Structure- and Cation-Dependent Mechanism of Interaction of Tricyclic Antidepressants with NMDA Receptor According to Molecular Modeling Data.

Some tricyclic antidepressants (TCAs), including amitriptyline (ATL), clomipramine (CLO), and desipramine (DES), are known to be effective for management of neuropathic pain. It was previously determined that ATL, CLO, and DES are capable of voltage-dependent blocking of NMDA receptors of glutamate (NMDAR), which play a key role in pathogenesis of neuropathic pain. Despite the similar structure of ATL, CLO, and DES, efficacy of their interaction with NMDAR varies significantly. In the study presented here, we applied molecular modeling methods to investigate the mechanism of binding of ATL, CLO, and DES to NMDAR and to identify structural features of the drugs that determine their inhibitory activity against NMDAR. Molecular docking of the studied TCAs into the NMDAR channel was performed. Conformational behavior of the obtained complexes in the lipid bilayer was simulated by the method of molecular dynamics (MD). A single binding site (upper) for the tertiary amines ATL and CLO and two binding sites (upper and lower) for the secondary amine DES were identified inside the NMDAR channel. The upper and lower binding sites are located along the channel axis at different distances from the extracellular side of the plasma membrane. MD simulation revealed that the position of DES in the lower site is stabilized only in the presence of sodium cation inside the NMDAR channel. DES binds more strongly to NMDAR compared to ATL and CLO due to simultaneous interaction of two hydrogen atoms of its cationic group with the asparagine residues of the ion pore of the receptor. This feature may be responsible for the stronger side effects of DES. It has been hypothesized that ATL binds to NMDAR less efficiently compared to DES and CLO due to its lower conformational mobility. The identified features of the structure- and cation-dependent mechanism of interaction between TCAs and NMDAR will help in the further development of effective and safe analgesic therapy.

Dibenzosuberyl substituted polyamines and analogs of clomipramine as effective inhibitors of trypanothione reductase; molecular docking, and assessment of trypanocidal activities.

Trypanothione reductase (TR) is an enzyme critical to the maintenance of the thiol redox balance in trypanosomatids, including the genera Trypanosoma and Leishmania that are parasites responsible for several serious diseases. Analogs of clomipramine were prepared since clomipramine is reported to inhibit TR and cure mice infected with trypanosomes, however its psychotropic activity precludes its use as an anti-trypanosomal therapeutic. The clomipramine analogs contained a tricyclic dibenzosuberyl moiety. Additionally a series of polyamines with N-dibenzosuberyl substituents were prepared. All compounds studied were competitive inhibitors of TR and showed trypanocidal activities against Trypanosoma brucei in vitro. The analogs of clomipramine were poor inhibitors of TR, whereas the polyamine derivatives were effective TR inhibitors with the most potent compound, N(4),N(8)-bis(dibenzosuberyl)spermine (7), having a Ki value of 0.26µM. However, compound (7) did not prolong the lives of mice infected with trypanosomes. Analysis of docking studies indicated: the tricyclic groups of inhibitors bind at four distinct hydrophobic regions in the active site of TR; the importance of the chlorine substituent of clomipramine in binding to TR; and binding of the dibenzosuberyl groups of (7) occur at separate and distinct hydrophobic regions within the active site of TR.

Study on the fluorescence quenching reaction of amitriptyline and clomipramine hydrochlorides with eosin Y and its analytical application.

Amitriptyline.HCl (AMI) and clomipramine.HCl (CMI) react with eosin Y (EY) in pH 3.8 NaAc-AcH buffer solution to form ion association complex which results in quenching of fluorescence of EY and appearance of a new resonance Rayleigh scattering (RSS) spectrum at 620 nm. The spectral characteristics of absorption, fluorescence and RSS spectra have been investigated. The factors influencing the reaction were studied and optimum conditions for the reaction have been determined. Based on fluorescence quenching, a simple and sensitive spectrofluorimetric method for determination of AMI and CMI has been developed. The fluorescence quenching intensity was measured at 550 nm using an excitation wavelength of 310 nm. The calibration graph was found to be rectilinear in the range 0.08-2.0 µg mL(-1) with detection limit of 0.017 µg mL(-1) for AMI and 0.06-2.0 µg mL(-1) with detection limit of 0.015 µg mL(-1) for CMI. The method can be satisfactorily applied to the determination of AMI and CMI in tablets without interference from commonly occurring exicipients. The recovery and RSD values obtained indicate good accuracy and precision of the method. The mechanism of the reaction and fluorescence quenching has also been discussed.

