Graphene oxide nanoribbons conjugated with 1, 2-distearoyl-sn-glycero-3 phosphoethanolamine-poly (ethylene glycol)-transferrin enhanced targeted delivery and cytotoxicity of raloxifene against breast cancer.

The clinical utility of raloxifene (RLX), a selective estrogen receptor modulator (SERM), has been compromised by severe side effects and unfavorable drug properties. To address these, a transferrin (Tf) conjugated graphene oxide nanoribbon (GONR) platform was tried for RLX. The stability of GONRs in biological media was improved by surface modification with 1, 2-Distearoyl-sn-glycero-3 phosphoethanolamine-Poly (ethylene glycol) (DSPE-PEG). The Tf molecule was covalently attached to DSPE-PEG (DPT) using EDC-NHS chemistry. The surface of GONR was then modified with DSPE-PEG (DP) or DPT and loaded with RLX (GDP-RLX and GDPT-RLX). The final formulations were characterized for drug loading and stability. The anticancer activities of pure RLX, GDP-RLX, and GDPT-RLX were evaluated and compared in all the in vitro and in vivo studies. In vitro cell line studies showed that GDPT-RLX have significantly high cytotoxicity, cellular uptake, apoptosis induction, G2/M phase arrest, anti-migration properties, and apoptotic protein expression, followed by GDP-RLX and RLX. Pharmacokinetics and tumor biodistribution were also found to be excellent with GDPT-RLX. The in vivo tumor therapy and tumor evaluation outcomes were also consistent with the in vitro data. The Tf conjugated GDPT-RLX represents a promising approach for targeted and sustained delivery of RLX with enhanced therapeutic efficacy.

Multivariate Analysis of Solubility Parameters for Drug-Polymer Miscibility Assessment in Preparing Raloxifene Hydrochloride Amorphous Solid Dispersions.

The success of obtaining solid dispersions for solubility improvement invariably depends on the miscibility of the drug and polymeric carriers. This study aimed to categorize and select polymeric carriers via the classical group contribution method using the multivariate analysis of the calculated solubility parameter of RX-HCl. The total, partial, and derivate parameters for RX-HCl were calculated. The data were compared with the results of excipients (N = 36), and a hierarchical clustering analysis was further performed. Solid dispersions of selected polymers in different drug loads were produced using solvent casting and characterized via X-ray diffraction, infrared spectroscopy and scanning electron microscopy. RX-HCl presented a Hansen solubility parameter (HSP) of 23.52 MPa1/2. The exploratory analysis of HSP and relative energy difference (RED) elicited a classification for miscible (n = 11), partially miscible (n = 15), and immiscible (n = 10) combinations. The experimental validation followed by a principal component regression exhibited a significant correlation between the crystallinity reduction and calculated parameters, whereas the spectroscopic evaluation highlighted the hydrogen-bonding contribution towards amorphization. The systematic approach presented a high discrimination ability, contributing to optimal excipient selection for the obtention of solid solutions of RX-HCl.

Bioavailability Enhancement of BCS Class II Raloxifene Hydrochloride by Inclusion Complex and Solid Dispersion Techniques.

OBJECTIVE: Raloxifene hydrochloride (RLX) is used extensively in the treatment of osteoporosis, only 2% of RLX's bioavailability remains after a significant first pass metabolism. Besides coming from BCS class II, RLX is not very soluble in water. Thus, the goal of the current study was to improve RLX solubility by creating an inclusion complex using ß cyclodextrin (ß-CD) as a carrier and solid dispersion with Poloxamer 407. METHODS: Inclusion complex and solid dispersion were made using a variety of techniques, including kneading, co-precipitation, and physical mixing and solid dispersion using different drug to carrier ratios (1:1, 1:2 and 1:3). RESULTS: Inclusion complex made using the co-precipitation method had shown 9-fold improvements in water solubility when compared with plain RLX. In order to assess the optimized complex's compatibility, thermal analysis, and crystallinity, X-ray diffraction, differential scanning calorimetry, and Fourier transform infrared spectroscopy were used. The XRD and DSC study's results indicated that RLX changed from a crystalline to an amorphous state. IC-6 exhibits effective water solubility based on the outcome. However, upon comparison of the two techniques, the ß-CD complexation method shown an impressive rise in drug solubility when compared to solid dispersion.

