Green Tea Catechins Decrease Solubility of Raloxifene In Vitro and Its Systemic Exposure in Mice.

PURPOSE: Green tea is a widely consumed beverage. A recent clinical study reported green tea decreased systemic exposure of raloxifene and its glucuronide metabolites by 34-43%. However, the underlying mechanism(s) remains unknown. This study investigated a change in raloxifene's solubility as the responsible mechanism. METHODS: The effects of green tea extract, (-)-epigallocatechin gallate (EGCG), and (-)-epigallocatechin (EGC) on raloxifene's solubility were assessed in fasted state simulated intestinal fluids (FaSSIF) and fed state simulated intestinal fluids (FeSSIF). EGCG and EGC represent green tea's main bioactive constituents, flavan-3-gallate and flavan-3-ol catechins respectively, and the tested concentrations (mM) match the µg/mg of each compound in the extract. Our mouse study (n = 5/time point) evaluated the effect of green tea extract and EGCG on the systemic exposure of raloxifene. RESULTS: EGCG (1 mM) and EGC (1.27 mM) decreased raloxifene's solubility in FaSSIF by 78% and 13%, respectively. Micelle size in FaSSIF increased with increasing EGCG concentrations (> 1000% at 1 mM), whereas EGC (1.27 mM) did not change micelle size. We observed 3.4-fold higher raloxifene solubility in FeSSIF compared to FaSSIF, and neither green tea extract nor EGCG significantly affected raloxifene solubility or micelle size in FeSSIF. The mice study showed that green tea extract significantly decreased raloxifene Cmax by 44%, whereas EGCG had no effect. Green tea extract and EGCG did not affect the AUC0-24 h of raloxifene or the metabolite-to-parent AUC ratio. CONCLUSIONS: This study demonstrated flavan-3-gallate catechins may decrease solubility of poorly water-soluble drugs such as raloxifene, particularly in the fasted state.

Advantage of ostarine over raloxifene and their combined treatments for muscle of estrogen-deficient rats.

PURPOSE: Selective androgen (ostarine, OST) and estrogen (raloxifene, RAL) receptor modulators with improved tissue selectivity have been developed as alternatives to hormone replacement therapy. We investigated the combined effects of OST and RAL on muscle tissue in an estrogen-deficient rat model of postmenopausal conditions. METHODS: Three-month-old Sprague Dawley rats were divided into groups: (1) untreated non-ovariectomized rats (Non-OVX), (2) untreated ovariectomized rats (OVX), (3) OVX rats treated with OST, (4) OVX rats treated with RAL, (5) OVX rats treated with OST and RAL. Both compounds were administered in the diet. The average dose received was 0.6 ± 0.1 mg for OST and 11.1 ± 1.2 mg for RAL per kg body weight/day. After thirteen weeks, rat activity, muscle weight, structure, gene expression, and serum markers were analyzed. RESULTS: OST increased muscle weight, capillary ratio, insulin-like growth factor 1 (Igf-1) expression, serum phosphorus, uterine weight. RAL decreased muscle weight, capillary ratio, food intake, serum calcium and increased Igf-1 and Myostatin expression, serum follicle stimulating hormone (FSH). OST + RAL increased muscle nucleus ratio, uterine weight, serum phosphorus, FSH and luteinizing hormone and decreased body and muscle weight, serum calcium. Neither treatment changed muscle fiber size. OVX increased body and muscle weight, decreased uterine weight, serum calcium and magnesium. CONCLUSION: OST had beneficial effects on muscle in OVX rats. Side effects of OST on the uterus and serum electrolytes should be considered before using it for therapeutic purposes. RAL and RAL + OST had less effect on muscle and showed endocrinological side effects on pituitary-gonadal axis.

Co-consuming green tea with raloxifene decreases raloxifene systemic exposure in healthy adult participants.

Green tea is a popular beverage worldwide. The abundant green tea catechin (-)-epigallocatechin gallate (EGCG) is a potent in vitro inhibitor of intestinal UDP-glucuronosyltransferase (UGT) activity (Ki ~2 µM). Co-consuming green tea with intestinal UGT drug substrates, including raloxifene, could increase systemic drug exposure. The effects of a well-characterized green tea on the pharmacokinetics of raloxifene, raloxifene 4'-glucuronide, and raloxifene 6-glucuronide were evaluated in 16 healthy adults via a three-arm crossover, fixed-sequence study. Raloxifene (60 mg) was administered orally with water (baseline), with green tea for 1 day (acute), and on the fifth day after daily green tea administration for 4 days (chronic). Unexpectedly, green tea decreased the geometric mean green tea/baseline raloxifene AUC0-96h ratio to ~0.60 after both acute and chronic administration, which is below the predefined no-effect range (0.75-1.33). Lack of change in terminal half-life and glucuronide-to-raloxifene ratios indicated the predominant mechanism was not inhibition of intestinal UGT. One potential mechanism includes inhibition of intestinal transport. Using established transfected cell systems, a green tea extract normalized to EGCG inhibited 10 of 16 transporters tested (IC50 , 0.37-12 µM). Another potential mechanism, interruption by green tea of gut microbe-mediated raloxifene reabsorption, prompted a follow-up exploratory clinical study to evaluate the potential for a green tea-gut microbiota-drug interaction. No clear mechanisms were identified. Overall, results highlight that improvements in current models and methods used to predict UGT-mediated drug interactions are needed. Informing patients about the risk of co-consuming green tea with raloxifene may be considered.