Recombinant laccase: II. Medical biosensor.

Langmuir-Blodgett (LB) technology was used to build a high-sensitivity enzyme-based biosensor for medical purposes. Recombinant fungal laccase from Rigidoporous lignosus, as previously described, was used to catalyze a widely used antidepressant in a micromolar range, namely, clomipramine. The topological properties of the laccase thin film were characterized via LB π-A isotherm and AFM (mean roughness 8.22 nm, compressibility coefficient 37.5 m/N). The sensitivity of the biosensor was investigated via UV spectroscopy, and linearity was found in the absorbance peak shift at 400 nm at drug concentration varying up to 20 uM. The enzyme kinetics was subsequently investigated with potentiometric and amperometric measurements, and we found electronic transfer of at least 1 electron, k(s) 0.57 s(-1), diffusion coefficient 3 × 10(-6) cm(2)/s, K(cat) 6825.92 min(-1), K(M) 4.1 uM, K(cat)/K(M) 2.8 × 10(7) mol(-1) s(-1), sensitivity of 440 nA/uM, maximum velocity 1706.48 nA/s, and response time less than 5 s. The amperometric and potentiometric measurements were repeated after a month, confirming the stability of the biosensor.

Reactive metabolites of desipramine and clomipramine: The kinetics of formation and reactivity with DNA.

Tricyclic antidepressants (TCAs), along with phenothyazines and some industrial chemicals, are shown to react with enzymes that exhibit peroxidase activity. These reactions result in the formation of reactive intermediates having unpaired electrons. The peroxidase oxidation and reactivity of two TCAs, desipramine and clomipramine, were investigated. As a model of peroxidase, horseradish peroxidase (HRP) was employed. The products of the peroxidase catalyzed oxidation of desipramine and clomipramine were identified as N-dealkylated compounds iminodibenzyl and 3-chloroiminodibenzyl using the GC/MS technique. Both drugs formed broad UV/vis absorption spectra in the presence of HRP and H(2)O(2), indicating the formation of a radical cations-reactive intermediate of the oxidation reaction. The dynamics of the formation of the desipramine intermediate was studied using UV/vis spectroscopy. The extinction coefficient was measured for the reactive intermediate, 7.80×10(3)M(-1)cm(-1), as well as the apparent Michaelis-Menten and catalytic constants, 4.4mM and 2.3s(-1), respectively. Both desipramine and clomipramine degraded DNA in the presence of HRP/H(2)O(2), as was revealed by agarose gel electrophoresis and PCI extraction. Manipulating the kinetic parameters of drug's radical formation and determining the extent of degradation to biomolecules could be potentially used for designing effective agents exhibiting specific reactivity.

EPR investigation of clomipramine interaction with phosphatidylcholine membranes in presence and absence of cholesterol.

The effects of tricylic antidepressant clomipramine (CLO) on the membrane properties of saturated dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine as well as on unsaturated egg yolk phosphatidylcholine liposomes were investigated by the electron paramagnetic resonance spin-labeling technique, in combination with the simulation of the spectra, taking into account that the membrane is heterogeneous and composed of the regions with different fluidity characteristics. Different spin labels, monitoring membrane properties in the upper and inner parts of the membrane, were used. In general, two spectral components, having different motional characteristics, were detected in all liposomes investigated. In liposomes with saturated chains, CLO decreased the phase-transition temperature, disordered the membrane, and increased polarity in the upper part of the membrane. However, less impact was observed in liposomes with unsaturated chains. In dipalmitoyl phosphatidylcholine liposomes, it also induced molecular rearrangements near the pretransition temperature. The presence of 30 mol% cholesterol increased the fluidizing effect of CLO and modified the lateral diffusion of nitroxide in the inner part of the membrane. A unique anomalous increase in diffusion of nitroxide, dependent on CLO concentration, was detected in the temperature region where the phosphatidylcholine membrane without cholesterol experiences the phase transitions. Since the changes in the central part of the membrane were even more pronounced than in the upper part of the membrane, it could be concluded that CLO incorporates into the membrane with its hydrophobic ring parallel to the phospholipid chains.

