Characterization of an Etoposide-Glutathione ... - Ingenta Connect

Current Drug Metabolism, 2006, 7, 897-911

897

Characterization of an Etoposide-Glutathione Conjugate Derived from Metabolic Activation by Human Cytochrome P450 Naiyu Zheng†,¶, Shaokun Pang†,‡, Tomoyuki Oe†, Carolyn A. Felix§, Suzanne Wehrli§ and Ian A. Blair†,* †

Center for Cancer Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6160, USA; Department of Pediatrics, University of Pennsylvania School of Medicine, Division of Oncology, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA; ¶Current address: Department of Bioanalytical Sciences, Bristol-Myers Squibb Company, One Squibb Drive, New Brunswick, NJ 08903, USA and ‡Current address: Takeda San Diego, Inc., 10410 Science Center Drive, San Diego, CA 92121, USA §

Abstract: Etoposide (VP-16), a DNA topoisomerase II poison widely used as an antineoplastic agent is also known to cause leukemia. One of its major metabolic pathways involves O-demethylation to etoposide catechol (etoposide-OH) by cytochrome P450 3A4 (CYP3A4). The catechol metabolite can undergo sequential one- and two-electron oxidations to form etoposide semi-quinone (etoposide-SQ) and etoposide quinone (etoposide-Q), respectively, which have both been implicated as cytotoxic metabolites. However, etoposide-Q is known to react with glutathione (GSH), which can protect DNA from oxidative damage by this reactive metabolite. In this study, etoposide-Q was reacted with GSH and the two etoposide-GSH conjugates were characterized. The major conjugate was etoposide-OH-6’-SG and the minor product was etoposide-OH-2’-SG. Etoposide-OH-6’-SG, which arose from Michael addition of GSH to etoposide-Q, was characterized by mass spectrometry and 2-D NMR. It was identified as the sole product from in vitro metabolism experiments using recombinant human CYP3A4 or liver microsomes incubated with etoposide in the presence of GSH. Etoposide-OH6’-SG was also detected from incubations of etoposide-OH and GSH alone. Therefore, the presence of etoposide-OH, which can be formed from etoposide metabolism by CYP3A4, is essential for formation of the GSH conjugate. The oxidation of etoposide-OH to a quinone intermediate is likely the precursor in the formation of etoposide-OH-6’-SG.

Key Words: Etoposide metabolism, glutathione conjugate, etoposide catechol, etoposide quinone, human liver microsomes, cytochrome P450, mass spectrometry, NMR spectroscopy.

Etoposide (VP-16, 4’-demethylepipodophyllotoxin-9-(4, 6-O-ethyliden--D-glucopyranoside), Fig. (1)) is a DNA topoisomerase II poison that is widely used as an antineoplastic agent. It is highly active against many adult and pediatric solid tumors and leukemias [1]. However, anticancer treatment with etoposide led to a form of secondary leukemia characterized by balanced chromosomal translocations as a treatment complication [2-5]. Approximately 35 % of etoposide administered to human subjects is excreted into urine as a parent drug [6,7] and less than 3 % is excreted into bile [8]. Several metabolites have been identified in human plasma and urine including a cis-(picro) lactone, hydroxy acid derivatives, 3-O-demethyletoposide, and etoposide glucuronide [9-14]. Etoposide glucuronide accounts for the disposition of 15 to 35% of administered etoposide [12,13]. Etoposide glucuronidation was studied using human liver microsomes [15]. UGT1A1 was shown to be the major enzyme responsible for glucuronide formation and there was large inter-individual variability between the different liver microsomes [15].

The etoposide catechol metabolite (etoposide-OH) arises through aromatic-O-demethylation (Fig. (1)) [14,16,17]. Cytochrome P450 3A4 (CYP3A4) was found to play a major role in O-demethylation of etoposide [14,17], and CYP3A5 was found to contribute to etoposide-OH formation from free drug at therapeutic concentrations [18]. Etoposide-OH can undergo sequential one-electron oxidations to form etoposide semi-quinone (etoposide-SQ) and then etoposide quinone (etoposide-Q) [19,20]. Metabolism of etoposide by liver microsomes results in covalent binding of the drug to microsomal proteins [21]. However, there are no reports that this leads to the inactivation of CYP3A4. A quinone metabolite could potentially be responsible for covalent binding to microsomal proteins and also to DNA [16,19,20,22,23]. Formation of a quinone metabolite could induce redox cycling with the concomitant production of reactive oxygen species [24], which would damage DNA directly or trigger lipid peroxidation and the formation of lipid hydroperoxides [25,26]. Lipid hydroperoxides can then undergo homolytic decomposition to form reactive bifunctional electrophiles, such as malondialdehyde, 4-hydroxy-2-nonenal, and 4-oxo-2-nonenal [26]. Thus, the parent drug and its etoposide metabolites are all potential genotoxins [27].

*Address correspondence to this author at the Center for Cancer Pharmacology, University of Pennsylvania School of Medicine, 854 BRB II/III, 421 Curie Boulevard, Philadelphia, PA 19104-6160; Tel: 215-573-9880; Fax: (215)-573-9889; E-mail: [email protected]

Our previous pharmacokinetic studies demonstrated a significant increase in the concentrations of etoposide-OH in the plasma or protein-free plasma in pediatric patients un-

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© 2006 Bentham Science Publishers Ltd.

898 Current Drug Metabolism, 2006, Vol. 7, No. 8

Zheng et al.

Lipid Hydroperoxides

g8

H3C

g6

g7

O O g4 HO

g5

Oxidative DNA Damage

HO. O

g1 O g3 g2 OH 5 11 10 4 O 6 3 C B D O A 2 7 12 9 1 O 8 O 1' 2' 3'

H3CO

E 4'

O2.-

O2--

OH

H3CO

O

H3CO

O

OH

O_

O

etoposide catechol (etoposide-OH)

etoposide simiquinone (etoposide-SQ)

etoposide quinone (etoposide-Q)

OCH3

etoposide

O2.-

O2

GSH

H3CO

OH

DNA Aducts Protein Adducts

HO.

