Gelatin microsphere coated Fe3O4@graphene quantum dots nanoparticles as a novel magnetic sorbent for ultrasound-assisted dispersive magnetic solid-phase extraction of tricyclic antidepressants in biological samples.

Gelatin microsphere-coated Fe3O4@graphene quantum dots (Fe3O4@GQD@GM) were designed and synthesized as a novel sorbent via ultrasonic-assisted dispersive magnetic solid-phase extraction (UA-DMSPE) method. The synthesized sorbent was identified and confirmed by FT-IR, XRD, VSM, and SEM techniques. UA-DMSPE was combined with corona discharge ion mobility spectrometry for trace determination of desipramine, sertraline, and citalopram. Effective parameters were considered and optimized. The proposed method, under optimal conditions, showed excellent linearity in different concentration ranges (2-700 ng mL-1, R2 > 0.995), repeatability (RSD < 5.1%), good sensitivity (LODs in the range 0.6-1.5 ng mL-1), high preconcentration factor (PF = 207-218), and acceptable relative recoveries (93.5-101.8%). Eventually, this method was used to determine tricyclic antidepressants in various biological samples. Schematic presentation of the microextraction and monitoring of TCAs by ultrasonic-assisted dispersive magnetic solid phase microextraction-ion mobility spectrometry producer.

Solubility-pH profile of desipramine hydrochloride in saline phosphate buffer: Enhanced solubility due to drug-buffer aggregates.

Although solubility-pH data for desipramine hydrochloride (DsHCl) have been reported previously, the aim of the present study was to critically examine the aqueous solubility-pH behavior of DsHCl in buffer-free and buffered solutions, in the presence of physiologically-relevant chloride concentration, using experimental practices recommended in the recently-published "white paper" (Avdeef et al., 2016). The computer program pDISOL-X was used to design the structured experiments (pH-RSF method), to process the data, and to refine the equilibrium constants. Low-to-high and high-to-low pH assays (using HCl, H3PO4, or NaOH to adjust pH) were performed on phosphate-buffered (0.12­0.15 M) saturated solutions of DsHCl in the pH 1.3-11.6 range. After equilibration (stirring 6 h, followed by 18 h stir-free sedimentation), filtration or centrifugation was used for phase separation. Concentration was measured using HPLC with UV/VIS detection. The 2:1 drug-phosphate solubility product (Ksp2:1 = [DsH+]2[HPO42-]) was determined from data in the pH 4-9 region. The free base of desipramine was prepared and used to determine the Ksp1:1 ([DsH+][H2PO4-]) in chloride-free acidified suspension. In addition, phosphate-free titrations were conducted to determine the intrinsic solubility, S0, and the 1:1 drug-chloride solubility product, KspDsHCl = [DsH+][Cl-]. Under the assay conditions, only the phosphate-free solutions showed some supersaturation near pHmax 8.0. In phosphate-containing solutions, pHmax was indicated at higher pH (8.8-9.6). Oils mixed with solids were observed to form in alkaline solutions (pH > 11). Notably, soluble drug-phosphate complexes appeared to form below pH 3.9 and above pHmax in saturated phosphate­containing saline solutions. This was indicated by the systematic pH shift to higher values in the log S-pH curve in alkaline solution than expected from the Henderson-Hasselbalch equation. For pH < 3.9, saturated phosphate-containing saline solutions exhibited elevated solubility, with drug-hydrochloride as the sole precipitate. Salt solubility products, intrinsic solubility, and complexation constants, which rationalized the data, were determined. Elemental, thermogravimetric (TGA), differential scanning calorimetric (DSC), and powder X-ray diffraction (PXRD) analyses were used to characterize the precipitates isolated from suspensions at different pH.

Calculating Kinetic Rates and Membrane Permeability from Biased Simulations.

