Polyphosphate kinase-1 regulates bacterial and host metabolic pathways involved in pathogenesis of Mycobacterium tuberculosis.

Inorganic polyphosphate (polyP) is primarily synthesized by Polyphosphate Kinase-1 (PPK-1) and regulates numerous cellular processes, including energy metabolism, stress adaptation, drug tolerance, and microbial pathogenesis. Here, we report that polyP interacts with acyl CoA carboxylases, enzymes involved in lipid biosynthesis in Mycobacterium tuberculosis. We show that deletion of ppk-1 in M. tuberculosis results in transcriptional and metabolic reprogramming. In comparison to the parental strain, the Δppk-1 mutant strain had reduced levels of virulence-associated lipids such as PDIMs and TDM. We also observed that polyP deficiency in M. tuberculosis is associated with enhanced phagosome-lysosome fusion in infected macrophages and attenuated growth in mice. Host RNA-seq analysis revealed decreased levels of transcripts encoding for proteins involved in either type I interferon signaling or formation of foamy macrophages in the lungs of Δppk-1 mutant-infected mice relative to parental strain-infected animals. Using target-based screening and molecular docking, we have identified raloxifene hydrochloride as a broad-spectrum PPK-1 inhibitor. We show that raloxifene hydrochloride significantly enhanced the activity of isoniazid, bedaquiline, and pretomanid against M. tuberculosis in macrophages. Additionally, raloxifene inhibited the growth of M. tuberculosis in mice. This is an in-depth study that provides mechanistic insights into the regulation of mycobacterial pathogenesis by polyP deficiency.

Raloxifene, a cannabinoid type-2 receptor inverse agonist, mitigates visual deficits and pathology and modulates microglia after ocular blast.

Visual deficits after ocular blast injury (OBI) are common, but pharmacological approaches to improve long-term outcomes have not been identified. Blast forces frequently damage the retina and optic nerves, and work on experimental animals has shown the pro-inflammatory actions of microglia can further exacerbate such injuries. Cannabinoid type-2 receptor (CB2) inverse agonists specifically target activated microglia, biasing them away from the harmful pro-inflammatory M1 state toward the helpful reparative M2 state. We previously found that treating mice with CB2 inverse agonists after traumatic brain injury, produced by either focal cranial air blast or dorsal cranial impact, greatly attenuated the visual deficits and pathology that otherwise resulted. Here we examined the consequences of single and repeat OBI and the benefit provided by raloxifene, an FDA-approved estrogen receptor drug that possesses noteworthy CB2 inverse agonism. After single OBI, although the amplitudes of the A- and B-waves of the electroretinogram and pupil light response appeared to be normal, the mice showed hints of deficits in contrast sensitivity and visual acuity, a trend toward optic nerve axon loss, and significantly increased light aversion, which were reversed by 2 weeks of daily treatment with raloxifene. Mice subjected to repeat OBI (5 blasts spaced 1 min apart), exhibited more severe visual deficits, including decreases in contrast sensitivity, visual acuity, the amplitudes of the A- and B-waves of the electroretinogram, light aversion, and resting pupil diameter (i.e. hyperconstriction), accompanied by the loss of photoreceptor cells and optic nerve axons, nearly all of which were mitigated by raloxifene. Interestingly, optic nerve axon abundance was strongly correlated with contrast sensitivity and visual acuity across all groups of experimental mice in the repeat OBI study, suggesting optic nerve axon loss with repeat OBI and its attenuation with raloxifene are associated with the extent of these two deficits while photoreceptor abundance was highly correlated with A-wave amplitude and resting pupil size, suggesting a prominent role for photoreceptors in these two deficits. Quantitative PCR (qPCR) showed levels of M1-type microglial markers (e.g. iNOS, IL1ß, TNFα, and CD32) in retina, optic nerve, and thalamus were increased 3 days after repeat OBI. With raloxifene treatment, the overall expression of M1 markers was more similar to that in sham mice. Raloxifene treatment was also associated with the elevation of IL10 transcripts in all three tissues compared to repeat OBI alone, but the results for the three other M2 microglial markers we examined were more varied. Taken together, the qPCR results suggest that raloxifene benefit for visual function and pathology was associated with a lessening of the pro-inflammatory actions of microglia. The benefit we find for raloxifene following OBI provides a strong basis for phase-2 efficacy testing in human clinical trials for treating ocular injury.

