Repurposing Estrogen Receptor Antagonists for the Treatment of Infectious Disease.

The concept of repurposing previously approved medications to the treatment of new indications by taking advantage of off-target effects has gained traction in recent years, particularly in areas of medicine that do not offer large profits to pharmaceutical firms. As infectious disease discovery research has declined among large pharmaceutical companies, the potential payoff of repurposing has become attractive. From these efforts, the triphenylethylene class of selective estrogen receptor modulators related to tamoxifen has shown activity against a wide range of medically important human pathogens, including bacteria, fungi, parasites, and viruses. Because it has activity against many pathogens affecting people in resource-limited areas of the world, TAM and related drugs may be particularly useful. Here, we review the in vitro, in vivo, and mechanistic studies of the anti-infective activity of tamoxifen, toremifene, clomiphene, and their analogs. We also discuss the pharmacologic properties of this privileged scaffold and its potential utility in treating infectious diseases.

Diethylenetriamine pentaacetic acid derivative of toremifene and in vitro evaluation in human breast cancer cell line MCF-7.

Cytotoxic and apoptotic effects of toremifene-diethylenetriamine pentaacetic acid (TOR-DTPA), formed by conjugation of TOR and DTPA, on the MCF-7 cell line were evaluated. TOR-DTPA was synthesized and qualified via gas chromatography-mass spectrometry system, thin layer chromatography, and high performance liquid chromatography methods. To screen the biological properties of TOR-DTPA at determined concentrations, our ongoing effort was to evaluate apoptotic and cytotoxic effects on the MCF-7 cell line. Trypan blue dye exclusion test, XTT, ELISA, and TUNEL assays were utilized to evaluate cytotoxicity and apoptosis. TOR-DTPA has no cytotoxic and limited apoptotic effect on the MCF-7 cell line according to the results of in vitro studies. It is concluded that the lack of obvious apoptotic and cytotoxic effects allows the already proposed ligand, TOR-DTPA, to be improved as a novel hydrophilic ligand for breast imaging.

Synthesis, radiolabeling and In Vivo tissue distribution of an anti-oestrogen glucuronide compound, (99m)Tc-TOR-G.

Toremifene (TOR) has been used as an anti-oestrogen drug for the treatment and prevention of human breast cancer. The aim of this study was the addition of the hydrophilic groups diethylenetriamine pentaacetic acid (DTPA) and glucuronic acid to the starting substance TOR and to label it with technetium-99m ((99m)Tc) radionuclide and to investigate radiopharmaceutical potential of the new compound. The synthesis reactions are completed in four steps, including enzymatic reaction, with the following substeps; preparation of microsomal fraction from Hutu 80 cell line and subsequent purification of UDP-glucuronyl transferase (UDPGT), estimation of protein quantity in microsomal samples and glucuronidation reaction. The results indicate that (99m)Tc-TOR-G may be proposed as a new anti-oestrogen glucuronide imaging agent for ovarian tumours.

DNA adduct formation in the livers of female Sprague-Dawley rats treated with toremifene or alpha-hydroxytoremifene.

Toremifene, an analogue of tamoxifen in which the ethyl side chain has been replaced with a 2-chloroethyl substituent, is used as a chemotherapeutic agent in postmenopausal women with advanced breast cancer. Toremifene is metabolized in a manner similar to that of tamoxifen, with alpha-hydroxytoremifene being a predominant metabolite in incubations in vitro. DNA adducts have been detected previously in liver DNA upon the administration of toremifene to rats; however, the identity of these adducts is unknown. In the present study, we have characterized the DNA adducts produced by alpha-hydroxytoremifene and have compared the extent of hepatic DNA adduct formation in rats administered toremifene, alpha-hydroxytoremifene, or tamoxifen. alpha-Hydroxytoremifene was synthesized, further activated by sulfation, and then reacted with salmon testis DNA. After enzymatic hydrolysis to deoxynucleosides, HPLC analysis indicated the formation of two major DNA adducts, which were characterized as (E)- and (Z)-alpha-(deoxyguanosin-N2-yl)toremifene on the basis of 1H NMR and mass spectral analyses. To assess the formation of toremifene DNA adducts in vivo, female Sprague-Dawley rats were treated intraperitoneally with toremifene, alpha-hydroxytoremifene, or tamoxifen. 32P-Postlabeling analyses of hepatic DNA from the tamoxifen-treated rats indicated three DNA adducts at a total level of 2,200 +/- 270 adducts/108 nucleotides. DNA adducts were not detected (<5 adducts/108 nucleotides) in the livers of rats treated with toremifene. Two DNA adducts, of which the major one coeluted with the 3',5'-bis-phosphate of (E)-alpha-(deoxyguanosin-N2-yl)toremifene, were present at a level of 57 +/- 12 adducts/108 nucleotides in hepatic DNA from rats administered alpha-hydroxytoremifene. The low level of hepatic DNA adduct formation observed with both toremifene and alpha-hydroxytoremifene, as compared to that with tamoxifen, may be due to the limited esterification of alpha-hydroxytoremifene and/or the poor reactivity of alpha-sulfoxytoremifene.

Toremifene metabolism in rat, mouse and human liver microsomes: identification of alpha-hydroxytoremifene by LC-MS.