Identification of hub genes and discovery of promising compounds in gastric cancer based on bioinformatics analysis.

Aim: To explore the mechanism of gastric carcinogenesis by mining potential hub genes and to search for promising small-molecular compounds for gastric cancer (GC). Materials & methods: The microarray datasets were downloaded from Gene Expression Omnibus database and the genes and compounds were analyzed by bioinformatics-related tools and software. Results: Six hub genes (MKI67, PLK1, COL1A1, TPX2, COL1A2 and SPP1) related to the prognosis of GC were confirmed to be upregulated in GC and their high expression was correlated with poor overall survival rate in GC patients. In addition, eight candidate compounds with potential anti-GC activity were identified, among which resveratrol was closely correlated with six hub genes. Conclusion: Six hub genes identified in the present study may contribute to a more comprehensive understanding of the mechanism of gastric carcinogenesis and the predicted potential of resveratrol may provide valuable clues for the future development of targeted anti-GC inhibitors.

Protective effects of 10,11-dihydro-5H-dibenzo[b,f]azepine hydroxamates on vascular cognitive impairment.

A series of 10,11-dihydro-5H-dibenzo [b,f]azepine hydroxamates (4-15) were synthesized, behaving as histone deacetylase inhibitors, and examined for their influence on vascular cognitive impairment (VCI), which correlated with dementia. The results revealed that (E)-3-(4-(((3-(3-chloro-10,11-dihydro-5H-dibenzo [b,f]azepin-5-yl)propyl)amino)methyl)phenyl)-N-hydroxy-acrylamide (13) increases cerebral blood flow (CBF), attenuates cognitive impairment, and improves hippocampal atrophy in in vivo study. It is also able to increase the level of histone acetylation (H3K14 or H4K5) in the cortex and hippocampus of chronic cerebral hypoperfusion (CCH) mice; as a result, it could be a potential HDAC inhibitor for the treatment of vascular cognitive impairment.

Photophysics and photochemistry of imipramine, desimipramine, and clomipramine in several solvents: a fluorescence, 266 nm laser flash, and theoretical study.

Imipramine (IPA) and its derivatives are used widely for the treatment of depression and other mental disorders. Although there are more than 20 FDA-approved antidepressant drugs, the search continues for better compounds with fewer deleterious side effects and higher efficacy. Over the past decade, several classes of antipsychotic drugs have been developed, which-in spite of their structural diversity-share an ability to modulate neurotransmission and to produce undesirable side effects. Phototoxicity is one of the most important side effects noted in treatment with tricyclic antidepressants (TCAs), but its mechanism has not yet been elucidated. To develop new knowledge regarding the relationship between the structure and the photophysics of these TCAs, we measured the photophysical properties of IPA, desimipramine (DIPA), and clomipramine (CIPA) in different solvents. The electronic configurations for the ground and the first excited singlet states were calculated using the AM1/RHF/CI and the AM1/RHF/HE semiempirical quantum theoretical methods, respectively. The ground-state properties are solvent-independent, while the emission maxima are red-shifted with increasing solvent polarity/polarizability. However, the fluorescence quantum yield is relatively low in all of the tested solvents (phif<0.02). The primary transient intermediates produced by 266 nm high-intensity laser photolysis are the solvated electron and the corresponding radical cation, with a negligible contribution of triplet-triplet absorption. The properties determined for the primary transients generated with a 266 nm laser flash are consistent with the photodamaging effects generated through a limited radical mechanism.

Conductometric study of antidepressant drug-cationic surfactant mixed micelles in aqueous solution.

