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.

In Vitro and In Vivo Pipeline for Validation of Disease-Modifying Effects of Systems Biology-Derived Network Treatments for Traumatic Brain Injury-Lessons Learned.

We developed a pipeline for the discovery of transcriptomics-derived disease-modifying therapies and used it to validate treatments in vitro and in vivo that could be repurposed for TBI treatment. Desmethylclomipramine, ionomycin, sirolimus and trimipramine, identified by in silico LINCS analysis as candidate treatments modulating the TBI-induced transcriptomics networks, were tested in neuron-BV2 microglial co-cultures, using tumour necrosis factor α as a monitoring biomarker for neuroinflammation, nitrite for nitric oxide-mediated neurotoxicity and microtubule associated protein 2-based immunostaining for neuronal survival. Based on (a) therapeutic time window in silico, (b) blood-brain barrier penetration and water solubility, (c) anti-inflammatory and neuroprotective effects in vitro (p < 0.05) and (d) target engagement of Nrf2 target genes (p < 0.05), desmethylclomipramine was validated in a lateral fluid-percussion model of TBI in rats. Despite the favourable in silico and in vitro outcomes, in vivo assessment of clomipramine, which metabolizes to desmethylclomipramine, failed to demonstrate favourable effects on motor and memory tests. In fact, clomipramine treatment worsened the composite neuroscore (p < 0.05). Weight loss (p < 0.05) and prolonged upregulation of plasma cytokines (p < 0.05) may have contributed to the worsened somatomotor outcome. Our pipeline provides a rational stepwise procedure for evaluating favourable and unfavourable effects of systems-biology discovered compounds that modulate post-TBI transcriptomics.

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.

Serum clomipramine and desmethylclomipramine levels in a CYP2C19 and CYP2D6 intermediate metabolizer.

Pharmacogenetics within psychiatry has the potential to aid in the dose and selection of medications. A substantial number of psychiatric medications are metabolized through either of the highly polymorphic drug-metabolizing enzymes CYP2D6 and CYP2C19. Of these, clomipramine is subject to metabolism by both CYP2C19 and CYP2D6, leaving individuals with deficiencies of these drug-metabolizing enzymes at risk of higher concentrations of the parent molecule. Herein, we present the case of a 29-year-old male with diagnoses of depression and obsessive compulsive disorder who had trialed and failed a dozen psychiatric medications, many of which are subject to metabolism by CYP2D6 and/or CYP2C19, and had most recently been taking clomipramine for approximately 2.5 years. Pharmacogenetic testing revealed this patient to be an intermediate metabolizer for both CYP2C19 (*1/*2) and CYP2D6 (*4/*41), which resulted in considerably elevated serum trough concentrations of clomipramine and its active metabolite desmethylclomipramine. This case provides a retrospective view of how the knowledge of an individual's pharmacogenetic test results can aid in their clinical care.

Pharmacokinetics of clomipramine during pregnancy.

PURPOSE: Clomipramine is one of the drugs for depression during pregnancy; however, pharmacokinetic data of clomipramine and its active metabolite desmethylclomipramine in this vulnerable period are lacking. In this study, we describe clomipramine and desmethylclomipramine concentrations including their ratios during pregnancy. Second, we describe Center for Epidemiologic Studies Depression scale (CES-D) scores during pregnancy. METHODS: During 13 pregnancies, every trimester and 3 months after pregnancy, the clomipramine and desmethylclomipramine concentrations were measured with LC-MSMS and the severity of depression was assessed by taking the CES-D score. All concentrations used in our calculations were in fact the ratio between actual plasma concentration (µg/l) and the actual dose (mg). We compared differences in ratios between trimesters by using the Friedman test. RESULTS: Studying 12 women and 13 pregnancies, we found no changes in mean clomipramine concentrations, a statistically significant decrease in mean desmethylclomipramine concentrations (p = 0.014) and a significant decrease in the ratio of desmethylclomipramine/clomipramine mean concentrations during pregnancy (p = 0.014) compared to the post-partum period. Sub-therapeutic concentrations of clomipramine and desmethylclomipramine were found in three patients during whole pregnancy. CONCLUSIONS: The mean concentrations of the pharmacologically active metabolite of clomipramine and desmethylclomipramine changes during pregnancy, where a decrease in mean concentrations was found during pregnancy. In case of recurrent disease, we recommend to control clomipramine and its metabolite concentrations, while both are active.

