxy-deramciclane II (M3) increased by about 40 to 45%. The rate of side chain cleavage was also elevated slightly, but significantly during the incubation with microsomes of isoniazid-treated animals. However, no change was observed in the production of N-desmethyl deramciclane (M4) and M9.
View larger version (14K):
[in this window]
[in a new window]
|
FIG. 2. HPLC analysis of deramciclane (A) or N-desmethyl deramciclane (B) and their metabolites in microsomal incubation media. Rat liver microsomes were incubated with 50 µM [14C]deramciclane (D) or 14C-N-desmethyl deramciclane (N), and after 30 min, the metabolites were extracted in dichloromethane. The evaporated organic phase was resolved in 100 µl of methanol, and 20 µl was loaded onto the HPLC column. The effluent was analyzed on the basis of radioactivity. Metabolites: M2, N-desmethyl 9-hydroxy-deramciclane; M3, N-desmethyl hydroxy-deramciclane II; M4, N-desmethyl deramciclane; M5, 9-hydroxy-deramciclane; M6, hydroxy-deramciclane II; M8, phenylborneol; M9 and M10, the structures are not identified.
|
|
View larger version (29K):
[in this window]
[in a new window]
|
FIG. 3. Biotransformation of deramciclane in liver microsomes from untreated and isoniazid-treated rats and by cDNA-expressed rat CYP2E1. Microsomes (1.5 mg/ml) were incubated with 50 µM deramciclane. The amount of metabolites produced after 30 min is plotted for microsomes of untreated and isoniazid-treated animals (A) as well as of lymphoblastoid cells expressing rat CYP2E1 (B). Significant difference from control rats (*, P < 0.05; **, P < 0.01). ND, not detectable.
|
|
The ability of cDNA-expressed rat CYP2E1 to metabolize deramciclane was also examined. CYP2E1 generated all metabolites that were produced by rat liver microsomes, except for M9 (Fig. 3B).
The Effect of Deramciclane on CYP2E1 Activities. Liver microsomes from isoniazid-treated rats were assayed for p-nitrophenol and chlorzoxazone hydroxylation activities of CYP2E1. Deramciclane was not able to decrease the activities of CYP2E1, even at a concentration of 200 µM. However, some of its metabolites produced during the 15- to 60-min preincubation time caused the reduction in p-nitrophenol and chlorzoxazone hydroxylation rate (Fig. 4A). After 60 min of preincubation with deramciclane, chlorzoxazone 6-hydroxylation activity decreased by 43%, whereas p-nitrophenol hydroxylation was almost totally blocked. The inhibition constant (Ki value) for chlorzoxazone 6-hydroxylation was estimated to be 39.9 ± 4.0 µM, referring to 30 min of preincubation with deramciclane.
View larger version (13K):
[in this window]
[in a new window]
|
FIG. 4. Inhibitory effect of deramciclane (A) and N-desmethyl deramciclane (B) on CYP2E1 activities as the function of preincubation time. Microsomes from isoniazid-treated rat liver were preincubated with 50 µM deramciclane or N-desmethyl deramciclane, and the reaction was started by the addition of p-nitrophenol (50 µM) or chlorzoxazone (50 µM). Control incubation refers to activity measured in the absence of deramciclane or N-desmethyl deramciclane.
|
|
To characterize the nature and the degree of the binding of deramciclane to microsomal P450, we measured the shift in optical spectra induced by its addition to microsomes. The binding of deramciclane in a manner similar to that of chlorzoxazone induced a type I spectral shift of microsomal P450. However, the association constants were estimated to be different. Ka values of 0.0183 and 0.0338 µM–1 were calculated for deramciclane and chlorzoxazone, respectively.
Biotransformation of N-Desmethyl Deramciclane by Microsomes of Isoniazid-Pretreated Rat Liver and cDNA-Expressed Rat CYP2E1. Since N-desmethyl deramciclane formation seemed to be one of the first steps in deramciclane metabolism, biotransformation of this metabolite was also investigated. Three metabolites of N-desmethyl deramciclane were detected by HPLC analysis of the extracts of the incubation media with rat liver microsomes (Fig. 2B). Two hydroxylated derivatives, N-desmethyl 9-hydroxy-deramciclane (M2) and N-desmethyl hydroxy-deramciclane II (M3), were generated from N-desmethyl deramciclane in a manner similar to deramciclane. Microsomal biotransformation of N-desmethyl deramciclane resulted in the formation of an additional metabolite, M10, that was not produced in a detectable amount during deramciclane metabolism. Pretreatment with isoniazid caused some increase in hydroxylation; the formation of N-desmethyl 9-hydroxy-deramciclane (M2) and N-desmethyl hydroxy-deramciclane II (M3) was elevated significantly, by 63% and 36%, respectively (Fig. 5A). The induction of CYP2E1 by isoniazid had no effect on the production of M10. On the other hand, cDNA-expressed CYP2E1 was able to generate all three metabolites that were formed by rat liver microsomes (Fig. 5B).