Conductivity measurements have been carried out on aqueous solutions of two antidepressant drugs (nortriptyline hydrochloride and clomipramine hydrochloride) with four cationic surfactants (monomeric: cetyltrimethylammonium bromide, tetradecylammonium bromide; dimeric: 1,5-pentanediyl-alpha-omega-bis(hexadecyldimethylammonium bromide), 1,4-butanediyl-alpha,omega-bis(hexadecyldimethylammonium bromide) as well as with sodium chloride. Counterions from NaCl adsorb to the charged headgroup of the drug molecules and reduce the repulsion, hence cmc decreases. cmc values decreased with the addition of surfactants indicating mixed micelle formation. Experimental mole fraction of surfactants in micelle (X1) and their ideal values (X1 id) also support this explanation. Interaction parameter, beta, and excess free energy of micellization are negative suggesting synergism in mixed state. Activity coefficients are less than unity which means non-ideal mixing.

Modulating cellular autophagy for controlled antiretroviral drug release.

AIM: Pharmacologic agents that affect autophagy were tested for their abilities to enhance macrophage nanoformulated antiretroviral drug (ARV) depots and its slow release. METHODS: These agents included URMC-099, rapamycin, metformin, desmethylclomipramine, 2-hydroxy-ß-cyclodextrin (HBC) and clonidine. Each was administered with nanoformulated atazanavir (ATV) nanoparticles to human monocyte-derived macrophages. ARV retention, antiretroviral activity and nanocrystal autophagosomal formation were evaluated. RESULTS: URMC-099, HBC and clonidine retained ATV. HBC, URMC-099 and rapamycin improved intracellular ATV retention. URMC-099 proved superior among the group in affecting antiretroviral activities. CONCLUSION: Autophagy inducing agents, notably URMC-099, facilitate nanoformulated ARV depots and lead to sustained release and improved antiretroviral responses. As such, they may be considered for development as part of long acting antiretroviral treatment regimens.

Synthesis of some tropane derivatives of anticipated activity on the reuptake of norepinephrine and/or serotonin.

A variety of tropane derivatives 14a-g were prepared via the reaction of the alcohol analogs 12a and 12b with substituted fluorobenzenes 13a-f. The prepared compounds were tested for their activity and selectivity toward the norepinephrine transporter (NET) and serotonin transporter (SERT) using yohimbine-induced mortality and 5-hydroxytryptophan-induced neurotoxicity in mice, respectively. All the tested compounds were found to be NE and 5-HT reuptake inhibitors except 14d which exhibited selective 5-HT reuptake inhibition activity.

Ultrasound-assisted dispersive magnetic solid phase extraction based on amino-functionalized Fe3O4 adsorbent for recovery of clomipramine from human plasma and its determination by high performance liquid chromatography: Optimization by experimental design.

The applicability of Amino-functionalized Fe3O4 nanoparticles (NPs) as an effective adsorbent was developed for the extraction and determination of clomipramine (CLP) in plasma sample by ultrasound-assisted dispersive magnetic solid phase extraction (UADM-SPE) and high-performance liquid chromatography-ultraviolet (HPLC-UV) detection. Fabrication of the Fe3O4@SiO2-NH2 magnetic nanoparticles confirmed by Fourier transform infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The effect of different extraction parameters (i.e. pH of the sample solution, the amount of magnetic nanoparticles (MNPs), sample volume, temperature and sonication time) on the extraction recovery of CLP were investigated by response surface methodology through central composite design (CCD). The optimum condition is obtained when the affecting parameters are set to: pH of the sample solution=9, the amount of MNPs=37mg, sample volume=23mL, 25°C temperature and sonication time=1min. Under the optimum condition, extraction recovery was 90.6% with relative standard deviation of 3.5%, and enrichment factor of 117. The linear range for determination of CLP was 0.017-0.70mgL-1 with a determination coefficient (R2) of 0.999. Limit of detection (LOD) and limit of quantification (LOQ) were 0.005 and 0.0167mgL-1, respectively. The established UADM-SPE-HPLC-UV method was rapid, simple and efficient for determination of CLP in human plasma samples.

