Reversible time-dependent inhibition of cytochrome P450 enzymes by duloxetine and inertness of its thiophene ring towards bioactivation
Highlights
► Duloxetine does not cause irreversible TDI of CYP1A2, 2B6, 2D6, 2C19 and 3A4/5. ► Thiophene ring of duloxetine does not undergo bioactivation. ► Hepatotoxicity is possibly related to epoxidation of duloxetine's naphthyl ring.
Introduction
Duloxetine (Cymbalta®) is a selective serotonin–norepinephrine reuptake inhibitor (SNRI) that has been approved to treat major depressive disorder and diabetic peripheral neuropathic pain in the US and Europe, generalized anxiety disorder in the US, and stress urinary incontinence in women in Europe. First approved in 2004, duloxetine is currently the second most commonly prescribed antidepressant with 14.6 million prescriptions written in the US in 2006 alone, second only to escitalopram. Duloxetine was approved in Singapore in 2006 to treat major depressive disorder, diabetic peripheral neuropathic pain, generalized anxiety and pain associated with fibromyalgia.
Between 2004 and 2008, post-marketing surveillance revealed over 400 cases of hepatotoxicity potentially caused by duloxetine, of which 58 were considered clinically significant. These cases prompted the manufacturer, Eli Lilly and Company, to revise the product label to include a warning that duloxetine ‘should ordinarily not be prescribed to a patient with substantial alcohol use or evidence of chronic liver disease’ (Vuppalanchi et al., 2010). Another report examined seven cases of drug-induced liver injury between 2006 and 2009, of which six were assessed as definitely or very likely to be attributed to duloxetine (Vuppalanchi et al., 2010). While a number of patients who suffered from duloxetine-induced hepatotoxicity had pre-existing liver disease, another case was reported where a patient without signs of pre-existing liver disease developed fatal fulminant hepatic failure six weeks after a dose increase of duloxetine (Hanje et al., 2006).
Duloxetine has been shown to be extensively and rapidly metabolized primarily by CYP1A2 and CYP2D6, where it undergoes phase I metabolism via hydroxylation of the naphthyl ring, followed by phase II conjugation forming glucuronide or sulfate conjugates. These metabolites are excreted primarily in the urine (Lantz et al., 2003). Duloxetine has been reported to be an inhibitor of CYP2D6 (Skinner et al., 2003) but not CYP1A2 (Lobo et al., 2008) in vivo. However, no attempt was made to elucidate the mechanism of inhibition of CYP2D6 by duloxetine. Additionally, duloxetine has been shown to be a time-dependent inhibitor (TDI) of CYP1A2, CYP2B6, CYP2C19 and CYP3A4/5 in vitro (Paris et al., 2009), however the study did not identify whether the TDI was reversible or irreversible.
The ability of duloxetine to cause TDI of various CYP isoforms might potentially account for its clinically observed hepatotoxicity. Irreversible TDI, in both forms of mechanism-based inhibition (MBI) and metabolite-intermediate complex are known to be responsible for various clinically significant drug–drug interactions (DDIs) by causing irreversible inhibition of CYP450. Irreversible TDI has also been implicated in hepatotoxicity. Tienilic acid has been shown to cause drug-induced hepatitis by covalently alkylating CYP2C9, triggering the formation of anti-LKM2 auto-antibodies which attack hepatocytes, resulting in immunoallergic hepatitis (Homberg et al., 1984). In addition, it has been reported that duloxetine is metabolized to reactive metabolites that are trapped by glutathione (Wu et al., 2010). Duloxetine possesses several potential toxicophores: a naphthyl ring and a thiophene ring (Fig. 1A), which could be biotransformed to reactive metabolites which may result in drug-induced toxicity. These observations provide the impetus to investigate the nature of the TDI of CYP450 by duloxetine and the potential of duloxetine in generating reactive metabolites so as to elucidate the mechanism of its clinically observed hepatotoxicity.