We present a simple approach to calculate the kinetic properties of lipid membrane crossing processes from biased molecular dynamics simulations. We demonstrate that by using biased simulations, one can obtain highly accurate kinetic information with significantly reduced computational time with respect to unbiased simulations. We describe how to conveniently calculate the transition rates to enter, cross, and exit the membrane in terms of the mean first passage times. To obtain free energy barriers and relaxation times from biased simulations only, we constructed Markov models using the dynamic histogram analysis method (DHAM). The permeability coefficients that are calculated from the relaxation times are found to correlate highly with experimentally evaluated values. We show that more generally, certain calculated kinetic properties linked to the crossing of the membrane layer (e.g., barrier height and barrier crossing rates) are good indicators of ordering drugs by permeability. Extending the analysis to a 2D Markov model provides a physical description of the membrane crossing mechanism.

Super paramagnetic core-shells anchored onto silica grafted with C8/NH2 nano-particles for ultrasound-assisted magnetic solid phase extraction of imipramine and desipramine from plasma.

In current work the Fe3O4 magnetic nano-particles anchored to core-shells of SiO2 which grafted by C8/NH2 dual mixed groups, have been synthesized. The magnetic nano-particles were characterized by scanning electron microscopy, X-ray diffraction spectroscopy, and zeta-potential reader. The resulted nano-particles have spherical structure with diameters in the range of 105 to 110 nm. A magnetic solid phase extraction method was developed for extraction of imipramine and desipramine from human plasma samples under ultrasonic conditions by using of prepared NPs as sorbent. The MNPs were dispersion in plasma under sonicated conditions, accumulated by an external magnetic field and washed with Briton-Robinson buffer-acetonitrile solution (0.05 mol l-1, pH = 5, 10%V/V). The drugs were removed by methanol and quantified by gas chromatography. The calibration curves (correlation coefficient > 0.99) for IMP and DES were linear in the concentration range of 0.005 to 5 and 0.01 to 4 µg ml-1, respectively. The LOD, LOQ, intra and inter-day precision values were measured too. The proposed Fe3O4/SiO2/C8/NH2 MNPs could be applied for 3.0 times.

UV-photodegradation of desipramine: Impact of concentration, pH and temperature on formation of products including their biodegradability and toxicity.

Desipramine (DMI) is a widely used tricyclic antidepressant, and it is the major metabolite of imipramine (IMI) and lofepramine (LMI); IMI and LMI are two of the most commonly used tricyclic antidepressants. If DMI enters the aquatic environment, it can be transformed by the environmental bacteria or UV radiation. Therefore, photolysis of DMI in water was performed using a simulated sunlight Xenon-lamp and a UV-lamp. Subsequently, the biodegradability of DMI and its photo-transformation products (PTPs) formed during its UV photolysis was studied. The influence of variable conditions, such as initial DMI concentration, solution pH, and temperature, on DMI UV photolysis behavior was also studied. The degree of mineralization of DMI and its PTPs was monitored. A Shimadzu HPLC-UV apparatus was used to follow the kinetic profile of DMI during UV-irradiation; after that, ion-trap and high-resolution mass spectrometry coupled with chromatography were used to monitor and identify the possible PTPs. The environmentally relevant properties and selected toxicity properties of DMI and the non-biodegradable PTPs were predicted using different QSAR models. DMI underwent UV photolysis with first-order kinetics. Quantum yields were very low. DOC values indicated that DMI formed new PTPs and was not completely mineralized. Analysis by means of high-resolution mass spectrometry revealed that the photolysis of DMI followed three main photolysis pathways: isomerization, hydroxylation, and ring opening. The photolysis rate was inversely proportional to initial DMI concentration. The pH showed a significant impact on the photolysis rate of DMI, and on the PTPs in terms of both formation kinetics and mechanisms. Although temperature was expected to increase the photolysis rate, it showed a non-significant impact in this study. Results from biodegradation tests and QSAR analysis revealed that DMI and its PTPs are not readily biodegradable and that some PTPs may be human and/or eco-toxic, so they may pose a risk to the environment.

Photolysis of the antidepressants amisulpride and desipramine in wastewaters: Identification of transformation products formed and their fate.

