Tissue Distribution and Systemic Toxicity Evaluation of Raloxifene Targeted Polymeric Micelles of Poly (Styrene-Maleic Acid)-Poly (Amide- Ether-Ester-Imide)-Poly (Ethylene Glycol) Loaded With Docetaxel in Breast Cancer Bearing Mice.

BACKGROUND: Due to the low water solubility of Docetaxel (DTX), it is formulated with ethanol and Tween 80 with lots of side effects. For this reason, special attention has been paid to formulate it in new drug nano-carriers. OBJECTIVE: The goal of this study was to evaluate the safety, antitumor activity and tissue distribution of the novel synthesized Raloxifene (RA) targeted polymeric micelles. METHODS: DTX-loaded RA-targeted polymeric micelles composed of poly(styrene-maleic acid)- poly(amide-ether-ester-imide)-poly(ethylene glycol) (SMA-PAEE-PEG) were prepared and their antitumor activity was studied in MC4-L2 tumor-bearing mice compared with non-targeted micelles and free DTX. Safety of the micelles was studied by Hematoxylin and Eosin (H&E) staining of tumors and major organs of the mice. The drug accumulation in the tumor and major organs was measured by HPLC method. RESULTS: The results showed better tumor growth inhibition and increased survival of mice treated with DTX-loaded in targeted micelles compared to the non-targeted micelles and free DTX. Histopathological studies, H&E staining of tumors and immunohistochemical examination showed the potential of DTX-loaded RA-targeted micelles to inhibit tumor cells proliferation. The higher accumulation of the DTX in the tumor tissue after injection of the micelles compared to the free DTX may indicate the higher uptake of the targeted micelles by the G-Protein-Coupled Estrogen Receptors (GPER). CONCLUSION: The results indicate that RA-conjugated polymeric micelles may be a strong and effective drug delivery system for DTX therapy and uptake of the drug into tumor cells, and overcome the disadvantages and side effects of conventional DTX.

Diesel exhaust particulate extracts inhibit transcription of nuclear respiratory factor-1 and cell viability in human umbilical vein endothelial cells.

Endothelial dysfunction precedes cardiovascular disease and is accompanied by mitochondrial dysfunction. Here we tested the hypothesis that diesel exhaust particulate extracts (DEPEs), prepared from a truck run at different speeds and engine loads, would inhibit genomic estrogen receptor activation of nuclear respiratory factor-1 (NRF-1) transcription in human umbilical vein endothelial cells (HUVECs). Additionally, we examined how DEPEs affect NRF-1-regulated TFAM expression and, in turn, Tfam-regulated mtDNA-encoded cytochrome c oxidase subunit I (COI, MTCO1) and NADH dehydrogenase subunit I (NDI) expression as well as cell proliferation and viability. We report that 17ß-estradiol (E(2)), 4-hydroxytamoxifen (4-OHT), and raloxifene increased NRF-1 transcription in HUVECs in an ER-dependent manner. DEPEs inhibited NRF-1 transcription, and this suppression was not ablated by concomitant treatment with E(2), 4-OHT, or raloxifene, indicating that the effect was not due to inhibition of ER activity. While E(2) increased HUVEC proliferation and viability, DEPEs inhibited viability but not proliferation. Resveratrol increased NRF-1 transcription in an ER-dependent manner in HUVECs, and ablated DEPE inhibition of basal NRF-1 expression. Given that NRF-1 is a key nuclear transcription factor regulating genes involved in mitochondrial activity and biogenesis, these data suggest that DEPEs may adversely affect mitochondrial function leading to endothelial dysfunction and resveratrol may block these effects.

Effects of estrogen, raloxifene, and levormeloxifene on the expression of Rho-kinase signaling molecules in urethral smooth muscle cells.

OBJECTIVES: To investigate the effects of estrogen, raloxifene, and levormeloxifene on the expression of Rho-kinase signaling molecules in urethral smooth muscle cells (USMCs). METHODS: USMCs were isolated from female rats. Expression of calponin and estrogen receptors α (ERα) was detected by immunofluorescence staining. Cells were treated with estrogen, raloxifene, or levormeloxifene at 0, 1, 10, and 100 nmol/L for 48 h and then processed for Western blotting with antibodies against RhoA, Rho kinase I and II (Rock-I and Rock-II), myosin light chain (MLC), phosphorylated MLC, and ß-actin. Protein expression was quantitated by densitometry, followed by statistical analysis with ß-actin as control. RESULTS: USMCs expressed calponin and ERα. Treatment of USMCs with estrogen, raloxifene or levormeloxifene resulted in decreased expression of RhoA, Rock-I, Rock-II, and p-MLC in a dosage-dependent manner. CONCLUSIONS: Estrogen, raloxifene, and levormeloxifene may affect urinary continence by inhibiting the expression of Rho-kinase signaling molecules.