Unraveling the Abnormal Molecular Mechanism of Suicide Inhibition of Cytochrome P450 3A4.

Suicide inhibition of the CYP3A4 enzyme by a drug inactivates the enzyme in the drug biotransformation process and often shows safety concerns about the drug. Despite extensive experimental studies, the abnormal molecular mechanism of a suicide inhibitor that forms a covalent bond with the residue far away from the catalytically active center of CYP3A4 inactivating the enzyme remains elusive. Here, the authors used molecular simulation approaches to study in detail how diquinone methide (DQR), the metabolite product of raloxifene, unbinds from CYP3A4 and inactivates the enzyme at the atomistic level. The results clearly indicate that in one of the intermediate states formed in its unbinding process, DQR covalently binds to Cys239, a residue far away from the catalytically active center of CYP3A4, and hinders the substrate from entering or leaving the enzyme. This work therefore provides an unprecedented way of clarifying the abnormal mechanism of suicide inhibition of the CYP3A4 enzyme.

Mg,Si-Co-Substituted Hydroxyapatite/Alginate Composite Beads Loaded with Raloxifene for Potential Use in Bone Tissue Regeneration.

Osteoporosis is a worldwide chronic disease characterized by increasing bone fragility and fracture likelihood. In the treatment of bone defects, materials based on calcium phosphates (CaPs) are used due to their high resemblance to bone mineral, their non-toxicity, and their affinity to ionic modifications and increasing osteogenic properties. Moreover, CaPs, especially hydroxyapatite (HA), can be successfully used as a vehicle for local drug delivery. Therefore, the aim of this work was to fabricate hydroxyapatite-based composite beads for potential use as local carriers for raloxifene. HA powder, modified with magnesium and silicon ions (Mg,Si-HA) (both of which play beneficial roles in bone formation), was used to prepare composite beads. As an organic matrix, sodium alginate with chondroitin sulphate and/or keratin was applied. Cross-linking of beads containing raloxifene hydrochloride (RAL) was carried out with Mg ions in order to additionally increase the concentration of this element on the material surface. The morphology and porosity of three different types of beads obtained in this work were characterized by scanning electron microscopy (SEM) and mercury intrusion porosimetry, respectively. The Mg and Si released from the Mg,Si-HA powder and from the beads were measured by inductively coupled plasma optical emission spectrometry (ICP-OES). In vitro RAL release profiles were investigated for 12 weeks and studied using UV/Vis spectroscopy. The beads were also subjected to in vitro biological tests on osteoblast and osteosarcoma cell lines. All the obtained beads revealed a spherical shape with a rough, porous surface. The beads based on chondroitin sulphate and keratin (CS/KER-RAL) with the lowest porosity resulted in the highest resistance to crushing. Results revealed that these beads possessed the most sustained drug release and no burst release effect. Based on the results, it was possible to select the optimal bead composition, consisting of a mixture of chondroitin sulphate and keratin.

Induced osteoblast differentiation by amide derivatives of stilbene: The possible osteogenic agents.

A series of amide derivatives of stilbene was synthesized and investigated for osteogenic activity. Out of sixteen, seven compounds viz19c, 19g, 19i, 24b, 25a, 25c and 26a showed significant osteoblast differentiation within 1 pM-1 µM concentrations. Amongst all, 26a was identified as most active molecule which presented effective mineralization of osteoblasts and expression of mRNA of osteogenic marker gene such as BMP-2, ALP, and Runx-2 at 1 pM. In estrogen-deficient balb/c mice, 26a showed significant osteogenic activity at 5 mg-kg-1 body weight dose. The protein expression study for estrogen receptors α and ß (ER-α & ER-ß) using mouse calvarial osteoblasts (MCOs) and molecular docking analyses showed preferential expression of ER-ß by 26a indicating the possibility of ER-ß mediated osteogenic activity of 26a.

Development and validation of ultra-high-performance liquid chromatography-mass spectrometry method for the determination of raloxifene and its phase II metabolites in plasma: Application to pharmacokinetic studies in rats.