CYP 3A4

6' 5'

Decomposition Products

etoposide catechol (etoposide-OH)

GSH S-transferase

GSH S-transferase COOH S H3CO

SG

NH2

OH

H3CO

OH

SG H

OH

H3CO

O

OH

etoposide-OH-6'-Cys

OH

etoposide-OH-6'-SG (glutathione conjugates)

COOH S H3CO

NHAc

OH

O SG =

H N

COOH NH2

S N H

OH

COOH

etoposide-OH-6'-NAc-Cys

Fig. (1). Metabolic pathway of etoposide oxidation and formation of glutathione conjugates.

dergoing multiple-day chemotherapy with etoposide on day 5 compared with day 1 [28]. These findings may be relevant to the risk of treatment complications, such as secondary leukemia, because etoposide-OH is a genotoxin. However, it is also possible that the genotoxicity of etoposide catechol and quinone metabolites are decreased in vivo through a detoxication pathway involving the formation of GSH conjugates (Fig. (1)). In fact, GSH (L--glutamyl-L-cysteinyl-glycine) has been shown to react with etoposide-Q and etoposide-SQ to partially protect single-stranded and doublestranded DNA through the formation of GSH conjugates [29]. GSH plays an important role in the metabolism of various xenobiotics. It is present in high concentrations in liver tissue (4 mM), and can react with electrophiles either nonenzymatically or enzymatically to form GSH conjugates [30]. GSH conjugates are then catabolized via mercapturic acid biosynthesis to form N-acetylcysteine (N-AcCys) con-

jugates (mercapturic acids) and excreted in urine (Fig. (1)) [30]. Therefore, the formation of GSH conjugates is one of the most important detoxification pathways for many different drugs [31,32]. The structures of potential etoposide-Q GSH conjugates have not yet been established. In this study, a major GSH conjugate of etoposide, etoposide-OH-6’-SG, and a minor conjugate, etoposide-OH-2’-SG, were characterized from the reaction products of etoposide-Q with GSH using LC/MS and NMR spectroscopy. Only the etoposideOH-6’-SG conjugate was formed when etoposide was incubated with human CYP3A4 or liver microsomes in the presence of GSH. MATERIALS AND METHODS Materials Etoposide and GSH (reduced form) were purchased from Sigma (St. Louis, MO). L-Ascorbic acid and ammonium

Characterization of an Etoposide-Glutathione Conjugate Derived

formate were obtained from Aldrich (Milwaukee, WI). HPLC-grade water and trifluoroacetic acid (TFA) were obtained from Fisher Scientific (Fair Lawn, NJ). HPLC-grade acetonitrile was obtained from B&J (Muskegon, MI). Human CYP3A4/P450 reductase Supersomes™ was purchased from Gentest Corp. (Woburn, MA). Human liver microsomes were purchased from In Vitro Technologies (Baltimore, MD). NMR The proton; two-dimensional 1H, 1H chemical shifts correlation spectroscopy (COSY); and long-range COSY NMR spectra of etoposide-OH and etoposide-OH-6’-SG were performed using a Bruker (Billerica, MA) DMX-400 NMR spectrometer operating at 400 MHz using the XWIN NMR version 2.5 software. One dimensional 1H spectra were obtained from roughly 5 mg of compound dissolved in 0.5 mL MeOH-d4 using a 5 mm o.d. inverse probe under the following conditions: sweep width (SW) 6775 Hz, 64k data points (0.1 Hz/point digital resolution), pulse width (PW) 45o, repetition time (TR) 8.8 s, and 32 transients (5 min). The combined water and exchangeable proton signal was saturated during the 4 s relaxation delay. The 2D experiments were acquired with the following conditions: SW2 and SW1 4125Hz (10 ppm centered at 4.9 ppm), 1024 data points in F2 and 256 experiments in F1, 24 transients. Data were zero filled once in F1. A sinebell window was applied before Fourier transformation. An additional delay of 60 ms was used for the long range J COSY. Chemical shifts were reported relative to MeOH-d4 (3.31 ppm), and the coupling constants (J) are given in Hertz (Hz). Mass Spectrometry The data were acquired on a Finnigan LCQ ion trap mass spectrometer (Thermo Electron, San Jose, CA) equipped with a Finnigan electrospray ionization (ESI) source. The mass spectrometer was operated in the positive ion mode with a potential of 4.50 kV applied to the ESI needle. Nitrogen was used as the sheath (60 units) and auxiliary (5 units) gas to assist with nebulization. The capillary temperature was held at 200 °C. Full scan analyses were performed in the range of m/z 100 to 900. Multiple collision-induced dissociation (CID) experiments coupled with multiple tandem mass spectrometry (MSn) were conducted using helium as the collision gas. All CID spectra were acquired under the optimal collision conditions determined individually for the analytes. Liquid Chromatography Reversed phase LC was performed using a Waters Alliance 2690 HPLC system (Milford, MA) on a YMC ODS-AQ column (150 mm 2.0 mm i.d., 120 Å, 3 μm; YMC, Inc., Wilmington, NC) preceded by a pre-column filter (2 μm; Alltech, Deerfield, IL). The mobile phases consisted of 5 mM HCOONH4 and 0.01% TFA water solution with 5% acetonitrile as A and with 90% acetonitrile as B. The analytes were eluted with a linear gradient at a flow rate of 0.2 mL/min. The gradient conditions were as follows: 20% B at 0 min, 20% B at 0.3 min, 100% B at 5 min, 100% B at 8 min, and 20% B at 9 min, followed by a 6 min equilibration time.