View larger version (18K):
[in this window]
[in a new window]
|
FIG. 5. Biotransformation of N-desmethyl deramciclane in liver microsomes from untreated and isoniazid-treated rat and by cDNA-expressed rat CYP2E1. Microsomes (1.5 mg/ml) were incubated with 50 µM N-desmethyl deramciclane. The amount of metabolites produced after 30 min is plotted for microsomes of untreated and isoniazid-treated animals (A) as well as of lymphoblastoid cells expressing rat CYP2E1 (B). Significant difference from control rats (*, P < 0.05; **, P < 0.01).
|
|
The Effect of N-Desmethyl Deramciclane on the Activities of CYP2E1. Similar to deramciclane, the rate of p-nitrophenol or chlorzoxazone hydroxylation also failed to be changed by N-desmethyl deramciclane alone, even at the highest concentration (200 µM). Although the activities of CYP2E1 were reduced after 15 to 60 min of preincubation with N-desmethyl deramciclane, the inhibitory profile was not exactly the same as that of deramciclane (Fig. 4B). After 30 min of preincubation with N-desmethyl deramciclane, chlorzoxazone 6-hydroxylase activity decreased by about 30%, whereas the loss of p-nitrophenol hydroxylase activity was 37%, and it was blocked by only 43% even after 60 min of preincubation.
|
Discussion
|
|---|
The knowledge of the metabolic pathway and the structures of the metabolites is essential for the interpretation of the pharmacological or toxicological behavior of a drug. Additionally, revealing the relative contribution of different P450 isoforms to the metabolism can predict some metabolic drug interactions, which are considered one of the reasons for adverse drug effects. All processes (induction or inhibition of drug-metabolizing enzymes) that can modify the hepatic metabolism constitute the source of drug interactions. Our present study made an attempt to identify P450 isoforms involved in the biotransformation of deramciclane and to determine its effect on the P450 enzyme, which plays the main role in its metabolism.
Although the results clearly demonstrated the implication of several P450 enzymes in the pathways of deramciclane metabolism both in rat and human, CYP2E1 was the main form involved in almost all metabolic steps. The conclusion was drawn from the following data: 1) inhibition of CYP2E1 by diethyl-dithiocarbamate (CYP2E1 inhibitor) decreased the total deramciclane metabolism and the formation of all metabolites; 2) hepatic microsomes of isoniazid-induced rats exhibited higher (by about 40%) metabolite production; and 3) cDNA-expressed CYP2E1 was able to generate all the metabolites except for M9. It should be mentioned that cDNA-expressed CYP2E1 was active in N-demethylation of deramciclane, but the formation of N-desmethyl deramciclane (M4) did not increase as a result of isoniazid treatment. N-Desmethyl deramciclane (M4), which is considered a primary derivative of the parent compound, can be hydroxylated at various positions (Monostory et al., 2005). We think that CYP2E1 induction may result in some elevation of N-demethylase activity, but hydroxylation as a second step causes consumption of N-desmethyl deramciclane at the same time. Our hypothesis is confirmed by the results of N-desmethyl deramciclane biotransformation: 1) hydroxylation of N-desmethyl deramciclane increased significantly in microsomes of isoniazid-treated rats; and 2) cDNA-expressed rat CYP2E1 was able to produce both hydroxylated derivatives of N-desmethyl deramciclane.