Design, synthesis and antiproliferative activity of raloxifene/histone deacetylase inhibitor hybrids in breast cancer.

Antiestrogen/histone deacetylase inhibitor (HDACi) hybrids were designed by merging structures of raloxifene with suberoylanilide hydroxamic acid, incorporating the HDACi unit into the phenolic ring of the antiestrogen. These hybrids were synthesized with a range of HDACi chain lengths and assessed for bifunctionality. Four hybrids, 21 (YW471), 22 (YW490), 27(YW486), and 28 (YW487) showed good potency both as antiestrogens in a BRET assay and in a fluorometric HDACi assay. The antiproliferative activity of the hybrids was demonstrated in both ER+ MCF7 and ER- MDA-MB-231 breast cancer cell lines.

Exploring Raloxifene-Based Metallodrugs: A Versatile Vector Combined with Platinum(II), Palladium(II) and Nickel(II) Dichlorides and Carborates against Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) poses challenges in therapy due to the absence of target expression such as estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Frequently, the treatment of TNBC involves the combination of several therapeutics. However, an enhanced therapeutic effect can be also achieved within a single molecule. The efficacy of raloxifene can be improved by designing a raloxifene-based hybrid drug bearing a 2,2'-bipyridine moiety (2). Integration of platinum(II), palladium(II), and nickel(II) complexes into this structure dramatically changed the cytotoxicity. The platinum(II) dichloride complex 3 did not demonstrate any activity, while palladium(II) and nickel(II) dichloride complexes 4 and 5 exhibited various cytotoxic behavior towards different types of hormone-receptor positive (HR+) cancer and TNBC cell lines. The replacement of the two chlorido ligands in 3-5 with a dicarbollide (carborate) ion [C2B9H11]2- resulted in reduced activity of compounds 6, 7, and 8. However, the palladacarborane complex 7 demonstrated higher selectivity towards TNBC. Furthermore, the mechanism of action was shifted from cytotoxic to explicitly cytostatic with detectable proliferation arrest and accelerated aging, characterized by senescence-associated phenotype of TNBC cells. This study provides valuable insights into the development of hybrid therapeutics against TNBC.

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.

Mechanism of Caspase-1 Inhibition by Four Anti-inflammatory Drugs Used in COVID-19 Treatment.

The inflammatory protease caspase-1 is associated with the release of cytokines. An excessive number of cytokines (a "cytokine storm") is a dangerous consequence of COVID-19 infection and has been indicated as being among the causes of death by COVID-19. The anti-inflammatory drug colchicine (which is reported in the literature to be a caspase-1 inhibitor) and the corticosteroid drugs, dexamethasone and methylprednisolone, are among the most effective active compounds for COVID-19 treatment. The SERM raloxifene has also been used as a repurposed drug in COVID-19 therapy. In this study, inhibition of caspase-1 by these four compounds was analyzed using computational methods. Our aim was to see if the inhibition of caspase-1, an important biomolecule in the inflammatory response that triggers cytokine release, could shed light on how these drugs help to alleviate excessive cytokine production. We also measured the antioxidant activities of dexamethasone and colchicine when scavenging the superoxide radical using cyclic voltammetry methods. The experimental findings are associated with caspase-1 active site affinity towards these compounds. In evaluating our computational and experimental results, we here formulate a mechanism for caspase-1 inhibition by these drugs, which involves the active site amino acid Cys285 residue and is mediated by a transfer of protons, involving His237 and Ser339. It is proposed that the molecular moiety targeted by all of these drugs is a carbonyl group which establishes a S(Cys285)-C(carbonyl) covalent bond.

Design and synthesis of marine sesterterpene analogues as novel estrogen receptor α degraders for breast cancer treatment.

