Mass Spectrometry Imaging Combined with Sparse Autoencoder Method Reveals Altered Phosphorylcholine Distribution in Imipramine Treated Wild-Type Mice Brains.

Mass spectrometry imaging (MSI) is essential for visualizing drug distribution, metabolites, and significant biomolecules in pharmacokinetic studies. This study mainly focuses on imipramine, a tricyclic antidepressant that affects endogenous metabolite concentrations. The aim was to use atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI)-MSI combined with different dimensionality reduction methods to examine the distribution and impact of imipramine on endogenous metabolites in the brains of treated wild-type mice. Brain sections from both control and imipramine-treated mice underwent AP-MALDI-MSI. Dimensionality reduction methods, including principal component analysis, multivariate curve resolution, and sparse autoencoder (SAE), were employed to extract valuable information from the MSI data. Only the SAE method identified phosphorylcholine (ChoP) as a potential marker distinguishing between the control and treated mice brains. Additionally, a significant decrease in ChoP accumulation was observed in the cerebellum, hypothalamus, thalamus, midbrain, caudate putamen, and striatum ventral regions of the treated mice brains. The application of dimensionality reduction methods, particularly the SAE method, to the AP-MALDI-MSI data is a novel approach for peak selection in AP-MALDI-MSI data analysis. This study revealed a significant decrease in ChoP in imipramine-treated mice brains.

A Radioactivity-mass Spectrometry Calibration Method Coupled with Biosynthesis to Generate a Metabolite Standard for Enzyme Kinetics Studies.

In early stages of drug development, the absence of authentic metabolite standards often results in semi-quantitative measurements of metabolite formation in reaction phenotyping studies using mass spectrometry (MS), leading to inaccuracies in the determination of enzyme kinetic parameters, such as the Michaelis constant (Km). Moreover, it is impossible to ascertain the maximum rate of enzyme-catalyzed reactions (kcat or Vmax). The use of radiolabeled parent compounds can circumvent this problem. However, radiometric detection exhibits significantly lower sensitivity compared to MS. To address these challenges, we have developed a stepwise approach that leverages biosynthesized radiolabeled and non-radiolabeled metabolites as standards, enabling accurate determination of Km, kcat or Vmax without the need for authentic metabolite standards. This approach, using the carbon-14 [14C] labeled metabolite to calibrate the unlabeled metabolite (14C calibration method), combines radiometric with LC-MS/MS detection to generate both [14C]-labeled and unlabeled metabolite standard curves to ensure that the sample concentrations measured are accurately quantitated. Two case studies were presented to demonstrate the utility of this method. We first compared the accuracy of the 14C calibration method to the use of authentic standards for quantitating imipramine metabolites. Next, we biosynthesized and quantitated the metabolites of BI 894416 using 14C calibration method and evaluated the enzyme kinetics of metabolite formation. The Km values of the metabolite formation demonstrated substantially improved accuracy compared to MS semi-quantitation. Moreover, the 14C calibration method offers a streamlined approach to prepare multiple metabolite standards from a single biosynthesis, reducing the time required for structure elucidation and metabolite synthesis.

Elucidating Gender-Specific Distribution of Imipramine, Chloroquine, and Their Metabolites in Mice Kidney Tissues through AP-MALDI-MSI.

Knowledge of gender-specific drug distributions in different organs are of great importance for personalized medicine and reducing toxicity. However, such drug distributions have not been well studied. In this study, we investigated potential differences in the distribution of imipramine and chloroquine, as well as their metabolites, between male and female kidneys. Kidneys were collected from mice treated with imipramine or chloroquine and then subjected to atmospheric pressure matrix-assisted laser desorption ionization-mass spectrometry imaging (AP-MALDI-MSI). We observed differential distributions of the drugs and their metabolites between male and female kidneys. Imipramine showed prominent distributions in the cortex and medulla in male and female kidneys, respectively. Desipramine, one of the metabolites of imipramine, showed significantly higher (*** p < 0.001) distributions in the medulla of the male kidney compared to that of the female kidney. Chloroquine and its metabolites were accumulated in the pelvis of both male and female kidneys. Interestingly, they showed a characteristic distribution in the medulla of the female kidney, while almost no distributions were observed in the same areas of the male kidney. For the first time, our study revealed that the distributions of imipramine, chloroquine, and their metabolites were different in male and female kidneys.

Methanolic Extract of Aerva javanica Leaves Prevents LPS-Induced Depressive Like Behavior in Experimental Mice.