Attenuation of pharmaceuticals due to natural sunlight is expected to be an important removal pathway in wastewater treatment plants using treatment lagoon systems. In this work, the photolysis of two antidepressants, amisulpride and desipramine, has been investigated in both ultrapure water and wastewater under simulated solar irradiation. Results showed that for amisulpride short irradiation times (t1/2 approximately 3h in pure water and 4h in wastewater) were adequate to degrade the parent compound while a longer exposure period was required for desipramine (t1/2 of approximately 36 h in pure water), although its degradation is enhanced almost three times by indirect photolysis in wastewaters. A significant number of transformation products (TPs) were identified for both pharmaceuticals by high-resolution mass spectrometry. In general, TPs formed are not persistent although acute toxicity tests for desipramine and its TPs showed an increase of the mixture toxicity after solar irradiation, suggesting that some TPs may be more toxic than the parent compound. In wastewaters collected from treatment lagoons, only amisulpride and one of its major TPs, TP 357, were detected. This indicates that long solar exposure times may be necessary for an effective elimination of these substances in lagoon systems or that photolysis may not be the main removal pathway for these particular compounds.

Antidepressant desipramine leads to C6 glioma cell autophagy: implication for the adjuvant therapy of cancer.

Depression is the most common psychiatric syndrome in cancer patients and adversely affects anti-cancer immune system and life quality of patients. Antidepressant desipramine (DMI) is clinically prescribed in the auxiliary treatment of cancer patients. Increasing evidences suggest that DMI has a broad spectrum of target-off biological effects, such as anticancer properties. Our previous study revealed that DMI at the clinical relevant concentrations could induce CHOP-dependent apoptotic death in C6 glioma cells. In this study, we further explored the pro-autophagic effect of DMI in C6 glioma cells and its underlying mechanism. Treatment with DMI could induce autophagic cell death characterized by the formation of autophagosome and the elevated level of autophagic protein Beclin-1 and cellular redistribution of marker LC3. Meanwhile, DMI inhibited the activation of PI3K-AKT-mTOR pathway which is considered as a negative regulator of autophagy. Furthermore, DMI activated PERK-eIF2α and ATF6 of endoplasmic reticulum (ER) stress pathway, while knockdown of PERK with the PERK-specific short interfering RNA (siRNA) could obviously attenuate the autophagy. The results strongly suggested that DMI could induce autophagy through the PERK-ER stress pathway in C6 glioma cells. Our findings provided new insights into another beneficial potential of antidepressant DMI in the adjuvant therapy of cancer.

Desolvation of BSA-ligand complexes measured using the quartz crystal microbalance and dual polarization interferometer.

By taking advantage of their unique difference in hydration sensitivity, we have shown that dual polarization interferometer (DPI) and quartz-crystal microbalance with dissipation monitoring (QCM-D) measurements can be used together to explore the degree of desolvation involved in the binding of small drug molecules to an immobilized bovine serum albumin film in real time. Results with DPI and QCM-D show significantly different mass values for three ligands of varying hydrophobicities that may be attributed to changes in the degree of hydration of the ligand-protein complexes in accordance with the physicochemical properties of the ligands. Furthermore, our data suggest that masses measured by QCM-D can be overwhelmed by changes in water content of ligand-protein, binary complexes, which has important consequences for future studies using mechanical resonators to study protein-binding events.

Analysis of region-specific changes in gene expression upon treatment with citalopram and desipramine reveals temporal dynamics in response to antidepressant drugs at the transcriptome level.