The Combination Sorafenib-raloxifene-loratadine as a Novel Potential Therapeutic Approach Against Human Liver Cancer.

BACKGROUND/AIM: Liver cancer is one of the malignancies with the highest mortality-to-incidence ratio worldwide. Therefore, novel therapeutic approaches are urgently needed. Combination therapy and drug repurposing can improve the response of the patients to therapy in several cancers. The aim of the present study was to merge these two strategies and evaluate whether the two-drug- or three-drug- combination of sorafenib, raloxifene, and loratadine improves the antineoplastic effect on human liver cancer cells in comparison to the single-drug effect. MATERIALS AND METHODS: The human liver cancer cell lines HepG2 and HuH7 were studied. The effect of sorafenib, raloxifene, and loratadine on the metabolic activity was determined using the MTT assay. The inhibitory concentrations (IC20 and IC50) were calculated from these results and used in the drug-combination experiments. Apoptosis and cell survival were studied by flow cytometry and using the colony formation assay, respectively. RESULTS: In both cell lines, sorafenib, raloxifene, and loratadine in two-drug and three-drug combinations significantly reduced metabolic activity and significantly increased the percentage of apoptotic cells compared to the single-drug effect. In addition, all the combinations significantly reduced the colony-forming capacity in the HepG2 cell line. Surprisingly, the effect of raloxifene on apoptosis was similar to that observed using the combinations. CONCLUSION: The triple combination sorafenib-raloxifene-loratadine may be a novel promising approach in the treatment of liver cancer patients.

Implications and Efficacy of Aromatase Inhibitors in Combination and Monotherapy for the Treatment of Lung Cancer.

BACKGROUND: Lung tumors express high levels of aromatase enzyme compared to surrounding normal tissue. Inhibition of aromatase has emerged as a recent therapeutic approach for the treatment of breast cancer. However, the role of aromatase inhibition in lung cancer treatment requires further investigation. METHODS: The anti-proliferative effects of aromatase inhibitors were evaluated by MTT assay. Cell migration was assessed using a wound healing assay. The mechanism of cell death was determined using the annexin VFITC/ propidium iodide staining flow cytometry method. The soft agar colony formation assay evaluated cells' capability to form colonies. RESULT: Exemestane and curcumin significantly inhibited the growth of lung cancer cell lines in a dose- and timedependent manner. The IC50 values after 48 hours of treatment with exemestane were 176, 180, and 120 µM in A549, H661, and H1299, respectively. Curcumin IC50 values after 48 hours were 80, 43, and 68 µM in A549, H661, and H1299, respectively. The combined treatment of exemestane or curcumin with cisplatin, raloxifene, and celecoxib resulted in a synergistic effect in the A549 lung cell line with a combination index of less than 1, suggesting synergism. Exemestane resulted in approximately 96% inhibition of wound closure at 100 µM, while curcumin resulted in approximately 63% inhibition of wound closure at 50 µM. Exemestane and curcumin inhibited the formation of cell colonies by reducing the number and size of formed colonies of A549, H661, and H1299 cell lines in a concentration dependent manner. Exemestane and curcumin had significantly induced apoptosis in A549 cells compared to control of untreated cells. CONCLUSION: Aromatase inhibition by exemestane or curcumin had significantly inhibited the growth of lung cancer cell lines, synergized with cisplatin, raloxifene, and celecoxib, suppressed lung cancer cell migratory potential, induced apoptosis, and reduced colony formation of lung cancer cells.

Repurposing Approved Drugs as Fluoroquinolone Potentiators to Overcome Efflux Pump Resistance in Staphylococcus aureus.

