Quantification of the aromatase inhibitor letrozole and its carbinol metabolite in mouse plasma by UHPLC-MS/MS.

A liquid chromatography - electrospray ionization-mass spectrometry (LC-ESI-MS) method was developed for the quantification of letrozole, a third-generation aromatase inhibitor, and its main carbinol metabolite (CM) in support of murine pharmacokinetic studies. Using polarity switching, simultaneous ESI-MS measurement of letrozole and CM was achieved in positive and negative mode, respectively. The assay procedure involved a one-step protein precipitation and extraction of all analytes from mouse plasma requiring only 5 µL of sample. Separation was optimized on an Accucore aQ column with gradient elution at a flow rate of 0.4 mL/min in 5 min. Two calibration curves per day over four consecutive measurement days showed satisfactory linear responses (r2 > 0.99) over concentration ranges of 5-1000 ng/mL and 20-2000 ng/mL for letrozole and CM, respectively. No matrix effect was found, and the mean extraction recoveries were 103-108 % for letrozole and 99.8-107 % for CM. Precision and accuracy within a single run and over four consecutive measurement days were verified to be within acceptable limits. Application of the developed method to preclinical pharmacokinetic studies in mice receiving oral letrozole at a dose 1 or 10 mg/kg revealed that the systemic exposure to letrozole was dose-, formulation-, and strain-dependent. These findings may inform the future design of preclinical studies aimed at refining the pharmacological profile of this clinically important drug.

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.

Preparation of smart PVP/HPMC based IPN hydrogel, its characterization and toxicity evaluation.

In this study, the interpenetrating polymeric network (IPN) were fabricated via free radical polymerization using polymers hydroxypropyl methylcellulose (HPMC), Polyvinylpyrrolidone (PVP) and monomer Methacrylic acid (MAA) and also investigated their influence by changing their concentrations. The developed polymeric network is crosslinked via N' N' -methylene bis-acrylamide (MBA). Different characterizations have been performed to analyze fabricated interpenetrating polymeric network structure i.e., Scanning Electron Microscopy (SEM), X-ray Powder Diffraction (XRD), Fourier-Transform Infrared Spectroscopy (FT-IR), Thermo-gravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC). Letrozole (LTZ) was loaded as a model drug in the developed system. Swelling dynamics as well as drug release behavior were thoroughly examined. FTIR studies corroborated the formation of interpenetrating polymeric network. SEM uncovered porous structure while TGA depicted enhanced thermal stability of polymeric network. PXRD depicted amorphous dispersion of LTZ. Swelling dynamics as well as LTZ release behavior from developed interpenetrating polymeric network hydrogels were dependent upon pH of the medium and concentration of pure reactants employed. Higuchi model was best fit to regression coefficient which indicated diffusion controlled mechanism of drug release. Acute oral toxicity study depicted no mortality or any signs relating to acute toxicity throughout the whole observed period. Hence, the designed interpenetrating polymeric network might turn out to be a safe and a potential carrier system for the delivery of LTZ in the treatment of breast cancer (BC).

Super Magnetic Niosomal Nanocarrier as a New Approach for Treatment of Breast Cancer: A Case Study on SK-BR-3 and MDA-MB-231 Cell Lines.