The aim of this study is to establish a reliable liquid chromatography-mass spectrometry method to simultaneously quantitate raloxifene, and its major metabolites, raloxifene-6-glucuronide, raloxifene-4'-glucuronide, and raloxifene-6-sulfate in rat plasma samples for pharmacokinetic studies. The separation of the analytes was achieved on a Waters BEH C18 column. Water (0.1% formic acid) and acetonitrile were used as the mobile phases for elution. A one-step protein precipitation using a mixture solvent was applied for plasma sample preparation. The method was validated following the FDA guidance. The results showed that the linear range were 1.95-1000 nM for raloxifene-6-glucuronide, and raloxifene-4'-glucuronide, 0.195-100 nM for raloxifene-6-sulfate, and 0.195-200 nM for raloxifene, respectively. The lower limit of quantification was 1.95, 1.95, 0.195, and 0.195 nM for raloxifene-6-glucuronide, raloxifene-4'-glucuronide, raloxifene-6-sulfate, and raloxifene, respectively. Only 20 µl of plasma sample was required since the method is sensitive. The intra- and interday variance is <15% and the accuracy is within 85-115%. The variance of matrix effect and recovery were <15%. The method was successfully applied in a pharmacokinetic study in rats with oral administration of raloxifene.

The Effect of Piperine Pro-Nano Lipospheres on Direct Intestinal Phase II Metabolism: The Raloxifene Paradigm of Enhanced Oral Bioavailability.

Phase II biotransformation reactions have been gaining more attention due to their acknowledged significance in drug bioavailability, drug development, and drug-drug interactions. However, the predominant role of phase I metabolism has always overshadowed phase II metabolism, resulting in insufficient data regarding its mechanisms. In this paper, we investigate the effect of an advanced lipid based formulation on the phase II metabolism process of glucuronidation, occuring in the enterocytes monolayer. The investigated formulation is a self-emulsifying drug delivery system, termed pro-nano lipospheres, which contains the natural absorption enhancer piperine. To evaluate the effect of this formulation on direct glucuronidation we chose the model molecule raloxifene. First, glucuronidation is the main clearance pathway of this compound without involvement of preceding mechanisms. Second, raloxifene's extensive glucuronidation site is primarily at the intestine. Raloxifene's oral bioavailability was determined in a series of pharmacokinetic experiments using the freely moving rat model. In order to test the effect of the formulation on the relevant UGT enzymes reported in the clinic, we used the in vitro method of UGT-Glo Assay. Coadministration of raloxifene and piperine pro-nano lipospheres to rats resulted in a 2-fold increase in the relative oral bioavailability of raloxifene. However, coadministration of raloxifene with blank pro-nano lipospheres had no effect on its oral bioavailability. In contrast to the difference found in vivo between the two vehicles, both formulations extended an inhibitory effect on UGT enzymes in vitro. Ultimately, these findings prove the ability of the formulation to diminish intestinal direct phase II metabolism which serves as an absorption obstacle for many of today's marketed drugs. Pro-nano lipospheres is a formulation that serves as a platform for the simultaneous delivery of the absorption enhancer and a required drug. The discrepancy found between the in vivo and in vitro models demonstrates that the in vitro method may not be sensitive enough to distinguish the difference between the formulations.

Glucose-Based Mesoporous Carbon Nanospheres as Functional Carriers for Oral Delivery of Amphiphobic Raloxifene: Insights into the Bioavailability Enhancement and Lymphatic Transport.

PURPOSE: Oral therapy with raloxifene (RXF), an amphiphobic drug for remedy of the postmenopausal osteoporosis and estrogen-dependent breast cancer, is less effective due to its poor bioavailability (2% or so). This work aimed to devise mesoporous carbon nanospheres (MCNs) for oral delivery of RXF and evaluate their performance in bioavailability enhancement and lymphatic transport. METHODS: Glucose-based MCNs were fabricated by hydrothermal reaction followed by high-temperature activation. RXF-loaded MCNs (RXF-MCNs) were prepared by solvent-diffusion/high-pressure homogenization and stabilized by phospholipid. RXF-MCNs were fully characterized by particle size, morphology, in vitro drug release and metabolism, in vivo pharmacokinetics and lymphatic transport, and ex vivo fluorescent imaging. RESULTS: The prepared RXF-MCNs were 230 nm around in particle size, showing high entrapment efficiency (95.35%) and satisfactory physical stability. The oral bioavailability of RXF was enhanced by 2.07 folds through MCNs compared with RXF suspensions in rats. It was shown that reduced intestinal metabolism due to entrapment into MCNs, active transcellular uptake and increased lymphatic transport were responsible for enhanced bioavailability as a result of transport improvement. CONCLUSIONS: The results suggest that MCNs are suitable nanocarriers for oral delivery of poorly bioavailable RXF.