Staphylococcus aureus is a versatile human commensal bacteria and pathogen that causes various community and hospital-acquired infections. The S. aureus efflux pump NorA which belongs to the major facilitator superfamily, confers resistance to a range of substrates. Many efflux pump inhibitors (EPIs) have been discovered, but none is clinically approved due to their undesirable toxicities. In this study, we have screened clinically approved drugs for possible NorA EPI-like activity. We identified six drugs that showed the best efflux pump inhibition in vitro, with a fractional inhibitory concentration index of ≤0.5, indicating synergism with hydrophilic fluoroquinolones. The mechanistic validation of efflux inhibitory potential was demonstrated in ethidium bromide-based accumulation and efflux inhibition assays. We further confirmed the functionality of EPIs by norfloxacin accumulation assay depicting more realistic proof of the conjecture. None of the EPIs disturbed membrane function or depleted the ATP synthesis levels in bacteria. Both raloxifene and pyrvinium displayed an increase in bactericidal activity of ciprofloxacin in time-kill kinetics, prolonged its post-antibiotic effect, and reduced the frequency of spontaneous resistant mutant development. The combination of EPIs with ciprofloxacin caused significant eradication of preformed biofilms. Moreover, in the murine thigh infection model, a single dose of pyrvinium combined with ciprofloxacin reduced the bacterial burden significantly compared to untreated control and ciprofloxacin alone, indicating the efficacy of the combination. Conclusively, this study represents approved drugs that can be repurposed and combined with antibiotics as NorA EPIs, having anti-biofilm properties to treat severe S. aureus infections at clinically relevant concentrations. IMPORTANCE Staphylococcus aureus is a frequent pathogen bacterium and the predominant cause of worsened nosocomial infections. Efflux pumps contribute to drug efflux and are reportedly associated with biofilm formation, thereby promoting difficult-to-treat biofilm-associated S. aureus infections. One strategy to combat these bacteria is to reduce active efflux and increase pathogen sensitivity to existing antibiotics. Repurposing approved drugs may solve the classical toxicity issues with previous efflux pump inhibitors and help reach sufficient plasma concentrations. We describe the in silico-based screening of FDA-approved drugs that identified six different molecules able to inhibit NorA pump (Major Facilitator Superfamily). Our study highlights that these compounds bind to and block the activity of the NorA pump and increase the sensitivity of S. aureus and methicillin-resistant S. aureus to fluoroquinolones. These drugs combined with fluoroquinolones significantly reduced the preformed biofilms and displayed significant efficacy in the murine thigh infection model when compared to untreated control and ciprofloxacin alone.

Turning scientific serendipity into discoveries in breast cancer research and treatment: a tale of PhD students and a 50-year roaming tamoxifen team.

PURPOSE: This retrospective, about a single "mobile" laboratory in six locations on two continents, is intended as a case study in discovery for trainees and junior faculty in the medical sciences. Your knowledge of your topic is necessary to expect the unexpected. HISTORICAL METHOD: In 1972, there was no tamoxifen, only ICI 46, 474, a non-steroidal anti-estrogen with little chance of clinical development. No one would ever be foolish enough to predict that the medicine, 20 years later, would achieve legendary status as the first targeted treatment for breast cancer, and millions of women would benefit from long-term adjuvant tamoxifen therapy. The secret of tamoxifen's success was a translational research strategy proposed in the mid 1970's. This strategy was to treat only patients with estrogen receptor (ER)-positive breast cancer and deploy 5 or more years of adjuvant tamoxifen therapy to prevent recurrence. Additionally, tamoxifen prevented mammary cancer in animals. Could the medicine prevent breast cancer in women? RESULTS: Tamoxifen and the failed breast cancer drug raloxifene became the first selective estrogen receptor modulators (SERMs): a new drug group, discovered at the University of Wisconsin, Comprehensive Cancer Center. Serendipity can play a fundamental role in discovery, but there must be a rigorous preparation for the investigator to appreciate the possibility of a pending discovery. This article follows the unanticipated discoveries when PhD students "get the wrong answer." The secret of success of my six Tamoxifen Teams was their technical excellence to create models, to decipher mechanisms, that drove the development of new medicines. Discoveries are listed that either changed women's health or allowed an understanding of originally opaque mechanisms of action of potential therapies. These advances in women's health were supported entirely by government-sponsored peer-reviewed funding and major philanthropy from the Lynn Sage Breast Cancer Foundation, the Avon Foundation, and the Susan G. Komen Breast Cancer Foundation. The resulting lives saved or extended, families aided in a time of crisis and the injection of billions of dollars into national economies by drug development, is proof of the value of Federal or philanthropic investment into unencumbered research aimed at saving millions of lives.

Osteoporosis - risk factors, pharmaceutical and non-pharmaceutical treatment.

Osteoporosis is a metabolic disease of the skeletal system which currently affects over 200 million patients worldwide. The WHO criteria define osteoporosis as low bone mineral density, with a T-score ≤ -2.5 found in the spine, the neck of the femur, or during a full hip examination. Osteoporosis considerably reduces a patient's quality of life. QoL should be carefully evaluated before fractures occur to enable the development of an appropriate treatment plan. The progression of osteoporosis may be significantly inhibited by following a proper diet, leading a healthy lifestyle, taking dietary supplements, and receiving appropriate treatment. Education and the prevention of the disease play a major role. Potentially modifiable risk factors for osteoporosis are vitamin D deficiency, smoking, alcohol consumption, low calcium intake, low or excessive phosphorus intake, protein deficiency or a high-protein diet, excessive consumption of coffee, a sedentary lifestyle or lack of mobility, and insufficient exposure to the sun. Pharmaceutical treatment for osteoporosis involves bisphosphonates, calcium and vitamin D3, denosumab, teriparatide, raloxifene, and strontium ranelate. Data indicates that 30%-50% of patients do not take their medication correctly. Other methods of treatment include exercise, kinesitherapy, treatment at a health resort, physical therapy, and diet.