The selective estrogen receptor modulator raloxifene inhibits cardiac delayed rectifier potassium currents and voltage-gated sodium current without QTc interval prolongation.

Raloxifene is widely used in the treatment of postmenopausal osteoporosis and also has been shown to be cardioprotective. The effect of raloxifene on cardiac ion channels is not fully understood. The present study investigated whether raloxifene could affect the cloned hERG channel (I(hERG)) and recombinant human cardiac KCNQ1/KCNE1 channel (I(Ks)) stably expressed in HEK 293 cells using a patch-clamp technique. Raloxifene blocked I(hERG) with an IC(50) of 1.1 µM and decreased I(Ks) (IC(50): 4.8 µM) without affecting activation kinetics. In addition, raloxifene significantly decreased I(Na) (IC(50): 2.8 µM) in guinea pig ventricular myocytes. However, this drug (1 µM) did not increase QRS and QTc interval in isolated guinea pig hearts. These results demonstrate that raloxifene, despite its inhibitory action on delayed rectifier potassium currents, does not prolong ECG QTc interval, suggesting that raloxifene is likely a safe selective estrogen receptor modulator with less cardiac toxicity.

Development and validation of a test for environmental estrogens: Checking xeno-estrogen activity by CXCL12 secretion in BREAST CANCER CELL LINES (CXCL-test).

Several methods have been developed to evaluate and quantify the effects of Endocrine disruptor chemicals (EDC). Nevertheless, most of these methods are time-consuming or not enough sensitive to detect EDC at the environmental range. To link the biological effect of tested EDC to natural protein secretion, we have developed a new screening method based on the secretion of the cytokine CXCL12 (or SDF-1, Stroma-cell Derived Factor 1), which plays a capital role in cell survival and migration. We have demonstrated that CXCL12 secretion is regulated by estrogenic compounds in a dose-dependent way in ER-positive breast cancer cell lines (MCF-7 and T47D). By combining cell culture and ELISA test, we used this up-regulation of CXCL12 secretion to test several major environmental contaminants. Our results showed that 17ß-estradiol (from 10(-11) M), 17α-ethynylestradiol (from 10(-12) M), genistein (from 10(-8) M) and bisphenol A (from 10(-6) M) dose-regulate CXCL12 secretion in T47D. In contrast, antiestrogens, raloxifen and 4-hydroxytamoxifen, had no effect on the CXCL12 secretion, but were able to inhibit E2 effect. Moreover, we used cell proliferation assays to evaluate the effect of these different compounds on the growth of T47D cells. We found strong correlation (P = 0.7) between proliferation and CXCL12 secretion. However CXCL12 secretion was as sensitive as cell proliferation assays but appeared more rapid. Thus, this bioassay named CXCL-test (for Checking Xeno-estrogen activity by CXCL12 secretion in breast cancer cell Lines) constitutes a fast and sensitive method for the detection of estrogenic compounds allowing in 14 h to achieve a detection limit of 10(-11) M of E2 (2.7 ng/L).

Lasofoxifene: a third-generation selective estrogen receptor modulator for the prevention and treatment of osteoporosis.

This article reviews lasofoxifene, a new-generation selective estrogen receptor modulator (SERM) that is currently in Phase III development for the prevention and treatment of osteoporosis in postmenopausal women. This compound selectively binds to both of the estrogen receptors with a high affinity and a median inhibitory concentration that is similar to that seen with estradiol and > or = 10-fold higher than those reported for other SERMs (raloxifene and tamoxifen). Lasofoxifene has a remarkably improved oral bioavailability with respect to other SERMs due to increased resistance to intestinal wall glucuronidation. In both preclinical and short-term studies, the compound showed a favourable safety profile and demonstrated a proven efficacy in preventing bone loss and lowering cholesterol levels. Dose modelling from Phase II studies allowed the selection of lasofoxifene 0.25 mg/day as the lowest fully effective dose.

Oxidation of raloxifene to quinoids: potential toxic pathways via a diquinone methide and o-quinones.

