Rat CYP2D2, not 2D1, is functionally conserved with human CYP2D6 in endogenous morphine formation

Abstract

The assumption that CYP2D1 is the corresponding rat cytochrome to human CYP2D6 has been revisited using recombinant proteins in direct enzyme assays. CYP2D1 and 2D2 were incubated with known CYP2D6 substrates, the three morphine precursors thebaine, codeine and (R)-reticuline. Mass spectrometric analysis showed that rat CYP2D2, not 2D1, catalyzed the 3-O-demethylation reaction of thebaine and codeine. In addition, CYP2D2 incubated with (R)-reticuline generated four products corytuberine, pallidine, salutaridine and isoboldine while rat CYP2D1 was completely inactive. This intramolecular phenol-coupling reaction follows the same mechanism as observed for CYP2D6. Michaelis-Menten kinetic parameters revealed high catalytic efficiencies for rat CYP2D2. These findings suggest a critical evaluation of other commonly accepted, however untested, CYP2D1 substrates.

1. Introduction

Human CYP2D6 is involved in the oxidation of about 30% of clinically relevant drugs including the morphine precursor codeine [1,2]. The enzyme has been connected to a phenomenon known as the debrisoquine/sparteine metabolism of drug oxidation which is caused by aberrant metabolism patterns due to the absence or deficiency of functional allelic variants of CYP2D6 [3]. At least 112 allelic variants of CYP2D6 have been described since the discovery of the genetic polymorphism resulting in a total of four individual phenotypes: poor, intermediate, extensive and ultrarapid metabolizers [4]. Considering the multitude of clinically relevant compounds that are metabolized by human CYP2D6, drug dosages tailored towards individual phenotypes are currently discussed [2].

In the past, an in vitro model to test possible CYP2D6 substrates utilized microsomal preparations of different rat strains that differentially express CYP2D1, the proposed rat orthologue to CYP2D6 [5]. Using this approach, liver microsomes obtained from Dark-Agouti rats deficient in CYP2D1 were found to convert the two morphine precursors, thebaine and codeine, less efficiently as microsomes from Sprague-Dawley rats expressing CYP2D1 [6,7].

Our interest in elucidating mammalian enzymes involved in mammalian morphine biosynthesis led us to test rat CYP2D1 in direct enzyme assays similar to the CYP2D6 approach described previously [8]. Surprisingly, rat CYP2D1 was not capable of catalyzing the 3-O-demethylation of thebaine and codeine nor the phenol-coupling reaction of (R)-reticuline. The rat genome was subsequently searched and five isoforms were identified: CYP2D2, CYP2D3, CYP2D4, CYP2D5 and CYP2D18 among which CYP2D2 was described as poorly expressed in the Dark-Agouti rat strain next to CYP2D1 with similar catalytic activities as observed for human CYP2D6 [9,10]. We obtained microsomes containing rat CYP2D2 from a commercial source and describe herein its enzymatic characterization using the three morphine precursors (R)-reticuline, thebaine and codeine.

2. Material and methods

2.1 Enzymes

CYP2D1 and CYP2D2 were purchased from BD Biosciences (Woburn, MA). For confirmation of amino acid sequences, the P450 enzymes were subjected to SDS-PAGE on a 10% (w/v) acrylamide gel. Protein bands at approximately 55–58 kDa were excised, eluted and sequenced using a 6520 QTOF with Chip Cube (Agilent, Santa Clara, CA).

2.2 Enzyme assays

Reactions were conducted in a total volume of 250 µl containing 100 mM potassium phosphate buffer (pH 7.4), 15 µg L-α-dilauroyl-sn-glycero-3-phosphocholine, 1 mM NADP⁺, 10 mM glucose 6-phosphate, 1 unit/ml glucose-6-phosphate dehydrogenase (yeast), 1000 units/ml catalase (bovine liver), 10 µg bovine erythrocyte superoxide dismutase, and 0.25–50 µM alkaloidal substrate. Reactions were started by adding 5–30 pmol P450 enzyme (BD Biosciences) and incubated for 10–120 min at 37 °C. Extraction of alkaloids was conducted as previously described [8]. Alkaloids were analyzed by LC-MS/MS. Kinetic parameters were estimated by non-linear regression with GraphPad Prism. The debrisoquine oxidation assay was prepared according to Hiroi et al. 2002 [10]. Reactions were conducted in a total volume of 250 µl containing 100 mM potassium phosphate buffer (pH 7.4), 0.8 mM NADPH, and 4 µM debrisoquine or alkaloidal substrate. Reactions were started by adding 10 pmol P450 enzyme (BD Biosciences), incubated for 10–120 min and stopped with 25 µl 10% trichloroacetic acid. After centrifugation, the supernatant was analyzed by LC-MS/MS.