Water extract of Er-xian decoction selectively exerts estrogenic activities and interacts with SERMs in estrogen-sensitive tissues.

ETHNOPHARMACOLOGICAL RELEVANCE: The increasing use of "kidney"-nourishing Traditional Chinese Medicine (TCM) like Er-xian decoction (EXD) for management of menopausal symptoms and osteoporosis has aroused concerns about their safety, and whether they interact with prescription drugs as both of them act via estrogen receptors (ERs) and regulate serum estradiol. AIM OF THE STUDY: The present study aimed to evaluate whether EXD selectively exerted estrogenic activities and interacted with Selective Estrogen Receptor Modulators (SERMs). MATERIALS AND METHODS: In vivo, mature ovariectomized (OVX) rats were administrated with EXD or combined treatment of EXD and SERMs for 12 weeks. The tissue-selective effect of EXD and its interaction of SERMs were studied in four estrogen sensitive tissues, bone, brain, breast and uterus. In vitro, the interaction of extracts of EXD-treated serum and SERMs in four ER-positive cell lines. RESULTS: In OVX rats, EXD selectively alleviated estrogen deficiency-induced changes in the bone and brain without inducing any estrogenic effects in the breast or uterus. Two-way ANOVA indicated the presence of interactions between EXD and SERMs in OVX rats but EXD did not significantly alter the tissue responses to SERMs in the bone, breast or brain. Indeed, the combined use of EXD and SERMs appeared to suppress the estrogenic effect of raloxifene and tamoxifen in the uterus. Extract of EXD-treated serum directly stimulated cell proliferation or differentiation in human osteosarcoma MG-63, neuroblastoma SHSY5Y, breast cancer MCF-7, and endometrial Ishikawa cells. Two-way ANOVA revealed that EXD-treated serum interacted with SERMs at various concentrations and altered the effects of tamoxifen in MG-63 and MCF-7 cells. CONCLUSIONS: EXD exerted estrogenic effects in a tissue-selective manner and interacted with SERMs. Combined treatment of EXD and SERMs did not hamper the beneficial effects of SERMs on the bone or brain but appeared to moderate the estrogenic effect of SERMs in the uterus.

Pinus roxburghii alleviates bone porosity and loss in postmenopausal osteoporosis by regulating estrogen, calcium homeostasis and receptor activator of nuclear factor-κB, osteoprotegerin, cathepsin bone markers.

OBJECTIVES: The study was aimed to evaluate the potential of hydroalcoholic extract of Pinus roxburghii (PRE) stem bark in post-menopausal osteoporosis and its underlying mechanisms. METHODS: In silico docking of the markers was done using AutoDock version 4.2. for molecular targets: receptor activator of nuclear factor-κB (RANK), osteoprotegerin (OPG) and Cathepsin. Female Wistar rats of bodyweight 200-250 g were employed and surgical ovariectomy (OVX) was performed. PRE was administered at a dose of 100 and 200 mg/kg whereas standard drug, raloxifene given at 1 mg/kg orally for eight weeks. KEY FINDINGS: PRE (20 and 40 µg/mL) significantly increased the cellular proliferation in osteoblastic UMR cell lines 11.58 and 15.09% respectively. Eight weeks after surgical removal of ovaries, a significant bone porosity was confirmed by modulation in bone breaking strength of tibia, lumber, and femur; bone mineral density (BMD), calcium, phosphorus, hydroxyproline levels in OVX group. Treatment with PRE 100 and 200 mg/kg significantly restored the bone loss. Real-time polymerase chain reaction (RT-PCR) analysis of molecular markers RANK, OPG and cathepsin and histology also confirmed the attenuation of bone loss. The quantification of quercetin, gallic acid, caffeic acid, catechin, tannic acid and ascorbic acid was done by high-performance liquid chromatography (HPLC) and high performance thin layer chromatography. CONCLUSIONS: P. roxburghii produced anti-osteoporotic effect possibly due to estrogenic modulation, and improved bone remodeling.

Therapeutic potential of annatto tocotrienol with self-emulsifying drug delivery system in a rat model of postmenopausal bone loss.

