Selective estrogen receptor modulator (SERM) lasofoxifene forms reactive quinones similar to estradiol

Abstract

The bioactivation of both endogenous and equine estrogens to electrophilic quinoid metabolites has been postulated as a contributing factor in carcinogenic initiation and/or promotion in hormone sensitive tissues. Bearing structural resemblance to estrogens, extensive studies have shown that many selective estrogen receptor modulators (SERMs) are subject to similar bioactivation pathways. Lasofoxifene (LAS), a third generation SERM which has completed phase III clinical trials for the prevention and treatment of osteoporosis, is currently approved in the European Union for this indication. Previously, Prakash et al. (Drug Metab. Dispos. (2008) 36, 1218-1226) reported that similar to estradiol, two catechol regioisomers of LAS are formed as primary oxidative metabolites, accounting for roughly half of the total LAS metabolism. However, the potential for further oxidation of these catechols to electrophilic o-quinones has not been reported. In the present study, LAS was synthesized and its oxidative metabolism investigated in vitro under various conditions. Incubation of LAS with tyrosinase, human liver microsomes, or rat liver microsomes in the presence of GSH as a trapping reagent resulted in the formation of two mono-GSH and two di-GSH catechol conjugates which were characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Similar conjugates were also detected in incubations with P450 3A4, P450 2D6, and P450 1B1 supersomes. Interestingly, these conjugates were also detected as major metabolites when compared to competing detoxification pathways such as glucuronidation and methylation. The 7-hydroxylasofoxifene (7-OHLAS) catechol regioisomer was also synthesized and oxidized either chemically or enzymatically to an o-quinone that was shown to form depurinating adducts with DNA. Collectively, these data show that analogous to estrogens, LAS is oxidized to catechols and o-quinones which could potentially contribute to in vivo toxicity for this SERM.

Figure 1

Representative LC chromatograms of 30 RM LAS (A) and 30 RM E₂ (B) incubated with tyrosinase (0.1 mg/mL) and GSH (1 mM) in 50 mM phosphate buffer (pH 7.4) for 30 min at 37 °C; Mass spectrometric analyses of OHLAS-SG1, OHLAS-SG2 (C), and OHLAS-diSG1, OHLAS-diSG2 (D).

Figure 2

Representative LC chromatograms of 30 RM LAS (A, B) or 30 RM E₂ (C, D) incubated with rat or human liver microsomes (1 nmol P450/mL) and GSH (1 mM) in the presence of a NADPH-generating system (1 mM NADP⁺, 5 mM MgCl₂, 5 mM isocitric acid, 0.2 unit/mL isocitrate dehydrogenase) in 50 mM phosphate buffer (pH 7.4) for 30 min at 37 °C.

Figure 3

Representative LC chromatograms of 30 ZM LAS (A, B, C) or 30 ZM E₂ (D, E, F) incubated with P450 3A4, P450 2D6, or P450 1B1 (10 pmol/mL) supersomes, along with GSH (1 mM) and a NADPH-generating system (1 mM NADP⁺, 5 mM MgCl₂, 5 mM isocitric acid, 0.2 unit/mL isocitrate dehydrogenase) in 50 mM phosphate buffer (pH 7.4) for 30 min at 37 °C.

Figure 4

Representative LC chromatograms of 30 RM LAS, rat liver microsomes (1 nmol P450/mL), and a NADPH-generating system (1 mM NADP⁺, 5 mM MgCl₂, 5 mM isocitric acid, 0.2 unit/mL isocitrate dehydrogenase), incubated in 50 mM phosphate buffer (pH 7.4) for 30 min at 37 °C along with either of the following: (A) UDPGA (1mM) and alamethicin (10 Rg/mg protein); (B) GSH (1 mM), UDPGA (1mM), and alamethicin (10 Rg/mg protein); (C) COMT (1mM) and SAM (0.3 mM); or (D) GSH (1 mM), COMT (1mM) and SAM (0.3 mM).

Figure 5

Representative LC chromatogram of 30 RM 7-OHLAS, tyrosinase (0.1 mg/mL), and dA (300 μm) incubated in 50 mM phosphate buffer (pH 7.4) for 30 min at 37 °C (A) and MS-MS fragmentation of 7-OHLAS-Ade (B).

Figure 6

Detection of 7-OHLAS-Ade by multiple reaction monitoring (MRM) at m/z 563 > 136 (A) and 7-OHLAS-Gua51 and 7-OHLAS-Gua-2 by MRM at m/z 579 > 152 (B).

Scheme 1

Proposed bioactivation of lasofoxifene compared to estradiol.

Scheme 2

Proposed mechanism for formation of DHN-7-OHLAS via tyrosinase oxidation; Formation of 2-OHE₂-p-Quinone methide A.