In the present study, a magnetic niosomal nanocarrier for co-delivery of curcumin and letrozole into breast cancer cells has been designed. The magnetic NiCoFe2O4 core was coated by a thin layer of silica, followed by a niosomal structure, allowing us to load letrozole and curcumin into the silica layer and niosomal layer, respectively, and investigate their synergic effects on breast cancer cells. Furthermore, the nanocarriers demonstrated a pH-dependent release due to the niosomal structure at their outer layer, which is a promising behavior for cancer treatment. Additionally, cellular assays revealed that the nanocarriers had low cellular uptake in the case of non-tumorigenic cells (i.e., MCF-10A) and related high viability but high cellular uptake in cancer cell lines (i.e., MDA-MB-231 and SK-BR-3) and related low viability, which is evidenced in their high cytotoxicity against different breast cancer cell lines. The cytotoxicity of the letrozole/curcumin co-loaded nanocarrier is higher than that of the aqueous solutions of both drugs, indicating their enhanced cellular uptake in their encapsulated states. In particular, NiCoFe2O4@L-Silica-L@C-Niosome showed the highest cytotoxicity effects on MDA-MB-231 and SK-BR-3 breast cancer cells. The observed cytotoxicity was due to regulation of the expression levels of the studied genes in breast cancer cells, where downregulation was observed for the Bcl-2, MMP 2, MMP 9, cyclin D, and cyclin E genes while upregulation of the expression of the Bax, caspase-3, and caspase-9 genes was observed. The flow cytometry results also revealed that NiCoFe2O4@L-Silica-L@C-Niosome enhanced the apoptosis rate in both MDA-MB-231 and SK-BR-3 cells compared to the control samples. The findings of our research show the potential of designing magnetic niosomal formulations for simultaneous targeted delivery of both hydrophobic and hydrophilic drugs into cancer cells in order to enhance their synergic chemotherapeutic effects. These results could open new avenues into the future of nanomedicine and the development of theranostic agents.

In Silico Molecular Interaction Studies of Chitosan Polymer with Aromatase Inhibitor: Leads to Letrozole Nanoparticles for the Treatment of Breast Cancer.

BACKGROUND: It takes a lot more studies to evaluate the molecular interaction of nanoparticles with the drug, their drug delivery potential and release kinetics. Thus, we have taken in silico and in vitro approaches into account for the evaluation of the drug delivery ability of the chitosan nanoparticles. OBJECTIVE: The present work was aimed to study the interaction of chitosan nanoparticles with appropriate aromatase inhibitors using in silico tools. Further, synthesis and characterization of chitosan nanoparticles having optimal binding energy and affinity between drug and polymer in terms of size, encapsulation efficiency were carried out. METHODS: In the current study, molecular docking was used to map the molecular interactions and estimation of binding energy involved between the nanoparticles and the drug molecules in silico. Letrozole is used as a model cytotoxic agent currently being used clinically; hence Letrozole loaded chitosan nanoparticles were formulated and characterized using photomicroscope, particle size analyzer, scanning electron microscope and fourier transform infra-red spectroscopy. RESULTS: Letrozole had the second-highest binding affinity within the core of chitosan with MolDock (-102.470) and Re-rank (-81.084) scores. Further, it was investigated that formulated nanoparticles were having superior drug loading capacity and high encapsulation efficiency. In vitro drug release study exhibited prolonged release of the drug from chitosan nanoparticles. CONCLUSION: Results obtained from the in silico and in vitro studies suggest that Letrozole loaded nanoparticles are ideal for breast cancer treatment.

Structural Recognition and Binding Pattern Analysis of Human Topoisomerase II Alpha with Steroidal Drugs: In Silico Study to Switchover the Cancer Treatment.

BACKGROUND AND OBJECTIVE: Topoisomerase TOP-IIA (TTOP-IIA) is widely used as a significant target for cancer therapeutics because of its involvement in cell proliferation. Steroidal drugs have been suggested for breast cancer treatment as aromatase enzymes inhibitors . TTOP-IIA inhibitors can be used as a target for the development of new cancer therapeutics. MATERIALS AND METHODS: In this study, we conducted a docking study on steroidal drugs Anastrozole (ANA), Letrozole (LET), and exemestane (EXE) with TTOP-IIA  to explore the therapeutic area of these drugs. RESULTS: The binding interaction of EXE drug had significant docking interaction which is followed by ANA and LET. Thus, all these drugs could be used to inhibit the TTOP-IIA mediated cell proliferation and could be a hope to treat the other types of cancers. Among all three tested steroidal drugs, EXE showed binding energy -7.05 kcal/mol, hydrogen bond length1.78289 Å and amino acid involved in an interaction was A: LYS723:HZ3 -: UNK1:O6. CONCLUSION: The obtained data showed the most significant binding interaction analyzed with the tested enzyme. Thus, in vitro laboratory experimentation and in vivo research are necessary to put forward therapeutic repositioning of these drugs to establish them as a broad spectrum potential anticancer drugs.
.