The evidence for the involvement of CYP2E1 in deramciclane metabolism suggested that the effect of deramciclane on the activity of CYP2E1 should be investigated. Although deramciclane was not able to affect directly p-nitrophenol or chlorzoxazone hydroxylation, the activities of CYP2E1, preincubation with the parent compound, resulted in the reduction of CYP2E1 activities. In other words, during deramciclane biotransformation, one or more metabolites must have been produced, which were potent inhibitors of CYP2E1. The fact that N-desmethyl deramciclane did not decrease directly the activity of CYP2E1 precludes the possibility of considering it as an inhibitor of CYP2E1. Additionally, the results confirmed that some metabolites with inhibitory effect were also formed after preincubation with N-desmethyl deramciclane, but the inhibitory profile was not the same as that observed in the case of deramciclane. It may be assumed that during the incubation with deramciclane, some other, and/or more potent, inhibitors were produced than those found during the biotransformation of N-desmethyl deramciclane. On the other hand, chlorzoxazone binding to microsomal P450 was found to be twice as strong as that of deramciclane, which was also supposed to contribute to the prevention of direct inhibition of CYP2E1-catalyzed chlorzoxazone 6-hydroxylation. It should also be noted that the Ka value for deramciclane measured in microsomes may cover the binding affinity toward several P450 enzymes catalyzing deramciclane metabolism.
A direct inhibitory effect of deramciclane on CYP2D6 has been reported by Laine et al. (2004). The in vivo drug interaction study demonstrated that the CYP2D6-dependent elimination of desipramine was prolonged during coadministration with deramciclane. Although, from this fact, the binding of deramciclane to CYP2D6 might be supposed, our results indicated that CYP2D6 did not play a specific and significant role in deramciclane metabolism.
Our present work has demonstrated that there is no exclusive P450 isoform in deramciclane biotransformation, but the role of CYP2E1 should be emphasized. CYP2E1 catalyzes almost all steps of deramciclane metabolism, and some of the metabolites are supposed to block the activity of CYP2E1.
|
Acknowledgments
|
|---|
We thank EGIS Pharmaceuticals Ltd. (Budapest, Hungary) for digital autoradiographic measurements and for providing deramciclane and authentic reference compounds used in this study.
|
Footnotes
|
|---|
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.105.003772.
ABBREVIATIONS: P450, cytochrome P450; HPLC, high-performance liquid chromatography; deramciclane, (1R,2S,4R)-(–)-2-phenyl-2-(2'-dimethylamino-ethoxy)-1,7,7-trimethyl-bicyclo[2.2.1]heptane; N-desmethyl-deramciclane (M4), (1R,2S,4R)-(–)-2-phenyl-2-(2'-methylamino-ethoxy)-1,7,7-trimethyl-bicyclo[2.2.1]heptane; 9-hydroxy-deramciclane (M5), (1R,2S,4R,7R)-2-(2'-dimethylamino-ethoxy)-2-phenyl-7-(hydroxy-methyl)-1,7-dimethylbicyclo[2.2.1]heptane; N-desmethyl 9-hydroxy-deramciclane (M2), (1R,2S,4R,7R)-2-(2'-methyl-amino-ethoxy)-2-phenyl-7-(hydroxy-methyl)-1,7-dimethyl-bicyclo[2.2.1]heptane; hydroxy-deramciclane II (M6), (1R,2S,4R)-(–)-2-phenyl-2-(2'-dimethylamino-ethoxy)-5 or 6-hydroxy-1,7,7-trimethyl-bicyclo[2.2.1]heptane; N-desmethyl hydroxy-deramciclane II (M3), (1R,2S,4R)-(–)-2-phenyl-2-(2'-methylamino-ethoxy)-5 or 6-hydroxy-1,7,7-trimethyl-bicyclo[2.2.1]heptane, phenylborneol, (1R,2S,4R)-(–)-2-hydroxy-2-phenyl-1,7,7-trimethyl-bicyclo[2.2.1]heptane.
Address correspondence to: Katalin Monostory, P.O. Box 17, Budapest, H-1525 Hungary. E-mail: monostor{at}chemres.hu
|
References
|
|---|
Berényi E, Blaskó G, Fekete M, and Nógrádi M (1990) EGYT-3886. Drugs Future 15: 1174–1175.
Gacsályi I, Gyertyán I, Petöcz L, and Budai Z (1988) Psychopharmacology of a new anxiolytic agent EGYT-3886. Pharmacol Res Commun 20 (Suppl I): 115–116.
Guengerich FP, Kim D-H, and Iwasaki M (1991) Role of human cytochrome P-450 IIE1 in the oxidation of many low molecular weight suspects. Chem Res Toxicol 4: 168–179.[CrossRef][Medline]
Kobayashi S, Murray S, Watson D, Sesardic D, Davies DS, and Boobis AR (1989) The specificity of inhibition of debrisoquine 4-hydoxylase activity by quinidine and quinine in the rat is the inverse of that in man. Biochem Pharmacol 38: 2795–2799.[CrossRef][Medline]
Kovács I, Maksay G, and Simonyi M (1989) Inhibition of high-affinity synaptosomal uptake of -aminobutyric acid by a bicyclo-heptane derivative. Arzneim-Forsch/Drug Res 39: 295–297.
