Introduction

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The antidepressant drug mirtazapine (1,2,3,4,10,14b-hexahydro-2-methylpyrazino[2,1-a]pyrido[2,3-c]benzazepine), marketed as REMERON SolTab, has a tetracyclic chemical structure (Fig. 1) that is unlike those of selective serotonin reuptake inhibitors, monoamine oxidase inhibitors, or tricyclic antidepressants. It also has a different mode of action. Mirtazapine acts as an antagonist of ₂-adrenergic autoreceptors and heteroreceptors, resulting in increased release of norepinephrine and serotonin (Puzantian, 1998). Therefore, it has been regarded as a noradrenergic and specific serotonergic antidepressant (Westenberg, 1999). Mirtazapine is also an antagonist of postsynaptic serotonin type 2 (5-HT₂)¹ and type 3 (5-HT₃), but not type 1 (5-HT₁) receptors, decreasing unwanted side effects (Stimmel et al., 1997). The drug has a high affinity for histamine H₁ receptors, causing it to act as a sedative (Puzantian, 1998).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Structures of S(+)- and R()-mirtazapine.

Mirtazapine is commercially available as a racemic mixture of the S(+)- and R()-enantiomers (Fig. 1). The S(+)-enantiomer is responsible for blocking ₂ activity and is the more potent 5-HT₂ antagonist (McGrath et al., 1998). The R()-enantiomer is responsible for the 5-HT₃ antagonist activity and contributes equally to the blocking of ₂-heteroreceptors (McGrath et al., 1998). Both enantiomers function as antihistamines (Stimmel et al., 1997).

Since it is assumed that metabolism of this drug takes place mainly in the liver (Sandker et al., 1994), metabolic studies have been undertaken using human liver and rat hepatocytes and human liver microsomes (Sandker et al., 1994; Dahl et al., 1997). Phase I and II human metabolites reported were mirtazapine-N-oxide, 8-hydroxymirtazapine, N-desmethylmirtazapine, mirtazapine N-glucuronide, mirtazapine N-sulfate, 8-hydroxymirtazapine glucuronide, 8-hydroxy-N-desmethylmirtazapine, 8-hydroxy-N-desmethylmirtazapine glucuronide, 8-hydroxymirtazapine sulfate, and 8-hydroxy-N-desmethylmirtazapine sulfate. The same metabolites are also produced in rat hepatocytes, with the exception of mirtazapine N-glucuronide (Sandker et al., 1994). 13-Hydroxymirtazapine has been reported in metabolism studies with mice (Delbressine and Vos, 1997). In human liver microsomal fractions, 8-hydroxymirtazapine, N-desmethylmirtazapine, and mirtazapine N-oxide were formed (Dahl et al., 1997). Pharmacokinetic data indicated that N-desmethylmirtazapine has activity similar to that of the parent drug; however, the activity is 5 to 10 times less than that of mirtazapine (Delbressine and Vos, 1997).

In human studies, the R()-enantiomer is preferentially conjugated to form a quaternary N-glucuronide directly, but the S(+)-enantiomer is preferentially transformed to 8-hydroxymirtazapine, followed by glucuronide conjugation (Delbressine et al., 1998). In the antidepressant drug mianserin, which differs from mirtazapine in that a carbon atom replaces the N-6 of mirtazapine, metabolism studies indicate that the R()-enantiomer is metabolized to demethylated products whereas the S(+)-enantiomer is metabolized to 8-hydroxy products (Heinig and Blaschke, 1993). The metabolism is mediated by cytochrome P450; enantiomeric differences are accredited to the effects of various CYP450 isoforms involved (Dahl et al., 1997; Delbressine et al., 1998).

The use of microorganisms as models of mammalian metabolism has been well documented (Zhang et al., 1995, 1996a,b; Moody et al., 1999, 2000). The fungus Cunninghamella elegans can metabolize a variety of xenobiotics in regio- and stereoselective manners that are often similar to those in mammalian enzyme systems (Davis, 1988; Clark and Hufford, 1991; Cerniglia, 1997; Rao and Davis, 1997). It generates useful quantities of metabolites that are the same as those produced by humans and animals, as well as novel metabolites. Milligram quantities of major and minor metabolites can be produced more cost effectively and in less time than those produced by experimental animals, cell cultures, or mammalian enzyme systems. It is important to determine alternative modes to produce compounds to be evaluated for toxicity and potential adverse effects. Since mirtazapine has been widely used in clinical human medicine and its metabolism has been well documented, however, less is known about the toxicity of the metabolites. Therefore, we have chosen mirtazapine as a model compound and investigated its metabolism by C. elegans to produce significant quantities of metabolites via microbial biotransformation for neurotoxicological evaluation.