Tocotrienol has been shown to prevent bone loss in animal models of postmenopausal osteoporosis, but the low oral bioavailability might limit its use. A self-emulsifying drug delivery system (SEDDS) could increase the bioavailability of tocotrienol. However, evidence of this system in improving the skeletal effects of tocotrienol is scanty. This study aims to evaluate the therapeutic efficacy of annatto tocotrienol with SEDDS in a rat model of postmenopausal bone loss. Ten-month-old female Sprague Dawley rats were randomized into six groups. The baseline group was euthanatized at the onset of the study. Four other groups underwent ovariectomy to induce estrogen deficiency. The sham underwent similar surgery procedure, but their ovaries were retained. Eight weeks after surgery, the ovariectomized rats received one of the four different regimens orally daily: (a) SEDDS, (b) annatto tocotrienol [60 mg/kg body weight (b.w.)] without SEDDS, (c) annatto-tocotrienol (60 mg/kg b.w.) with SEDDS, (d) raloxifene (1 mg/kg b.w.). After eight weeks of treatment, blood was collected for the measurement of delta-tocotrienol level and oxidative stress markers. The rats were euthanized and their bones were harvested for the evaluation of the bone microstructure, calcium content and strength. Circulating delta-tocotrienol level was significantly higher in rats receiving annatto tocotrienol with SEDDS compared to the group receiving unformulated annatto-tocotrienol (p < 0.05). Treatment with unformulated or SEDDS-formulated annatto tocotrienol improved cortical bone thickness, preserved bone calcium content, increased bone biomechanical strength and increased antioxidant enzyme activities compared with the ovariectomized group (p < 0.05). Only SEDDS-formulated annatto tocotrienol improved trabecular microstructure, bone stiffness and lowered malondialdehyde level (p < 0.05 vs the ovariectomized group). The improvement caused by annatto tocotrienol was comparable to raloxifene. In conclusion, SEDDS improves the bioavailability and skeletal therapeutic effects of annatto tocotrienol in a rat model of postmenopausal bone loss. This formulation should be tested in a human clinical trial to validate its efficacy.

Raloxifene, identified as a novel LSD1 inhibitor, suppresses the migration of renal cell carcinoma.

Aim: As an important epigenetic modulator, histone lysine-specific demethylase 1 (LSD1) has been proved to be associated with the progression of renal cell carcinoma (RCC). Discovering novel LSD1 inhibitors offers therapeutic potential for RCC treatment. Methods & Results: We identified raloxifene as a novel LSD1 inhibitor (IC50 = 2.08 µM) through small compound library screening. Molecular docking indicated raloxifene might bind LSD1 in the flavin adenine dinucleotide (FAD) binding cavity in a reversible manner. Cell viability and migration assays showed raloxifene could suppress the proliferation and migration of RCC cells bearing overexpressed LSD1. Conclusion: Our findings indicated that LSD1 might be a promising therapeutic target for RCC and that raloxifene could serve as a lead compound for further anti-RCC metastasis drug discovery.

Evaluation of inhibitors of intestinal UDP-glucuronosyltransferases 1A8 and 1A10 using raloxifene as a substrate in Caco-2 cells: Studies with four flavonoids of Scutellaria baicalensis.

UDP glucuronosyltransferases (UGTs) of the gastrointestinal tract play a crucial role in protection against the toxic effects of xenobiotics in the environment. UGTs such as UGT1A8 and UGT1A10 are predominantly expressed in gastrointestinal tissues. In this study, we examined the phase II metabolism of raloxifene in differentiated Caco-2 monolayers by inducing UGT1A8 and UGT1A10 expression in these cells. The present study evaluated the following four flavonoids of Scutellaria baicalensis as model herbal compounds: scutellarein, salvigenin, baicalein, and wogonin. All test compounds, endpoint substrates, and their metabolites were quantified using liquid chromatography and high-resolution mass spectrometry. The transepithelial electrical resistance values for the individual compounds were comparable regardless of whether they were measured individually. Salvigenin significantly inhibited UGT1A8 and UGT1A10 activities in a concentration-dependent manner. All individual compounds except scutellarein inhibited UGT1A8 and UGT1A10 activity at a concentration of 100 µM. In addition, all individual flavonoids at 100 µM, except wogonin, significantly increased the amount of raloxifene in the basolateral chambers. The positive control, canagliflozin, significantly inhibited both UGT1A8 and UGT1A10 activities. These findings suggest that the Caco-2 assay can be utilized for identifying UGT1A8 and UGT1A10 inhibitors and indicate the potential of salvigenin for enhancing the pharmacological effects of UGT substrate drugs.

The short-term effects of estradiol, raloxifene, and a phytoestrogen in women with perimenopausal depression.