Anti-tumoral effect of desmethylclomipramine in lung cancer stem cells.

Lung cancer is the most feared of all cancers because of its heterogeneity and resistance to available treatments. Cancer stem cells (CSCs) are the cell population responsible for lung cancer chemoresistance and are a very good model for testing new targeted therapies. Clomipramine is an FDA-approved antidepressant drug, able to inhibit in vitro the E3 ubiquitin ligase Itch and potentiate the pro-apoptotic effects of DNA damaging induced agents in several cancer cell lines. Here, we investigated the potential therapeutic effect of desmethylclomipramine (DCMI), the active metabolite of Clomipramine, on the CSCs homeostasis. We show that DCMI inhibits lung CSCs growth, decreases their stemness potential and increases the cytotoxic effect of conventional chemotherapeutic drugs. Being DCMI an inhibitor of the E3 ubiquitin ligase Itch, we also verified the effect of Itch deregulation on CSCs survival. We found that the siRNA-mediated depletion of Itch induces similar anti-proliferative effects on lung CSCs, suggesting that DCMI might exert its effect, at least in part, by inhibiting Itch. Notably, Itch expression is a negative prognostic factor in two primary lung tumors datasets, supporting the potential clinical relevance of Itch inhibition to circumvent drug resistance in the treatment of lung cancer.

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.

Clomipramine demethylation rate is important on the outcome of obsessive-compulsive disorder treatment.

The aim of this study was to investigate the influence of demethylation rate on the outcome of obsessive-compulsive disorder patients treated with clomipramine. Eighteen patients meeting the DSM-IV criteria for obsessive-compulsive disorder received 150-300 mg of clomipramine daily in a single-blind design for 12 weeks. The patients were evaluated with the Clinical Global Impression scale and the Yale-Brown Obsessive-Compulsive Scale (YBOCS). Clinical assessment and serum measurements of clomipramine and desmethylclomipramine were carried out at baseline and after 3, 6, 8, 10, and 12 weeks. A greater improvement in Clinical Global Impression scale rating was associated with a lower desmethylclomipramine/daily dose and the total clomipramine and desmethylclomipramine/daily dose. Moreover, an improved response on the YBOCS-obsession score was associated with higher serum levels of clomipramine and the total clomipramine and desmethylclomipramine/daily dose. Patients with a greater reduction in baseline YBOCS rating had a lower desmethylclomipramine/clomipramine ratio. These data suggest that a lower demethylation rate correlates with better clinical outcome.

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.

NET occupancy by clomipramine and its active metabolite, desmethylclomipramine, in non-human primates in vivo.

RATIONALE: Norepinephrine transporter (NET) is one of the key targets for antidepressants such as combined serotonin and norepinephrine reuptake inhibitors as well as some of the tricyclic antidepressants. Clomipramine, a tricyclic antidepressant, has been reported to have an active metabolite, desmethylclomipramine, which has high affinity for NET in vitro. However, the NET occupancy of clomipramine and desmethylclomipramine has not fully been evaluated in vivo. OBJECTIVES: In this positron emission tomography (PET) study, we investigate NET occupancy by clomipramine and desmethylclomipramine, respectively, in non-human primates with a selective radioligand for NET, (S,S)-[(18)F]FMeNER-D(2). METHODS: PET measurements were performed with (S,S)-[(18)F]FMeNER-D(2) at baseline and after the intravenous administration of clomipramine and desmethylclomipramine, respectively. NET binding was calculated with the simplified reference tissue model using the caudate as reference region. NET occupancy was calculated as the difference in NET binding between the baseline and pretreatment condition. The relationship between NET occupancy and dose/plasma concentration was evaluated using hyperbolic functions. RESULTS: NET occupancy by both clomipramine and desmethylclomipramine increased in a dose and plasma concentration-dependent manner. The mean Kd values, expressed as the dose or plasma concentration at which 50% of NET was occupied, were 0.44 mg/kg and 24.5 ng/ml for clomipramine and 0.11 mg/kg and 4.4 ng/ml for desmethylclomipramine. CONCLUSIONS: Not only desmethylclomipramine but also clomipramine was demonstrated to occupy NET in the non-human primate in vivo. It can thus be assumed that NET occupancy during clinical treatment with clomipramine is a combined effect of unchanged clomipramine and its main metabolite desmethylclomipramine.