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For preparative HPLC, separations were performed using a Hitachi L-6200 intelligent pump on a YMC ODS-AQ column (150 mm 10 mm i.d., 120 Å, 5 μm; YMC, Inc., Wilmington, NC) at a flow rate of 3 mL/min. HPLC chromatograms were obtained by using a Hitachi L-4200 UV detector at 289 nm. The mobile phases consisted of 5% acetonitrile in water as A and 90% acetonitrile in water as B. The linear gradient conditions were as follows: 20% B at 0 min, 100% B at 10 min, 100% B at 12 min, and 20% B at 15 min, followed by a 5 min equilibration time. Synthesis of Etoposide-Q and Etoposide-OH Etoposide-Q was synthesized by oxidation of etoposide with sodium metaperiodate (NaIO4) as described by Nemec [33]. Etoposide-OH was synthesized by reduction of etoposide-Q with sodium borohydride (NaBH4) using a modification of previously described methods [14, 29]. The detailed synthetic procedure was described previously [34]. 1H-NMR for etoposide-OH (400 MHz, MeOH-d4, ): 6.95 (s, 1H, 5H), 6.49 (s, 1H, 8-H), 5.95 (d, J = 1.2 Hz, 1H, A-Ha), 5.94 (d, J = 1.2 Hz, 1H, A-Hb), 6.43 (d, J =1.6 Hz, 1H, 2’-H), 5.86 (d, J =1.6 Hz, 1H, 6’-H), 4.49 (d, J = 5.2 Hz, 1H, 1-H), 4.98 (d, J = 3.6 Hz, 1H, 4-H), 4.75 (q, J = 5.0 Hz, 1H, g7-H), 4.63 (d, J = 7.6 Hz, 1H, g1-H), 4.41 (dd, J = 8.8, 10.8 Hz, 1H, 11Ha), 4.26 (t, J = 8.4 Hz, 1H, 11-Hb), 4.16 (dd, J = 4.8,10.4 Hz, 1H, g6e-H), 3.41 (dd, J = 5.2, 14.0 Hz, 1H, 2-H), 3.57 (t, J = 9.6 Hz, 1H, g6a-H), 3.76 (s, 3H, 3’-OCH3), 3.52 (t, J = 9.2 Hz, 1H, g3-H), 3.22-3.30 (m, 3H, remaining protons), 2.97 (m, 1H, 3-H), 1.31 (d, J = 4.8 Hz, 3H, g8-H). Synthesis of Etoposide-OH-6’-SG and Etoposide-OH-2’SG A solution of GSH (reduced form, 3.4 mg, 11 μmol) in 1.1 mL of water was added to etoposide-Q (3.0 mg, 5.25 μmol) in water (0.3 mL) and 2.2 mL of potassium phosphate buffer solution (0.18 M, pH 4.3). The reaction mixture was kept at room temperature for 15 min. HPLC analysis of the reaction mixture on a YMC ODS-AQ column (150 mm 2.0 mm i.d., 120 Å, 3 μm) showed two products, the major product etoposide-OH-6’-SG with retention time of 4.05 min and the minor product etoposide-OH-2’-SG with retention time of 3.46 min. After separation on a semi-preparative YMC ODS-AQ column (150 mm x 10 mm i.d., 120 Å, 5 μm), etoposide-OH-6’-SG was obtained as amorphous powder with >99% purity. Etoposide-OH-6’-SG had UV absorption in a methanol solution at max = 291 nm. 1H-NMR of etoposide-OH-6’-SG (400 MHz, MeOH-d4, ): 6.87 (s, 1H, 5-H), 6.35 (s, 1H, 8-H), 5.93 (d, J = 7.6 Hz, 2H, A-H), 5.82 (s, 1H, 2’-H), 5.55 (d, J = 7.2 Hz, 1H, 1-H), 5.03 (d, J = 2.4 Hz, 1H, 4-H), 4.76 (q, J = 4.8 Hz, 1H, g7-H), 4.57 (d, 1H, g1-H), 4.40 (m, 1H, -Cys-H), 4.35 (m, 2H, 11-H), 4.18 (dd, J = 4.2, 10.2 Hz, 1H, g6e-H), 3.85 (dd, J = 18 Hz, 2H, GlyH), 3.65 (dd, J = 7.2, 14.0 Hz, 1H, 2-H), 3.65 (m, 1H, -GluH), 3.60 (m, 1H, g6a-H), 3.57 (s, 3H, 3’-OCH3), 3.53 (m, 1H, g3-H), 3.47 (m, 1H, -Cys-Ha), 3.20-3.35 (m, 4H, remaining protons), 3.01 (m, J = 4.0 Hz, 1H, -Cys-Hb), 2.55 (dd, J = 14.0 Hz, 2H, -Glu-H), 2.10 (m, 2H, -Glu-H), 1.31 (d, 3H, g8-H). ESI-MS (m/z): 880 [M+H]+ (C38H45O19N3S).


Zheng et al.

above. In another experiment, 34 μM of etoposide was replaced by etoposide-OH, and the rest of the procedures were the same as above.

Microsomal and CYP3A4 Incubation A solution (250 μL) containing 50 mM Tris buffer (pH 7.4), 3 mM MgCl2, 68 μM etoposide, 2 mM GSH and 50 pmol CYP3A4 was incubated at 37 C for 5 min, then NADPH (final concentration 1.4 mM) was added. The mixture in an open vial was incubated in a 37 C water bath with agitation for 60 min. At the end of incubation, 800 μL of acetonitrile was added to the incubation mixture to stop the reaction. The incubation mixture was centrifuged, the protein precipitate was discarded, and the supernatant was dried with a stream of nitrogen gas. The resulting residue was reconstituted into 22% aqueous acetonitrile (200 μL). An aliquot of the solution (20 μL) was analyzed by LC/multi-stage tandem mass spectrometry (MSn). In the control sample, NADPH was replaced by water, and the rest of the components were the same as described above. For the in vitro test on human liver microsomes, an incubation mixture (250 μL) containing 50 mM Tris buffer (pH 7.4), 34 μM etoposide, 1 mM GSH, 0.5 mg microsomes, and 1.0 mM NADPH was used. The procedure was the same as that for CYP3A4.