	Materials and Methods

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Chemicals. Racemic mirtazapine was a gift from Organon Pharmaceuticals, Inc. (West Orange, NJ). Optically pure R()- and S(+)-mirtazapine were gifts from Organon NV (Oss, The Netherlands). NMR solvents were purchased from Isotec, Inc. (Miamisburg, OH). Solvents were HPLC grade; all other chemicals were reagent grade and the highest purity available.

Microbial Culture and Biotransformation Conditions. Cultures of C. elegans, American Type Culture Collection 9245 (Manassas, VA), were maintained on potato dextrose agar slants and stored at 4°C. The spores and/or mycelia were aseptically transferred to potato dextrose agar plates and allowed to grow for 72 h at room temperature. The mycelia from five plates were blended with 90-ml sterile physiological saline solution for 5 min. Ten-milliliters aliquots of the homogenate were used to inoculate 250-ml Erlenmeyer flasks containing 50-ml Sabouraud dextrose broth. The cultures were incubated for 72 h at 27°C on a rotary shaker operating at 120 rpm and then 6 mg of mirtazapine dissolved in 30 µl of N,N-dimethylformamide was added to each flask. Control flasks consisted of culture broth without mirtazapine and only sterile medium with mirtazapine. Flasks with inoculum were extracted at 4, 8, 24, 48, 72, 96, 120, 144, and 168 h to determine the optimum time for extraction. The incubation time for maximum metabolite formation was determined to be 168 h. The data represent the average of three separate experiments with replicated batch cultures. The standard deviation was no more than 5%. In a separate experiment, six flasks were inoculated with C. elegans as described above, then three of the flasks were treated with 6 mg of R()-mirtazapine and three with 6 mg of S(+)-mirtazapine.

Extraction and Isolation of Metabolites. After 168 h of incubation, the culture broth was filtered through glass wool into a separatory funnel and extracted with three equal volumes of ethyl acetate. The organic extracts were dried over sodium sulfate and evaporated to dryness in vacuo at 34°C on a Buchi 011 rotary evaporator (Brinkmann Instruments, Westbury, NY). The residue was dissolved in 5 ml of methanol and concentrated to approximately 100 µl for HPLC analysis.

Identification of Metabolites. In a separate experiment, seven flasks were treated with a racemic mixture of R() and S(+)-mirtazapine prepared as above and extracted at 24, 48, 72, 96, 120, 144, and 168 h to be analyzed by HPLC. The percentages of metabolites formed were estimated by comparing their peak areas at 168 h with consideration of their molar absorption coefficients (L. P. C. Delbressine, personal communication).

	Results

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The HPLC chromatograms of ethyl acetate extracts of the 168-h incubation of C. elegans with racemic mirtazapine and the optical isomers are shown in Fig. 2. The biotransformation pathways are presented in Fig. 3. Although N-desmethylmirtazapine and 8-hydroxymirtazapine have been previously reported (Delbressine et al., 1998), we provide unequivocal structural proof of these and the other metabolites.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Reversed-phase HPLC chromatogram of (A) racemic mirtazapine, (B) S(+)-mirtazapine, and (C) R()-mirtazapine and metabolites produced by C. elegans after 168 h of incubation. [I] N-desmethyl-8-hydroxymirtazapine, [II] N-desmethyl-13-hydroxymirtazapine, [III] mirtazapine N-oxide, [IV] N-desmethylmirtazapine, [V] 13-hydroxymirtazapine, [VI] 12-hydroxymirtazapine, [VII] 8-hydroxymirtazapine, and [VIII] mirtazapine.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Proposed biotransformation pathway of mirtazapine incubated with C. elegans.

The chromatographic, mass spectral, and ¹H NMR data of mirtazapine and its metabolites are given in Table 1. Peak I eluted at 19.6 min. The DEP/EI mass spectrum showed an apparent molecular ion at m/z 267 [M^+·] and significant ions at m/z 237, 225, 212, 211 (base peak), 210, and 196. The ions were consistent with N-demethylation and hydroxylation of an aromatic ring. The ¹H NMR spectrum showed the absence of a methyl singlet and downfield shifts of the aliphatic proton resonances. In the aromatic region, the four resonances of the benzene-type ring were present. Since the two resonances of the ring containing one nitrogen atom exhibited meta coupling (J_7,9 = 2.8 Hz), the hydroxyl substitution was determined to be at C-8. The metabolite was identified as N-desmethyl-8-hydroxymirtazapine.