2.3 LC-MS/MS analysis

Enzyme activities were analyzed with a 4000 QTRAP LC-TIS-MS-MS (Agilent) system using the multiple reaction monitoring (MRM) in positive ionization mode. Compound dependent parameters (collision energy, declustering potential, quantifier MRM, qualifier MRM, dwell time) are listed in Table 1. For the analysis of incubations containing codeine and thebaine, separation of (10 µl) samples was achieved by using a Gemini C18 octadecylsilane HPLC column (Phenomenex, 5 µm, 150 mm × 2 mm) combined with a C18 guard column (Phenomenex, 4 mm × 2 mm). The mobile phase total flow was set to 0.1 ml/min with binary gradient elution, using solvent A (0.2% acetic acid) and B (99.8% CH₃CN, 0.2% acetic acid) (all v/v). The gradient started with 100% A for 5 min and was increased to 60% B over 20 min. Elution was continued for 5 min at 60% B. A detailed description of the LC-MS/MS analysis for reaction mixtures containing reticuline has been published previously [8]. Incubations containing debrisoquine were analyzed using an Eclipse XDB-C18 HPLC column combined with a microfilter unit (Sigma, 1/16 OD tubing). The mobile phase total flow was set to 0.2 ml/min with binary gradient elution, using solvent A (0.1% formic acid) and B (CH₃CN). The gradient started with 95% A for 2 min and was increased to 100% B over 8 min. Elution was continued for 2 min at 100% B. Identification of analytes was based on retention times, MRM transition ratios, and comparison with the expected values for standards. Quantitation was performed by constructing standard curves for each analyte and integrating the peak area of the quantifier MRM transition with Analyst 1.4.1 (Applied Biosystems, MDS SCIEX Instruments).

Table 1.

Compound dependent parameters for the LC-MS/MS method.

Analyte	Collision energy (V)	Declustering potential (V)	Quantifier MRM transition	Qualifier MRM transition	Dwell time (ms)
Reticuline	35	45	330 → 192	330 → 137	50
Corytuberine	40	50	328 → 265	328 → 282	50
Pallidine	40	50	328 → 211	328 → 237	50
Salutaridine	40	50	328 → 211	328 → 237	50
Isoboldine	40	50	328 → 265	328 → 237	50
Thebaine	35	40	312 → 251	312 → 281	50
Oripavine	35	40	298 → 218	298 → 249	50
Codeine	40	47	300 → 215	300 → 225	50
Morphine	45	45	286 → 165	286 → 201	50
Debrisoquine	35	70	176 → 134	176 → 117	100
Hydroxydebrisoquine	35	70	192 → 130	192 → 115	100

3. Results and discussion

Microsomal studies suggested rat P450 enzyme CYP2D1 as functionally conserved with human CYP2D6 and an enzymatic catalyst for the 3-O-demethylation reaction of thebaine and codeine [6,7]. Thus, CYP2D1 was obtained from a commercial source and tested in incubations containing these morphine precursors. Since the 3-O-demethylation of thebaine, m/z 312, or codeine, m/z 300, results in a mass loss of 14 mass units, specific multiple reaction monitoring (MRM) for oripavine, m/z 298, and morphine, m/z 286, was selected. Minor product formation of m/z 298 was observed in incubations with CYP2D1 and thebaine while codeine was not converted by CYP2D1 (Fig. 1).

Fig. 1.

Conversion of thebaine and codeine by rat CYP2D1. (A) MRM for the substrate thebaine, m/z 312, after incubation with CYP2D1. (B) MRM for the demethylated product, m/z 298, after incubation of thebaine with CYP2D1. (C) MRM for the substrate codeine, m/z 300, after incubation with CYP2D1. (D) MRM for the demethylated product, m/z 286, after incubation of codeine with CYP2D1. Illustrated is the sum of selected MRM. Incubations were carried out for 120 min at 37°C.