Aim: Depression is a chronic recurrent neuropsychiatric disorder associated with inflammation. This study explored the pharmacological activities of Aerva javanica leaves crude extract (Aj.Cr) on lipopolysaccharide (LPS)-induced depressive-like behavior in experimental mice. Methods: Aj.Cr was evaluated for its phenolic and flavonoid contents, bioactive potential, amino acid profiling and enzyme inhibition assays using different analytical techniques followed by in-silico molecular docking was performed. In addition, three ligands identified in HPLC analysis and standard galantamine were docked to acetyl cholinesterase (AchE) enzyme to assess the ligand interaction along with their binding affinities. In in-vivo analysis, mice were given normal saline (10 mL/kg), imipramine (10 mg/kg) and Aj.Cr (100, 300, and 500 mg/kg) orally for 14-consecutive days. On the 14th day, respective treatment was given 30-minutes before intra-peritoneal administration of (0.83 mg/kg) LPS. Open field, forced swim and tail suspension tests were performed 24-hours after LPS injection, followed by a sucrose preference test 48-hours later. Serum corticosterone levels, as well as levels of nitric oxide (NO), malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), tumor necrosis factor-alpha (TNF-), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), brain-derived neurotrophic factor (BDNF) and catecholamines were determined in brain tissues. Results: In-vitro results revealed that crude extract of Aj.Cr possesses anti-depressant agents with solid antioxidant potential. In-vivo analysis showed that LPS significantly increased depressive-like behavior followed by alteration in serum and tissue biomarkers as compared to normal control (p < 0.001). While imipramine and Aj.Cr (100, 300, and 500 mg/kg) treated groups significantly (p<0.05) improved the depressive-like behavior and biomarkers when compared to the LPS group. Conclusion: The mitigation of LPS-induced depressive-like behavior by Aj.Cr may be linked to the modulation of oxidative stress, neuro-inflammation and catecholamines due to the presence of potent bioactive compounds exerting anti-depressant effects.

Cancer cell autophagy, reprogrammed macrophages, and remodeled vasculature in glioblastoma triggers tumor immunity.

Glioblastoma (GBM) is poorly responsive to therapy and invariably lethal. One conceivable strategy to circumvent this intractability is to co-target distinctive mechanistic components of the disease, aiming to concomitantly disrupt multiple capabilities required for tumor progression and therapeutic resistance. We assessed this concept by combining vascular endothelial growth factor (VEGF) pathway inhibitors that remodel the tumor vasculature with the tricyclic antidepressant imipramine, which enhances autophagy in GBM cancer cells and unexpectedly reprograms immunosuppressive tumor-associated macrophages via inhibition of histamine receptor signaling to become immunostimulatory. While neither drug is efficacious as monotherapy, the combination of imipramine with VEGF pathway inhibitors orchestrates the infiltration and activation of CD8 and CD4 T cells, producing significant therapeutic benefit in several GBM mouse models. Inclusion up front of immune-checkpoint blockade with anti-programmed death-ligand 1 (PD-L1) in eventually relapsing tumors markedly extends survival benefit. The results illustrate the potential of mechanism-guided therapeutic co-targeting of disparate biological vulnerabilities in the tumor microenvironment.

Differences in Cerebral Distribution between Imipramine and Paroxetine via Membrane Transporters at the Rat Blood-Brain Barrier.

PURPOSE: The present study aimed to elucidate the transport properties of imipramine and paroxetine, which are the antidepressants, across the blood-brain barrier (BBB) in rats. METHODS: In vivo influx and efflux transport of imipramine and paroxetine across the BBB were tested using integration plot analysis and a combination of brain efflux index and brain slice uptake studies, respectively. Conditionally immortalized rat brain capillary endothelial cells, TR-BBB13 cells, were utilized to characterize imipramine and paroxetine transport at the BBB in vitro. RESULTS: The in vivo influx clearance of [3H]imipramine and [3H]paroxetine in rats was determined to be 0.322 mL/(min·g brain) and 0.313 mL/(min·g brain), respectively. The efflux clearance of [3H]imipramine and [3H]paroxetine was 0.380 mL/(min·g brain) and 0.126 mL/(min·g brain), respectively. These results suggest that the net flux of paroxetine, but not imipramine, at the BBB in vivo was dominated by transport to the brain from the circulating blood. The uptake of imipramine and paroxetine by TR-BBB13 cells exhibited time- and temperature-dependence and one-saturable kinetics with a Km of 37.6 µM and 89.2 µM, respectively. In vitro uptake analyses of extracellular ion dependency and the effect of substrates/inhibitors for organic cation transporters and transport systems revealed minor contributions to known transporters and transport systems and the difference in transport properties in the BBB between imipramine and paroxetine. CONCLUSIONS: Our study showed the comprehensive outcomes of imipramine and paroxetine transport at the BBB, implying that molecular mechanism(s) distinct from previously reported transporters and transport systems are involved in the transport.