Raloxifene was approved in 1997 by the FDA for the treatment of osteoporosis in postmenopausal women, and it is currently in clinical trials for the chemoprevention of breast cancer. Before widespread use as a chemopreventive agent in healthy women, the potential cytotoxic mechanisms of raloxifene should be investigated. In the current study, raloxifene was incubated with GSH and either rat or human liver microsomes, and the metabolites and GSH conjugates were characterized using liquid chromatography-tandem mass spectrometry. Raloxifene was converted to raloxifene diquinone methide GSH conjugates, raloxifene o-quinone GSH conjugates, and raloxifene catechols. For comparison, three raloxifene catechols were synthesized and characterized. In particular, 7-hydroxyraloxifene was found to oxidize to the 6,7-o-quinone. As compared with raloxifene diquinone methide, which has a half-life of less than 1 s in phosphate buffer, the half-life of raloxifene 6,7-o-quinone was much longer at t(1/2) = 69 +/- 2.5 min. The stability offered by raloxifene 6,7-o-quinone implies that it may be more toxic than raloxifene diquinone methide. Cytotoxicity studies in the human breast cancer cell lines S30 and MDA-MB-231 showed that 7-hydroxyraloxifene was more toxic than raloxifene in both cell lines. These results suggest that raloxifene could be metabolized to electrophilic and redox active quinoids, which have the potential to cause toxicity in vivo.

Nitrosation, nitration, and autoxidation of the selective estrogen receptor modulator raloxifene by nitric oxide, peroxynitrite, and reactive nitrogen/oxygen species.

The regulation of estrogenic and antiestrogenic effects by selective estrogen receptor modulators (SERMs) provides the basis for use in long-term therapy in cancer chemoprevention and postmenopausal osteoporosis. However, the evidence for carcinogenic properties within this class requires study of potential pathways of toxicity. There is strong evidence for the elevation of cellular levels of NO in tissue treated with SERMs, including the benzothiophene derivative, raloxifene, in part via up-regulation of nitric oxide synthases. Therefore, the reactions of 17beta-estradiol (E(2)), raloxifene, and an isomer with NO, peroxynitrite, and reactive nitrogen/oxygen species (RNOS) generated from NO(2)(-)/H(2)O(2) systems were examined. Peroxynitrite from bolus injection or slow release from higher concentrations of 3-morpholinosydnonimine (SIN-1) reacted with the benzothiophenes and E(2) to give aromatic ring nitration, whereas peroxynitrite, produced from the slow decomposition of lower concentrations of SIN-1, was relatively unreactive toward E(2) and yielded oxidation and nitrosation products with raloxifene and its isomer. The oxidation and nitrosation products formed were characterized as a dimer and quinone oxime derivative. Interestingly, the reaction of the benzothiophenes with NO in aerobic solution efficiently generated the same oxidation products. Stable quinone oximes are not unprecedented but have not been previously reported as products of RNOS-mediated metabolism. The reaction of glutathione (GSH) with the quinone oxime gave both GSH adducts from Michael addition and reduction to the corresponding o-aminophenol. The ready autoxidation of raloxifene, observed in the presence of NO, is the first such observation on the reactivity of SERMs and is potentially a general phenomenon of significance to SERM chemical toxicology.

Toxicity of antiestrogens.

The object of this article is to review briefly the preclinical and clinical safety of some antiestrogens. Tamoxifen, toremifene, droloxifene, and idoxifene are polyphenylethylene antiestrogens, whereas the pure antiestrogen, ICI 182,780 or faslodex, as well as raloxifene, is of a different structure. Tamoxifen has been shown to be genotoxic in several studies. It induces unscheduled DNA synthesis in rat hepatocytes and micronuclei in MCL-5 a cells in vitro. Tamoxifen also induces aneuploidy in rat liver in vivo and chromosome aberrations and micronuclei in mouse bone marrow. Toremifene has also shown to be genotoxic, but to a far lower extent, by inducing micronuclei in MCL-5 a cells in vitro and by inducing aneuploidy in rat liver in vivo. Tamoxifen has been shown to be hepatocarcinogenic in the rat in at least four independent long-term studies. The initiation of tumors in the rat is the result of metabolic activation by cytochrome P450 isoenzymes to an electrophile(s) that binds irreversibly to DNA. The other antiestrogens have not been shown to be carcinogenic in rodents. In several independent clinical studies, the risk of endometrial cancer has increased among tamoxifen-treated women. After reviewing the available data, the International Agency for Research on Cancer concluded that there was sufficient evidence to show that tamoxifen is a class I human carcinogen. The increased risk for endometrial cancer occurs predominantly among women who are 50 years old or older and who have been treated with tamoxifen. It is not yet clear whether the uterine tumor formation is a result of genetic mechanisms, analogous to those seen in the rat liver or due to the estrogen agonist action of tamoxifen. However, the other antiestrogens with a more or less similar intrinsic estrogenic potential have not been shown to be carcinogenic in humans.

