Estrogen Receptor/ERR

Previous studies demonstrated that sulfate conjugation is involved in the metabolism of three commonly used breast cancer drugs, tamoxifen, raloxifene and fulvestrant. The current study was designed to systematically identify the human cytosolic sulfotransferases (SULTs) that are capable of sulfating raloxifene, fulvestrant, and two active metabolites of tamoxifen, afimoxifene and endoxifen. A systematic analysis using 13 known human SULTs revealed SULT1A1 and SULT1C4 as the major SULTs responsible for the sulfation of afimoxifene, endoxifen, raloxifene and fulvestrant. Kinetic parameters of these two human SULTs in catalyzing the sulfation of these drug compounds were determined. Sulfation of afimoxifene, endoxifen, raloxifene and fulvestrant under metabolic conditions was examined using HepG2 human hepatoma cells and MCF-7 breast cancer cells. Moreover, human intestine, kidney, liver, and lung cytosols were examined to verify the presence of afimoxifene/endoxifen/raloxifene/fulvestrant-sulfating activity.[3]

Methods: Premenopausal women aged at least 18 years experiencing moderate to severe symptoms were randomized to receive placebo, 2 mg, or 4 mg of Afimoxifene daily delivered as a transdermal hydroalcoholic gel for 4 menstrual cycles. The primary efficacy parameter was change in mean pain intensity as measured by the Visual Analog Scale (VAS) for the seven worst pain score days within a cycle from baseline to the fourth cycle.[4]
Results: After 4 cycles of treatment, statistically significant improvements relative to placebo were measured in mean VAS score in the 4-mg Afimoxifene group (-12.71 mm [95% confidence interval, -0.96 to -24.47; P = 0.034]). Patient global assessment of pain, physician's assessment of pain, tenderness on palpation, and nodularity following 4 cycles of treatment were significantly more likely to show improvements in the 4-mg group, compared with placebo (P = 0.010 [pain]; P = 0.012 [tenderness]; P = 0.017 [nodularity]). Overall, Afimoxifene was well tolerated with few adverse events and no drug-related SAE occurred in any group. There were no changes in menstrual pattern or plasma hormone levels and no breakthrough vaginal bleeding in patients treated with Afimoxifene.[4]

Previous studies demonstrated that sulfate conjugation is involved in the metabolism of three commonly used breast cancer drugs, tamoxifen, raloxifene and fulvestrant. The current study was designed to systematically identify the human cytosolic sulfotransferases (SULTs) that are capable of sulfating raloxifene, fulvestrant, and two active metabolites of tamoxifen, afimoxifene and endoxifen. A systematic analysis using 13 known human SULTs revealed SULT1A1 and SULT1C4 as the major SULTs responsible for the sulfation of afimoxifene, endoxifen, raloxifene and fulvestrant. Kinetic parameters of these two human SULTs in catalyzing the sulfation of these drug compounds were determined. Sulfation of afimoxifene, endoxifen, raloxifene and fulvestrant under metabolic conditions was examined using HepG2 human hepatoma cells and MCF-7 breast cancer cells. Moreover, human intestine, kidney, liver, and lung cytosols were examined to verify the presence of afimoxifene/endoxifen/raloxifene/fulvestrant-sulfating activity[3].

Using the doxorubicin-sensitive K562 cell line and the resistant derivative lines KD30 and KD225 as models, we found that acquisition of multidrug resistance (MDR) is associated with enhanced FOXO3a activity and expression of ABCB1 (MDR1), a plasma membrane P-glycoprotein that functions as an efflux pump for various anticancer agents. Furthermore, induction of ABCB1 mRNA expression on doxorubicin treatment of naive K562 cells was also accompanied by increased FOXO3a activity. Analysis of transfected K562, KD30, and KD225 cells in which FOXO3a activity can be induced by 4-hydroxytamoxifen (Afimoxifene) showed that FOXO3a up-regulates ABCB1 expression at protein, mRNA, and gene promoter levels. Conversely, silencing of endogenous FOXO3a expression in KD225 cells inhibited the expression of this transport protein. Promoter analysis and chromatin immunoprecipitation assays showed that FOXO3a regulation of ABCB1 expression involves binding of this transcription factor to the proximal promoter region. Moreover, activation of FOXO3a increased ABCB1 drug efflux potential in KD30 cells, whereas silencing of FOXO3a by siRNA significantly reduced ABCB1 drug efflux ability. Together, these findings suggest a novel mechanism that can contribute towards MDR, involving FOXO3a as sensor for the cytotoxic stress induced by anticancer drugs. Although FOXO3a may initially trigger a program of cell cycle arrest and cell death in response to doxorubicin, sustained FOXO3a activation promotes drug resistance and survival of cells by activating ABCB1 expression.[2]