Section snippets
Chemicals
HPLC grade acetonitrile (ACN) was purchased from Tedia Company Inc. (Fairfield, OH). Water was purified using a Milli-Q water purification system (Millipore, Bedford, MA, USA). Potassium phosphate monobasic (ACS grade) was purchased from Mallinckrodt Baker (Phillipsburg, NJ, USA). Buproprion hydrochloride and S-mephenytoin were purchased from Enzo Life Sciences International Inc. (Plymouth Meeting, PA, USA). Galantamine, paracetamol (APAP), debrisoquine sulfate, carbamazepine, furafylline,
Time-dependent inhibition studies
The TDI assay was validated using furafylline, a known MBI of CYP1A2. As the concentration of furafylline increased, the residual activity of CYP1A2 decreased (indicating the inhibition is concentration-dependent). Within each inhibitor concentration, the residual activity also decreased as preincubation time increased (indicating the inhibition is time-dependent). Supplementary data, Fig. 1A shows a plot illustrating a composite of both effects, where the slopes of the lines became
Distinguishing reversible and irreversible TDI
Irreversible TDI has been demonstrated to be responsible for many clinically significant DDIs, as the inhibition of CYP enzymes is of a permanent nature, and the drug metabolizing capacity can only be restored by the synthesis of fresh enzymes. While it has been shown that duloxetine causes TDI of several CYP isoforms (Paris et al., 2009), it was not certain whether the observed TDI demonstrated was of a reversible or irreversible nature as the design of the in vitro HLM assays did not
Conclusion
We demonstrated that duloxetine does not exhibit irreversible TDI of CYP1A2, CYP2B6, CYP2C19, CYP2D6 and CYP3A4/5 and confirmed that duloxetine only exhibits reversible TDI with CYP1A2, CYP2B6, CYP2C19 and CYP3A4/5. It was also ascertained that the thiophene moiety in duloxetine does not undergo phase 1 bioactivation in the form of epoxidation, S-oxidation or ring opening to generate reactive metabolites. The hepatotoxicity of duloxetine is possibly not related to the phenomenon of irreversible
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgements
This work was supported by the National University of Singapore (NUS) grant (R-148-000-138-305 to Eric Chun Yong Chan) and Department of Pharmacy Final Year Project grant (R-148-000-003-001). Duloxetine hydrochloride was a generous gift by Nosch Labs Pte. Ltd. The authors would like to thank Professor Sidney Nelson at the University of Washington for providing constructive suggestions to improve our paper.
References (20)
- et al.
First evidence that cytochrome P450 may catalyze both S-oxidation and epoxidation of thiophene derivatives
Biochem. Biophys. Res. Commun.
(2005) - et al.
Case report: fulminant hepatic failure involving duloxetine hydrochloride
Clin. Gastroenterol. Hepatol.
(2006) - et al.
Pharmacokinetics of duloxetine hydrochloride enteric-coated tablets in healthy Chinese volunteers: a randomized, open-label, single- and multiple-dose study
Clin. Ther.
(2009) - et al.
Biotransformation reactions of five-membered aromatic heterocyclic rings
Chem. Res. Toxicol.
(2002) - et al.
Negative ion tandem mass spectrometry for the detection of glutathione conjugates
Chem. Res. Toxicol.
(2005) - et al.
Metabolism and disposition of the thienopyridine antiplatelet drugs ticlopidine, clopidogrel, and prasugrel in humans
J. Clin. Pharmacol.
(2010) - et al.
Ticlopidine as a selective mechanism-based inhibitor of human cytochrome P450 2C19
Biochemistry
(2001) - et al.
A new anti-liver-kidney microsome antibody (anti-LKM2) in tienilic acid-induced hepatitis
Clin. Exp. Immunol.
(1984) - et al.
Approaches for minimizing metabolic activation of new drug candidates in drug discovery
Handb. Exp. Pharmacol.
(2010) - et al.
Metabolism, excretion, and pharmacokinetics of duloxetine in healthy human subjects
Drug Metab. Dispos.
(2003)