RATIONALE: The notion that the onset of action of antidepressant drugs (ADs) takes weeks is widely accepted; however, the sequence of events necessary for therapeutic effects still remains obscure. OBJECTIVE: We aimed to evaluate a time-course of ADs-induced alterations in the expression of 95 selected genes in 4 regions of the rat brain: the prefrontal and cingulate cortices, the dentate gyrus of the hippocampus, and the amygdala. METHODS: We employed RT-PCR array to evaluate changes during a time-course (1, 3, 7, 14, and 21 days) of treatments with desipramine (DMI) and citalopram (CIT). In addition to repeated treatment, we also conducted acute treatment (a single dose of drug followed by the same time intervals as the repeated doses). RESULTS: Time-dependent and structure-specific changes in gene expression patterns allowed us to identify spatiotemporal differences in the molecular action of two ADs. Singular value decomposition analysis revealed differences in the global gene expression profiles between treatment types. The numbers of characteristic modes were generally smaller after CIT treatment than after DMI treatment. Analysis of the dynamics of gene expression revealed that the most significant changes concerned immediate early genes, whose expression was also visualized by in situ hybridization. Transcription factor binding site analysis revealed an over-representation of serum response factor binding sites in the promoters of genes that changed upon treatment with both ADs. CONCLUSIONS: The observed gene expression patterns were highly dynamic, with oscillations and peaks at various time points of treatment. Our study also revealed novel potential targets of antidepressant action, i.e., Dbp and Id1 genes.

Platinum anticancer agents and antidepressants: desipramine enhances platinum-based cytotoxicity in human colon cancer cells.

A unique synergistic effect on platinum drug cytotoxicity is noted in the presence of the tricyclic antidepressant desipramine. Desipramine is used for treating neuropathic pain, particularly in prostate cancer patients. The clinically used drugs cisplatin (cis-[PtCl(2)(NH(3))(2)]), oxaliplatin [1,2-diaminocyclohexaneoxalatoplatinum(II)], and the cationic trinuclear agent BBR3464 [{trans-PtCl(NH(3))(2)}(2)-µ-(trans-Pt(NH(3))(2)(H(2)N(CH(2))(6)NH(2))(2))](4+), which has undergone evaluation in phase II clinical trials for activity in lung and ovarian cancers, were evaluated. Surprisingly, desipramine greatly augments the cytotoxicity of all the platinum-based chemotherapeutics in HCT116 colorectal carcinoma cell lines. Desipramine enhanced cellular accumulation of cisplatin, but had no effect on the accumulation of oxaliplatin or BBR3464, suggesting that enhanced accumulation could not be a consistent means by which desipramine altered the platinum-drug-mediated cytotoxicity. The desipramine/cisplatin combination resulted in increased levels of p53 as well as mitochondrial damage, caspase activation, and poly(ADP ribose) polymerase cleavage, suggesting that desipramine may synergize with cisplatin more than with other platinum chemotherapeutics partly by activating distinct apoptotic pathways. The study argues that desipramine may be a means of enhancing chemoresponsiveness of platinum drugs and the results warrant further investigation. The results emphasize the importance of understanding the differential pharmacological action of adjuvants employed in combinations with cancer chemotherapeutics.

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.

Evaluation of the potential of surface enhancement Raman spectroscopy for detection of tricyclic psychotropic drugs. Case studies on imipramine and its metabolite.

The potential use of surface Raman enhanced spectroscopy (SERS) for confirmatory identification and the semi-quantitative analysis of selected tricyclic antidepressants (TCAs) is examined utilizing a conventional silver colloid. Raman and SERS spectra of aqueous solutions of imipramine (Imi) and its metabolite, desipramine (Des), were recorded as the function of concentration using NIR excitation. A good linear correlation is observed for the dependence of the SERS signal at 684 cm(-1) (R(2) = 0.9997) on Imi concentration over the range of 0.75-7.5 µM. The limit of detection of imipramine in the silver colloidal solution is 0.98 µM. SERS spectra of Imi and Des were also recorded for blood plasma samples without prior purification as well as after the use of standard solid phase extraction. All spectra show the characteristic spectral profile of the molecules and moreover, stronger signal enhancement is observed for Imi in the "raw" samples as opposed to Imi extracted from a biological matrix.

Functional Inhibitors of Acid Sphingomyelinase (FIASMAs): a novel pharmacological group of drugs with broad clinical applications.

