Genetic polymorphism of cytochrome P450 2D6 determines oestrogen receptor activity of the major infertility drug clomiphene via its active metabolites

Abstract

Clomiphene citrate is the most used drug for the treatment of female infertility, a common condition in western societies and developing countries. Despite dose escalation, up to 30% of women do not respond. Since clomiphene shares structural similarities with tamoxifen, which is predominantly bioactivated by the polymorphic cytochrome P450 (CYP) 2D6, we systematically explored clomiphene metabolism and action in vitro and in vivo by pharmacogenetic, -kinetic and -dynamic investigations. Human liver microsomes were incubated with clomiphene citrate and nine metabolites were identified by mass spectrometry and tested at the oestrogen receptor for their antagonistic capacity. (E)-4-hydroxyclomiphene and (E)-4-hydroxy-N-desethylclomiphene showed strongest inhibition of the oestrogen receptor activity with 50% inhibitory concentrations of 2.5 and 1.4 nm, respectively. CYP2D6 has been identified as the major enzyme involved in their formation using recombinant CYP450 isozymes as confirmed by inhibition experiments with CYP monoclonal antibodies. We correlated the CYP2D6 genotype of 30 human liver donors with the microsomal formation rate of active metabolites and observed a strong gene-dose effect. A healthy female volunteer study confirmed our in vitro data that the CYP2D6 polymorphism substantially determines the formation of the active clomiphene metabolites. Comparison of the C(max) of (E)-4-hydroxyclomiphene and (E)-4-hydroxy-N-desethylclomiphene showed 8 and 12 times lower concentrations in subjects with non-functional CYP2D6 alleles. Our results highlight (E)-4-hydroxyclomiphene and (E)-4-hydroxy-N-desethylclomiphene as the active clomiphene metabolites, the formation of which strongly depends on the polymorphic CYP2D6 enzyme. Our data provide first evidence of a biological rationale for the variability in the response to clomiphene treatment.

Pharmacologically Active Drug Metabolites: Impact on Drug Discovery and Pharmacotherapy

“…), clomiphene is metabolized by hydroxylation at the para position of one of the phenyl rings by CYP2D6 to yield a metabolite, 4-hydroxyclomiphene ( Fig. 11), that has two orders of magnitude greater potency at binding to the estrogen receptor (Ruenitz et al, 1983;Mürdter et al, 2012). Thus, CYP2D6 extensivemetabolizer (EM) and poor-metabolizer (PM) subjects exhibit different responses, with the EM subjects expected to enjoy greater efficacy due to 7-fold greater exposure (Mürdter et al, 2012).…”

“…11), that has two orders of magnitude greater potency at binding to the estrogen receptor (Ruenitz et al, 1983;Mürdter et al, 2012). Thus, CYP2D6 extensivemetabolizer (EM) and poor-metabolizer (PM) subjects exhibit different responses, with the EM subjects expected to enjoy greater efficacy due to 7-fold greater exposure (Mürdter et al, 2012). Clomiphene also undergoes N-deethylation, but N-desethylclomiphene has similarly low activity to the parent drug.…”

Recent examples on the clinical relevance of the CYP2D6 polymorphism and endogenous functionality of CYP2D6

“…Drug-drug interaction Many potent inhibitors including mechanism-based inhibitors (e.g., paroxetine) leading to complete loss of functionality ("phenocopy" of PM status) [32][33][34][35]38] Food interaction Possible but usually of less extent [36,37] Brought to you by | New York University Bobst Library Technical Services Authenticated Download Date | 6/11/15 4:06 PM in the thalamic blood flow and in the activation of various brain areas linked to working memory and emotion [53,75]. Interestingly, also a variant of the pseudogene CYP2D7 can be expressed in an active form in the brain [76] without any functional correlate thus far.…”

“…However, although this thought is a well-defined example, recently the value of preemptive genotyping for tamoxifen therapy has been questioned [35], and even the lack of any relationship between the CYP2D6 genotype or phenotype and clinical outcome has been reported [36]. The metabolism of the structurally related clomiphene, a drug for the treatment of anovulation, is also dependent on CYP2D6 [37], and recently a correlation between the occurrence of rash and lower CYP2D6 activity has been reported for gefitinib, an EGFR tyrosine kinase inhibitor [38].…”

Role and pharmacologic significance of cytochrome P‐450 2D6 in oxidative metabolism of toremifene and tamoxifen

“…The metabolism of another triphenylethylene SERM clomiphene closely parallels that of TAM. 27 The ethyl side chain of TAM is replaced by a chlorine atom in clomiphene, which is sterically accommodated in the docking solution with a similar catalytically relevant orientation to TAM (Supporting Information Fig. 2).…”