Targeted protein degradation using small molecules is an intriguing strategy for drug development. The marine sesterterpene compound MHO7 had been reported to be a potential ERα degradation agent. In order to further improve its biological activity, two series of novel MHO7 derivatives with long side chains were designed and identified as novel selective estrogen receptor down-regulators (SERDs). The growth inhibition activity of the novel SERD compounds were significantly affected by the type and length of the side chain. Most of the derivatives were significantly more potent than MHO7 against both drug-sensitive and drug-resistant breast cancer cells. Among them, compound 16a, with IC50 values of 0.41 µM against MCF-7 cell lines and 9.6-fold stronger than MHO7, was the most potential molecule. A whole-genome transcriptomic analysis of MCF-7 cells revealed that the mechanism of 16a against MCF-7 cell was similar with that of MHO7. The estrogen signaling pathway was the most affected among the disturbed genes, but the ERα degradation activity of 16a was observed higher than that of MHO7. Other effects of 16a were confirmed similar with MHO7, which means that the basic mechanisms of the derivatives are the same with the ophiobolin backbone, i.e. the degradation of ERα is mediated via proteasome-mediated process, the induction of apoptosis and the cell cycle arrest at the G1 phase. Meanwhile, a decrease of mitochondrial membrane potential and an increase of cellular ROS were also detected. Based on these results, as a novel modified ophiobolin derived compound, 16a may warrant further exploitation as a promising SERD candidate agent for the treatment of breast cancer.

Searching for an ideal SERM: Mining tamoxifen structure-activity relationships.

The repurposing of old drugs for new treatments has recently garnered increased attention in the face of new diseases and declining productivity of the pharmaceutial industry. This report draws attention to potential opportunities hiding in plain sight within the SAR of off-patent drugs. Herein we explore the untapped potential of Selective Estrogen Receptor Modulators (SERMs). SERMs are a class of molecules that have been highly influential in the treatment of estrogen receptor-positive breast cancers. However, the most commonly prescribed SERM, tamoxifen, has been found to increase the risk of endometrial cancer. Another SERM, raloxifene, does not increase incidence of endometrial cancer, but has been abandoned as a breast cancer treatment. We report the design, synthesis, and evaluation of an unexplored tamoxifen substitution pattern which mimics the geometry of raloxifene to confer its favorable pharmacodynamics. This substitution pattern was found to maintain excellent binding affinity to estrogen receptor-α.

Identifying FDA-approved drugs with multimodal properties against COVID-19 using a data-driven approach and a lung organoid model of SARS-CoV-2 entry.

BACKGROUND: Vaccination programs have been launched worldwide to halt the spread of COVID-19. However, the identification of existing, safe compounds with combined treatment and prophylactic properties would be beneficial to individuals who are waiting to be vaccinated, particularly in less economically developed countries, where vaccine availability may be initially limited. METHODS: We used a data-driven approach, combining results from the screening of a large transcriptomic database (L1000) and molecular docking analyses, with in vitro tests using a lung organoid model of SARS-CoV-2 entry, to identify drugs with putative multimodal properties against COVID-19. RESULTS: Out of thousands of FDA-approved drugs considered, we observed that atorvastatin was the most promising candidate, as its effects negatively correlated with the transcriptional changes associated with infection. Atorvastatin was further predicted to bind to SARS-CoV-2's main protease and RNA-dependent RNA polymerase, and was shown to inhibit viral entry in our lung organoid model. CONCLUSIONS: Small clinical studies reported that general statin use, and specifically, atorvastatin use, are associated with protective effects against COVID-19. Our study corroborrates these findings and supports the investigation of atorvastatin in larger clinical studies. Ultimately, our framework demonstrates one promising way to fast-track the identification of compounds for COVID-19, which could similarly be applied when tackling future pandemics.

Interrogating the Inhibition Mechanisms of Human Aldehyde Oxidase by X-ray Crystallography and NMR Spectroscopy: The Raloxifene Case.

Human aldehyde oxidase (hAOX1) is mainly present in the liver and has an emerging role in drug metabolism, since it accepts a wide range of molecules as substrates and inhibitors. Herein, we employed an integrative approach by combining NMR, X-ray crystallography, and enzyme inhibition kinetics to understand the inhibition modes of three hAOX1 inhibitors-thioridazine, benzamidine, and raloxifene. These integrative data indicate that thioridazine is a noncompetitive inhibitor, while benzamidine presents a mixed type of inhibition. Additionally, we describe the first crystal structure of hAOX1 in complex with raloxifene. Raloxifene binds tightly at the entrance of the substrate tunnel, stabilizing the flexible entrance gates and elucidating an unusual substrate-dependent mechanism of inhibition with potential impact on drug-drug interactions. This study can be considered as a proof-of-concept for an efficient experimental screening of prospective substrates and inhibitors of hAOX1 relevant in drug discovery.

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.

Assessing the viability of carbamoylethyl pullulan-g-stearic acid based smart polymeric micelles for tumor targeting of raloxifene.