Acid sphingomyelinase (ASM) is an important lipid-metabolizing enzyme cleaving sphingomyelin to ceramide, mainly within lysosomes. Acid ceramidase (AC) further degrades ceramide to sphingosine which can then be phosphorylated to sphingosine-1-phosphate. Ceramide and its metabolite sphingosine-1-phosphate have been shown to antagonistically regulate apoptosis, cellular differentiation, proliferation and cell migration. Inhibitors of ASM or AC therefore hold promise for a number of new clinical therapies, e.g. for Alzheimer's disease and major depression on the one hand and cancer on the other. Inhibitors of ASM have been known for a long time. Cationic amphiphilic substances induce the detachment of ASM protein from inner lysosomal membranes with its consecutive inactivation, thereby working as functional inhibitors of ASM. We recently experimentally identified a large number of hitherto unknown functional inhibitors of ASM and determined specific physicochemical properties of such cationic amphiphilic substances that functionally inhibit ASM. We propose the acronym "FIASMA" (Functional Inhibitor of Acid SphingoMyelinAse) for members of this large group of compounds with a broad range of new clinical indications. FIASMAs differ markedly with respect to molecular structure and current clinical indication. Most of the available FIASMAs are licensed for medical use in humans, are minimally toxic and may therefore be applied for disease states associated with increased activity of ASM.

Discovery of a potent, selective, and less flexible selective norepinephrine reuptake inhibitor (sNRI).

The design, synthesis, and SAR of a series of ring-constrained norepinephrine reuptake inhibitors are described. A substantially rigid inhibitor with potent functional activity at the transporter (IC(50)=8 nM) was used to develop a model for the distance and orientation of key features necessary for interaction with the norepinephrine transporter (NET).

Determination of fluoxetine in plasma by gas chromatography-mass spectrometry using stir bar sorptive extraction.

This article presents a method employing stir bar sorptive extraction (SBSE) with in situ derivatization, in combination with either thermal or liquid desorption on-line coupled to gas chromatography-mass spectrometry for the analysis of fluoxetine in plasma samples. Ethyl chloroformate was employed as derivatizing agent producing symmetrical peaks. Parameters such as solvent polarity, time for analyte desorption, and extraction time, were evaluated. During the validation process, the developed method presented specificity, linearity (R(2)>0.99), precision (R.S.D.<15%), and limits of quantification (LOQ) of 30 and 1.37 pg mL(-1), when liquid and thermal desorption were employed, respectively. This simple and highly sensitive method showed to be adequate for the measurement of fluoxetine in typical and trace concentration levels.

Fast urinary screening for imipramine and desipramine using on-line solid-phase extraction and selective derivatization.

A continuous-flow configuration based on sequential solid-phase extraction and derivatization is proposed for the screening of urine samples for imipramine and related metabolites. For the first time, a 50/50 (v/v) methanol/nitric acid mixture is used as both eluent and derivatizing reagent. Sample aliquots are injected into the flow manifold and driven by a water stream to an RP-C(18) column where the drugs are quantitatively retained. Following clean-up step with 40/60 (v/v) methanol/water, the eluent/derivatizing reagent is injected and passed through the sorbent column, eluted drugs reacting with nitric acid to form a blue dye that is monitored at 600 nm. The global signal thus obtained for the antidepressants can be used to estimate their total concentration in the samples without the need to individually quantify the analytes. This total index can be used for timely decision-making in case of overdosage. The proposed method is sensitive and selective; thus, typical interferents such as endogenous and diet compounds have no substantial effect on the analytical signal. This allows imipramine and its metabolites to be determined at therapeutic levels in urine samples.

LeuT-desipramine structure reveals how antidepressants block neurotransmitter reuptake.