Peak II (t_R = 20.5 min) had a DEP/EI mass spectrum with the same significant ions as in N-desmethyl-8-hydroxymirtazapine. In the 2.00 to 5.00 ppm region, the NMR spectrum was essentially the same as that of N-desmethyl-8-hydroxymirtazapine. The aromatic region showed a change in the benzene-type ring resonances for protons 11 through 14. Irradiation of the resonance at 4.29 ppm (H14b) produced a nuclear Overhauser enhancement of the meta-coupled doublet (J_12,14 = 2.6 Hz) at 6.56 ppm (H14). Irradiation of the resonance at 3.40 ppm (H10, axial) produced an enhancement of the doublet at 7.04 ppm, H11. Subsequent homonuclear decoupling and the NOE experiments proved the hydroxyl substitution at C-13, and the metabolite was identified as N-desmethyl-13-hydroxymirtazapine.

Peak III eluted at 22.2 min. The DEP/EI mass spectrum showed an apparent molecular ion at m/z 281 [M^+·] and significant ions, 264, 263, 222, 221, 208, 195 (base peak), 194, 193, 180, and 167. All resonances were present in the NMR spectrum. The aromatic region and the H10 resonances were nearly the same as those of mirtazapine whereas the piperazine ring resonances were shifted downfield. Since this is consistent with N-oxidation (Zhang et al., 1996a, 1997; Moody et al., 1999), the metabolite was identified as mirtazapine N-oxide.

The DEP/EI mass spectrum of peak IV (t_R = 24.8 min) showed an apparent molecular ion at m/z 251 [M^+·] and significant fragment ions at 221, 196, 195 (base peak), 194, 180, and 111. The H1, H3, and H4 resonances of the NMR spectrum were shifted downfield from those of mirtazapine, but the shifts were less than those of mirtazapine N-oxide. Because the other resonances were almost identical to those of mirtazapine, and the methyl resonance was absent the metabolite was identified as N-desmethylmirtazapine.

Peak V eluted at 26.8 min. The DEP/EI mass spectrum showed the addition of an oxygen atom to mirtazapine, with significant ions at m/z 281 [M^+·], 224, 211 (base peak), 210, 196, 119, and 71. The NMR spectrum revealed a hydroxyl substitution on the benzene-type ring. The metabolite was identified as 13-hydroxymirtazapine, using homonuclear decoupling and NOE experiments.

Peaks VI and VII eluted closely together; the retention times were 30.2 and 30.3 min, respectively. Peak VI had DEP/EI mass spectral ions at m/z 281 [M^+·], 237, 224, 211 (base peak), and 71, whereas peak VII had ions at m/z 281 [M^+·], 224, 211 (base peak), 210, 196, 119, and 71. Although each compound was enriched with the other, resonance overlap of the aromatic protons in the NMR spectra was minimal. The coupling pattern of the aromatic resonances of peak VI showed a single substitution on the ring without the nitrogen. Irradiation of the resonance at 3.39 ppm (H10, axial) produced an NOE at the meta-coupled doublet (J_11,13 = 2.6 ppm) at 6.55 ppm (H11) and irradiation of the resonance at 4.16 ppm (H14b) produced an NOE at the doublet at 7.00 ppm (H14). The coupling pattern and chemical shifts of peak VII were practically identical to those of N-desmethyl-8-hydroxymirtazapine. The metabolites were identified as 12-hydroxymirtazapine and 8-hydroxymirtazapine, respectively.

Mirtazapine, peak VIII, eluted at 34.1 min. Its DEP/EI mass and NMR spectra were identical to the compound provided by Organon Pharmaceuticals, Inc. The CD spectra of optically pure R()-mirtazapine, optically pure S(+)-mirtazapine, racemic mirtazapine, and peak VIII were measured. As shown in Fig. 4, the R()-mirtazapine had a CD spectrum with a positive Cotton effect at 216 nm (ellipticity = 45 m°) and negative Cotton effects at 253 nm (ellipticity = 44 m°) and 288 nm (ellipticity = 25 m°), respectively. The CD spectrum of S(+)-mirtazapine was the mirror image of that of the R()-mirtazapine enantiomer, with Cotton effects at 216, 253, and 288 nm and ellipticities of 45, 44, and 25 m°, respectively (Fig. 4). As expected, the racemic mirtazapine did not show any Cotton effects on the CD spectrum, resulting in a line overlapped with the baseline (data not shown). The CD spectrum of peak VIII at the same concentration was measured. As shown in Fig. 4, the CD spectrum is similar to that of R()-mirtazapine, with the Cotton effects shown at 216 and 253 nm, but with a much smaller ellipticity (4.5 and 4.5 m°, respectively). The Cotton effect at 288 nm is too small to be measured. The results indicated that the residual parent mirtazapine is slightly rich in the R()-enantiomer. Based on the ellipticities at 216 and 253 nm of the pure R()-mirtazapine and the parent mirtazapine, the optical purity of the parent mirtazapine is 10%. Therefore, the amounts of R()-enantiomer and S(+)-enantiomer in the residual parent compound are 55 and 45%, respectively.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   CD spectra of optically pure R()-mirtazapine (), optically pure S(+)-mirtazapine (- - - - -), and the residual parent mirtazapine (- · - · - · - · -).

