Potentiation of Brain Bioavailability Using Thermoreversible Cubosomal Formulation.

The aim of the present study was to develop and evaluate intranasal formulations of the thermoreversible fluoxetine cubosomal in situ gel. This gel was intended for permeation and bioavailability enhancement to target the brain effectively by bypassing the blood-brain barrier (BBB). Fluoxetine-loaded cubosomes were prepared by the homogenization method followed by the cold method approach to develop in situ gel. Fluoxetine-loaded cubosomes displayed a higher encapsulation efficiency (82.60 ± 1.25%) than fluoxetine. This might be due to the solubilizing activity of the polymer to cause partitioning of the lipophilic drug into the aqueous phase during the change from the cubic gel phase to cubosomes. In vitro analysis of fluoxetine-loaded cubosomal in situ gel showed a sustained release profile (93.22 ± 2.47%) due to limited diffusion of fluoxetine. The formation of strong affinity bonds of the drug with GMO (drug transporter) decreased the drug release in comparison to that with fluoxetine-loaded cubosomes (90.68 ± 1.74%). The ex vivo drug release profile revealed the drug release of 96.31 ± 2.88% by the end of 24 h. This is attributed to the higher capability of the intranasal cubosomal in situ gel to prolong the retention and enable better permeation through the nasal mucosa. In male Wistar rats, in vivo biodistribution studies for cubosomal in situ gel administered via the intranasal route at a dose of 3.5 mg/kg demonstrated an increase in pharmacokinetic parameters like the AUC (406 ± 75.35 µg/mL), Cmax (368.07 ± 0.23 µg/mL), Tmax (4 h), and t1/2 (14.06 h). The mucoadhesive nature of the in situ gel led to an increase in the residence time of the gel in the nasal mucosa. The biodistribution study of intranasal in situ cubosomal gel improved the bioavailability 2.21-fold in comparison to that with the cubosomal dispersion but 2.83-fold in comparison to that with the drug solution. Therefore, fluoxetine-loaded cubosomal in situ gel proved as a promising carrier for effective transportation of fluoxetine via the intranasal route with significant brain bioavailability.

Protein synergistic action-based development and application of a molecularly imprinted chiral sensor for highly stereoselective recognition of S-fluoxetine.

In order to improve the recognition performance of MIPs sensors in chiral drug enantiomers, a novel a highly selective molecular recognition method based on protein-assisted immobilization of chiral molecular conformation was developed. S-fluoxetine (S-FLX) as the target chiral molecule, human serum albumin (HSA), which has a high affinity and strong interactions with S-FLX, was screened from 11 proteins to serve as an auxiliary recognition unit for the fixation of chiral conformation. By incorporating HSA into the preparation of molecularly imprinted polymers (MIPs), the natural chirality and high stereoselectivity of the protein were leveraged for the induction and fixation of the stereo conformation of S-FLX, refinement of internal structures of the imprinted cavities. The sensor exhibited excellent chiral recognition ability and high detection sensitivity. The changes of probe signal intensity of the MIPs/HSA sensor were positively correlated with the logarithmic concentration of S-FLX in the range of 1.0 × 10-16-1.0 × 10-11 mol L-1, where a detection limit of 6.43 × 10-17 mol L-1 was achieved (DL = 3δb/K). The selectivity of MIPs/HSA sensor in recognizing S-FLX was increased by 18.5 times and the sensitivity was increased by 2.6 times after the incorporation of HSA. The developed sensor was successfully used for the analysis of S-FLX in fluoxetine hydrochloride capsules.

Fluoxetine and sertraline based multitarget inhibitors of cholinesterases and monoamine oxidase-A/B for the treatment of Alzheimer's disease: Synthesis, pharmacology and molecular modeling studies.

For the potential therapy of Alzheimer's disease (AD), cholinesterases (ChE) and monoamine oxidase (MAO) are key enzymes that regulate the level of acetylcholinesterase (AChE)/butyrylcholinesterase (BChE) and monoamines. The aim of current research is the synthesis of multi-target compounds that can concomitantly inhibit ChEs and MAO. A series of fluoxetine and sertraline hybrids was designed and evaluated as multi-target inhibitors of ChEs and hMAO. In-vitro enzyme inhibition studies demonstrated that a number of compounds displayed excellent inhibition in submicromolar to nanomolar range. However, compounds 17, 22, 38-40 possess excellent concomitant inhibitory activity against ChEs and hMAO-A/B enzymes and thus emerged as optimal multi-target hybrids. In-vivo acute toxicity study showed the safety of synthesized compounds up to 2000 mg/kg dose. The examinations of brain tissue in Swiss albino mice suggested that selected most active MAO-B inhibitors 17 and 22 have a propensity to block the MAO-B activity that could be responsible for their neurodegenerative effect in mice. The in-vitro inhibitory manner of interaction of these multipotent compounds on all four targets were confirmed by molecular docking investigations.