Synthesis, Docking Studies and Biological Activity of New Benzimidazole- Triazolothiadiazine Derivatives as Aromatase Inhibitor.

In the last step of estrogen biosynthesis, aromatase enzyme catalyzes the conversion of androgens to estrogens. Aromatase inhibition is an important way to control estrogen-related diseases and estrogen levels. In this study, sixteen of benzimidazole-triazolothiadiazine derivatives have been synthesized and studied as potent aromatase inhibitors. First, these compounds were tested for their anti-cancer properties against human breast cancer cell line (MCF-7). The most active compounds 5c, 5e, 5k, and 5m on MCF-7 cell line were subject to further in vitro aromatase enzyme inhibition assays to determine the possible mechanisms of action underlying their activity. Compound 5e showed slight less potent aromatase inhibitory activity than that of letrozole with IC50 = 0.032 ± 0.042 µM, compared to IC50 = 0.024 ± 0.001 µM for letrozole. Furthermore, compound 5e and reference drug letrozole were docked into human placental aromatase enzyme to predict their possible binding modes with the enzyme. Finally, ADME parameters (absorption, distribution, metabolism, and excretion) of synthesized compounds (5a-5p) were calculated by QikProp 4.8 software.

Simultaneous quantification of abemaciclib and letrozole in rat plasma: method development, validation and pharmacokinetic application.

Treatment through a combination of drugs involving cyclin D-dependent kinase inhibitors like abemaciclib and aromatase inhibitor like letrozole proved to be a potential therapeutic regimen and first-line treatment in estrogen receptor-positive breast cancer. In this study, we developed a simple and simultaneous RP-HPLC bioanalytical method for quantifying abemaciclib and letrozole in rat plasma. Abemaciclib and letrozole were separated on Zorbax Eclipse C18 column employing a gradient elution method comprising 10 mM ammonium acetate (pH 5) and acetonitrile as mobile phase. The method was found to have acceptable selectivity, accuracy (97.20-118.17%), precision (1.10-9.39%) and stability in the validation experiment performed as per the US Food and Drug Administration guidelines. The method sensitivity was low at a concentration level of 100 ng/ml. The applicability of the method has been verified through a single-dose oral pharmacokinetic study in rat. The developed method will be useful to quantitate the analytes in rat plasma samples of different preclinical studies including their pharmacokinetic drug-drug interactions in the future. To date, no method has been reported for the quantification of abemaciclib and letrozole simultaneously in any type of biological matrices. Therefore, this study makes a definite significant contribution in the field of bioanalytical research.

Design and development of letrozole nanoemulsion: A comparative evaluation of brain targeted nanoemulsion with free letrozole against status epilepticus and neurodegeneration in mice.

The target of the current study is to formulate letrozole loaded nanoemulsion (LET-NE) for the direct nose to brain delivery to reduce peripheral effects of letrozole (LET). LET-NE is compared against intraperitoneally administered free LET in kainic acid (KA) induced status epilepticus (SE) in mice. LET loaded nanoemulsion (LET-NE) was prepared by aqueous microtitration method using Triacetin, Tween 80 and PEG-400 as the oil phase, surfactant, and co-surfactant. Nanoemulsion was studied for droplet size, polydispersity index (PDI), zeta potential, percentage transmittance, drug content, surface morphology. TEM images of developed formulation demonstrated spherical droplets with a mean diameter of 95.59 ±â€¯2.34 nm, PDI of 0.162 ±â€¯0.012 and zeta potential of -7.12 ±â€¯0.12 mV respectively. In in-vitro and ex-vivo drug release, LET-NE showed prolonged drug release profile as compared to suspension. SE was induced by KA (10 mg/kg, i.p.) in Swiss albino mice. Behavioral seizure monitoring, biochemical estimations, and histopathological examination were performed. The onset time of SE was significantly enhanced and % incidence of SE was reduced by intranasal administration of LET-NE as compared to KA and LET administered intraperitoneally. Biochemical estimations revealed that LET-NE effectively decreased levels of 17-ß estradiol while the levels of 5α-Dihydrotestosterone (5α-DHT) and 3α-androstanediol (3α-Diol) were significantly increased in the hippocampus. In cresyl violet staining LET-NE showed better protection of the hippocampus from neurotoxicity induced by KA as compared to LET. Also, in gamma scintigraphy of mouse brain, intranasal administration of nanoemulsion exhibited the presence of high concentration of LET. The study demonstrates the anticonvulsant and neuroprotective effect of LET-NE probably by inhibition of aromatization of testosterone into 17-ß estradiol, proconvulsant, and diverting the pathway into the synthesis of testosterone metabolites, 3α-Diol with known anticonvulsant and neuroprotective action. Brain targeting of LET-NE showed better anticonvulsant and neuroprotective action than LET.