Structural features underlying raloxifene's biophysical interaction with bone matrix.

Raloxifene, a selective estrogen receptor modulator (SERM), reduces fracture risk at least in part by improving the mechanical properties of bone in a cell- and estrogen receptor-independent manner. In this study, we determined that raloxifene directly interacts with the bone tissue. Through the use of multiple and complementary biophysical techniques including nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy (FTIR), we show that raloxifene interacts specifically with the organic component or the organic/mineral composite, and not with hydroxyapatite. Structure-activity studies reveal that the basic side chain of raloxifene is an instrumental determinant in the interaction with bone. Thus, truncation of portions of the side chain reduces bone binding and also diminishes the increase in mechanical properties. Our results support a model wherein the piperidine interacts with bone matrix through electrostatic interactions with the piperidine nitrogen and through hydrophobic interactions (van der Waals) with the aliphatic groups in the side chain and the benzothiophene core. Furthermore, in silico prediction of the potential binding sites on the surface of collagen revealed the presence of a groove with sufficient space to accommodate raloxifene analogs. The hydroxyl groups on the benzothiophene nucleus, which are necessary for binding of SERMs to the estrogen receptor, are not required for binding to the bone surface, but mediate a more robust binding of the compound to the bone powder. In conclusion, we report herein a novel property of raloxifene analogs that allows them to interact with the bone tissue through potential contacts with the organic matrix and in particular collagen.

Raloxifene glucuronidation in liver and intestinal microsomes of humans and monkeys: contribution of UGT1A1, UGT1A8 and UGT1A9.

1. Raloxifene is an antiestrogen that has been marketed for the treatment of osteoporosis, and is metabolized into 6- and 4'-glucuronides by UDP-glucuronosyltransferase (UGT) enzymes. In this study, the in vitro glucuronidation of raloxifene in humans and monkeys was examined using liver and intestinal microsomes and recombinant UGT enzymes (UGT1A1, UGT1A8 and UGT1A9). 2. Although the K(m) and CL(int) values for the 6-glucuronidation of liver and intestinal microsomes were similar between humans and monkeys, and species differences in Vmax values (liver microsomes, humans > monkeys; intestinal microsomes, humans < monkeys) were observed, no significant differences were noted in the K(m) or S50, Vmax and CL(int) or CLmax values for the 4'-glucuronidation of liver and intestinal microsomes between humans and monkeys. 3. The activities of 6-glucuronidation in recombinant UGT enzymes were UGT1A1 > UGT1A8 >UGT1A9 for humans, and UGT1A8 > UGT1A1 > UGT1A9 for monkeys. The activities of 4'-glucuronidation were UGT1A8 > UGT1A1 > UGT1A9 in humans and monkeys. 4. These results demonstrated that the profiles for the hepatic and intestinal glucuronidation of raloxifene by microsomes were moderately different between humans and monkeys.

Milk Thistle Constituents Inhibit Raloxifene Intestinal Glucuronidation: A Potential Clinically Relevant Natural Product-Drug Interaction.

Women at high risk of developing breast cancer are prescribed selective estrogen response modulators, including raloxifene, as chemoprevention. Patients often seek complementary and alternative treatment modalities, including herbal products, to supplement prescribed medications. Milk thistle preparations, including silibinin and silymarin, are top-selling herbal products that may be consumed by women taking raloxifene, which undergoes extensive first-pass glucuronidation in the intestine. Key constituents in milk thistle, flavonolignans, were previously shown to be potent inhibitors of intestinal UDP-glucuronosyl transferases (UGTs), with IC50s ≤ 10 µM. Taken together, milk thistle preparations may perpetrate unwanted interactions with raloxifene. The objective of this work was to evaluate the inhibitory effects of individual milk thistle constituents on the intestinal glucuronidation of raloxifene using human intestinal microsomes and human embryonic kidney cell lysates overexpressing UGT1A1, UGT1A8, and UGT1A10, isoforms highly expressed in the intestine that are critical to raloxifene clearance. The flavonolignans silybin A and silybin B were potent inhibitors of both raloxifene 4'- and 6-glucuronidation in all enzyme systems. The Kis (human intestinal microsomes, 27-66 µM; UGT1A1, 3.2-8.3 µM; UGT1A8, 19-73 µM; and UGT1A10, 65-120 µM) encompassed reported intestinal tissue concentrations (20-310 µM), prompting prediction of clinical interaction risk using a mechanistic static model. Silibinin and silymarin were predicted to increase raloxifene systemic exposure by 4- to 5-fold, indicating high interaction risk that merits further evaluation. This systematic investigation of the potential interaction between a widely used herbal product and chemopreventive agent underscores the importance of understanding natural product-drug interactions in the context of cancer prevention.