OBJECTIVE: We examined the short-term efficacies of three estrogen-like compounds under placebo-controlled conditions in women with perimenopause-related depression (PMD). METHODS: Women with PMD were randomized in a double-blind parallel design to one of four treatments: transdermal 17-beta estradiol (TE) (100 mcg/d); oral raloxifene (60 mg/d); a proprietary phytoestrogen compound, Rimostil (1,000 mg twice/d); or placebo for 8 weeks. The main outcome measures were the Center for Epidemiology Studies Depression Scale, 17-item Hamilton Rating Scale for Depression (HRSD), and the Beck Depression Inventory completed at each clinic visit. Secondary outcomes included a visual analogue self-rating completed at each clinic visit, and daily self-ratings of hot flush severity. Cognitive tests were performed at pretreatment baseline and at the end of the trial. In the primary analysis, we obtained four repeated measures in each woman in the four treatment arms. Analyses were done with SAS Version 9.4 software (SAS Institute, Inc, Cary, NC), using PROC MIXED (for mixed models). All models included the following four explanatory variables, regardless of whether they were statistically significant: 1) treatment group (TE, raloxifene, Rimostil, placebo); 2) week (W2, W4, W6, W8); 3) treatment group-by-week interaction; and 4) baseline value of the measure being analyzed. The inclusion of additional variables was evaluated individually for each outcome measure. RESULTS: Sixty-six women were randomized into the trial, four women dropped out of the trial, and 62 women were included in the final data analysis. No effect of treatment group was observed in either the Center for Epidemiology Studies Depression Scale (P = 0.34) or Beck Depression Inventory (P = 0.27) scores; however, there was a difference in HRSD scores between treatment groups (P = 0.0037) that pair-wise comparisons of the combined weekly scores in each treatment demonstrated TE's beneficial effects on HRSD scores compared with Rimostil (P = 0.0005), and less consistently with placebo (P = 0.099). The average (SD) of the baseline scores for each treatment group on the HRSD was as follows: TE-15.3 (4.5), raloxifene-16.0 (3.7), Rimostil-14.0 (2.7), and placebo-15.2 (3.0). Whereas the HRSD scores after 8 weeks of treatment (least-square means) were TE-5.2(1.1), raloxifene-5.8(1.2), Rimostil-11.2(1.4), and placebo-7.8(1.1). No differences were observed between raloxifene and either TE or placebo in any scale score. HRSD scores in women assigned to TE were improved compared with those on Rimostil during weeks 6 and 8 (P values = 0.0008, 0.0011, respectively). Cognitive testing at week 8 showed that none of the three active treatment groups performed better than placebo. CONCLUSIONS: This study did not identify significant therapeutic benefits of TE, Rimostil, or raloxifene compared with placebo in PMD. However, improvements in depression ratings were observed between TE compared with Rimostil. Thus, our findings do not support the role of ERbeta compounds in the treatment of PMD (and indeed could suggest a more important role of ERalpha).

Raloxifene is a Female-specific Proteostasis Therapeutic in the Spinal Cord.

Several neurodegenerative disorders are characterized by proteasome dysfunctions leading to protein aggregations and pathogenesis. Since we showed that estrogen receptor alpha (ERα) activates the proteasome, drugs able to stimulate ERα in the central nervous system (CNS) could hold potential for therapeutic intervention. However, the transcriptional effects of selective estrogen receptor modulators (SERMs), such as tamoxifen and raloxifene, can be tissue specific. A direct comparison of the effects of different SERMs on gene transcription in the CNS has never been performed. Here, we report an RNA-seq analysis of the spinal cord treated with estrogen, tamoxifen, or raloxifene. We find stark SERM and sex-specific differences in gene expression profiles in the spinal cord. Notably, raloxifene, but not estrogen or tamoxifen, modulates numerous deubiquitinating enzymes, proteasome subunits and assembly factors, and these effects translate into decreased protein aggregates. In the SOD1-G93A mouse model of amyotrophic lateral sclerosis, we found that even a low dose of raloxifene causes a significant decrease in mutant SOD1 aggregates in the spinal cord, accompanied by a delay in the decline of muscle strength in females, but not in males. These results strongly indicate SERM-selective as well as sex-specific effects, and emphasize the importance of sex as a biological variable to be considered for the careful selection of specific SERM for use in clinical trials for neurodegenerative diseases.

Antiviral drugs targeting endosomal membrane proteins inhibit distant animal and human pathogenic viruses.

The endocytic pathway is a common strategy that several highly pathogenic viruses use to enter into the cell. To demonstrate the usefulness of this pathway as a common target for the development of broad-spectrum antivirals, the inhibitory effect of drug compounds targeting endosomal membrane proteins were investigated. This study entailed direct comparison of drug effectiveness against animal and human pathogenic viruses, namely Ebola (EBOV), African swine fever virus (ASFV), and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A panel of experimental and FDA-approved compounds targeting calcium channels and PIKfyve at the endosomal membrane caused potent reductions of entry up to 90% in SARS-CoV-2 S-protein pseudotyped retrovirus. Similar inhibition was observed against transduced EBOV glycoprotein pseudovirus and ASFV. SARS-CoV-2 infection was potently inhibited by selective estrogen receptor modulators in cells transduced with pseudovirus, among them Raloxifen inhibited ASFV with very low 50% inhibitory concentration. Finally, the mechanism of the inhibition caused by the latter in ASFV infection was analyzed. Overall, this work shows that cellular proteins related to the endocytic pathway can constitute suitable cellular targets for broad range antiviral compounds.

Hyperphosphorylated tau aggregation and cytotoxicity modulators screen identified prescription drugs linked to Alzheimer's disease and cognitive functions.