C. elegans transformed 91% of a racemic mixture of R()- and S(+)-mirtazapine to form the major metabolites, N-desmethyl-13-hydroxymirtazapine, mirtazapine N-oxide, N-desmethylmirtazapine, 13-hydroxymirtazapine, 12-hydroxymirtazapine, and 8-hydroxymirtazapine, as well as the minor metabolite, N-desmethyl-8-hydroxymirtazapine (Fig. 2A). Incubation of C. elegans with optically pure R() and S(+)-mirtazapine showed that all seven metabolites were formed from the S(+)-enantiomer, with mirtazapine N-oxide as the major metabolite (Fig. 2B). The R() enantiomer formed N-desmethyl-8-hydroxymirtazapine, mirtazapine N-oxide, N-desmethylmirtazapine, and 8-hydroxymirtazapine. 8-Hydroxymirtazapine was the major metabolite (Fig. 2C).

	Discussion

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C. elegans metabolized mirtazapine via the same routes as those reported in human and animal studies, namely by 8-hydroxylation, N-oxidation, demethylation, and 13-hydroxylation. Two novel fungal metabolites, 12-hydroxymirtazapine and 13-hydroxy-N-desmethylmirtazapine, were also found. The reactions that form 8-hydroxymirtazapine, 12-hydroxymirtazapine, and 13-hydroxymirtazapine can be explained by the National Institutes of Health shift mechanism catalyzed by a cytochrome P450 monooxygenase (Cerniglia et al., 1983). In this case, mirtazapine 7,8- or 8,9-epoxide and mirtazapine 12,13-epoxide would be the expected intermediates. Metabolites corresponding to 12-hydroxymirtazapine, 13-hydroxymirtazapine, and N-desmethylmirtazapine have been reported from C. elegans with structure analogs of mirtazapine, including amitriptyline, azatadine, amoxapine, cyproheptadine, doxepin, and cyclobenzaprine (Zhang et al., 1995, 1996a,b, 1997; Moody et al., 1999, 2000). In humans, 8-hydroxymirtazapine is formed by cytochrome P450 isoform CYP2D6 or CYP1A2, and the N-desmethyl and N-oxide compounds are formed by CYP3A (Delbressine and Vos, 1997). C. elegans has cytochrome P450 activity that is responsible for the hydroxylation, N-oxidation, and N-demethylation of several tricyclic antidepressants (Zhang et al., 1995, 1996a, 1997). Recently Wang et al. (2000) found that the cytochrome P450 in C. elegans belongs to the CYP51 family.

In the time course measurements, N-desmethylmirtazapine and 8-hydroxymirtazapine were formed 48 h after adding mirtazapine (data not shown). At 96 h, 8-hydroxymirtazapine and 13-hydroxymirtazapine had become the major metabolites, with 12-hydroxymirtazapine and N-desmethylmirtazapine as minor metabolites. This implies N-desmethylmirtazapine was initially formed and then transformed later to N-desmethyl hydroxy compounds. It is also possible that the hydroxylation reaction occurred prior to the demethylation reaction. N-Desmethyl-13-hydroxymirtazapine and N-desmethyl-8-hydroxymirtazapine were probably formed by one of these subsequent reaction mechanisms. It would be reasonable to expect N-desmethyl-12-hydroxymirtazapine to be formed as well; however, if it did form, it did so in amounts too small to be detected using our analytical method.

Delbressine et al. (1998) reported that, in human metabolism, the R()-enantiomer of mirtazapine is converted mainly to a quarternary N-glucuronide conjugate, while the S(+)-enantiomer forms 8-hydroxymirtazapine that is then converted to the corresponding glucuronide. C. elegans also metabolized the enantiomers differently. The S(+)-enantiomer was converted to the same metabolites as the racemic mixture, whereas the R()-enantiomer formed neither 12- nor 13-substituted metabolites. The absence of those metabolites may partially explain the enrichment of the R()-enantiomer present in the residual parent compound, as shown by the CD measurements (Fig. 4). The R()-enantiomer produced a larger percentage of 8-hydroxy compounds. Both enantiomers formed mirtazapine N-oxide and N-desmethylmirtazapine, but the amounts differed. This was not unexpected, since fungi may form metabolites that have the opposite absolute configurations as those formed by rat liver microsomes, as shown with other types of substrates (Cerniglia et al., 1984; McMillan et al., 1987; Cerniglia et al., 1990).

The ability of C. elegans to mimic mammalian metabolism, to perform novel biotransformations, and to produce usable amounts of material clearly demonstrates that this microbial system represents an attractive alternative to the use of mammalian systems or chemical synthesis.