Targeted screen for human UDP-glucuronosyltransferases inhibitors and the evaluation of potential drug-drug interactions with zafirlukast.

Inhibition of drug metabolizing enzymes is a major mechanism in drug-drug interactions (DDIs). A number of cases of DDIs via inhibition of UDP-glucuronosyltranseferases (UGTs) have been reported, although the changes in pharmacokinetics are relatively small in comparison with drugs that are metabolized by cytochrome P450s. Most of the past studies have investigated hepatic UGTs, although recent studies have revealed a significant contribution of UGTs in the small intestine to drug clearance. To evaluate potential DDIs caused by inhibition of intestinal UGTs, we assessed inhibitory effects of 578 compounds, including drugs, xenobiotics, and endobiotics, on human UGT1A8 and UGT1A10, which are major contributors to intestinal glucuronidation. We identified 29 inhibitors by monitoring raloxifene glucuronidation with recombinant UGTs. All of the inhibitors potently inhibited UGT1A1 activity, as well. We found that zafirlukast is a potent general inhibitor of UGT1As and a moderate inhibitor of UGT2Bs because it monitors 4-methylumbelliferone glucuronidation by recombinant UGTs. However, zafirlukast did not potently inhibit diclofenac glucuronidation, suggesting that the inhibitory effects might be substrate specific. Inhibitory effects of zafirlukast on some UGT substrates were further investigated in human liver and human small intestine microsomes in order to evaluate potential DDIs. The R values (the ratios of intrinsic clearance with and without an inhibitor) revealed that zafirlukast has potential to cause clinical DDIs in the small intestine. Although we could not identify specific UGT1A8 and UGT1A10 inhibitors, zafirlukast was identified as a general inhibitor for UGTs in vitro. The present study suggests that the inhibition of UGT in the small intestine would be an underlying mechanism for DDIs.

Sulfation of afimoxifene, endoxifen, raloxifene, and fulvestrant by the human cytosolic sulfotransferases (SULTs): A systematic analysis.

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.

Regulation of UDP-glucuronosyltransferase 1A1 expression and activity by microRNA 491-3p.

The UDP-glucuronosyltransferase (UGT) 1A enzymes are involved in the phase II metabolism of many important endogenous and exogenous compounds. The nine UGT1A isoforms exhibit high interindividual differences in expression, but their epigenetic regulation is not well understood. The purpose of the present study was to examine microRNA (miRNA) regulation of hepatic UGT1A enzymes and determine whether or not that regulation impacts enzymatic activity. In silico analysis identified miRNA 491-3p (miR-491-3p) as a potential regulator of the UGT1A gene family via binding to the shared UGT1A 3'-untranslated region common to all UGT1A enzymes. Transfection of miR-491-3p mimic into HuH-7 cells significantly repressed UGT1A1 (P < 0.001), UGT1A3 (P < 0.05), and UGT1A6 (P < 0.05) mRNA levels. For UGT1A1, this repression correlated with significantly reduced metabolism of raloxifene into raloxifene-6-glucuronide (ral-6-gluc; P < 0.01) and raloxifene-4'-glucuronide (ral-4'-gluc; P < 0.01). In HuH-7 cells with repressed miR-491-3p expression, there was a significant increase (~80%; P < 0.01) in UGT1A1 mRNA and a corresponding increase in glucuronidation of raloxifene into ral-6-gluc (50%; P < 0.05) and ral-4'-gluc (22%; P < 0.01). Knockdown of endogenous miR-491-3p in HepG2 cells did not significantly alter UGT1A1 mRNA levels but did increase the formation of ral-6-gluc (50%; P < 0.05) and ral-4'-gluc (34%; P < 0.001). A significant inverse correlation between miR-491-3p expression and both UGT1A3 (P < 0.05) and UGT1A6 (P < 0.01) mRNA levels was observed in a panel of normal human liver specimens, with a significant (P < 0.05) increase in UGT1A3 and UGT1A6 mRNA levels observed in miR-491-3p nonexpressing versus expressing liver specimens. These results suggest that miR-491-3p is an important factor in regulating the expression of UGT1A enzymes in vivo.

