Preparation, characterization and in vitro evaluation of microemulsion of raloxifene hydrochloride.

Raloxifene hydrochloride (RLX) is a selective estrogen receptor modulator which is orally used for treatment of osteoporosis and prevention of breast cancer. The drug has low aqueous solubility and bioavailability. The aim of the present study is to formulate and characterize oil-in-water microemulsion systems for oral delivery of RLX. To enhance the drug aqueous solubility, microemulsion based on sesame oil was prepared. Sesame oil and Tween 80 were selected as the drug solvent oil and surfactant, respectively. In the first and second formulations, Edible glycerin and Span 80 were applied as co-surfactant, respectively. Pseudo-ternary phase diagrams showed that the best surfactant/co-surfactant ratios in the first and second formulations were 4:1 and 9:1, respectively. The particle size of all free drug-loaded and drug loaded samples were in the range of 31.25 ± 0.3 nm and 60.9 ± 0.1 nm, respectively. Electrical conductivity coefficient and refractive index of all microemulsion samples confirmed the formation of oil-in-water type of microemulsion. In vitro drug release profile showed that after 24 hours, 46% and 63% of the drug released through the first formulation in 0.1% (w/v) Tween 80 in distilled water as a release medium and phosphate buffer solution (PBS) at pH = 5.5, respectively. These values were changed to 57% and 98% for the second formulation. Results confirmed that the proposed microemulsion system containing RLX could improve and control the drug release profile in comparison to conventional dosage form.

Testosterone attenuates and the selective estrogen receptor modulator, raloxifene, potentiates amphetamine-induced locomotion in male rats.

Although sex steroids are known to modulate brain dopamine, it is still unclear how testosterone modifies locomotor behaviour controlled, at least in part, by striatal dopamine in adolescent males. Our previous work suggests that increasing testosterone during adolescence may bias midbrain neurons to synthesise more dopamine. We hypothesised that baseline and amphetamine-induced locomotion would differ in adult males depending on testosterone exposure during adolescence. We hypothesised that concomitant stimulation of estrogen receptor signaling, through a selective estrogen receptor modulator (SERM), raloxifene, can counter testosterone effects on locomotion. Male Sprague-Dawley rats at postnatal day 45 were gonadectomised (G) or sham-operated (S) prior to the typical adolescent testosterone increase. Gonadectomised rats were either given testosterone replacement (T) or blank implants (B) for six weeks and sham-operated (i.e. intact or endogenous testosterone group) were given blank implants. Subgroups of sham-operated, gonadectomised and gonadectomised/testosterone-replaced rats were treated with raloxifene (R, 5mg/kg) or vehicle (V), daily for the final four weeks. There were six groups (SBV, GBV, GTV, SBR, GBR, GTR). Saline and amphetamine-induced (1.25mg/kg) locomotion in the open field was measured at PND85. Gonadectomy increased amphetamine-induced locomotion compared to rats with endogenous or with exogenous testosterone. Raloxifene increased amphetamine-induced locomotion in rats with either endogenous or exogenous testosterone. Amphetamine-induced locomotion was negatively correlated with testosterone and this relationship was abolished by raloxifene. Lack of testosterone during adolescence potentiates and testosterone exposure during adolescence attenuates amphetamine-induced locomotion. Treatment with raloxifene appears to potentiate amphetamine-induced locomotion and to have an opposite effect to that of testosterone in male rats.

Estrogens, selective estrogen receptor modulators, and a selective estrogen receptor down-regulator inhibit endothelial production of tissue factor pathway inhibitor 1.