Effects of pH and Ascorbic Acid on the Formation of Etoposide-OH-6’-SG from Etoposide-OH To each of the three GSH solutions (GSH reduced form, 1 mM, 20 μL) was added etoposide-OH (1.75 mM, 5.8 μL). Then to each of the three resulting mixtures was added one of three phosphate buffers (0.5 M, 40 μL) with different pHs (pH = 4.3, 7.0 and 9.4), respectively. After adding water (134.2 μL) for each, the resulting solutions were kept at room temperature for 60 min. The incubation products were analyzed by using LC/MSn as described above. These experiments were repeated, but ascorbic acid (20 mM, 20 μL) in water (114.2 μL) was used instead of water (134.2 μL). The incubation products were analyzed by using LC/MS as described above. RESULTS Reaction of Etoposide-Q with GSH

Formation of Etoposide-OH-6’-SG from Etoposide and Etoposide-OH by Denatured Liver Microsomes

The reaction of etoposide-Q with GSH was completed immediately (< 1 min) as indicated by the bleaching of the red-brown color of the quinone form. LC/MS analysis of the reaction mixture revealed the formation of etoposide-OH. The result showed that the reaction at higher pH produced relatively high yield of the conjugates; however, the conjugate decomposed at high pH, so the reaction was controlled at pH 4.0-7.0. GSH reacted with etoposide-Q by Michael addition. As shown in Fig. (2), there are two possible prod-

Human liver microsomes were denatured by heating at 100 oC for 20 min and cooling down at room temperature. A 250-μL incubation mixture containing 50 mM Tris buffer (pH 7.4), 34 μM etoposide, 1.0 mM GSH, and 0.5 mg denatured liver microsomes was incubated at 37 C for 5 min, then NADPH (final concentration 1.0 mM) was added. The incubation products were analyzed by LC/MS as described

H3C

O O HO

O O OH

O O O O 6'

2'

GSH

GSH H3CO

O O

H3C

O O HO

etoposide-Q O O OH

H3C

O O HO

O O OH

O

O O

O

O O

6'

H3CO

O SG

OH OH

etoposide-OH-6'-SG

Fig. (2). Two possible reaction products of etoposide-Q reacted with GSH.

O GS 2'

H3CO

OH OH

etoposide-OH-2'-SG



ucts: 1) etoposide-OH-6’-SG formed from the reaction of GSH with etoposide-Q at the 6’ position through 1, 6 addition; 2) etoposide-OH-2’-SG formed from the reaction of GSH with etoposide-Q at the 2’ position through 1, 4 addition. As predicted, two GSH conjugates were detected by LC/MS/MS analysis. The major product, whose structure was confirmed later to be etoposide-OH-6’-SG, had a retention time at 4.05 min (Fig. (3)). The minor product, which was identified later as etoposide-OH-2’-SG, had a retention time at 3.46 min (Fig. (4)).

901

LC/MS Analysis of Etoposide-OH-6’-SG Conjugate

at m/z 751 and m/z 805. The product ion at m/z 751 resulted from the loss of 129, a fragment of -glutamate moiety, which is a typical fragmentation for GSH [35]. Another product ion at m/z 805 was due to the loss of the glycine moiety. LC/MS2 analysis on the CID of m/z 880 (Fig. (3C)) indicated the formation of an intense product ion at m/z 751, along with very weak product ions at m/z 805, 734, 674, 545, 399 and 303 (Fig. (3C)). The product ions m/z 751 and 805 were explained above. The product ion m/z 674 was due to the loss of glucopyranoside moiety in the etoposide-OH-6’SG. The proposed fragmentation pathways for the formation of m/z 734, 545, 399 and 303 are shown in Fig. (3).

The GSH conjugates were separated as minor and major peaks on HPLC and gave two identical MH+ ions at m/z 880 (Figs. (3) and (4)). LC/MS analysis of the major conjugate, etoposide-OH-6’-SG, is shown in Fig. (3). The full scan mass spectrum of etoposide-OH-6’-SG (Fig. (3B)) indicated the formation of an MH+ ion at m/z 880, and two weak ions

CID of m/z 751 (MS3) gave rise to the product ions at m/z 734, 605, 545, 473, 417, 399 and 303 (base peak ion), respectively (Fig. (5)). The proposed fragmentation diagrams for the CID of m/z 751 are shown in Fig. (5). As discussed below, the proposed structure for etoposide-OH-6’-SG from LC/MSn analysis has been confirmed by NMR analysis.

H3C

O O HO

O OH

O 751

O

674 545

734

COOH HN H N

O O O

NH2 COOH

O O 6'

399 S 303

H3CO

805

OH OH

Fig. (3). LC/MSn analysis of the major glutathione conjugate, etoposide-OH-6’-SG. (A) Reconstructed total ion current (TIC) chromatogram for etoposide-OH-6’-SG at 4.05 min. (B) Positive ESI full scan mass spectrum. (C) Positive ESI MS/MS spectrum of the protonated molecule at m/z 880.


Zheng et al.

H3C O 751

HOOC NH2

O O HO

O O

OH

545

NH

674

O O

H N

O

399

HOOC O

O

NH4+ 624

805

2'

S H3CO

OH OH

Fig. (4). LC/MSn analysis of the minor glutathione conjugate, etoposide-OH-2’-SG. (A) Reconstructed total ion current (TIC) chromatogram for etoposide-OH-2’-SG at 3.46 min. (B) Positive ESI full scan mass spectrum. (C) Positive ESI MS/MS spectrum of the protonated molecule at m/z 880.