However, MS/MS data of the generated product m/z 298 were not identical to the MS/MS obtained for the standard oripavine. It was therefore concluded that the observed mass loss of 14 was due to a possible CYP2D1-catalyzed demethylation of thebaine at the ring nitrogen. Indeed, further comparison and mass spectrometric characterization suggested the CYP2D1 product as N-demethylated thebaine (Fig. 2). To confirm that CYP2D1 was still active in incubations lasting as long as 120 min, the rat P450 enzyme was incubated with the reference substrate, debrisoquine, as described previously [10]. Indeed, as shown in Fig. 3A and B debrisoquine was hydroxylated by CYP2D1 as previously described [9,10,15]. Using the same incubation conditions, it was again confirmed that CYP2D1 was not active with the morphine precursors. Since CYP2D1 did not show similar catalytic activities to human CYP2D6 in incubations with the alkaloidal substrates, we searched the rat genome for other possible candidates capable of catalyzing the 3-O-demethylation reaction of thebaine or codeine. Five isoforms were identified CYP2D2, CYP2D3, CYP2D4, CYP2D5 and CYP2D18 among which rat CYP2D2, next to CYP2D1, was described as poorly expressed in the Dark-Agouti rat strain [9]. Moreover, studies with cDNA-expressed rat CYP isoforms elucidated that CYP2D2, CYP2D4 and CYP2D18, but not CYP2D1, catalyzed the hydroxylation of tyramine to dopamine with similar kinetic characteristics for CYP2D2 compared with human CYP2D6 [11]. While progesterone hydroxylation properties of CYP2D4 and CYP2D6 were alike [10,12], CYP2D2 and CYP2D6 resembled each other in their catalytic activities for the hydroxylation of bufuralol, debrisoquine and propranolol, which are common CYP2D substrates [10]. Thus, recombinant CYP2D2 was obtained from a commercial source and tested first with debrisoquine to confirm activity. Similar to rat CYP2D1, CYP2D2 converted debrisoquine to hydroxydebrisoquine in incubations as long as 120 min (Fig. 3C,D). Moreover, as shown in Fig. 4 CYP2D2 was highly active with both morphine precursors, thebaine and codeine, using the same incubation conditions. The reaction products at m/z 298 and m/z 286 showed identical MS/MS spectra upon comparison to oripavine and morphine standards, respectively (Fig. 4).

Fig. 2.

Identification of product, m/z 298, formed from thebaine by CYP2D1. (A) MS/MS of CYP2D1 product, m/z 298. (B) MS/MS of northebaine.

Fig. 3.

Conversion of debrisoquine by rat CYP2D1 and CYP2D2. (A) MRM for the substrate debrisoquine, m/z 176, after incubation with CYP2D1. (B) MRM for the product hydroxydebrisoquine, m/z 192, after incubation of debrisoquine with CYP2D1. (C) MRM for the substrate debrisoquine, m/z 176, after incubation with CYP2D2. (D) MRM for the product hydroxydebrisoquine, m/z 192, after incubation of debrisoquine with CYP2D2. Illustrated is the sum of selected MRM. Incubations were carried out for 120 min at 37°C.

Fig. 4.

Conversion of thebaine and codeine by rat CYP2D2. (A) MRM for the substrate thebaine, m/z 312, after incubation with CYP2D2. (B) MRM for the demethylated product, m/z 298, after incubation of thebaine with CYP2D2. (C) MS/MS of m/z 298 formed from thebaine by CYP2D2. (D) MS/MS of oripavine. (E) MRM for the substrate codeine, m/z 300, after incubation of CYP2D2. (F) MRM for the demethylated product, m/z 286, after incubation of CYP2D2 with codeine. (G) MS/MS of m/z 286 formed from codeine by CYP2D2. (H) MS/MS of morphine. Illustrated is the sum of selected MRM. Incubations were carried out for 120 min at 37°C. Note that substrates were completely converted to respective reaction products.

After confirming 3-O-demethylating activity for CYP2D2 with thebaine and codeine, the phenol coupling reaction of (R)-reticuline was examined and enzymatic activities of rat CYP2D1 and CYP2D2 towards this third morphine precursor were compared. Since the mechanism of the phenol-coupling reaction of (R)-reticuline, m/z 330, leads to a total loss of 2 mass units, the MRM for m/z 328 was monitored. Fig. 5 illustrates that while CYP2D1 was inactive, CYP2D2 formed the four expected products corytuberine, pallidine, salutaridine and isoboldine. Using (R)-reticuline as substrate, an optimal pH of 7.4 was determined for CYP2D2. The relative distribution of the four phenol-coupled products was the same after 10 min and 120 min of incubation as well as at any pH between 5 and 9.