---

Tamoxifen is widely used to treat estrogen receptor (ER)-positive breast cancer. Recent findings that tamoxifen and its derivative 4-dehydroxy-tamoxifen (OHT/Afimoxifene) can exert ER-independent cytotoxic effects have prompted the initiation of clinical trials to evaluate its use in ER-negative malignancies. For example, tamoxifen and OHT exert cytotoxic effects in malignant peripheral nerve sheath tumors (MPNSTs) where estrogen is not involved. In this study, we gained insights into the ER-independent cytotoxic effects of OHT by studying how it kills MPNST cells. Although caspases were activated following OHT treatment, caspase inhibition provided no protection from OHT-induced death. Rather, OHT-induced death in MPNST cells was associated with autophagic induction and attenuated by genetic inhibition of autophagic vacuole formation. Mechanistic investigations revealed that OHT stimulated K-Ras degradation through autophagy induction, which is critical for survival of MPNST cells. Similarly, we found that OHT induced K-Ras degradation in breast, colon, glioma and pancreatic cancer cells. Our findings describe a novel mechanism of autophagic death triggered by tamoxifen and OHT in tumor cells that may be more broadly useful clinically in cancer treatment.[5]

Tamoxifen is a widely used chemotherapeutic agent, which has been associated with prolongation of the QT interval. Other studies have reported that acute exposure to tamoxifen can reduce cardiac K(+) currents. However, in vivo tamoxifen is largely metabolized and most of its activity is attributable to its major metabolite, 4-hydroxytamoxifen (4OH-tamoxifen). Accordingly, in the present study, we performed voltage-clamp experiments to directly investigate the effects of 4OH-tamoxifen on the repolarizing K(+) currents in adult mouse ventricular myocytes in order to determine whether the effects of tamoxifen on repolarization could be ascribed to 4OH-tamoxifen. K(+) currents were recorded before and after acute exposure to 4OH-tamoxifen (0.5, 1 and 10microM). 4OH-tamoxifen reduced the density of the Ca(2+)-independent transient outward (I(to)), the ultrarapid delayed rectifier (I(Kur)) and the inward rectifier (I(K1)) K(+) currents (by up to 43%, 41% and 26%, respectively) but had no significant effect on the steady-state outward K(+) current (I(ss)). Voltage dependence of steady-state inactivation and reactivation time of I(to) and I(Kur) were not affected by 4OH-tamoxifen. Experiments using the pure estrogen receptor antagonist, ICI 182,780 and the inhibitor of gene transcription, actinomycin D, were undertaken to assess the involvement of estrogen receptor. Administered alone these compounds did not affect the density of K(+) currents. Moreover, pretreatment of the cells with ICI 182,780 or actinomycin D did not prevent the inhibitory response to 4OH-tamoxifen. Overall, 4OH-tamoxifen reduced K(+) currents in mouse ventricle and this effect is unrelated to gene transcription and does not involve interaction of the drug with estrogen receptor[6].

---

Ventricular myocytes were isolated from adult female CD-1 mice (2–3 months) by enzymatic dissociation as previously described. (Fiset et al., 1997, Trépanier-Boulay et al., 2001). All experiments were conducted in accordance with the Canadian Council Animal Care guidelines. The animals were heparinized (1 U/kg; i.p.) 20 min prior to sacrifice, anaesthetized by inhalation of isoflurane and then killed by cervical dislocation. The heart was rapidly excised and retrogradely perfused on a modified Langendorff apparatus through the aorta (2 ml/min) with the following solutions: (1) 5 min with Tyrode solution containing (in mM): 130 NaCl; 5.4 KCl; 1 CaCl2; 1 MgCl2; 0.33 Na2HPO4; 10 HEPES, 5.5 glucose (pH adjusted to 7.4 with NaOH); (2) 10 min with Tyrode solution with zero Ca2+; (3) 20 min with Tyrode solution containing 0.03 mM Ca2+, 20 mM taurine, 0.1% bovine serum albumin (BSA; Fraction V, Sigma Chemicals Co., St. Louis, MO, USA) and 73.7 U/ml type II collagenase (Worthington Co. Ltd., Freehold, NJ, USA); and (4) 5.5 min with Kraftbrühe (KB) solution containing (in mM) 100 K+-glutamate; 10 K+-aspartate; 25 KCl; 10 KH2PO4, 2 MgSO4; 20 creatine base; 0.5 EGTA; 5 HEPES; 0.1% BSA, 20 glucose (pH to 7.4 with KOH) (Isenberg and Klockner, 1982). During cell isolation solutions were maintained at 37 ± 1 °C and were equilibrated with 100% O2. At the end of the perfusion the right ventricular free wall was dissected from the heart and placed in “KB” solution. The ventricular tissue was triturated gently with a Pasteur pipette for 10–15 min to free individual ventricular myocytes. Rod-shape single myocytes were then collected and stored in “KB” solution at 4 °C until use[6].