The neurodegenerative Alzheimer's disease (AD) affects more than 30 million people worldwide. There is thus far no cure or prevention for AD. Aggregation of hyperphosphorylated tau in the brain correlates with the cognitive decline of patients of AD and other neurodegenerative tauopathies. Intracerebral injection of tau aggregates isolated from tauopathy brains causes similar pathology in the recipient mice, demonstrating the pathogenic role of abnormally phosphorylated tau. Compounds controlling the aggregation of hyperphosphorylated tau therefore are probable modulators for the disease. Here we report the use of recombinant hyperphosphorylated tau (p-tau) to identify potential tauopathy therapeutics and risk factors. Hyperphosphorylation renders tau prone to aggregate and to impair cell viability. Taking advantage of these two characters of p-tau, we performed a screen of a 1280-compound library, and tested a selective group of prescription drugs in p-tau aggregation and cytotoxicity assays. R-(-)-apomorphine and raloxifene were found to be p-tau aggregation inhibitors that protected p-tau-treated cells. In contrast, a subset of benzodiazepines exacerbated p-tau cytotoxicity apparently via enhancing p-tau aggregation. R-(-)apomorphine and raloxifene have been shown to improve cognition in animals or in humans, whereas benzodiazepines were linked to increased risks of dementia. Our results demonstrate the feasibility and potential of using hyperphosphorylated tau-based assays for AD drug discovery and risk factor identification.

Self-assembled lecithin-chitosan nanoparticles improve the oral bioavailability and alter the pharmacokinetics of raloxifene.

In this study, we report the development and evaluation of soy lecithin-chitosan hybrid nanoparticles to improve the oral bioavailability of raloxifene hydrochloride. The nanoparticles were formed by interaction of negatively charged soy lecithin with positively charged chitosan. The ratio of soy lecithin to chitosan was critical for the charge, and hence the size of the nanoparticles. The optimal soy lecithin to chitosan ratio was 20:1 to obtain nanoparticles with particle size of 208 ± 3 nm, a ζ-potential of 36 ± 2 mV and an entrapment efficiency of 73 ± 3%. The nanoparticles were also characterized by differential scanning calorimetry and FT-IR spectrophotometer. In-vitro drug release was assessed using dialysis bag method in pH 7.4 buffer. The drug loaded nanoparticles did not cause significant reduction in the cell viability at low doses. Pharmacokinetic studies in female Wistar rats showed significant improvement (~4.2 folds) in the oral bioavailability of the drug when loaded into nanoparticles. Further, the modified everted gut sac study showed that these nanoparticles are taken up by active endocytic processes in the intestine. The ex-vivo mucoadhesion studies proved that the nanoparticles get bound to the mucus layer of the intestine, which in turn correlates with reduced excretion of the drug in faeces. In conclusion, the proposed nanoparticles appear promising for effective oral delivery of poorly bioavailable drugs like raloxifene hydrochloride.

Denosumab, raloxifene, romosozumab and teriparatide to prevent osteoporotic fragility fractures: a systematic review and economic evaluation.

BACKGROUND: Fragility fractures are fractures that result from mechanical forces that would not ordinarily result in fracture. OBJECTIVES: The objectives were to evaluate the clinical effectiveness, safety and cost-effectiveness of non-bisphosphonates {denosumab [Prolia®; Amgen Inc., Thousand Oaks, CA, USA], raloxifene [Evista®; Daiichi Sankyo Company, Ltd, Tokyo, Japan], romosozumab [Evenity®; Union Chimique Belge (UCB) S.A. (Brussels, Belgium) and Amgen Inc.] and teriparatide [Forsteo®; Eli Lilly and Company, Indianapolis, IN, USA]}, compared with each other, bisphosphonates or no treatment, for the prevention of fragility fracture. DATA SOURCES: For the clinical effectiveness review, nine electronic databases (including MEDLINE, EMBASE and the World Health Organization International Clinical Trials Registry Platform) were searched up to July 2018. REVIEW METHODS: A systematic review and network meta-analysis of fracture and femoral neck bone mineral density were conducted. A review of published economic analyses was undertaken and a model previously used to evaluate bisphosphonates was adapted. Discrete event simulation was used to estimate lifetime costs and quality-adjusted life-years for a simulated cohort of patients with heterogeneous characteristics. This was done for each non-bisphosphonate treatment, a strategy of no treatment, and the five bisphosphonate treatments previously evaluated. The model was populated with effectiveness evidence from the systematic review and network meta-analysis. All other parameters were estimated from published sources. An NHS and Personal Social Services perspective was taken, and costs and benefits were discounted at 3.5% per annum. Fracture risk was estimated from patient characteristics using the QFracture® (QFracture-2012 open source revision 38, Clinrisk Ltd, Leeds, UK) and FRAX® (web version 3.9, University of Sheffield, Sheffield, UK) tools. The relationship between fracture risk and incremental net monetary benefit was estimated using non-parametric regression. A probabilistic sensitivity analysis and scenario analyses were used to assess uncertainty. RESULTS: Fifty-two randomised controlled trials of non-bisphosphonates were included in the clinical effectiveness systematic review and an additional 51 randomised controlled trials of bisphosphonates were included in the network meta-analysis. All treatments had beneficial effects compared with placebo for vertebral, non-vertebral and hip fractures, with hazard ratios varying from 0.23 to 0.94, depending on treatment and fracture type. The effects on vertebral fractures and the percentage change in bone mineral density were statistically significant for all treatments. The rate of serious adverse events varied across trials (0-33%), with most between-group differences not being statistically significant for comparisons with placebo/no active treatment, non-bisphosphonates or bisphosphonates. The incremental cost-effectiveness ratios were > £20,000 per quality-adjusted life-year for all non-bisphosphonate interventions compared with no treatment across the range of QFracture and FRAX scores expected in the population eligible for fracture risk assessment. The incremental cost-effectiveness ratio for denosumab may fall below £30,000 per quality-adjusted life-year at very high levels of risk or for high-risk patients with specific characteristics. Raloxifene was dominated by no treatment (resulted in fewer quality-adjusted life-years) in most risk categories. LIMITATIONS: The incremental cost-effectiveness ratios are uncertain for very high-risk patients. CONCLUSIONS: Non-bisphosphonates are effective in preventing fragility fractures, but the incremental cost-effectiveness ratios are generally greater than the commonly applied threshold of £20,000-30,000 per quality-adjusted life-year. STUDY REGISTRATION: This study is registered as PROSPERO CRD42018107651. FUNDING: This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 24, No. 29. See the NIHR Journals Library website for further project information.