Comparative sorption of clomipramine, viloxazine, and maprotiline hydrochlorides in polyvinylchloride bags and glass vials.

The sorption of clomipramine, viloxazine, and maprotiline hydrochlorides was studied comparatively in polyvinylchloride (PVC) bags and glass flasks for up to 72 hours. When stored in glass flasks, no decrease in concentration was observed for any of the three drugs, either in dextrose or sodium chloride isotonic solution. When stored in PVC bags, a slight loss (about 7% within 72 hours), was noted for clomipramine hydrochloride in both isotonic solutions, but not for the other two drugs.

Synthesis and antidepressant activities of some 3,5-diphenyl-2-pyrazolines.

Ten new 3,5-diphenyl-2-pyrazoline derivatives were synthesised by reacting 1,3-diphenyl-2-propen-1-one with hydrazine hydrate. The chemical structures of the compounds were proved by means of their IR, 1H-NMR spectroscopic data and microanalyses. The antidepressant activities of these compounds were evaluated by the 'Porsolt Behavioural Despair Test' on Swiss-Webster mice. 3-(4-Methoxyphenyl)-5-(3,4-dimethoxyphenyl)-2-pyrazoline, 3-(4-methoxyphenyl)-5-(2-chloro-3,4-dimethoxyphenyl)-2-pyrazoline and 3-(4-chlorophenyl)-5-(2-chloro-3,4-dimethoxyphenyl)-2-pyrazoline reduced 41.94-48.62% immobility times at 100 mgkg(-1) dose level. In addition, it was found that 4-methoxy and 4-chloro substituents on the phenyl ring at position 3 of the pyrazoline ring increased the antidepressant activity; the replacement of these groups by bromo and methyl substituents decreased activity in mice.

Membrane permeation by multidrug-resistance-modulators and non-modulators: effects of hydrophobicity and electric charge.

This study was designed to test the hypothesis that lipophilic cationic drugs with only roughly similar structures mediate the reversal of multidrug-resistance (MDR) by interacting with membrane phospholipids. The permeation properties of MDR-modulators and non-modulators were studied by quantifying their ability to induce the leakage of Sulphan blue through the membrane of negatively charged unilamellar liposomes. Of the 22 compounds under investigation, only those bearing a net positive electric charge per molecule (z) > or = 0.2 induced dye leakage. All these efficient drugs are well-known MDR-modulators: calcium-channel blockers (propranolol, verapamil, diltiazem and dipyridamole), calmodulin antagonists (clomipramine and thioridazine) and antiparasitic agents (mepacrine, thioacridine derivatives and quinine). The non-modulators tested, including antineoplastic agents and steroids, did not induce any membrane permeation. The permeation process was a co-operative one (1.1 < Hill coefficient < 4.1) and the permeation doses inducing 50% dye leakage (PD50) were 1.9-11.2 mM. The permeation ability of the MDR-modulators (log(1/PD50)) increased significantly with octanol-buffer distributions per unit net electric charge ((logD)/z). The results provide evidence that a complex interplay occurs between the electric charge and the lipophilicity of the MDR-modulators when a dye leakage is induced through model membranes, and probably also when the MDR is reversed in leukaemic cells.

Polyelectrolyte/amphiphile interaction studied by surface tension measurements.

The interaction of kappa-carrageenan with three positively charged drug molecules with amphiphile character has been examined using surface tension measurements. The surface tension was measured by the pendant drop method which makes possible the determination at an apparent steady state which is important for polymeric systems. The results are compared with adsorption isotherms from dialysis equilibrium. The surface tension data, show that the presence of kappa-carrageenan in the amphiphile solutions leads to an increased and pronounced lowering of the surface tension in a low concentration range of amphiphile. It is also shown that not only the hydrophobicity of the amphiphile but also the structure of the polyelectrolyte (charge density and helix-coil structure) largely determine the extent of interaction.

Comparative study of the sorption of clomipramine and viloxazine hydrochlorides in Stedim 6 and PVC bags.