A Comprehensive Physicochemical, In Vitro and Molecular Characterization of Letrozole Incorporated Chitosan-Lipid Nanocomplex.

PURPOSE: The aim of this study is to show a new mesomicroscopic insight into Letrozole (LTZ) loaded nanocomplexes and their ex vivo characteristics as a drug delivery system. METHODS: The LTZ loaded hybrid chitosan-based carrier was fabricated using a modified ionic crosslinking technique and characterized in more detail. To understand the mechanism of LTZ action encapsulated in the hybrid polymer-lipid carrier, all-atom molecular dynamics simulations were also used. RESULTS: The physicochemical properties of the carrier demonstrated the uniform morphology, but different drug loading ratios. In vitro cytotoxic activity of the optimized carrier demonstrated IC50 of 67.85 ± 0.55 nM against breast cancer cell line. The ex vivo study showed the positive effect of nanocomplex on LTZ permeability 7-10 fold greater than the free drug. The molecular dynamic study also confirmed the prsence of hydrophobic peak of lipids at a distance of 5 Å from the center of mass of LTZ which proved drug entrapment in the core of nanocomplex. CONCLUSIONS: The hybrid nanoparticle increased the cytotoxicity and tissue permeability of LTZ for oral delivery. This study also confirmed the atomic mesostructures and interaction of LTZ in the core of hybrid polymer-lipid nanoparticles.

Preparation and characterization of letrozole-loaded poly(d,l-lactide) nanoparticles for drug delivery in breast cancer therapy.

Letrozole (LTZ), an aromatase inhibitor used for the treatment of hormonally-positive breast cancer in postmenopausal women, has poor water solubility, rapid metabolism, and a range of side effects. In this study, polymer-based nanoparticles (NPs) incorporating the drug have been designed and characterized, aimed to control the release, potentially maximize the therapeutic efficiency, and minimize the side effects of the drug. LTZ was incorporated into poly(d,l-lactide) (PDLLA) NPs by employing the emulsion-solvent evaporation technique using a range of drug concentrations. Loaded drug and drug-polymer interactions were studied using X-ray diffraction and NPs morphology was evaluated using scanning electron microscopy (SEM). Particle size distribution (PSD) and zeta potential of the NPs were analyzed using dynamic light scattering (DLS) and laser Doppler velocimetry (LDV), respectively. Drug content and release profile studies were carried out and determined using ultra performance liquid chromatography (UPLC). The yield of LTZ-PDLLA NPs reached as high as 85%. The NPs were spherical and smooth, regardless of LTZ concentration in the formulation. However, particle size increased from 241.6 ± 1.2 to 348.7 ± 6.1 nm upon increasing LTZ concentration from 0 to 30% w/w, with entrapment efficiencies reaching up to 96.8%. Drug release from the polymeric matrix was best described by Higuchi model with a predominant diffusion-based mechanism. More than 15, 46, and 86% of LTZ was released in a controlled fashion over 30 d from the 10, 20, and 30% LTZ-PDLLA NPs, respectively. Overall, LTZ-PDLLA NPs were designed with appropriate size and surface charge, high drug loading, superior entrapment efficiency, and prolonged release profile.

Preparation and optimization of monodisperse polymeric microparticles using modified vibrating orifice aerosol generator for controlled delivery of letrozole in breast cancer therapy.