LC/MS Analysis of Etoposide-OH-2’-SG Conjugate Etoposide-OH-2’-SG was identified as a minor product as indicated in the HPLC chromatogram (Fig. (4A)). The full scan mass spectrum of the minor conjugate, etoposide-OH2’-SG, showed the MH+ ion at m/z 880 as well as a product ion at m/z 878 not found in the MS spectrum of the major product, etoposide-OH-6’-SG (Fig. (4B)). The CID of product ion m/z 880 (MS2) for etoposide-OH-2’-SG yielded a product ion at m/z 749, in addition to a base peak at m/z 751 (Fig. (4C)). The fragment ions m/z 878 and 749, known as “M-2” type fragment ions, were previously reported to distinguish the structural isomers of GSH conjugates of estrone and estradiol [36]. These “M-2” type fragment ions (m/z 878, 749) provided unique spectroscopic information to distinguish the structure of etoposide-OH-2’-SG from that of etoposide-OH-6’-SG as shown in Fig. (2). Structurally, the “M-2" type fragment ions are formed from two-electron oxidations of the GSH conjugate (Fig. (6)). The formation of m/z 878 and m/z 749 ions suggested that the sulfur group in etoposide-OH-2’-SG was in para- position to a hydroxy group because S-O para-quinones could be easily formed

when in this configuration. Thus, etoposide-OH-2’-SG was formed by the addition of glutathione to etoposide quinone at the C-2’ position. CID of m/z 751 (MS3) for etoposide-OH-2’-SG showed the formation of a product ion at m/z 399 with an intensity higher than m/z 303 (Fig. (7)). However, the product ion at m/z 303 was observed as a base peak in etoposide-OH-6’-SG (Fig. (5)). The more intense peak at m/z 399 for etoposideOH-2’-SG was due to the C-S bond in the cysteine moiety between the - methylene and sulfur group in para- position to the hydroxy group, which can be fragmented more easily than in etoposide-OH-6’-SG (Fig. (6)). These all were unique characteristics that could distinguish etoposide-OH2’-SG from the major conjugate, etoposide-OH-6’-SG. A full scan mass spectrum of etoposide-OH-2’-SG showed another ion at m/z 624, which was the ammonium adduct of the fragmented etoposide-OH-2’-SG shown in Fig. (4). The proposed fragmentation pathways for the corresponding mass spectra for etoposide-OH-2’-SG are shown in Fig. (4), Fig. (6) and Fig. (7), respectively.



O

O

O

O O O H S

H3CO

903

O O

NH2

O O

H N

COOH

O

OH

SH

SH

H3CO

OH

H3CO

O

O

OH m/z 545

O

m/z 303

m/z 399

m/z 734 OH O

O HO

O

O

O

O O OH O

O

O

O H S

SH

O

O O HO O

O

O

H3CO

H3C

O OH

O O

-NH3

O

H3C

OH

H3CO

OH

OH

OH

m/z 417

m/z 473

O

O H

O H S

NH2

O H N

COOH

SH

O OH

H3CO

O

H3CO

O OH

OH m/z 751

m/z 605

Fig. (5). Positive ESI MS3 spectrum of the product ion of m/z 751 from etoposide-OH-6’-SG and proposed fragmentation pathway for m/z 751.

NMR Analysis of Etoposide-OH-6’-SG Conjugate Before the structure of etoposide-OH-6’-SG was elucidated, NMR experiments on etoposide-OH were carried out. The proton signals of etoposide-OH were assigned based on the proton chemical shifts, coupling constants (Table 1) and 1 H-1H COSY correlation (Fig. (8)). Structurally, etoposideOH was different from etoposide with a hydroxyl group at C-5’ as compared with the methoxy group at C-5’ for etoposide (Fig. (1)). Unlike etoposide, the protons at C-2’ and C-6’ for etoposide-OH were not equivalent to each other. The signals at 5.86 ppm and 6.43 ppm for etoposideOH were assigned to H-6’ and H-2’ protons, respectively, because H-6’ was shifted upfield by the ortho hydroxy group at C-5’ due to its -donor effect [37]. In addition, the COSY spectrum of etoposide-OH showed the correlation between the H-2’ proton at 6.43 ppm and 3’-OCH3 protons at 3.76 ppm (Fig. (8)), which suggested that the H-2’ proton was

adjacent to the 3’-OCH3 protons. The remaining proton signals (Table 1) were quite similar to those for etoposide reported previously [38]. Theoretically, the reaction between etoposide-Q and GSH by Michael addition could produce two conjugates, with an addition at C-2’ for one product and at C- 6’ for another (Fig. (2)). Similar regiochemistry of the reaction is well known in the conjugates of estrogen quinone with thiol nucleophiles [39]. Since the structures of both conjugates from the reaction between etoposide-Q and GSH contained a etoposide-OH moiety, the NMR analysis of etoposide-OH provided a useful spectroscopic reference for comparison with the spectroscopic data of etoposide-OH-6’SG. In order to confirm the conjugation site of GSH to etoposide-Q, the major product (etoposide-OH-6’-SG) was purified and analyzed by 1H and 1H-1H COSY spectroscopy. The 1H NMR assignments for etoposide-OH-6’-SG are


H3C

O O HO

Zheng et al.