The impact of fluoxetine and pH values on relaxation of the ternary lipid monolayers.

Fluoxetine (FLX), used in the clinic to treat depression, is a well-known cationic amphiphilic antidepressant. To get a deeper insight into the effect of FLX on Langmuir monolayers, in this study the stability and relaxation of 1,2-dioctadecanoyl-sn-glycero-3-phophocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/cholesterol (DSPC/POPC/CHOL) monolayers without and with FLX at different pH values were studied. The experiments involved surface pressure-area (π-A) measurements, mean molecular area-time (A-t) measurements, and atomic force microscope (AFM) analysis. It was found that intermolecular interactions decreased after the addition of FLX in the subphase but increased with increasing pH values. The relaxation of the ternary lipid monolayers with FLX was dominated by dissolution steps, and the dissolution rates decreased with increasing pH values. These findings can be easily confirmed by the analysis of thermodynamic parameters calculated for the investigated films. The data obtained in this study help to understand the effect of drugs on the ternary lipid monolayers from the molecular point of view.

Label-free high-throughput screening assay for the identification of norepinephrine transporter (NET/SLC6A2) inhibitors.

The human norepinephrine transporter (NET) is an established drug target for a wide range of psychiatric disorders. Conventional methods that are used to functionally characterize NET inhibitors are based on the use of radiolabeled or fluorescent substrates. These methods are highly informative, but pose limitations to either high-throughput screening (HTS) adaptation or physiologically accurate representation of the endogenous uptake events. Recently, we developed a label-free functional assay based on the activation of G protein-coupled receptors by a transported substrate, termed the TRACT assay. In this study, the TRACT assay technology was applied to NET expressed in a doxycycline-inducible HEK 293 JumpIn cell line. Three endogenous substrates of NET-norepinephrine (NE), dopamine (DA) and epinephrine (EP)-were compared in the characterization of the reference NET inhibitor nisoxetine. The resulting assay, using NE as a substrate, was validated in a manual HTS set-up with a Z' = 0.55. The inhibitory potencies of several reported NET inhibitors from the TRACT assay showed positive correlation with those from an established fluorescent substrate uptake assay. These findings demonstrate the suitability of the TRACT assay for HTS characterization and screening of NET inhibitors and provide a basis for investigation of other solute carrier transporters with label-free biosensors.

Tuning the activity of known drugs via the introduction of halogen atoms, a case study of SERT ligands - Fluoxetine and fluvoxamine.

The selective serotonin reuptake inhibitors (SSRIs), acting at the serotonin transporter (SERT), are one of the most widely prescribed antidepressant medications. All five approved SSRIs possess either fluorine or chlorine atoms, and there is a limited number of reports describing their analogs with heavier halogens, i.e., bromine and iodine. To elucidate the role of halogen atoms in the binding of SSRIs to SERT, we designed a series of 22 fluoxetine and fluvoxamine analogs substituted with fluorine, chlorine, bromine, and iodine atoms, differently arranged on the phenyl ring. The obtained biological activity data, supported by a thorough in silico binding mode analysis, allowed the identification of two partners for halogen bond interactions: the backbone carbonyl oxygen atoms of E493 and T497. Additionally, compounds with heavier halogen atoms were found to bind with the SERT via a distinctly different binding mode, a result not presented elsewhere. The subsequent analysis of the prepared XSAR sets showed that E493 and T497 participated in the largest number of formed halogen bonds. The XSAR library analysis led to the synthesis of two of the most active compounds (3,4-diCl-fluoxetine 42, SERT Ki = 5 nM and 3,4-diCl-fluvoxamine 46, SERT Ki = 9 nM, fluoxetine SERT Ki = 31 nM, fluvoxamine SERT Ki = 458 nM). We present an example of the successful use of a rational methodology to analyze binding and design more active compounds by halogen atom introduction. 'XSAR library analysis', a new tool in medicinal chemistry, was instrumental in identifying optimal halogen atom substitution.

Antidepressant drugs act by directly binding to TRKB neurotrophin receptors.