The role of encapsulation by ß-cyclodextrin in the interaction of raloxifene with macromolecular targets: a study by spectroscopy and molecular modeling.

We report the binding of the drug raloxifene with Calf thymus DNA (ctDNA) and bovine serum albumin (BSA) in the presence and absence of ß-cyclodextrin (ß-CD) and explain the influence of ß-cyclodextrin on the binding of the drug to macromolecules. UV-Vis absorption, fluorescence, proton nuclear magnetic resonance and two-dimensional rotating-frame nuclear overhauser effect spectroscopic techniques are used to study the stoichiometry and the binding strength of the complexes. Molecular modeling is used in combination with other techniques to propose the structure of the inclusion complex and the interaction with ctDNA. The Stern-Volmer quenching constants of the interaction of raloxifene with ctDNA in aqueous and in ß-CD solution are compared. The competition for binding of ctDNA with raloxifene and Methylene Blue is studied. The apparent binding constant and the number of binding sites for the binding of raloxifene with BSA in aqueous solution are significantly different from those in the presence of ß-CD. The influence of ß-CD on the binding of the small molecules with biological macromolecules is discussed. We infer that the binding strengths between raloxifene and macromolecules, viz., ctDNA and BSA are influenced by the ß-CD encapsulation. These results may suggest new ways to tune the drug binding to biomacromolecules by encapsulating specific moieties of drugs.

Identification of raloxifene as a novel CB2 inverse agonist.

The purpose of the current study was to apply a high throughput assay to systematically screen a library of food and drug administration (FDA)-approved drugs as potential ligands for the cannabinoid receptor 2 (CB2). A cell-based, homogenous time resolved fluorescence (HTRF) method for measuring changes in intracellular cAMP levels was validated and found to be suitable for testing ligands that may act on CB2. Among the 640 FDA-approved drugs screened, raloxifene, a drug used to treat/prevent post-menopausal osteoporosis, was identified for the first time to be a novel CB2 inverse agonist. Our results demonstrated that by acting on CB2, raloxifene enhances forskolin-stimulated cAMP accumulation in a concentration-dependant manner. Furthermore, our data showed that raloxifene competes concentration-dependently for specific [(3)H]CP-55,940 binding to CB2. In addition, raloxifene pretreatment caused a rightward shift of the concentration-response curves of the cannabinoid agonists CP-55,940, HU-210, and WIN55,212-2. Raloxifene antagonism is most likely competitive in nature, as these rightward shifts were parallel and were not associated with any changes in the efficacy of cannabinoid agonists on CB2. Our discovery that raloxfiene is an inverse agonist for CB2 suggests that it might be possible to repurpose this FDA-approved drug for novel therapeutic indications for which CB2 is a target. Furthermore, identifying raloxifene as a CB2 inverse agonist also provides important novel mechanisms of actions to explain the known therapeutic effects of raloxifene.

Magnetic Nanoplatform With Novel Potential for the Treatment of Bone Pathologies: Drug Loading and Biocompatibility on Blood and Bone Cells.

Magnetic iron oxide nanoparticles (MNPs) coated with citric acid (MG@CA) are proposed as raw materials for the treatment of bone diseases. Citric acid (CA) was selected as coating due to its role in the stabilization of apatite nanocrystals and as a signaling agent for osteoblast activation. Raloxifene (Ral), curcumine (Cur) and methylene blue (MB) were employed as model drugs as therapeutic agents for bone diseases. Characterization of raw and drug loaded nanosystems was conducted in order to elucidate the mechanisms governing interactions between therapeutics and the magnetic platform. Biocompatibility studies were performed on red blood cells (RBCs) from peripheral human blood. Cytotoxicity was evaluated on endothelial cells (ECs); and viability was studied for bone cells exposed at concentrations of 1, 10 and 100 [Formula: see text]/mL of the magnetic nano-platform. MG@CA exhibited proper physicochemical properties for the applications intended within this work. It presented satisfactory biocompatibility on peripheral red blood cells. Only doses of 100 [Formula: see text]/mL induced a decrease in metabolic activity of ECs and MC3T3-E1 cells. Drug adsorption efficiency was estimated as 62.0, 15.0 and 54.0 % for Ral, Cur and MB and drug loading capability of 12.0, 20.0 and 13.6%, respectively.