Regional distribution of clomipramine and desmethylclomipramine in rat brain and peripheral organs on chronic clomipramine administration.

The tricyclic antidepressant (TCA) clomipramine has been widely used in psychiatry for over 40 years. More recently, its therapeutic potential as an antineoplastic drug has been identified. However, there are no prior data on regional distribution in the brain of clomipramine and its primary metabolite (desmethylclomipramine) after chronic oral administration. The aim of this study was to determine the concentrations of clomipramine and desmethylclomipramine in different rat-brain regions and to compare those with levels in plasma and peripheral organs after chronic oral treatment of Sprague Dawley rats (15 mg/kg) for 14 days. The levels of both parent TCA and metabolite were analysed by high-performance liquid chromatography in six brain regions (cortex, hypothalamus, hippocampus, striatum, brainstem and cerebellum), five peripheral organs and in plasma. Our data show that the cerebral cortex had the highest concentration of clomipramine (2.9 microg/mg), with successively lower concentrations in the hypothalamus, striatum, cerebellum, hippocampus and brainstem. Of the peripheral organs, the lungs and liver, had the highest levels of clomipramine, while in the heart, only the metabolite was detected. The plasma concentration (0.17 microg/ml or 0.48 microM) was comparable to that in the hippocampus and cerebellum (approximately 0.20 microg/mg). The differential distribution of clomipramine in different brain regions and the regional variation in clomipramine to desmethylclomipramine ratios have implications for the use of clomipramine in psychiatry and neuro-oncology.

Desmethylclomipramine induces the accumulation of autophagy markers by blocking autophagic flux.

Alterations in the autophagic pathway are associated with the onset and progression of various diseases. However, despite the therapeutic potential for pharmacological modulators of autophagic flux, few such compounds have been characterised. Here we show that clomipramine, an FDA-approved drug long used for the treatment of psychiatric disorders, and its active metabolite desmethylclomipramine (DCMI) interfere with autophagic flux. Treating cells with DCMI caused a significant and specific increase in autophagosomal markers and a concomitant blockage of the degradation of autophagic cargo. This observation might be relevant in therapy in which malignant cells exploit autophagy to survive stress conditions, rendering them more susceptible to the action of cytotoxic agents. In accordance, DCMI-mediated obstruction of autophagic flux increased the cytotoxic effect of chemotherapeutic agents. Collectively, our studies describe a new function of DCMI that can be exploited for the treatment of pathological conditions in which manipulation of autophagic flux is thought to be beneficial.

CE of tricyclic antidepressant clomipramine and metabolites: electromigration and wall adsorption.

CE of tricyclic antidepressants clomipramine and its metabolites demethylclomipramine, didemethylclomipramine and 8-hydroxyclomipramine resulted in partly extremely tailing peaks in bare fused-silica capillaries. Especially at high pH of the BGE this behavior was not unexpected as adsorption of the cationic analytes onto the negatively charged wall due to electrostatic attraction can be supposed. Less expected was the observation that peak tailing could not be overcome neither by using a capillary with dynamic coating with cationic CTAB added to the BGE, nor by the usage of a capillary permanently coated with polyvinyl alcohol (PVA), both operated at acidic pH. As this tailing was even more pronounced than with bare fused silica, and was suppressed upon addition of MeCN to the BGE, another source of adsorption than pure ion-ion interaction seems plausible. In the bare silica capillary the mobility, mu, of the analytes followed roughly the pH dependence of a monoacidic base, but two deviations from the sigmoid theoretical curve were evident: (i) even at low pH the mobilities were not constant; they decreased in contrary with pH over the entire range; (ii) the apparent pK(a) values of two analytes, derived at the pH with halve the mobility at low pH, are significantly smaller than the thermodynamic pK(a). Upon modifying the expression for mu = f(pH), and considering the pH dependence of the negative charge density at the wall by an additional term which takes chromatographic retention into account, an equation was derived which enables the description of the observed electromigration of the analytes as function of pH, pK(a) of analytes and surface silanol groups, actual mobility of analytes, distribution coefficient (or retention factor) due to adsorption including its pH dependence. The interplay of electrophoretic movement and residual adsorptive retention allowed to resolve the analytes finally in an uncoated capillary, namely at pH 7.65 (30 mM ionic strength), whereas at the cost of the robustness of the separation system.