The stability of two antidepressant drugs, clomipramine and viloxazine hydrochlorides, was studied as was their possible sorption on Stedim 6, a new multilayer polyethylene-lined film, which was considered comparatively to polyvinyl chloride (PVC) and glass surfaces. Appropriate amounts of the drugs were added to 500 ml of 5% dextrose and 0.9% sodium chloride solutions in Stedim 6 and PVC bags, and in glass flasks, in order to obtain the concentrations currently used in clinical practice. All the containers were stored at room temperature in daylight for 72 hours. Samples were taken at various times and evaluated for remaining drug concentrations by UV spectrometry. The two drugs appeared stable under the given conditions. No concentration decrease was observed in glass flasks. Viloxazine hydrochloride showed an excellent compatibility with the PVC bags, but a slight concentration decrease (about 6-7% in 72 hours) was observed for clomipramine hydrochloride, depending on the contact duration. The compatibility of the two drugs with the new material Stedim 6 was found to be perfect. The behavioral differences observed between the two drugs with regard to PVC are explained in terms of differences of lipophilicity of the drugs. Those observed for a given drug with regard to the two materials are due to the crystalline structure of polyethylene and the amorphous one of PVC.

Isolation and identification of the photodegradation products of the photosensitizing antidepressant drug clomipramine. Phototoxicity studies on erythrocytes.

The isolation and identification of the photodegradation products of clomipramine (CIP) in phosphate buffered saline (PBS pH 7.4 and 6.0) solution and methanol under aerobic conditions were studied. Six compounds were identified and four of them were isolated and characterized by spectroscopic methods. A radical mechanism with the participation of the solvent is proposed for the photodegradation of CIP which undergoes homolytic cleavage of the carbon-chlorine bond and also photooxidation of the amine group. CIP was able to induce photohemolysis when it was irradiated in PBS pH 7.4 and in PBS pH 6.0 containing a suspension of human red blood cells (RBCs). The photohemolysis experiments in the presence of additives DABCO and GSH showed nearly total inhibition of drug-induced photohemolysis. The efficient inhibition of photohemolysis by the radical scavenger GSH compared with the inhibition show by DABCO suggests a moderate effect by singlet oxygen. Clomipramine-N-oxide was the unique photoproduct able to induce hemolysis and photohemolysis when it was incubated and irradiated with RBCs for 1 h. A mechanism involving singlet oxygen, radicals and photoproducts is suggested for the reported phototoxicity.

High throughput screening for inhibitors of the HECT ubiquitin E3 ligase ITCH identifies antidepressant drugs as regulators of autophagy.

Inhibition of distinct ubiquitin E3 ligases might represent a powerful therapeutic tool. ITCH is a HECT domain-containing E3 ligase that promotes the ubiquitylation and degradation of several proteins, including p73, p63, c-Jun, JunB, Notch and c-FLIP, thus affecting cell fate. Accordingly, ITCH depletion potentiates the effect of chemotherapeutic drugs, revealing ITCH as a potential pharmacological target in cancer therapy. Using high throughput screening of ITCH auto-ubiquitylation, we identified several putative ITCH inhibitors, one of which is clomipramine--a clinically useful antidepressant drug. Previously, we have shown that clomipramine inhibits autophagy by blocking autophagolysosomal fluxes and thus could potentiate chemotherapy in vitro. Here, we found that clomipramine specifically blocks ITCH auto-ubiquitylation, as well as p73 ubiquitylation. By screening structural homologs of clomipramine, we identified several ITCH inhibitors and putative molecular moieties that are essential for ITCH inhibition. Treating a panel of breast, prostate and bladder cancer cell lines with clomipramine, or its homologs, we found that they reduce cancer cell growth, and synergize with gemcitabine or mitomycin in killing cancer cells by blocking autophagy. We also discuss a potential mechanism of inhibition. Together, our study (i) demonstrates the feasibility of using high throughput screening to identify E3 ligase inhibitors and (ii) provides insight into how clomipramine and its structural homologs might interfere with ITCH and other HECT E3 ligase catalytic activity in (iii) potentiating chemotherapy by regulating autophagic fluxes. These results may have direct clinical applications.