The potential renoprotective effect of Raloxifene in renal ischemia-reperfusion injury in a male rat model.

Renal ischemia-reperfusion injury is caused by a temporary reduction in oxygen-carrying blood flow to the kidney, followed by reperfusion. During ischemia, kidney tissue damage induces overproduction of reactive oxygen species, which produces oxidative stress. The blood flow restoration during the reperfusion period causes further production of reactive oxygen species that ends with apoptosis and cell death. This study aimed to investigate the potential renoprotective effects of Raloxifene on bilateral renal ischemia-reperfusion injury in rats by looking into kidney function biomarkers, urea and creatinine, inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1ß). Additionally, antioxidant markers such as total antioxidant capacity (TAC) and the pro-apoptotic marker caspase-3 were assessed. Histopathological scores were also employed for evaluation. Our experimental design involved 20 rats divided into four groups: the sham group underwent median laparotomy without ischemia induction, the control group experienced bilateral renal ischemia for 30 minutes followed by 2 hours of reperfusion, the vehicle group received pretreatment with a mixture of corn oil and dimethyl sulfoxide (DMSO) before ischemia induction, and the Raloxifene-treated group was administered Raloxifene at a dose of 10 mg/kg before ischemia induction, followed by ischemia-reperfusion. Urea and creatinine, TNF-α, IL-1ß, and caspase-3 in the Raloxifene group were significantly lower compared to the control and vehicle groups. On the other hand, TAC levels in the Raloxifene group were significantly higher than in the control and vehicle groups. This study concluded that Raloxifene had a renoprotective impact via multiple actions as an anti-inflammatory, anti-apoptotic, and antioxidant agent.

Use of a multistaged time-dependent inhibition assay to assess the impact of intestinal metabolism on drug-drug interaction potential.

In early discovery, compounds are often eliminated because of their potential to undergo metabolic activation and/or cytochrome P450 time-dependent inactivation (TDI). The blockbuster drug raloxifene is an example of a compound that would have been eliminated in the current paradigm. Despite raloxifene's in vitro bioactivation and TDI of CYP3A4, it is well tolerated in patients with no drug-drug interactions. This discordance is attributed to its presystemic glucuronidation, thereby decreasing the amount of unchanged raloxifene available for CYP3A inactivation. The current study used raloxifene as a model to assess the effect of hepatic and intestinal glucuronidation on the kinetic parameters of CYP3A4 inactivation. Therefore, a simple multistaged time-dependent inactivation using UDP-glucuronosyltransferase-enabled and -absent reactions was built to understand the impact of the gut metabolism on inactivation potential. The results of these experiments demonstrated a 2.7-fold change in inactivation efficiency of CYP3A4. Incorporation of these results into a simulated midazolam drug-drug interaction study showed very little change in the pharmacokinetic parameters of the victim drug. In contrast, the absence of glucuronidation resulted in a 4.1-fold increase in the area under the curve (AUC) of midazolam, when in the presence of raloxifene, hence providing an understanding of the impact of intestinal glucuronidation on raloxifene's time-dependent inhibition of CYP3A4 and also providing a validation of a simple in vitro experiment to assess the influence of gut metabolism on time-dependent inhibitors at the discovery phase.

A multiplexed screening method for agonists and antagonists of the estrogen receptor protein.

The estrogen receptor (ER) is regarded as a significant drug target because of its important physical and pathological function. In this article, we describe a novel screening method to obtain agonists and antagonists of ER. ER was immobilized onto an aldehyde-modified glass slide. The affinity of Cy3-labeled estradiol for ER protein microarrays was then determined. Two libraries, one containing 29 synthetic compounds and the other with 384 natural products that served as a model, were screened to find new ligands for ER. The IC(50) values obtained for tamoxifen and raloxifene were consistent with those found in the literature (4.85 × 10(-7) M versus 1.74~4.23 × 10(-7) M and 7.58 × 10(-8) M versus 0.89~5.84 × 10(-8) M, respectively). Finally, 65 active ligands (5 synthetic compounds and 60 natural products) of ER were identified. This novel method gave identical results to a conventional fluorescence polarization assay, thus verifying the accuracy of this simultaneous multireceptor screening method based on protein microarrays. The presented method is sensitive, accurate, and reliable, and shows great potential for use in high-throughput drug-screening research.