Laine K, De Bruyn S, Björklund H, Rouru J, Hanninen J, Scheinin H, and Anttila M (2004) Effect of the novel anxiolytic drug deramciclane on cytochrome P450 2D6 activity as measured by desipramine pharmacokinetics. Eur J Clin Pharmacol 59: 893–898.[CrossRef][Medline]
Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265–275.[Free Full Text]
Maurel P (1996) The CYP3A family, in Cytochromes P450: Metabolic and Toxicological Aspects (Ioannides C ed) pp 241–270, CRC Press, Boca Raton, FL.
Maurel P (1998) Drug metabolizing enzymes. Curr Opin Crit Care 4: 213–218.
Miners JO, Smith KJ, Robson RA, McManus ME, Veronese ME, and Birkett DJ (1988) Tolbutamide hydroxylation by human liver microsomes. Kinetic characterisation and relationship to other cytochrome P450 dependent xenobiotic oxidations. Biochem Pharmacol 37: 1137–1144.[CrossRef][Medline]
Monostory K, Köhalmy K, Ludányi K, Czira G, Holly S, Ürmös I, Klebovich I, Vereczkey L, and Kóbori L (2005) Biotransformation of deramciclane in primary hepatocytes of rat, mouse, rabbit, dog, and human. Drug Metab Dispos 33: 1708–1716.[Abstract/Free Full Text]
Ono S, Hatanaka T, Hotta H, Satoh T, Gonzalez FJ, and Tsutsui M (1996) Specificity of substrates and inhibitor probes for cytochrome P450s: evaluation of in vitro metabolism using cDNA-expressed human P450s and human liver microsomes. Xenobiotica 26: 681–693.[Medline]
Peter R, Böcker R, Beaune PH, Iwasaki M, Guengerich FP, and Yang CS (1990) Hydroxylation of chlorzoxazone as a specific probe for human liver cytochrome P-450IIE1. Chem Res Toxicol 3: 566–573.[CrossRef][Medline]
Reinke LA and Moyer MJ (1985) p-Nitrophenol hydroxylation: a microsomal oxidation which is highly inducible by ethanol. Drug Metab Dispos 13: 548–552.[Abstract]
Rendic S and Di Carlo FJ (1997) Human cytochrome P450 enzymes: a status report summarizing their reactions, substrates, inducers and inhibitors. Drug Metab Rev 29: 413–580.[Medline]
Roos PH, Golub-Ciosk B, Kallweit P, Kauczinski D, and Hanstein WG (1993) Formation of ligand and metabolite complexes as a means for selective quantitation of cytochrome P450 isozymes. Biochem Pharmacol 45: 2239–2250.[CrossRef][Medline]
Ryan DE, Ramanathan L, Iida S, Thomas PE, Hanui M, Shively JE, Lieber CS, and Levin W (1985) Characterization of a major form of rat hepatic microsomal cytochrome P-450 induced by isoniazid. J Biol Chem 260: 6385–6393.[Abstract/Free Full Text]
Siess M-H, Leclerc J, Canivenc-Lavier M-C, Rat P, and Suschetet M (1995) Heterogenous effects of natural flavonoids on monooxygenase activities in human and rat liver microsomes. Toxicol Appl Pharmacol 130: 73–78.[CrossRef][Medline]
Tamási V, Vereczkey L, Falus A, and Monostory K (2003) Some aspects of interindividual variations in the metabolism of xenobiotics. Inflamm Res 52: 322–333.[Medline]
van der Hoeven TA and Coon MJ (1974) Preparation and properties of partially purified cytochrome P-450 and reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase from rabbit liver microsomes. J Biol Chem 249: 6302–6310.[Abstract/Free Full Text]
![[Feedback]](/icons/banner/feedbackACT.gif)
![[For Subscribers]](/icons/banner/subscriberACT.gif)
![[Archive]](/icons/banner/archiveACT.gif)
![[Search]](/icons/banner/searchACT.gif)
![[Contents]](/icons/banner/tocACT.gif)
Top
Abstract
Materials and Methods