It is unclear how binding of antidepressant drugs to their targets gives rise to the clinical antidepressant effect. We discovered that the transmembrane domain of tyrosine kinase receptor 2 (TRKB), the brain-derived neurotrophic factor (BDNF) receptor that promotes neuronal plasticity and antidepressant responses, has a cholesterol-sensing function that mediates synaptic effects of cholesterol. We then found that both typical and fast-acting antidepressants directly bind to TRKB, thereby facilitating synaptic localization of TRKB and its activation by BDNF. Extensive computational approaches including atomistic molecular dynamics simulations revealed a binding site at the transmembrane region of TRKB dimers. Mutation of the TRKB antidepressant-binding motif impaired cellular, behavioral, and plasticity-promoting responses to antidepressants in vitro and in vivo. We suggest that binding to TRKB and allosteric facilitation of BDNF signaling is the common mechanism for antidepressant action, which may explain why typical antidepressants act slowly and how molecular effects of antidepressants are translated into clinical mood recovery.

Radical scavenging activity of natural antioxidants and drugs: Development of a combined machine learning and quantum chemistry protocol.

Many natural substances and drugs are radical scavengers that prevent the oxidative damage to fundamental cell components. This process may occur via different mechanisms, among which, one of the most important, is hydrogen atom transfer. The feasibility of this process can be assessed in silico using quantum mechanics to compute ΔGHAT ○. This approach is accurate, but time consuming. The use of machine learning (ML) allows us to reduce tremendously the computational cost of the assessment of the scavenging properties of a potential antioxidant, almost without affecting the quality of the results. However, in many ML implementations, the description of the relevant features of a molecule in a machine-friendly language is still the most challenging aspect. In this work, we present a newly developed machine-readable molecular representation aimed at the application of automatized ML algorithms. In particular, we show an application on the calculation of ΔGHAT ○.

Antibacterial activity of fluoxetine-loaded starch nanocapsules.

This work proposes the development of a starch-based drug carrier for fluoxetine (FLX) delivery and evaluate the improvement of the drug antibacterial activity. The starch nanocapsules were prepared via interfacial polyaddition reaction presenting a core-shell morphology, based on polyurethane linkage, with a particle size in the range 250-300 nm. Furthermore, FLX-loaded nanocapsules were evaluated regarding antibacterial potential against Staphylococcus aureus (ATCC® 6538P ™) and its clinical strains of methicillin-resistant. As expected, the FLX-loaded presented lower minimum inhibitory concentration (MIC) values, in the range of 190-95 µg mL-1, against all isolated microorganisms in comparison to FLX, 255 µg mL-1. According to results, the FLX-loaded starch nanocapsules have successfully improved drug antibacterial activity, generating promising perspectives on the field of the hydrophilic drug delivery systems.

Simultaneous quantification of vortioxetine, fluoxetine and their metabolites in rat plasma by UPLC-MS/MS.

In this study, a specific and quick ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was fully developed and validated for simultaneous measurement of the rat plasma levels of vortioxetine (VOR), Lu AA34443 (the major metabolite of VOR), fluoxetine and its metabolite norfluoxetine with diazepam as the internal standard (IS). After a simple protein precipitation with acetonitrile for sample preparation, the separation of the analytes were performed on an Acquity UPLC BEH C18 (2.1 × 50 mm, 1.7 µm) column, with acetonitrile and 0.1% formic acid in water as mobile phase by gradient elution. The detection was achieved on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring mode via an electrospray ionization source. Good linearity was observed in the calibration curve for each analyte. The data of precision, accuracy, matrix effect, recovery and stability all conformed to the bioanalytical method validation of acceptance criteria of US Food and Drug Administration recommendations. The newly developed UPLC-MS/MS method allowed simultaneous quantification of VOR, fluoxetine and their metabolites for the first time and was successfully applied to a pharmacokinetic study in rats.

Effect of fluoxetine at different concentrations on the adsorption behavior of Langmuir monolayers.

Fluoxetine (FLX), approved for the treatment of depression and anxiety by the FDA in 2002, is an amphiphilic antidepressant. In general, amphiphilic drugs have high membrane permeability. Therefore, the interactions between these drugs and monolayers have been widely concerned. In this study, the adsorption of FLX on dipalmitoylphosphatidylcholine (DPPC) monolayers at different concentrations and surface pressures have been investigated by pressure-area isotherms (π-A), adsorption curves, compression-expansion curves, and atomic force microscopy (AFM). Our data showed that the adsorption behavior was related to the surface pressures and FLX concentrations in the subphase. The FLX that was added in the subphase under lower surface pressure (π = 10 mN/m) was easily adsorbed on DPPC monolayers. The stability of the monolayers was strong. The adsorption of FLX on DPPC monolayers and the stability decreased when π = 20 mN/m. In addition, the adsorption behavior and stability increased with increasing FLX concentrations. The AFM images of the monolayers confirmed the results of fitted adsorption curves. This study will be critical to our understanding of the interactions between drugs and lipid monolayers.