O O HO

H3C

O O

O

H3C

O

OH

OH

O

O O

H O O N

HOOC NH2

O H S 2'

N H

HOOC

H O O N

H3CO

OH

O HS

H3CO

OH

H3CO

OH

m/z 880

m/z 624

-2H

O O HO

H3C

O O

O O HO

O

O

O

O OH

OH

O

O O

O O

H O O N

O H O O N

HOOC O

NH2 HOOC

OH OH

m/z 751

-2H

HOOC

O O

O

OH

H3C

O OH

H S 2'

H2N

NH4+

O

O

O HOOC

O O HO

S

N H

H2N

H3CO m/z 878 (M-2 Pattern)

OH

O S

H3CO 2'

m/z 749 (M-2 Pattern)

O OH

H3CO

O

HS

O

m/z 399 (Intense Peak)

OH

Fig. (6). Proposed “M-2” fragmentation pathway and typical fragment ions for etoposide-OH-2’-SG observed from mass spectral analysis.

shown in Table 2. They are deduced from 1H-1H COSY and long-range COSY spectrum (Fig. (9)). In 1H-1H COSY spectrum, the characteristic couplings for etoposide-OH-6’-SG were observed between the protons -Cys-H (1 proton at 4.40 ppm) and -Cys-H (2 protons at 3.01 ppm and 3.47 ppm, respectively), -Glu-H (1 proton at 3.65 ppm) and Glu-H (2 protons at 2.10 ppm), -Glu-H and -Glu-H (2 protons at 2.55 ppm). A proton signal (d,d, AB coupling) at 3.85 ppm was assigned to the two protons at the position of the Gly residue. The integration of the signals for etoposide-OH6’-SG indicated the absence of one proton signal in the Ering (Fig (9)), as compared with that for etoposide-OH (Fig (8)). This meant that the conjugate was formed by Michael addition of the thiol to the aromatic E-ring. Structurally, both C-2’ and C-6’ carbons were possible conjugation sites for GSH. However, the signal at 5.82 ppm was assigned to H-2’ due to its correlation with 3’-OCH3 protons at 3.57 ppm in the long-range COSY spectrum (Fig. (9)). No signal corresponding to the H-6’ proton was detected. Therefore, the Michael addition site of GSH was at the C-6’ carbon of the E-ring. The signal of H-2’ in etoposide-OH-6’-SG was shifted upfield, which might be due to the -donor effect of sulfur group on the benzene ring [37]. This effect also

caused the 3’-OCH3 protons at 3.57 ppm to be shifted upfield, as compared with 3.76 ppm for etoposide-OH. The assignments for the rest of the protons could be easily deduced from the 1H-1H COSY spectrum and long-range COSY spectrum. The chemical shifts and coupling constant in the 1H NMR spectrum of etoposide-OH-6’-SG are shown in Table 2. Thus, the NMR data of etoposide-OH-6’-SG confirmed the mass spectral data that the Michael addition site of GSH was at the C-6’ carbon of the E-ring. These data also indirectly suggested that etoposide-OH-2’-SG must be formed from Michael addition of GSH to etoposide-Q at the C-2’ carbon of the E-ring. Formation of Etoposide-OH-6’-SG from Etoposide in vitro by Human CYP3A4 or Human Liver Microsomes In the presence of GSH, etoposide was incubated with recombinant human CYP3A4 or human liver microsomes, respectively. As shown in Fig. (10A) and Fig. (10B), LC/MS chromatograms obtained from human CYP3A4 or human liver microsomal incubations showed an intense peak at 3.99 min and 4.00 min, respectively. The retention times for both peaks were very close to that of the etoposide-OH-6’-SG standard sample (Fig. (10C)). A full scan mass spectral



O

O

O O

H O O N

HOOC

H2N

m/z 528

O O

O O

O

H S 2'

-NH3

HS

HS

H3CO

905

OH

H3CO

OH

H3CO

O

O OH

OH m/z 399

m/z 545

m/z 303

m/z 734 -NH3 OH

H3C

O

O O HO

H3C

O O OH

O O

O H S H

HOOC

H3CO

OH

O O OH

O O

O O

O O HO

H O O N H2N

OH

m/z 473

O O

O H S 2'

O HS

H3CO

OH

H3CO

O

OH

OH

m/z 751

m/z 605

Fig. (7). Positive ESI MS3 spectrum of the product ion of m/z 751 from etoposide-OH-2’-SG and proposed fragmentation pathway for m/z 751.

analysis showed an intense ion of MH+ at m/z 880 for both HPLC peaks, respectively (data not shown). No “M-2” type fragment ion at m/z 878 was found. As shown in Fig. (10A) and Fig. (10B), CID of m/z 880 (MS2) for both peaks revealed the same mass spectral characteristics as those of the etoposide-OH-6’-SG standard sample (Fig. (10C)). Also, no “M-2” type fragment ion at m/z 749 was found in the CID spectra of m/z 880 (MS2). The mass spectral data suggested that the product was etoposide-OH-6’-SG, not etoposideOH-2’-SG. These results confirmed that only etoposide-OH6’-SG was generated in the in vitro tests on both CYP34A and human liver microsomes.

Effects of Enzyme, pH and Ascorbic Acid on in vitro Formation of Etoposide-OH-6’-SG Etoposide or etoposide-OH was incubated with denatured microsomes in the presence of GSH. When etoposide was used, no GSH conjugate or etoposide-OH was formed. However, when etoposide-OH was used, etoposide-OH-6’-SG was formed (Fig. (10D)). Additional experiments were carried out in which etoposide-OH was incubated with GSH in the absence or presence of ascorbic acid at pH 4.3, 7.0, or 9.4. These experiments were performed in the absence of CYP3A4 or human liver microsomes. The results indicated that etoposide-OH-6’-SG could be formed from the reaction


Zheng et al.

of etoposide-OH with GSH under all pH conditions, but the yield of etoposide-OH-6’-SG increased with the increase in pH (Fig. (11), solid bar). In the presence of the powerful reducing agent, ascorbic acid, the formation of etoposideOH-6’-SG was remarkably suppressed even at higher pH (Fig. (11), open bar). Table 1.