How do psychostimulants enter the human brain? Analysis of the role of the proton-organic cation antiporter.

BACKGROUND: Although psychostimulants apparently do cross the BBB, it is poorly understood how these hydrophilic and positively charged molecules can pass the blood-brain barrier (BBB). That may be mediated by a genetically still uncharacterized H+/OC antiporter with high activity at the BBB. METHODS: We studied the uptake of 16 psychostimulants and hallucinogens with hCMEC/D3 cells using the prototypic inhibitor imipramine (cis-inhibition), exchange transport with diphenhydramine and clonidine (trans-stimulation), proton dependency of the uptake, and we characterized the concentration-dependent uptake. RESULTS: Cell uptake of methylenedioxyamphetamines, amphetamines and dimethyltryptamine (DMT) were strongly inhibited (to about 10% of the controls) by imipramine and diphenhydramine, whereas uptake of cathine was only weakly inhibited and mescaline not significantly. Amphetamine, methylamphetamine, para-Methoxy-N-methylamphetamine (PMMA), Methylenedioxymethamphetamine (MDMA), phentermine and DMT exhibited the highest exchange after preloading with diphenhydramine with only 5.5%, 5.2%, 7.8%, 6%, 1.9%, 7.6% remaining in the cells. Less and no exchange were seen with cathine and mescaline, respectively. Dependence on intracellular pH was most pronounced with the methylendioxyamphetamines while uptake of cathine, DOI and cocaine were only moderately affected and mescaline not at all. CONCLUSION: Except for mescaline, all psychostimulants studied here were substrates of the H+/OC antiporter, implicating a strong need for a better characterization of this transport protein.

An approach to evaluate metabolite-related phototoxicity with combined use of photochemical properties and skin deposition.

Some chemicals have been reported to cause metabolite-related phototoxicity, and this study aimed to verify the applicability of photosafety assessment based on photochemical and pharmacokinetic properties to evaluate the metabolite-related phototoxicity risk. The phototoxic risk of imipramine (IMI) and its metabolite, desipramine (DMI), was evaluated by photochemical and pharmacokinetic analyses. IMI and DMI were found to have similar photoreactivities based on the generation of reactive oxygen species. The skin concentrations of IMI and DMI reached maximal levels at approximately 1 and 4 h, respectively, after oral administration of IMI (10 mg/kg), and DMI showed high skin deposition compared with IMI. According to the results, DMI was identified as a contributor to phototoxicity induced by orally-taken IMI. In in vivo phototoxicity testing, ultraviolet A irradiation from 3 to 6 h after oral administration of IMI (100 mg/kg) caused more potent phototoxic reactions compared with that from 0 to 3 h, and DMI yielded by metabolism of IMI would be associated with phototoxic reactions caused by orally-administered IMI. In addition to the data on IMI, a parent chemical, photochemical and pharmacokinetic profiling of its metabolite, DMI, led to reliable phototoxicity prediction of orally-administered IMI. Thus, characterization of the photosafety of metabolites would generate reliable information on the phototoxicity risk of parent chemicals, and the proposed strategy may facilitate comprehensive photosafety assessment of drug candidates in pharmaceutical development.

Indomethacin, a nonselective cyclooxygenase inhibitor, does not interact with MTEP in antidepressant-like activity, as opposed to imipramine in CD-1 mice.

The contribution of metabotropic glutamate receptors (mGlu receptors) in depression is well known and tested worldwide. Our previous study showed the involvement of the cyclooxygenase-2 (COX-2) pathway in behavioral changes mediated by an antagonist of metabotropic glutamate receptor subtype 5 (mGlu5 receptor) 3-[(2-methyl-1,3-tiazol-4-yl)ethynyl]-pyridine (MTEP). Among others, we have found that chronic concomitant administration of a COX-2 inhibitor and sub-effective dose of MTEP accelerates antidepressant-like activity of MTEP. This paper seeks to explore whether the same effect would be observed with the use of a non-selective COX inhibitor 2-[1-(4-chlorobenzoyl)-5-methoxy-2-methylindol-3-yl]acetic acid (indomethacin). To that end, we have employed experimental procedure implemented in the earlier research. MTEP and indomethacin or MTEP + indomethacin were used chronically for 7 or 14 days. Then, the Porsolt test, tail suspension test and locomotor activity test were performed. Imipramine was used as a reference compound, as its action is connected with mGlu5 receptor. We found that, in contrast to COX-2 inhibition, indomethacin - acting both through COX-1 and COX-2 - did not release antidepressant-like potential of MTEP. The opposite effect was shown when imipramine was used.