Studies on the interfacial behavior of DPPC/DPPG mixed monolayers in the presence of fluoxetine.

In this study, the interfacial behavior of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPC/DPPG) mixed monolayers with fluoxetine (FLX) in the subphase was investigated by a combination of the Langmuir-Blodgett technique and atomic force microscopy (AFM). It was found that DPPC/DPPG mixed monolayers showed different interfacial behaviors before and after addition of FLX in the subphase. The electrostatic interaction between FLX and lipids molecules destroys the homogeneity of the mixed monolayers and changes the arrangement of lipids molecules at the interface after addition of FLX in the subphase, thereby leading to an increase of compressibility and miscibility and a decrease in the stability of the mixed monolayers. The surface morphology of the mixed monolayers observed by AFM was different between without and with FLX in the subphase, indicating the penetration of FLX into the mixed monolayers. The present study has provided detailed information for further understanding the interactions of drugs with membrane lipids in other lipid monolayers.

Surface Interactions during the Removal of Emerging Contaminants by Hydrochar-Based Adsorbents.

The aim of this work was to test activated carbons derived from hydrochars produced from sunflower stem, olive stone and walnut shells, as adsorbents for emerging contaminants in aqueous solution, namely fluoxetine and nicotinic acid. The adsorption capacity was determined by the chemical nature of the adsorbents, namely the presence of specific functional groups and their positive or negative ionization in aqueous solutions and also by steric factors. The activated carbons produced by air showed a higher adsorption capacity of fluoxetine, whilst the samples produced by carbon dioxide activation were more useful to remove nicotinic acid. In general, surface acidity was advantageous for fluoxetine adsorption and detrimental for nicotinic acid removal. The adsorption mechanisms involved in each case were discussed and related to the adsorbents characteristics. The maximum adsorption capacity, Q0, given by the Langmuir model was 44.1 and 91.9 mg g-1 for fluoxetine and nicotinic acid adsorption, respectively.

Synthesis and antiviral effect of novel fluoxetine analogues as enterovirus 2C inhibitors.

Enteroviruses (EV) are a group of positive-strand RNA (+RNA) viruses that include many important human pathogens (e.g. poliovirus, coxsackievirus, echovirus, numbered enteroviruses and rhinoviruses). Fluoxetine was identified in drug repurposing screens as potent inhibitor of enterovirus B and enterovirus D replication. In this paper we are reporting the synthesis and the antiviral effect of a series of fluoxetine analogues. The results obtained offer a preliminary insight into the structure-activity relationship of its chemical scaffold and confirm the importance of the chiral configuration. We identified a racemic fluoxetine analogue, 2b, which showed a similar antiviral activity compared to (S)-fluoxetine. Investigating the stereochemistry of 2b revealed that the S-enantiomer exerts potent antiviral activity and increased the antiviral spectrum compared to the racemic mixture of 2b. In line with the observed antiviral effect, the S-enantiomer displayed a dose-dependent shift in the melting temperature in thermal shift assays, indicative for direct binding to the recombinant 2C protein.

Evaluation of anti-depressant effects of phthalazinone-based triple-acting small molecules against 5-HT2A, 5-HT2C, and the serotonin transporter.

Development of highly effective, safe, and fast-acting anti-depressants is urgently required for the treatment of major depressive disorder. It has been suggested that targeting 5-HT2A and 5-HT2C in addition to inhibition of serotonin reuptake may be beneficial in generating anti-depressant agents with better pharmacology and less adverse effects. We have developed phthalazinone-based compounds that potently bind to 5-HT2A, 5-HT2C, and the serotonin transporter. The representative compounds 11j and 11l displayed strong binding affinities against these targets, and showed favorable toxicity profiles as determined by hERG binding and CYP inhibition assays. Furthermore, these compounds presented promising anti-depressant effects comparable to fluoxetine and also synergistic effects with fluoxetine in forced swimming test, which implicates these compounds can be developed to help the treatment of major depressive disorder.

Fast-Acting Black-Phosphorus-Assisted Depression Therapy with Low Toxicity.

A black phosphorus (BP)-nanosheet-based drug-delivery system containing a therapeutic drug (Fluoxetine, Flu) is synthesized. According to subsequent behavioral, biochemical, and electrophysiological analysis, BP-Flu, after irradiated with near-infrared light (808 nm), can significantly reduce the therapy time of depression. Meanwhile, the inherent biotoxicity of Flu is also alleviated.

Inhibitory and Stimulatory Effects of Selective Serotonin Reuptake Inhibitors on Cytochrome P450 2D6-mediated Dopamine Formation from p-Tyramine.