1

H NMR Assignments for Etoposide-OH (400 MHz, in MeOH-d4)

Assigned H Chemical shift (ppm) Multiplicity H-coupled [J (Hz)] g8

1.31(3H)

d

H-g7 (4.8)

3

2.97

m

2, 4, 11a, b

m

g1, g3

6’-SG) was found in the experiments with human liver microsomes or recombinant CYP3A4. Similarly, only one major conjugate was formed when etoposide-OH was incubated with boiled human microsomes in the presence of GSH (Fig. (10D)). This suggests that the regioselectivity of GSH conjugation was due to relative amounts of catechol and GSH that were present. Under normal physiological conditions, GSH is present in large excess (typically 4 mM) over etoposide-OH (typically nM), and only the 6’-isomer will be formed. In contrast, no GSH conjugate or etoposide-OH was formed from incubation of etoposide with denatured microsomes alone. This was due to the absence of enzymes to carry out the necessary O-demethylation of etoposide to form etoposide-OH. g8

a

H3C

g7

g6

O

g2

3.22

g4

3.22 a

t

g3 (9.0)

O g4 HO

g5

3.30 a

m

g6a, g6e

O

g3

3.52

t

g2, g4 ( 9.2)

g3

3.76 (3H)

s

g2

A

g1 O

OH 5

O 3'-OCH3

g5 O

10

6 B 7 8

9

2

3.57 3.41

t dd

g5 (9.6)

2'

1, 3 (5.2, 14.0)

3'

H3CO

a

g6e

4.16

dd

g5, g6a (4.8, 10.4)

11a

4.26

t

3 (8.4)

11b

4.41

dd

3, 11a (10.8, 8.8 )

g1

4.63

d

g2 (7.6)

g7

4.75

q

g8 (5.0)

4

4.98

d

3 (3.6)

1

4.49

d

2 (5.2)

6'

5.86

d

2' (1.6)

2'

6.43

d

6' (1.6)

A

a 5.94

d

Ab (1.2)

b 5.95

d

Aa (1.2)

8

6.49

s

5

6.95

s

D

O 12

O 1'

g6a

11

4 C 3 2 1

E 4'

6' 5'

OH

OH

Overlapping signals

DISCUSSION Etoposide is converted to etoposide-OH by CYP3A4mediated demethylation, then the catechol undergoes sequential one-electron oxidations to form etoposide-SQ and etoposide-Q (Fig. (1)) [14-16,19,21,22]. In order to determine whether the GSH conjugate could be formed as a result etoposide metabolism, the parent drug was incubated with human CYP3A4 or liver microsomes in the presence of GSH. With both in vitro systems, the formation of etoposideOH-6’-SG was observed by LC/MS (Figs. (10A, 10B)). Interestingly, only one major GSH conjugate (etoposide-OH-

Fig. (8). 1H-1H two-dimensional COSY NMR spectrum of etoposide-OH (400 MHz, in MeOH-d4) and the structure of etoposide-OH.


Table 2.

907

Chemical Shifts and Coupling Constant of the Protons of Etoposide-OH-6’-SG Assigned on the Basis of 1H-1H COSY Spectrum (400 MHz, MeOH-d4) Assigned H

Chemical shift (ppm)

No. of protons

Multiplicity

H-coupled [J(Hz)]

g8

1.31

3H

d

4.8

-Glu-H

2.10

2H

broad,m

Glu-H

2.55

2H

broad,m

14.0

-Cys-Ha

3.01

4.0

1H

m

3

3.20

a

1H

m

g2

3.28 a

1H

m

3.32

a

2H

m

-Cys-Hb

3.47

a

1H

m

g3

3.53 a

1H

m

3.57

a

3H

s

g6a

3.60

a

1H

m

-Glu-H

3.65 a

1H

m

2

3.65 a

1H

dd

14.0, 7.2

Gly-H

3.85

2H

d, d (AB)

18.0

g6e

4.18

1H

dd

10.2, 4.2

11

4.35

2H

m

-Cys-H

4.40

1H

m

g1

4.57

1H

d

g7

4.76

1H

q

4.8

4

5.03

1H

d

2.4

1

5.55

1H

d

7.2

2'

5.82

1H

s

A

5.93

2H

d (AB)

8

6.35

1H

s

5

6.87

1H

s

g4, g5

3’-OCH3

a


7.6

Overlapping signals

The formation of etoposide-OH-6’-SG from incubation of etoposide-OH with denatured microsomes in the presence of GSH suggests that once etoposide-OH is present, it can form either etoposide-SQ or etoposide-Q through autooxidation, which leads to the formation of etoposide-OH-6’SG. However, the oxidation of etoposide-OH to etoposide-Q in the presence of GSH (acting as a reducing agent) seems unlikely because our results showed that etoposide-Q could be reduced to etoposide-OH in the presence of GSH. Therefore, etoposide-SQ is more likely to be the intermediate involved in the formation of etoposide-OH-6’-SG. Since etoposide-SQ can be generated from etoposide-OH under neutral and basic pH conditions [20,40], or at pH >7.4 as shown by another report [20], we speculate that the mecha-

nism for the formation of etoposide-OH-6’-SG from etoposide-OH involves the initial formation of etoposide-SQ. It was reported previously that etoposide was oxidized to a phenoxyl radical, then to etoposide-Q in a tyrosinase-catalyzed system [41-43]. However, CYP3A4-mediated formation of etoposide-GSH-conjugates appears to involve intermediate formation of etoposide-OH, which then undergoes auto-oxidation to etoposide-SQ, which in turn forms a conjugate with GSH. In order to test whether or not etoposide-OH-6’-SG can also be formed directly from etoposide-OH without the intermediate formation of etoposide-SQ, experiments were carried out at pH 4.3, 7.0, and 9.4. We assumed little etoposide-SQ would be generated at pH = 4.3. Results indi-