Tricyclic antidepressants exert their pharmacological effect-inhibiting the reuptake of serotonin, norepinephrine, and dopamine-by directly blocking neurotransmitter transporters (SERT, NET, and DAT, respectively) in the presynaptic membrane. The drug-binding site and the mechanism of this inhibition are poorly understood. We determined the crystal structure at 2.9 angstroms of the bacterial leucine transporter (LeuT), a homolog of SERT, NET, and DAT, in complex with leucine and the antidepressant desipramine. Desipramine binds at the inner end of the extracellular cavity of the transporter and is held in place by a hairpin loop and by a salt bridge. This binding site is separated from the leucine-binding site by the extracellular gate of the transporter. By directly locking the gate, desipramine prevents conformational changes and blocks substrate transport. Mutagenesis experiments on human SERT and DAT indicate that both the desipramine-binding site and its inhibition mechanism are probably conserved in the human neurotransmitter transporters.

High-resolution 2D NMR spectroscopy of bicelles to measure the membrane interaction of ligands.

Magnetically aligned bicelles are increasingly being used as model membranes in solution- and solid-state NMR studies of the structure, dynamics, topology, and interaction of membrane-associated peptides and proteins. These studies commonly utilize the PISEMA pulse sequence to measure dipolar coupling and chemical shift, the two key parameters used in subsequent structural analysis. In the present study, we demonstrate that the PISEMA and other rotating-frame pulse sequences are not suitable for the measurement of long-range heteronuclear dipolar couplings, and that they provide inaccurate values when multiple protons are coupled to a 13C nucleus. Furthermore, we demonstrate that a laboratory-frame separated-local-field experiment is capable of overcoming these difficulties in magnetically aligned bicelles. An extension of this approach to accurately measure 13C-31P and 1H-31P couplings from phospholipids, which are useful to understand the interaction of molecules with the membrane, is also described. In these 2D experiments, natural abundance 13C was observed from bicelles containing DMPC and DHPC lipid molecules. As a first application, these solid-state NMR approaches were utilized to probe the membrane interaction of an antidepressant molecule, desipramine, and its location in the membrane.

Linking tricyclic antidepressants to ionotropic glutamate receptors.

Although tricyclic antidepressants have been in existence since the 1940s when they were discovered upon screening iminodibenzyl derivatives for other potential therapeutic uses, their mechanism of action has remained unclear [A. Goodman Gilman, T.W. Rall, A.S. Nies, P. Taylor, Goodman and Gilman's The Pharmacological Basis of Therapeutics, eighth ed., Pergamon Press, New York, 1990]. In addition to their ability to hinder the reuptake of biogenic amines, there is mounting evidence that the tricyclic antidepressants inhibit glutamate transmission. Here, intrinsic tryptophan fluorescence spectroscopy is used to document the binding of desipramine, a member of the tricyclic antidepressant family, to a well-defined extracellular glutamate binding domain (S1S2) of the GluR2 subunit of the amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor. The binding is distinct from those of other known effectors of the receptor, including the endogenous sulfated neurosteroids pregnenolone sulfate and 3alpha-hydroxy-5beta-pregnan-20-one sulfate, and is consistent with a conformational change upon binding that is allosterically transmitted to the channel region of the receptor.

Effects of the molecular structure of two amphiphilic antidepressant drugs on the formation of complexes with human serum albumin.

Interactions of two amphiphilic antidepressant drugs, imipramine and desipramine hydrochlorides, with the blood protein human serum albumin (HSA) were investigated to gain an understanding of the effects of drug molecular structure on the complex formation of drug-protein molecules. To elucidate the mechanisms of such effects, the protein-antidepressant interactions in aqueous buffered solutions of pH 3.0 and 5.5 (isoelectric point of HSA = 4.9) were investigated using conductivity, zeta potential, and dynamic light scattering. An increase of the critical micelle concentration of both antidepressants was detected as a consequence of extensive binding to the protein. From zeta-potential measurements, the Gibbs energies of adsorption of the drugs onto the protein were derived using the proposed models of Kayes and Ottewill and Watanabe. Measurements of the hydrodynamic radii of HSA-antidepressant complexes as a function of the drug concentration have shown a gradual increase of size of a saturation rather than a denaturation process of the protein. A larger drug adsorption at pH 5.5 than at pH 3.0 was also observed, as a consequence of a more important specific binding at the former pH.