Selectivity of (±)-citalopram at nicotinic acetylcholine receptors and different inhibitory mechanisms between habenular α3ß4* and α9α10 subtypes.

The inhibitory activity of (±)-citalopram on human (h) α3ß4, α4ß2, and α7 nicotinic acetylcholine receptors (AChRs) was determined by Ca2+ influx assays, whereas its effect on rat α9α10 and mouse habenular α3ß4* AChRs by electrophysiological recordings. The Ca2+ influx results clearly establish that (±)-citalopram inhibits (IC50's in µM) hα3ß4 AChRs (5.1 ±â€¯1.3) with higher potency than that for hα7 (18.8 ±â€¯1.1) and hα4ß2 (19.1 ±â€¯4.2) AChRs. This is in agreement with the [3H]imipramine competition binding results indicating that (±)-citalopram binds to imipramine sites at desensitized hα3ß4 with >2-fold higher affinity than that for hα4ß2. The electrophysiological, molecular docking, and in silico mutation results indicate that (±)-citalopram competitively inhibits rα9α10 AChRs (7.5 ± 0.9) in a voltage-independent manner by interacting mainly with orthosteric sites, whereas it inhibits a homogeneous population of α3ß4* AChRs at MHb (VI) neurons (7.6 ± 1.0) in a voltage-dependent manner by interacting mainly with a luminal site located in the middle of the ion channel, overlapping the imipramine site, which suggests an ion channel blocking mechanism. In conclusion, (±)-citalopram inhibits α3ß4 and α9α10 AChRs with higher potency compared to other AChRs but by different mechanisms. (±)-Citalopram also inhibits habenular α3ß4*AChRs, supporting the notion that these receptors are important endogenous targets related to their anti-addictive activities.

Imipramine administered before the first of two forced swim sessions results in reduced immobility in the second session 24 h later.

A previous study investigating the effects of dopamine receptor antagonists administered before the first of two 24-h apart forced swim test (FST) sessions, provided evidence suggesting that evaluation of response efficacy - dependent on dopamine D2-like receptors - might play a role in setting the balance between active behaviours and immobility in this test. Regardless of the underlying mechanisms, the observation that the effects of drugs in the first session have consequences in the second session might be relevant for a better understanding of the FST in behavioural/functional terms. Thus, the first objective of this study was to investigate the consequences in the second session of the administration of the prototypic antidepressant drug imipramine before the first of two sessions. A second objective was to investigate the effect of dopamine D1-like and D2-like receptor blockade on the effects of imipramine. Imipramine (20 mg/kg) was administered 24-h, 6-h and 30-min before the first of two FST sessions performed 24-h apart. SCH 23390 (0.01, 0.04 mg/kg) or raclopride (0.0125, 0.25 mg/kg) were administered 30-min before the first session. Imipramine increased active behaviours both in the first and in the second session. Raclopride attenuated and SCH 23390 potentiated imipramine effects only in the first session and to a limited extent. These results show that imipramine administration before the first of two FST sessions induces an increase in active behaviours in the second session, and suggest that this effect is the consequence of the behavioural effects of imipramine in the first session.

Selective drug metabolite trace analysis by very high-volume injections and heartcut two-dimensional (2D)-ultrahigh performance liquid chromatography (UHPLC).

The application of two-dimensional liquid chromatography (2D-LC) is gradually growing also in the area of metabolite profiling and identification. The current contribution describes a heartcut 2D-UHPLC configuration that is applied in support of drug metabolism studies in development. The setup applies four LC columns: two analytical UHPLC columns to perform the first and second dimension separations, which are both preceded by a short HPLC column operated as trapping column. The first HPLC column allows a significant online preconcentration by large volume injection. The second short HPLC column is placed between the first and second dimension columns and enables the selection of orthogonal conditions in the second dimension independent of the first dimension making the heartcutting 2D approach more generic. The value of the setup was demonstrated with selective ultraviolet chromatograms obtained for the two major hydroxylated metabolites of atorvastatin separating them from a very high biological background, originating from an injection of 4 mL feces extract, by heartcut 2D-LC. In a second application, the main metabolite of imipramine was baseline separated from some minor metabolites that were co-eluting in the first dimension, allowing accurate and sensitive quantification. A quantification limit in the attogram/mL range was achieved thanks to the injection of 200 mL diluted urine, corresponding to 100 mL urine on column.