Pharmacokinetics of clomipramine and desmethylclomipramine after single-dose intravenous and oral administrations in cats.

A cross-over study was performed in six adult spayed cats to determine the pharmacokinetics of clomipramine and its metabolite, desmethylclomipramine (DCMP) after intravenous (0.25 mg/kg) and oral (0.5 mg/kg) single-dose administrations. Plasma clomipramine and DCMP were measured by high-performance liquid chromatography at regular intervals for up to 30 h. Intravenous clomipramine best fit a two-compartmental model yielding an elimination rate constant of 0.037-0.09 h(-1) from which a mean half-life of 12.3 h was calculated. Mean clomipramine AUC(0--infinity) (ngxh/mL), clearance (L/hxkg), V(ss) (L/kg) and MRT (h) values were 652.5, 0.393, 5.0, and 13.5, respectively. Compartmental modeling for clomipramine, after oral administration, and DCMP after both administrations, produced wide parameter estimates and plots of residuals indicated poor goodness of fit. Noncompartmental analysis yielded mean AUC(0--30 h) (ngxh/mL), C(max) (ng/mL) and T(max) (h) of 948.3, 87.5 and 6.2 for clomipramine, and 613.8, 34.8, and 12.8 for DCMP respectively after oral administration. Clomipramine bioavailability was 90%. The present study showed marked pharmacokinetic variability for clomipramine and DCMP through biphasic absorption and potential genetic variability in clomipramine metabolism. It was concluded that population pharmacokinetics would allow better characterization of clomipramine variability that may explain the variability in clinical response noted in cats.

Placental passage of tricyclic antidepressants.

BACKGROUND: The use of antidepressants during pregnancy continues to garner considerable attention, though there are limited investigations that have sought to quantify fetal exposure. METHODS: Maternal and umbilical cord sera were collected at delivery from ten women taking nortriptyline and seven taking clomipramine. Placental passage was calculated as the ratio of umbilical cord to maternal serum concentration. Obstetrical outcome data were gathered from subjects at delivery. RESULTS: The placental passage ratio of nortriptyline and its active metabolite, cis-10-hydroxynortriptyline, were .68 +/- .40, 1.40 +/- 2.40, respectively. Clomipramine and desmethylclomipramine ratios were .60 +/- .50, .80 +/- .60. Obstetrical complications, such as pre-term delivery and pregnancy induced hypertension, were increased compared to the national average. CONCLUSIONS: The in vivo ratios of umbilical cord to maternal serum drug concentrations demonstrate considerable fetal exposure and differ greatly from previous results utilizing ex vivo perfusion.

Simultaneous analysis of clozapine, clomipramine and their metabolites by reversed-phase liquid chromatography.

1. The authors present here a sensitive and rapid reversed-phase liquid chromatographic method which enables the simultaneous analysis in plasma of two different drugs and their metabolites: the atypical neuroleptic clozapine and the tricyclic antidepressant clomipramine. 2. Samples and the internal standard (dibenzepine) were extracted through automated solid-phase procedure, evaporated dryness and injected into the chromatograph. Mobile phase was a mixture of water and acetonitrile (63:37, v:v) containing TEMED and triethylamine. The total chromatographic time was of 14 min and analyte peaks were detected by means of an ultraviolet spectrophotometer preset at 254 nm. 3. Results revealed an assay sensitivity of 5 microg/L for clozapine or norclozapine and of 10 microg/L for clomipramine and desmethylclomipramine. Recoveries for these drugs and their metabolites were more than 60% and their coefficient of variation (within day and day-to-day) ranged from 1.3 % to 2.5 %. In spiked plasma, within day and day-to-day coefficients of variability (CV) were less than 5%. The simultaneous evaluation of these two drugs with adequate sensitivity and precision makes it particularly useful for therapeutic drug monitoring during mono- or polypharmacotherapy.