BACKGROUND: Hormone therapy, oral contraceptives, and tamoxifen increase the risk of thrombotic disease. These compounds also reduce plasma content of tissue factor pathway inhibitor-1 (TFPI), which is the physiological inhibitor of the tissue factor pathway of coagulation. The current aim was to study if estrogens and estrogen receptor (ER) modulators may inhibit TFPI production in cultured endothelial cells and, if so, identify possible mechanisms involved. METHODS: Human endothelial cell cultures were treated with 17beta-estradiol (E2), 17alpha-ethinylestradiol (EE2), tamoxifen, raloxifene, or fulvestrant. Protein levels of TFPI in cell media and cell lysates were measured by an enzyme-linked immunosorbent assay, and TFPI mRNA levels were assessed by quantitative PCR. Expression of ERalpha was analysed by immunostaining. RESULTS: All compounds (each in a concentration of 10 nM) reduced TFPI in cell medium, by 34% (E2), 21% (EE2), 16% (tamoxifen), and 28% (raloxifene), respectively, with identical inhibitory effects on cellular TFPI levels. Expression of TFPI mRNA was principally unchanged. Treatment with fulvestrant, which was also associated with down-regulation of secreted TFPI (9% with 10 nM and 26% with 1000 nM), abolished the TFPI-inhibiting effect of raloxifene, but not of the other compounds. Notably, the combination of 1000 nM fulvestrant and 10 nM raloxifene increased TFPI secretion, and, conversely, 10 nM of either tamoxifen or raloxifene seemed to partly (tamoxifen) or fully (raloxifene) counteract the inhibitory effect of 1000 nM fulvestrant. The cells did not express the regular nuclear 66 kDa ERalpha, but instead a 45 kDa ERalpha, which was not regulated by estrogens or ER modulators. CONCLUSION: E2, EE2, tamoxifen, raloxifene, and fulvestrant inhibited endothelial production of TFPI by a mechanism apparently independent of TFPI transcription.

A synthetic peptide with estrogen-like activity derived from a phage-display peptide library.

We describe a novel approach to develop peptides with estrogen like activity using a monoclonal antibody specific to estradiol (mAb E2-15) for the affinity selection of phage displayed peptides from a combinatorial peptide library. Based on the sequences of the selected phage, we synthesized a 15-mer linear peptide LPALDPTKRWFFETK which was derivatized to a 23 mer cyclic peptide CAELPALDPTKRWFFETKPPPPC. Both peptides displayed estrogen-like activity according to the following criteria:(i) in inhibiting the binding of [3H]estradiol to mAb E2-15 and to estrogen receptor (ER)alpha; (ii) in inducing transcriptional activity in MCF7 human breast cancer cells transfected with an estrogen receptor element luciferase construct and (iii) in causing an increase in creatine kinase specific activity in rat tissues in vivo. This approach can be employed to design peptide mimetic for other hormones as well.

Neuroendocrine effects of raloxifene hydrochloride in postmenopausal women.

Raloxifene is a selective estrogen modulator able to exert an estrogen-like action on some target tissues and a specific antiestrogenic action on the uterus and breast. In ovariectomized rats, it has been shown to stimulate the beta-endorphin and allopregnanolone concentrations of the anterior and neurointermediate pituitary lobes, the hypothalamus and the hippocampus. The present study aimed to evaluate, in 12 healthy postmenopausal women, the effect of 60 mg/day raloxifene hydrochloride administration for 6 months on plasma beta-endorphin and allopregnanolone levels, and on the dynamic changes of both beta-endorphin and allopregnanolone secretion after the administration of: (1) clonidine, an alpha 2-presynaptic adrenergic agonist; (2) naloxone, an opioid receptor antagonist; and (3) fluoxetine, a serotonin selective reuptake inhibitor. The administration of raloxifene significantly increased both circulating beta-endorphin and allopregnanolone concentrations, at both the third and sixth months of treatment (p < 0.01). Clonidine, fluoxetine and naloxone administration before therapy was not able to stimulate the release of beta-endorphin, but the response was completely restored after raloxifene administration. Before therapy, clonidine and naloxone tests were accompanied by a significant rise in allopregnanolone secretion; the same changes were observed after raloxifene administration, but with significantly higher allopregnanolone concentrations at each time considered. While the fluoxetine test before therapy failed to increase the release of allopregnanolone, the same test after 6 months of raloxifene administration was characterized by a significant release of allopregnanolone at 60 and 90 minutes. The present data indicate that raloxifene has an estrogen-like effect on neuroendocrine pathways in postmenopausal women.