Letrozole (LTZ) is effective for the treatment of hormone-receptor-positive breast cancer in postmenopausal women. In this work, and for the first time, using vibrating orifice aerosol generator (VOAG) technology, monodisperse poly-ε-caprolactone (PCL), and poly (D, L-Lactide) (PDLLA) LTZ-loaded microparticles were prepared and found to elicit selective high cytotoxicity against cancerous breast cells with no apparent toxicity on healthy cells in vitro. Plackett-Burman experimental design was utilized to identify the most significant factors affecting particle size distribution to optimize the prepared particles. The generated microparticles were characterized in terms of microscopic morphology, size, zeta potential, drug entrapment efficiency, and release profile over one-month period. Long-term cytotoxicity of the microparticles was also investigated using MCF-7 human breast cancer cell lines in comparison with primary mammary epithelial cells (MEC). The prepared polymeric particles were monodispersed, spherical, and apparently smooth, regardless of the polymer used or the loaded LTZ concentration. Particle size varied from 15.6 to 91.6 µm and from 22.7 to 99.6 µm with size distribution (expressed as span values) ranging from 0.22 to 1.24 and from 0.29 to 1.48 for PCL and PDLLA based microparticles, respectively. Upon optimizing the manufacture parameters, span was reduced to 0.162-0.195. Drug entrapment reached as high as 96.8%, and drug release from PDLLA and PCL followed a biphasic zero-order release using 5 or 30% w/w drug loading in the formulations. Long-term in vitro cytotoxicity studies indicated that microparticles formulations significantly inhibited the growth of MCF-7 cell line over a prolonged period of time but did not have toxic effects on the normal breast epithelial cells.

Extraction and determination of trace amounts of three anticancer pharmaceuticals in urine by three-phase hollow fiber liquid-phase microextraction based on two immiscible organic solvents followed by high-performance liquid chromatography.

An automated three-phase hollow fiber liquid-phase microextraction based on two immiscible organic solvents followed by high-performance liquid chromatography with UV-Vis detection method was applied for the extraction and determination of exemestane, letrozole, and paclitaxel in water and urine samples. n-Dodecane was selected as the supported liquid membrane and its polarity was justified by trioctylphosphine oxide. Acetonitrile was used as an organic acceptor phase with desirable immiscibility having n-dodecane. All the effective parameters of the microextraction procedure such as type of the organic acceptor phase, the supported liquid membrane composition, extraction time, pH of the donor phase, hollow fiber length, stirring rate, and ionic strength were evaluated and optimized separately by a one variable at-a-time method. Under the optimal conditions, the linear dynamic ranges were 1.8-200 (R2  = 0.9991), 0.9-200 (R2  = 0.9987) and 1.2-200 µg/L (R2  = 0.9983), and the limits of detection were 0.6, 0.3, and 0.4 µg/L for exemestane, letrozole, and paclitaxel, respectively. To evaluate the capability of the proposed method in the analysis of biological samples, three different urinary samples were analyzed under the optimal conditions. The relative recoveries of the three pharmaceuticals were in the range of 91-107.3% for these three analytes.

Metabolomics Reveals that Dietary Xenoestrogens Alter Cellular Metabolism Induced by Palbociclib/Letrozole Combination Cancer Therapy.

Recently, the palbociclib/letrozole combination therapy was granted accelerated US FDA approval for the treatment of estrogen receptor (ER)-positive breast cancer. Since the underlying metabolic effects of these drugs are yet unknown, we investigated their synergism at the metabolome level in MCF-7 cells. As xenoestrogens interact with the ER, we additionally aimed at deciphering the impact of the phytoestrogen genistein and the estrogenic mycotoxin zearalenone. A global metabolomics approach was applied to unravel metabolite and pathway modifications. The results clearly showed that the combined effects of palbociclib and letrozole on cellular metabolism were far more pronounced than that of each agent alone and potently influenced by xenoestrogens. This behavior was confirmed in proliferation experiments and functional assays. Specifically, amino acids and central carbon metabolites were attenuated, while higher abundances were observed for fatty acids and most nucleic acid-related metabolites. Interestingly, exposure to model xenoestrogens appeared to counteract these effects.