BACKGROUND: Fragility fractures are fractures that result from mechanical forces that would not ordinarily result in fracture, known as low-level (or 'low-energy') trauma. Some people are at particularly high risk of fragility fractures. The first treatment used is often a bisphosphonate, but non-bisphosphonate treatments are alternatives. AIMS: We aimed to determine how effective non-bisphosphonates {denosumab [Prolia®; Amgen Inc., Thousand Oaks, CA, USA], raloxifene [Evista®; Daiichi Sankyo Company, Ltd, Tokyo, Japan], romosozumab [Evenity®; Union Chimique Belge (UCB) S.A. (Brussels, Belgium) and Amgen Inc.] and teriparatide [Forsteo®; Eli Lilly and Company, Indianapolis, IN, USA]} are at preventing fractures, whether or not treatment has any risks for patients and whether or not the clinical benefits are achieved at a reasonable cost. METHODS: We have systematically identified and examined trials that assessed the clinical effects of non-bisphosphonates. For each clinical outcome, we have combined data from multiple trials to estimate the clinical effectiveness of each non-bisphosphonate treatment. We combined data from published sources in an economic model to estimate lifetime costs and clinical benefits for each non-bisphosphonate and compared these with the estimated costs and clinical outcomes for untreated patients and patients treated with bisphosphonates. RESULTS: All non-bisphosphonates reduced the risk of vertebral fractures compared with no treatment. For fractures at the hip or at any non-vertebral site, all of the non-bisphosphonates reduced the average number of fractures, but, for some non-bisphosphonates, we could not exclude the possibility that this was a chance finding. The chance of patients experiencing serious side effects was generally similar regardless of whether patients took non-bisphosphonates, bisphosphonates or placebo (a dummy pill). Blood clots were more common in patients taking raloxifene than in those taking placebo, but these were still a rare outcome (fewer than 1 in 100). The benefits of denosumab, teriparatide and romosozumab are few compared with their costs. For raloxifene, the risks generally outweigh the benefits. Treatment with bisphosphonates is likely to represent better value for money than treatment with non-bisphosphonates.

Spray-dried raloxifene submicron particles for pulmonary delivery: Development and in vivo pharmacokinetic evaluation in rats.

Raloxifene hydrochloride (RH) is a selective oestrogen receptor modulator used for the treatment of osteoporosis. Even though 60% of an oral dose is quickly absorbed via the gastrointestinal tract, the absolute bioavailability of RH is only 2-3% in humans due to extensive first-pass metabolism. Various approaches to improve RH bioavailability have been reported over the past few years; however, none have focused on the development of products for pulmonary administration. Therefore, in this study, submicron particles containing RH were produced for pulmonary administration with the aim to limit first-pass metabolism. Powders were produced by vibrational atomisation spray drying with a high process yield (>80%). The drug content was between 440 and 890 mg·g-1, and powders had a high encapsulation efficiency (>95%), mean particle size of 400-700 nm, low residual moisture (<2%) and spherical shape. These powders showed an improved drug dissolution rate compared to the raw RH material. Moreover, they presented high dose uniformity (95-100%), a high in vitro respirable fraction (>55%) and adequate mass median aerodynamic diameter for pulmonary delivery (<5 µm). The pharmacokinetic study in male Wistar rats demonstrated an absolute bioavailability of 47.20% after pulmonary administration of the particles. Therefore, these submicron-sized powders are promising for pulmonary RH delivery as a dry powder medicine.