The in vitro metabolism of toremifene has been studied in liver microsomal preparations from rat, mouse and human sources using high-performance liquid chromatography-electrospray ionisation mass spectrometry (HPLC-ESIMS). The metabolites detected were N-desmethyltoremifene (m/z 392), 4-hydroxytoremifene (m/z 422), 4'-hydroxytoremifene (m/z 422) and toremifene N-oxide m/z 422). In addition, a new polar metabolite with a protonated molecule at m/z 422 has been detected in all three species. The compound was identified by tandem MS-MS as alpha-hydroxytoremifene, an analogue of alpha-hydroxytamoxifen. The results showed that alpha-hydroxylation is a common feature of tamoxifen and toremifene metabolism and that alpha-hydroxytamoxifen is unlikely to be the reactive metabolite responsible for the hepatocarcinogenesis in rat, as widely believed.

Mechanism of lower genotoxicity of toremifene compared with tamoxifen.

An increased incidence of endometrial cancer has been reported in breast cancer patients taking tamoxifen (TAM) and in healthy women participating in the TAM chemoprevention trials. Because TAM-DNA adducts are mutagenic and detected in the endometrium of women treated with TAM, TAM adducts are suspected to initiate the development of endometrial cancer. Treatment with TAM has been known to promote hepatocarcinoma in rats, but toremifene (TOR), a chlorinated TAM analogue, did not. TAM adducts are primarily formed via sulfonation of the alpha-hydroxylated TAM metabolites. To explore the mechanism of the lower genotoxicity of TOR, the formation of DNA adducts induced by TOR metabolites was measured using (32)P-postlabeling/ high-performance liquid chromatography analysis and compared with that of TAM metabolites. When alpha-hydroxytoremifene was incubated with DNA, 3'-phosphoadenosine 5'-phosphosulfate, and either rat or human hydroxysteroid sulfotransferase, the formation of DNA adducts was two orders of magnitude lower than that of alpha-hydroxytamoxifen. alpha-hydroxytoremifene was a poor substrate for rat and human hydroxysteroid sulfotransferases. In addition, the reactivity of alpha-acetoxytoremifene, a model activated form of TOR, with DNA was much lower than that of alpha-acetoxytamoxifen. Thus, TOR is likely to have lower genotoxicity than TAM. TOR may be a safer alternative by avoiding the development of endometrial cancer.

Intrinsic reactivity of tamoxifen and toremifene metabolites with DNA.

The antiestrogen tamoxifen is known to cause liver cancer in rats. This may be due to the formation of abundant DNA adducts in rat liver. A likely precursor to some of the tamoxifen adducts in rats is alpha-hydroxytamoxifen. It is not clear whether the rat data are relevant to human exposure. In the present study, we show that one of the major metabolites in humans reacts with double-stranded DNA in vitro in the absence of any metabolizing enzymes or activating chemicals. At least two distinct adduct spots resulting from 4-hydroxy-N-desmethyltamoxifen (metabolite Bx) were detected by 32P postlabeling and thin layer chromatography. The adduct level increases dramatically when metabolite Bx is irradiated with UV light to fuse into a phenanthrene ring system. 4-hydroxy-N-desmethyltoremifene, which differs from Bx by a single chlorine atom, forms fewer DNA adducts without irradiation but similar amounts after irradiation. These results suggest that the chlorine atom may interfere with drug-DNA interactions which facilitate adduct formation.

Antioxidant properties of the triphenylethylene antiestrogen drug toremifene.

The aim of the present study was to investigate antioxidativity of the triphenylethylene antiestrogen toremifene. Toremifene and its structural analogues were studied for their ability to inhibit chain reactions of lipid peroxidation and to act as scavengers of free radicals in vitro, and the effects of toremifene were compared to those of the estrogens, tamoxifen and known antioxidants. Moreover, the in vivo antioxidativity of toremifene was tested in a long-term experiment with rats. The ability of toremifene to prevent lipid peroxidation was assayed in two different test systems. In the first assay (initiated with ascorbate/ADP-FeCl3, detection by the formation of TBA-reactive material) toremifene was found to act as an efficient membrane antioxidant with an IC50-value (18 microM) comparable to that of tamoxifen (26 microM) and alpha-tocopherol (43 microM). Toremifene derivatives 4-hydroxytoremifene (IC50 = 8 microM) and Fc 1159 (IC50 = 31 microM), as well as diethylstilbestrol (IC50 = 17 microM) were also active while estradiol showed only weak antioxidativity (IC50 = 300 microM) in this test system. In the other assay (peroxidation initiated with t-butylhydroperoxide, detection by luminol-enhanced chemiluminescence) toremifene prevented lipid peroxidation only at high concentrations (IC50 = 450 microM) but the metabolite 4-hydroxytoremifene (IC50 = 0.18 microM), estradiol (IC50 = 4.6 microM) and diethylstilbestrol (IC50 = 1.7 microM) showed potent antioxidant activity. The potency of 4-hydroxytoremifene even exceded that of alpha-tocopherol (IC50 = 2.0 microM) and butylated hydroxyanisole (IC50 = 1.1 microM). Toremifene was found to have some superoxide anion but no peroxyl radical scavenging activity. Interestingly, diethylstilbestrol turned out to be a potent scavenger of peroxyl radicals. Treatment of female Sprague-Dawley rats with toremifene (12 or 48 mg/kg) was found to decrease serum levels of lipid peroxides. This was seen at various time points (2 days, 5 weeks, 6 and 12 months) during long-term administration of toremifene to rats, and results obtained with two different methods (diene conjugation, TBA-reactive material) gave similar results. The present study thus showed that (i) like steroidal estrogens and tamoxifen toremifene is a potent membrane antioxidant in vitro, (ii) the antioxidant action of toremifene is not due to scavenging of free radicals and, importantly, (iii) toremifene acts antioxidatively also in living organisms in vivo.