Fig. 5.

Test for phenol-coupling activities of CYP2D1 or CYP2D2. (A) MRM for m/z 330 after incubation of CYP2D1 with (R)-reticuline. (B) MRM for m/z 328 after incubation of CYP2D1 with (R)-reticuline. (C) MRM for m/z 330 after incubation of CYP2D2 with (R)-reticuline. (D) MRM for m/z 328 after incubation of CYP2D2 with (R)-reticuline (pallidine 8.92 min, corytuberine 10.25 min, salutaridine 12.58 min and isoboldine 13.44 min). Illustrated is the sum of selected MRM for all four products. Incubations were carried out for 120 min at 37°C.

Michaelis-Menten kinetic parameters revealed that, similar to human CYP2D6, rat CYP2D2 shows a higher K_m value for thebaine and codeine (29–46 µM) in comparison with (R)-reticuline (0.6–1.0 µM) (Table 2). However, catalytic efficiencies for all three substrates became similar due to lower maximum rates (k_cat) for the conversion of (R)-reticuline. Overall catalytic efficiencies for thebaine, codeine and (R)-reticuline were at least an order of magnitude higher than for human CYP2D6 (Table 2 and [8]).

Table 2.

Catalytic parameters for rat P450 2D2.

Substrate	Product	Km, µM	kcat, pmol/min/pmol P450	kcat//Km , mM−1 s−1
(R)-reticuline	Corytuberine	1.0±0.18	0.03±0.001	0.42
	Pallidine	0.8±0.10	0.86±0.03	17.9
	Salutaridine	0.7±0.12	2.26±0.11	53.9
	Isoboldine	0.6±0.10	0.69±0.03	19.3
Thebaine	Oripavine	42±9.0	63±7.6	25.0
Codeine	Morphine	29±4.6	14±1.1	8.0

Including this work, four enzymes are known to catalyze the intramolecular formation of the critical C12–C13 bridge in salutaridine during morphine biosynthesis [8,13,14]. Table 3 compares the relative capacity of these four cytochrome P450s to catalyze the formation of phenol-coupled products from reticuline. While plant salutaridine synthase only generates salutaridine and does not accept the (S)-enantiomer as substrate, the mammalian P450 enzymes appear to be more promiscuous. Among all, rat CYP2D2 is the most efficient mammalian P450 enzyme to produce the critical morphine precursor.

Table 3.

Relative capacity of salutaridine synthase, CYP2D6, CYP3A4 and CYP2D2 to catalyze the oxidative phenol-coupling of (R)- and (S)-reticuline.

enzyme	(−)-corytuberine / (+)-corytuberine (%)	(+)-pallidine / (−)-pallidine (%)	salutaridine / sinoacutine (%)	(+)-isoboldine / (−)-isoboldine (%)
salutaridine synthase [11,12]	nda / nda	nda/ nda	100/ nda	nda / nda
CYP2D6 [8]	4 / 2	23 / 17	7 / 5	66 / 76
CYP2D6 [8]	4 / 2	23 / 17	7 / 5	66 / 76
CYP3A4 [8]	8 / 12	22 / 17	34 / 35	36 / 36
CYP2D2 [this work]	1 / 4	20 / 34	74 / 56	5 / 4

^and…not detected

In conclusion, new enzymatic activities were found for rat CYP2D2 that have been incorrectly proposed for CYP2D1. A similar correction of CYP2D1/CYP2D2 has previously been made for the drug debrisoquine [9,15]. These findings become particularly important for studies relying on results based on comparison of enzyme activities between different rat strains [6,7,16–20]. Thus, the metabolism of some drugs, such as metoprolol, dextromethophan or 3,4-methylenedioxymethamphetamine, might need further evaluation.

• Activity of rat CYP2D1 and 2D2 with three morphine precursors was tested.

• Thebaine and codeine were 3-O-demethylated by CYP2D2, not 2D1.

• CYP2D2 generated four phenol-coupled products from (R)-reticuline.

• CYP2D1 was completely inactive with (R)-reticuline.

• CYP2D2 showed high catalytic efficiencies for the three human CYP2D6 substrates.