---

[1]. en.wikipedia.org/wiki/Afimoxifene
[2]. Doxorubicin activates FOXO3a to induce the expression of multidrug resistance gene ABCB1 (MDR1) in K562 leukemic cells. Mol Cancer Ther. 2008 Mar;7(3):670-8.
[3].J Pharmacol Sci. 2015 Jul;128(3):144-9.
[4]. Breast Cancer Res Treat. 2007 Dec;106(3):389-97.
[5]. Cancer Res. 2013 Jul 15; 73(14): 4395–4405.
[6]. Eur J Pharmacol. 2010 Mar 10;629(1-3):96-103.

Many women experience symptoms of cyclical mastalgia, such as breast pain, tenderness, and nodularity. Tamoxifen and other drugs have been used to alleviate cyclical mastalgia symptoms; however, their use is associated with potentially serious side effects. The current study compared the safety and efficacy of two doses of a topical gel containing 4-hydroxytamoxifen (Afimoxifene, formerly known as 4-OHT) with placebo gel for the treatment of moderate to severe cyclical mastalgia.[4]
After 4 months of treatment, daily topical breast application of Afimoxifene resulted in statistically significant improvements in signs and symptoms of cyclical mastalgia across patient- and physician-rated scales with excellent tolerability and safety.

---

Afimoxifene is a tertiary amino compound that is tamoxifen in which the phenyl group which is in a Z- relationship to the ethyl substituent is hydroxylated at the para- position. It is the active metabolite of tamoxifen. It has a role as an antineoplastic agent, an estrogen receptor antagonist and a metabolite. It is a tertiary amino compound and a member of phenols. It is functionally related to a tamoxifen. ChEBI
Afimoxifene (4-Hydroxytamoxifen, trade name TamoGel) is a new estrogen inhibitor under investigation for a variety of estrogen-dependent conditions, including cyclic breast pain and gynecomastia. TamoGel is formulated using Enhanced Hydroalcoholic Gel (EHG) Technology. This technology enables percutaneous delivery of drugs that cannot be delivered orally. It is being developed by Ascent Therapeutics. DrugBank

---

4-Hydroxytamoxifen is a natural product found in Penicillium aurantiacobrunneum with data available. LOTUS - the natural products occurrence database
Afimoxifene is a tamoxifen metabolite with both estrogenic and anti-estrogenic effects. Afimoxifene has a higher affinity for the estrogen receptor than tamoxifen, and functions as an antagonist in breast cancer cells. NCI Thesaurus (NCIt)
4-Hydroxytamoxifen (Afimoxifene) is a metabolite of Tamoxifen. Afimoxifene (4-hydroxytamoxifen) is a selective estrogen receptor modulator which is the active metabolite of tamoxifen. Afimoxifene is a transdermal gel formulation and is being developed by Ascend Therapeutics, Inc. under the trademark TamoGel. (Wikipedia) Toxin and Toxin Target Database (T3DB)

---

Metabolism / Metabolites: 4-Hydroxytamoxifen has known human metabolites that include Tamoxifen 4-O-sulfate, Endoxifen, 3,4-Dihydroxy-Tamoxifen, and Tamoxifen 4-O-glucuronide.
4-Hydroxytamoxifen is a known human metabolite of tamoxifen.
Mechanism of Action: Afimoxifene binds to estrogen receptors (ER), inducing a conformational change in the receptor. This results in a blockage or change in the expression of estrogen dependent genes.

C26H29NO2

387.51

387.21983

C, 80.59; H, 7.54; N, 3.61; O, 8.26

68392-35-8

4-Hydroxytamoxifen;68047-06-3;(E)-4-Hydroxytamoxifen;174592-47-3

449459

Typically exists as white to off-white solids at room temperature

1.092

135-144°C

5.7

32.7

OC1=CC=C(/C(C2=CC=C(OCCN(C)C)C=C2)=C(C3=CC=CC=C3)/CC)C=C1

TXUZVZSFRXZGTL-OCEACIFDSA-N

InChI=1S/C26H29NO2/c1-4-25(20-8-6-5-7-9-20)26(21-10-14-23(28)15-11-21)22-12-16-24(17-13-22)29-19-18-27(2)3/h5-17,28H,4,18-19H2,1-3H3/b26-25+

4-[1-[4-[2-(dimethylamino)ethoxy]phenyl]-2-phenyl-1-buten-1-yl]-phenol

(E/Z)-4-Hydroxytamoxifen; 4 hydroxytamoxifene ; 4Hydroxytamoxifen; paraHydroxytamoxifen; 4Monohydroxytamoxifen; Hydroxytamoxifen; Afimoxifene; Tamogel 4Hydroxytamoxifen; trans4Hydroxytamoxifen; Tamoxifen metabolite B; 4hydroxytamoxifen.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~83.33 mg/mL (~215.04 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.37 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.08 mg/mL (5.37 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)