Proliferative lesions of ovarian granulosa cells and reversible hormonal changes induced in rats by a selective estrogen receptor modulator.

This study assessed the effects of raloxifene, a selective estrogen receptor modulator (SERM), on ovarian morphology and circulating hormone levels in rats. Female Fischer-344 rats (65/group) were given dietary raloxifene for 6 months at average daily doses of 0, 15, 75, and 365 mg/kg. Morphologic evaluation of ovaries was conducted on 25 rats/group at the end of the treatment period and from 20 rats per group after 1 and 3 months withdrawal from treatment. Plasma hormone analyses were conducted on 10 rats pergroup at the end of the treatment period and aftereach withdrawal period. Treatment with raloxifene for 6 months resulted in disruption of the hypothalamic-pituitary-ovarian axis, manifested by increased plasma concentrations of luteinizing hormone (LH) and estradiol-17beta (E2), and failure of ovulation, manifested by ovarian follicular prominence (retained anovulatory follicles), lack of corpora lutea (CL), and depressed plasma progesterone (P4). Many (56% to 80%) rats in all raloxifene treated groups had focal, minimal to slight hyperplasia of granulosa cells within individual retained follicles. A few treated rats in the mid- and high-dose groups (2 of 25 and 3 of 25, respectively) had more extensive focal proliferation of granulosa cells. These foci were approximately 3 to 6 mm in overall size and were characterized by moderate papillary proliferation of large granulosa cells associated with cystic spaces, often with hemorrhage. In 4 of the 5 rats with this focal cystic granulosa cell hyperplasia, the remainder of the involved ovary and the contralateral ovary were atrophic. After 1 or 3 months of drug withdrawal, most previously treated rats examined had morphologic evidence of ovarian cyclic changes. including developing follicles, various stages of CL, and normal plasma levels of LH, E2, and P4. Continued lack of cyclic changes was limited to 4 of 20 rats from the low-dose group after 1 month of recovery and to 1 low dose rat after 3 months. Intrafollicular granulosa cell hyperplasia was not seen in rats in the reversibility phase. Areas of prior focal cystic granulosa cell hyperplasia were represented by focal sclerosis that included hemorrhage and/or hemosiderin. The foci of sclerosis were associated with cystic spaces after 1 month and were solid after 3 months. A granulosa cell tumor, approximately 12-13 mm diameter, was present in a high-dose rat in the 3-month reversibility group. This tumor effaced 1 ovary and was characterized by proliferative granulosa cells, usually in papillary formations and cords within cystic spaces. This rat had atrophy of the uninvolved ovary, excessive plasma levels of E2 and prolactin, and high P4 levels considering the absence of CL. The results of this study indicate that ovarian granulosa cells in rats are susceptible to proliferative changes when stimulated chronically with excessive trophic hormones. Most of these proliferative changes were reversible upon cessation of the hormonal stimulation. However, the proliferative lesion in one treated rat progressed to apparent autonomous (neoplastic) growth.

Proliferative lesions of ovarian granulosa cells and reversible hormonal changes induced in rats by a selective estrogen receptor modulator.