908 Current Drug Metabolism, 2006, Vol. 7, No. 8 g8

H3C

Zheng et al.

g7

g6

O O g4 HO

g5 O g3

O A

O

g2

g1 O

OH

5

10

6 B 7 9

8

2' 3'

H3CO

4 C 3 2 1

11 D

O

H Gly O N COOH O NH2 Cys 1' 6' S N COOH Glu H E 5' 12

4'

OH

OH

Fig. (9). 1H-1H long range COSY NMR spectrum of etoposide-OH-6’-SG (400 MHz, in MeOH-d4) and the structure of etoposide-OH-6’-SG.

cated that etoposide-OH-6’-SG could be formed from the reaction of etoposide-OH with GSH under all pH conditions, but the yield of etoposide-OH-6’-SG decreased significantly with the decrease in pH (Fig. (11), solid bar). This is probably because less etoposide-SQ was generated at lower pH, and as a result, less etoposide-OH-6’-SG was formed. This suggests that etoposide-OH is spontaneously oxidized to etoposide-SQ and then conjugated with GSH. This hypothesis is supported further by another experiment, in which etoposide-OH was mixed with GSH in the presence of ascorbic acid, a more powerful reducing agent [41]. The formation

of etoposide-OH-6’-SG was remarkably suppressed even at higher pH (Fig. (11), open bar). This suggests that when a one-electron auto-oxidation of etoposide-OH to etoposideSQ is inhibited by the addition of a more powerful reducing agent, the formation of etoposide-OH-6’-SG is suppressed. Therefore, the formation of etoposide-SQ may be a necessary precursor in the formation of etoposide-OH-6’-SG. A previous report showed that etoposide-SQ was responsible for the ortho-quinone-induced ss MX174 DNA inactivation [20]. Etoposide-SQ also binds irreversibly to microsomal proteins [45]. The present study has demonstrated that



909

Fig. (10). Formation of etoposide-OH-6’-SG in the in vitro tests indicated by LC/MS/MS analysis. (A) Incubation of etoposide and human CYP3A4 in the presence of GSH. (B) Incubation of etoposide and human liver microsomes in the presence of GSH. (C) Etoposide-OH-6’SG standard. (D) Incubation of denatured human liver microsomes and etoposide-OH in the presence of GSH.

etoposide-SQ can react with GSH to form etoposide-OH-6’SG conjugate in vitro. As a result, the free radicals normally generated by etoposide-SQ through redox cycling cannot arise. Therefore, the formation of etoposide-OH-6’-SG could protect DNA from damage caused by etoposide and its cytotoxic metabolites. In summary, two GSH conjugates were identified in the chemical reaction of etoposide-Q with GSH. The major conjugate was identified as etoposide-OH-6’-SG, which was formed from Michael addition of the sulfhydryl group of GSH to the C-6’ position on the E-ring in etoposide-Q. The minor product, on the other hand, was identified as etoposide-OH-2’-SG which was formed from Michael addition of GSH at the C-2’ position on the E-ring of etoposide-Q. Interestingly, only etoposide-OH-6’-SG was detected in the in vitro experiments in which human CYP3A4 or liver micro-

somes were incubated with etoposide in the presence of GSH. The same GSH conjugate was also detected after incubations of etoposide-OH with GSH in the absence of CYP3A4 or liver microsomes. Therefore, the presence of etoposide-OH, which can be formed from etoposide metabolism by CYP3A4, is essential for generation of the GSH conjugate. The oxidation of etoposide-OH to the corresponding semiquinone (etoposide-SQ) appears to be a major pathway that leads to the formation of etoposide-OH-6’-SG. These results provide new insights on etoposide as a potential genotoxin. Further studies on the relevance of etoposide-OH to secondary leukemia are needed; especially in the context of determining leukemia risk based on polygenetic determinants of etoposide metabolism and specific CYP 3A4 and GST polymorphisms [46].


Zheng et al.

NOE

= Nuclear Overhauser effect

P450

= Cytochrome P450

TIC

= Total ion chromatogram

REFERENCES [1] [2]

[3] [4] [5] [6] [7]

Fig. (11). Formation of etoposide-OH-6’-SG from etoposide-OH at pH 4.3, 7.0 and 9.4 in the reducing conditions with GSH in the presence of ascorbic acid (empty bar) or absence of ascorbic acid (solid bar).

ACKNOWLEDGEMENTS This work was supported by NIH Grants CA 80175 and CA 77683. We thank Dr. Michael Pollack for editorial assistance.

[8] [9] [10] [11] [12]

[13]

ABBREVIATIONS

[14]

CID

= Collision induced dissociation

[15]

COSY

=

CYP3A4

= Cytochrome P450 3A4

[17]

Cys

= Cysteine

[18]

dd

= Doublet of doublets

ddd

= Doublet of doublets of doublets

ESI

= Electrospray ionization

etoposide

= 4’-demethylepipodophyllotoxin-9-(4,6O-ethylidene--D-glucopyranoside)

1

H,1H-2D correlation spectroscopy

etoposide-OH = Etoposide catechol

[16]

[19]

[20] [21] [22]

etoposide-Q

= Etoposide quinone

etoposideOH-2’-SG

= Etoposide catechol-2’-glutathione conjugate

[23]

etoposideOH-6’-SG

= Etoposide catechol-6’-glutathione conjugate

[24]

etoposide-SQ = Etoposide semi-quinone

[25]

GSH

= Glutathione or L--glutamyl-L-cysteinylglycine

[26] [27]

LC/MS

= Liquid chromatography/mass spectrometry

[28]

m

= Multiplet

MSn

= Multiple tandem mass spectrometry

[30]

NMR

= Nuclear magnetic resonance

[31]

[29]

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Received: December 20, 2005

Revised: July 10, 2006

Accepted: August 2, 2006


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