PURPOSE: The effects of selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine and paroxetine on dopamine formation from p-tyramine, mediated by cytochrome P450 (CYP) 2D6.2 (Arg296Cys, Ser486Thr) and CYP2D6.10 (Pro34Ser, Ser486Thr), were compared with their effects on CYP2D6.1 (wild type)-mediated dopamine formation, to investigate the influence of a CYP2D6 polymorphism on neuroactive amine metabolism in the brain. METHODS: The Michaelis constants (Km) and maximal velocity (Vmax) values of dopamine formation mediated by CYP2D6.1, CYP2D6.2, and CYP2D6.10 (expressed in recombinant Escherichia coli), and inhibition constants (Ki) of the SSRIs toward dopamine formation catalyzed by the CYP2D6 variants were estimated. RESULTS: The Km values for CYP2D6.2 and CYP2D6.10 decreased at lower fluoxetine concentrations, while the Vmax values for all CYP2D6 variants increased, indicating that fluoxetine stimulated dopamine formation. Conversely, paroxetine competitively inhibited dopamine formation mediated by CYP2D6.1, CYP2D6.2, and CYP2D6.10 with Ki values of 0.47, 1.33, and 31.3 µM, respectively. CONCLUSIONS: These results suggest that the inhibition/stimulation of CYP2D6-mediated dopamine formation by these SSRIs would be affected by CYP2D6 polymorphisms in the brain.

Molecular determinants of chemical modulation of two-pore domain potassium channels.

The K+ ion channels comprising the two-pore domain (K2P) family have specific biophysical roles in generating the critical regulatory K+ current. Ion flow through K2P channels and, implicitly, channel regulation is mediated by diverse metabolic and physical inputs such as mechanical stimulation, interaction with lipids or endogenous regulators, intra- or extracellular pH, and phosphorylation, while their function can be finely tuned by chemical compounds. In the latter category, some drug-channel interactions can lead to side effects or have clinical action, while identifying novel chemical modulators of K2Ps is an area of intense research. Due to their cellular and therapeutic importance, much attention was turned to these channels in recent years and several experimental approaches have pinpointed the molecular determinants of K2P chemical modulation. Given their unique structural features and properties, chemical modulators act on K2P channels in multiple and diverse ways. In this review, the particularities of K2P modulation by chemical compounds, such as binding modality, affinity, or position, are identified, synthesized, and linked to structural and functional properties in order to refer to how activators and blockers modify channel function and vice versa, focusing on specificity related to protein structure (and its modification) and cross-linking information among different subfamilies.

CNTs coated charcoal as a hybrid composite material: Adsorption of fluoxetine probed by zebrafish embryos and its potential for environmental remediation.

Although traditional water treatment systems can remove various substances from wastewater, these conventional systems fail to remove many chemical molecules that pose potential ecological and health risks. Carbon nanotubes (CNTs) appear attractive to adsorption of many substances, but CNTs adsorbed with toxic substances becomes a nanocomposite still more toxic. Here, we employ zebrafish embryos as biosensor to examine how a hybrid micro/nanostructured carbonaceous material (HMNC) derived from a combination of activated carbon (AC) with hydrophilic carbon nanotubes (CNTs) can remediate wastewater contaminated with the pharmaceutical fluoxetine hydrochloride (FLX). AC and HMNC are practically non-toxic to zebrafish embryos (LC50 > 1000 mg.L-1). HMNC addition to culture medium containing FLX significantly reduces sublethal effects and lethality. Interaction between FLX and HMNC involves chemical adsorption such that embryo co-exposure to HMNC adsorbed with FLX in the range of concentrations evaluated herein does not elicit any behavioral changes in zebrafish.

An In Vitro Study of Aromatic Stacking of Drug Molecules.

In this paper, drug-drug chemical interactions between two different aromatic compounds were studied by mass spectrometry. Specifically, we examined non-covalent complexes (NCX) between paclitaxel, a chemotherapeutic compound, and medications widely used in palliative care for depression, psychosis, and anxiety. It is unknown whether psychotropic medications directly interact with paclitaxel. Here, we use a simple and rapid electrospray ionization mass spectrometry in vitro assay, which has been predictive in the case of neuropeptides, to measure the relative strength of non-covalent interactions. This chemical interaction is most likely due to the overlap of aromatic rings of π-orbitals between paclitaxel and five commonly used medications: diazepam, clonozepam, sertraline, fluoxetine, and haloperidol. Molecular modeling illustrates that differences in the stability of the NCXs are likely due to the distance between the aromatic rings present in both the paclitaxel and antidepressant medications. Graphical Abstract.