Population pharmacokinetics of clomipramine, desmethylclomipramine, and hydroxylated metabolites in patients with depression receiving chronic treatment: model evaluation.

Because metabolites play a major role in the clinical response to clomipramine, the objective of the current study was to develop a population model and evaluate its performance to describe the pharmacokinetic profiles of clomipramine (C) and its active metabolites desmethylclomipramine (DC), 8-hydroxy-clomipramine (OHC) and 8-hydroxy-desmethylclomipramine (OHDC). A first sample of 14 patients served for development of a 2-molecule C and DC model, which was shown to provide reasonable estimates of AUC-based clearances, as well as precise estimation of interindividual variability. Simulated data, generated to mimic a semi-rich sampling design and chronic treatment with clomipramine, indicated that clearance estimation was feasible under routine treatment conditions. A second sample of 30 patients, recruited prospectively and followed for a median 4-week period, was used to extend the 2-molecule model to a 4-molecule model. Goodness-of-fit assessment revealed that model-predicted concentrations were reasonably close to observed concentrations for a majority of patients. Interindividual variability was 50% to 60% for hydroxylation and desmethylation clearances, and residual variability was 30%. The proposed model incorporates much of what is known about the metabolism of clomipramine and may valuably integrate the influence of genetic and environmental factors on each metabolic pathway.

Steady state concentrations of clomipramine and its major metabolite desmethylclomipramine in rat brain and serum after oral administration of clomipramine.

Twenty male Sprague-Dawley rats received five oral doses of clomipramine 20 mg/kg at 4-h intervals. The animals were decapitated 1, 2, 3, 5 and 12 h after the last dose for determination of clomipramine and desmethylclomipramine in serum and frontal cerebral cortex. Time dependent concentrations of clomipramine and desmethylclomipramine paralleled in serum and brain. Half-lives were similar in serum and brain with 7.8 versus 6.2 h and 5.5 versus 5.0 h for clomipramine and desmethylclomipramine, respectively. Absolute concentrations, however, were markedly higher in brain than in serum - 12.5 fold for clomipramine and 7.4 fold for desmethylclomipramine. The data indicate that serum and brain concentrations of clomipramine and its demethylated metabolite are rapidly exchanged between blood and brain. Assuming that blood and brain kinetics in man and rat are comparable, it is concluded that monitoring blood concentrations of clomipramine and desmethylclomipramine is a useful way to evaluate brain concentrations.

Simultaneous plasma level analysis of clomipramine, N-desmethylclomipramine, and fluvoxamine by reversed-phase liquid chromatography.

A simple reversed-phase liquid chromatographic method enabling the simultaneous analysis in plasma of the tricyclic antidepressant clomipramine, its demethylated metabolite, and the selective serotonin reuptake inhibitor fluvoxamine, was developed. The drugs and dibenzepine, the internal standard, were extracted from 1 mL plasma through an automated solid-phase procedure, eluted in a total chromatographic time of approximately 14 min and detected by means of an ultraviolet spectrophotometer preset at 254 nm. An assay sensitivity of 10 microg/L was observed for all analytes. Recoveries for these drugs and their metabolites ranged between 65% and 98% and their coefficient of variation (within-day and day-to-day) between 1.9% and 2.9%. In spiked plasma, within-day and day-to-day imprecision data were less than 5%. The simultaneous determination of clomipramine, N-desmethylclomipramine, and fluvoxamine with adequate sensitivity and accuracy may be useful for the monitoring of drug treatment in depression and obsessive-compulsive disorder, where combinations of such drugs are employed.