The present investigation entails the synthesis of smart pullulan polymeric micelles for evaluating its tumor targeting potential. For this purpose, two step polymerization synthesis reactions were conducted. In the first step, carbamoylethylation occurs by reaction of the free alcoholic moieties at 6th position of glucopyranose unit of pullulan with acrylamide in presence of alkali to obtain carbamoylethyl pullulan (CmP). In the second step, CmP undergoes graft polymerization with stearic acid (SA) to obtain CmP-g-stearic acid diblock co-polymer (CmP-g-SA) as evident from FTIR and NMR analysis. The XpRD spectra showed crystalline nature that was further confirmed by SEM indicating rough and poly-porous morphology. The QbD based optimized formulations of raloxifene HCl (RLX) loaded polymeric micelles (RLX PMs) exhibited pH-dependent release profile with added advantage of 1.2 times reduction in percentage hemolysis giving substantial compatibility with erythrocytes. In vivo pharmacokinetic performance of RLX PMs suggested enhanced mean residence time and volume of distribution. Besides, the biodistribution study of RLX PMs manifested enhanced entry of RLX in mammary carcinoma tissues as compared to normal tissues suggested that CmP-g-SA based micelles enhanced the anti-tumor activity of RLX. Overall, the findings pointed toward the biocompatibility of CmP-g-SA as a potential carrier system for the delivery of RLX.

Design, synthesis, and biological evaluation of new raloxifene analogues of improved antagonist activity and endometrial safety.

Raloxifene agonism of estrogen receptor (ER) in post-menopausal endometrium is not negligible. Based on a rational drug design workflow, we synthesized 14 analogues of raloxifene bearing a polar group in the aromatic ring of the basic side chain (BSC) and/or changes in the bulkiness of the BSC amino group. Analogues with a polar BSC aromatic ring and amino group substituents of increasing volume displayed increasing ER antagonism in Ishikawa cells. Analogues with cyclohexylaminoethoxy (13a) or adamantylaminoethoxy BSC (13b) lacking a polar aromatic ring displayed high ER-binding affinity and ER antagonism in Ishikawa cells higher than raloxifene and similar to fulvestrant (ICI182,780). The endometrial surface epithelium of immature female CD1 mice injected with 13b was comparable to that of vehicle-treated mice, while that of mice treated with estradiol, raloxifene or 13b in combination with estradiol was hyperplastic. These findings indicate that raloxifene analogues with a bulky BSC amino group could provide for higher endometrial safety treatment of the menopausal syndrome.

Melt Dispersion Adsorbed onto Porous Carriers: An Effective Method to Enhance the Dissolution and Flow Properties of Raloxifene Hydrochloride.

The objective of the present investigation is to enhance the dissolution and flow properties of raloxifene hydrochloride (RXH), a biopharmaceutical classification system class II drug. Melt dispersion of RXH with polyethylene glycol (PEG) 6000 was prepared by the fusion method. The melt dispersion was then adsorbed onto a porous adsorbent, Neusilin, by the melt adsorption method. Response surface methodology was employed to establish the design space for formulation variables such as the ratio of RXH to PEG 6000 in melt dispersion and amount of porous adsorbent to melt dispersion. Differential scanning calorimetry, scanning electron microscopy, X-ray diffraction, Fourier-transform infrared spectroscopy, and accelerated stability techniques were utilized to characterize formulations. Negative Gibbs free energy values indicated spontaneous solubilization of RXH in PEG 6000. The time required for 80% of drug release from optimized formulation was <20 min compared with plain RXH. Accelerated stability studies confirmed the stabilization of amorphous melt dispersion in nanopores (nanoconfinement) of inorganic silicate Neusilin. Melt dispersion, adsorbed on porous carriers, is a promising technique to improve the dissolution characteristic as well as flow properties of drug molecules.

Chitosan-Raloxifene nanoparticle containing doxorubicin as a new double-effect targeting vehicle for breast cancer therapy.