This study assessed the effects of raloxifene. a selective estrogen receptor modulator (SERM), on ovarian morphology and circulating hormone levels in rats. Female Fischer-344 rats (65/group) were given dietary raloxifene for 6 months at average daily doses of 0, 15, 75, and 365 mg/kg. Morphologic evaluation of ovaries was conducted on 25 rats/group at the end of the treatment period and from 20 rats per group after 1 and 3 months withdrawal from treatment. Plasma hormone analyses were conducted on 10 rats per group at the end of the treatment period and after each withdrawal period. Treatment with raloxifene for 6 months resulted in disruption of the hypothalamic-pituitary-ovarian axis, manifested by increased plasma concentrations of luteinizing hormone (LH) and estradiol-17beta (E2), and failure of ovulation, manifested by ovarian follicular prominence (retained anovulatory follicles), lack of corpora lutea (CL), and depressed plasma progesterone (P4). Many (56% to 80%) rats in all raloxifene treated groups had focal, minimal to slight hyperplasia of granulosa cells within individual retained follicles. A few treated rats in the mid- and high-dose groups (2 of 25 and 3 of 25, respectively) had more extensive focal proliferation of granulosa cells. These foci were approximately 3 to 6 mm in overall size and were characterized by moderate papillary proliferation of large granulosa cells associated with cystic spaces, often with hemorrhage. In 4 of the 5 rats with this focal cystic granulosa cell hyperplasia, the remainder of the involved ovary and the contralateral ovary were atrophic. After 1 or 3 months of drug withdrawal, most previously treated rats examined had morphologic evidence of ovarian cyclic changes, including developing follicles, various stages of CL, and normal plasma levels of LH, E2, and P4. Continued lack of cyclic changes was limited to 4 of 20 rats from the low-dose group after 1 month of recovery and to 1 low dose rat after 3 months. Intrafollicular granulosa cell hyperplasia was not seen in rats in the reversibility phase. Areas of prior focal cystic granulosa cell hyperplasia were represented by focal sclerosis that included hemorrhage and/or hemosiderin. The foci of sclerosis were associated with cystic spaces after 1 month and were solid after 3 months. A granulosa cell tumor, approximately 12-13 mm diameter, was present in a high-dose rat in the 3-month reversibility group. This tumor effaced 1 ovary and was characterized by proliferative granulosa cells, usually in papillary formations and cords within cystic spaces. This rat had atrophy of the uninvolved ovary, excessive plasma levels of E2 and prolactin, and high P4 levels considering the absence of CL. The results of this study indicate that ovarian granulosa cells in rats are susceptible to proliferative changes when stimulated chronically with excessive trophic hormones. Most of these proliferative changes were reversible upon cessation of the hormonal stimulation. However, the proliferative lesion in one treated rat progressed to apparent autonomous (neoplastic) growth.

The reversible effects of raloxifene on luteinizing hormone levels and ovarian morphology in mice.

Raloxifene is a selective estrogen receptor modulator that has estrogen agonist effects on bone and serum lipids and estrogen antagonist effects on breast and uterine tissues. This study assessed the effects of raloxifene hydrochloride (HCl) treatment on circulating luteinizing hormone (LH) levels and ovarian morphology in sexually mature, 15-week-old, female CD-1 mice. Mice were maintained on diets providing average daily doses of 0 or 233 mg/kg raloxifene for 2 weeks (Study 1) or 0, 7.9, or 236 mg/kg raloxifene for 4 weeks (Study 2). At the end of the treatment period, blood samples were collected every 2 hours for 24 h in Study 1 (5 mice per group) and at 10:00 a.m. and 10:00 p.m. in Study 2 (8 mice per group). Serum LH levels were measured by radioimmunoassay. Ovarian histomorphology was evaluated in the 10 mice per group (Study 1) and the 8 mice per group (Study 2). For the reversibility phase (Study 2), mice were fed untreated diets for 3 weeks; serum LH levels and ovarian histomorphology were then assessed. Raloxifene treatment at 233 mg/kg/day for 2 weeks (Study 1) significantly elevated circulating LH levels by 4- to 7-fold compared with control. Raloxifene-treated mice had elevated LH levels sustained over the 24-h sampling period and did not exhibit the preovulatory LH surge evident in some control mice at the 4:00 p.m., 6:00 p.m., and 8:00 p. m. time points. Mice treated with 236 mg/day raloxifene for 4 weeks (Study 2) had elevated LH levels (4.4-fold compared to control), whereas mice exposed to 7.9 mg/kg/day raloxifene had a slight, nonsignificant increase in LH (2-fold compared to control). In both dose groups, LH levels were indistinguishable from controls 3 weeks after raloxifene treatment was discontinued. The ovaries in six of the eight mice treated with 7.9 mg/kg/day raloxifene had dilated and/or anovulatory follicles. One mouse in this group had a single hemorrhagic follicle; however, corpora lutea distribution was normal, indicating that ovulation was occurring. Raloxifene-treated mice in Study 1 and mice treated with a comparable raloxifene dose (236 mg/day) in Study 2 had histomorphological changes in the ovary indicative of arrested follicular maturation, including anovulatory hemorrhagic follicles, some developing follicles, and very few corpora lutea. At the end of the reversibility phase, hemorrhagic follicles were no longer evident and follicular maturation and corpora lutea distribution were normal. Raloxifene treatment in mice produces a dose-dependent, sustained elevation in serum LH levels and is associated with changes in ovarian follicular morphology. These changes are reversible upon discontinuation of raloxifene treatment.