BACKGROUND: treatment of breast cancer as one of the most common cancers in the world remains an important area of drug development based on nanoparticulate systems. Effective targeted therapy of affected cells based on ligand conjugate biocompatible polymeric nanoparticles is an attractive perspective in this context. OBJECTIVE: In this study, a novel double effect nanoparticle based on Chitosan-Raloxifene conjugate was prepared for adjuvant therapy (hormone and chemo therapy) and drug targeting to breast cancer cells via estrogen receptor (ER). METHODS: Chitosan-raloxifene conjugate was synthesized. Related nanoparticles containing doxorubicin (DOX) were prepared and characterized. Experimental design study was performed to determine the optimum levels of variables in the preparation of nanoparticle. Drug loading, release, nanoparticle stability, and the effect of nanoparticles on cell viability were evaluated. Further, inhibition tests were performed to demonstrate that the function of these novel nanoparticles is mediated via ER. RESULTS: Chitosan-raloxifene conjugate was successfully synthesized. The prepared nanoparticles showed sizes within 25-35 nm, more than 95% drug loading, about 60% of drug release and desired stability after 24 h. XTT assay on MCF-7 cell line illustrated that these nanoparticles could inhibit the cellular growth up to 60%. The results from inhibition tests revealed that prepared nanoparticles can inhibit cell growth via ER blocking. CONCLUSION: This study introduced chitosan-raloxifene nanoparticles containing doxorubicin as a novel targeting agent for adjuvant therapy of breast cancer. Graphical abstract.

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.

A matrix dispersion based on phospholipid complex system: preparation, lymphatic transport, and pharmacokinetics.

Raloxifene hydrochloride (RH) suffers from low oral bioavailability due to its low water-solubility and first-pass metabolism. Therefore, a novel phospholipid complex of RH (RHPC) and a matrix dispersion based on phospholipid complex (RHPC-MD) were successfully prepared and optimized. Several methods were used to validate the formation of RHPC and RHPC-MD, such as differential scanning calorimetry, X-ray diffraction, scanning electron microscopy, transmission electron microscopy, infrared spectroscopy, particle size, and zeta potential, meanwhile, their octanol-water partition coefficient, solubility, and dissolution in vitro were also evaluated. To investigate the absorption mechanism of RHPC in vivo, the RHPC was administered to the chylomicron flow blockage rat model. Interestingly, as we expected, a significant reduction in RHPC absorption (67%) (**p< .01) in presence of cycloheximide (CXI) inhibitor was observed, thus confirming the RHPC could be absorbed by lymphatic transport in vivo. Pharmacokinetic studies revealed that the relative oral bioavailability of RHPC as well as RHPC-MD was 223% and 329%, respectively, when comparing with the commercial RH tablets. These outcomes suggested that the current study provided an attractive formulation to enhance the oral bioavailability of RH and stimulated to further research the absorption mechanism of RHPC in vivo.

Sonochemical synthesis and fabrication of neodymium sesquioxide entrapped with graphene oxide based hierarchical nanocomposite for highly sensitive electrochemical sensor of anti-cancer (raloxifene) drug.

The ultrasound-assisted synthesis of a novel neodymium sesquioxide nanoparticles (Nd2O5 NPs) decorated graphene oxide (GO) nanocomposite under ultrasonic probe (Ultrasonic processor model-PR 1000; frequency-30 kHz; power of 100 W/cm2) has been reported. After then, SEM, TEM, XRD, EDX and electrochemical impedance spectroscopy characterized was analyzed using Nd2O5 NPs@GO nanomaterial. Furthermore, the nanomaterial modified GCE (glassy carbon electrode) shows excellent electrochemical sensing performance towards anti-cancer drug. Raloxifene is one of the important anti-cancer drug. Moreover, the fabricated electrochemical sensor has showed a wide linear range for raloxifene between 0.03 and 472.5 µM and nanomolar detection limit (18.43 nM). In addition, the Nd2O5 NPs@GO modified sensor has been applied to the determination of raloxifene in human blood and urine samples.

Discovery of a Bradykinin B2 Partial Agonist Profile of Raloxifene in a Drug Repurposing Campaign.

Covid-19 urges a deeper understanding of the underlying molecular mechanisms involved in illness progression to provide a prompt therapeutical response with an adequate use of available drugs, including drug repurposing. Recently, it was suggested that a dysregulated bradykinin signaling can trigger the cytokine storm observed in patients with severe Covid-19. In the scope of a drug repurposing campaign undertaken to identify bradykinin antagonists, raloxifene was identified as prospective compound in a virtual screening process. The pharmacodynamics profile of raloxifene towards bradykinin receptors is reported in the present work, showing a weak selective partial agonist profile at the B2 receptor. In view of this new profile, its possible use as a therapeutical agent for the treatment of severe Covid-19 is discussed.