Behavioral and metabolic effects of S-adenosylmethionine and imipramine in the Flinders Sensitive Line rat model of depression.

Depression is associated with dysregulation of methyl group metabolism such as low S-adenosylmethionine (SAM). We previously reported that Flinders Sensitive Line (FSL) rats, an animal model of depression, had lower concentrations of liver SAM than the control rats, Flinders Resistant Line (FRL) rats. The present study investigated if SAM supplementation may correct liver SAM and behavioral abnormalities in this model. Moreover, we compared one-carbon (C1) metabolites, neurotransmitters, and gastrointestinal (GI) transit in SAM-treated versus imipramine (IMI)-treated animals. FSL rats received vehicle, IMI, SAM, or IMI + SAM (n = 9-10 per group) once daily through oral gavage for 4 weeks; FRL rats received vehicle. Behavior was assessed using standard tests for locomotion, cognition, and depressive-like behavior. Monoamine neurotransmitters and C1 metabolites were measured using UHPLC-ECD and UPLC-MS/MS, respectively. Compared to FRL rats, FSLs had lower liver SAM, higher plasma serotonin, lower hippocampal dopamine and serotonin turnover, and faster GI transit. Behaviorally, FSL rats showed impaired cognitive performance as well as increased depressive-like behavior compared to FRLs. Coadministration of IMI and SAM seemed to have adverse effects on spatial memory. SAM or IMI administration did not reverse C1 metabolites, neurotransmitters, or GI transit in FSLs. Despite low liver SAM in FSL rats, orally administered SAM did not show antidepressant effects in this specific animal model of depression.

Short-term exposure to tricyclic antidepressants delays righting time in marine and freshwater snails with evidence for low-dose stimulation of righting speed by imipramine.

Active pharmaceutical ingredients such as tricyclic antidepressants (TCAs) are contaminants of emerging concern which are commonly detected in wastewater effluent and which can disrupt the behavior of non-target organisms. In aquatic snails, the righting response is a critical behavior that has been shown to be inhibited by exposure to SSRI-type antidepressants. We exposed marine and freshwater snails to three tricyclic antidepressants (clomipramine, amitriptyline, and imipramine) for 1 h and measured righting response time. In the marine mud snail (Ilyanassa obsoleta), all three TCAs significantly increased righting time at concentrations as low as 156 µg/L. Similarly, in the freshwater snail Leptoxis carinata, all three TCAs increased righting time at concentrations as low as 263 µg/L. However, exposure to imipramine from 15.8 to 316 µg/L resulted in significantly faster righting time. Such low-dose stimulation and high-dose inhibition are characteristics of a hormetic response. We discuss the possible physiological mechanism of action of TCAs and other antidepressants on snail behavior, and the occurrence of non-monotonic, hormetic dose responses to human pharmaceuticals in the aquatic environment.

The evaluation of effect of selected metal ions on the efficiency of passive and active transport of imipramine. / Ocena wplywu wybranych jonów metali na efektywnosc transportu biernego i aktywnego imipraminy.

OBJECTIVES: The aim of the study was to evaluate the effect of zinc as well as magnesium or copper ions on the efficacy of passive transport of imipramine hydrochloride in in vitro model. According to results from passive transport, the next aim of the studies was to check the efficiency of active transport of imipramine hydrochloride in the presence or absence of zinc ions. METHODS: The passive transport study was conducted in specially designed capsules, while CaCo-2 cell lines were used in active transport evaluation. Zinc, magnesium and copper content was determined by F-AAS method. The analysis of imipramine hydrochloride was performed using HPLC method. RESULTS: Mean concentrations of zinc, magnesium, and copper ions obtained in this experiment were as follows: 2.98, 1.34 and 3.52 mg/L, respectively. The presence of zinc ions increased the efficiency of active transport of imipramine hydrochloride by 39%. Furthermore, the transport of zinc ions in the presence of imipramine hydrochloride was 27% greater than that of the zinc-containing solutions without imipramine hydrochloride. The extending of the time of experiment from 30 to 60 minutes resulted in an increase in transport efficiency of more than 10% in both cases. CONCLUSIONS: The efficiency of passive and active transport of imipramine hydrochloride is influenced by the presence of Mg, Zn and Cu ions. The passive transport of imipramine hydrochloride after 90 minutes of experiment was the most effective in the presence of copper and zinc ions. Further studies conducted on the CaCo-2 cell line indicated a clear positive interaction of imipramine - zinc.

Irinotecan Alters the Disposition of Morphine Via Inhibition of Organic Cation Transporter 1 (OCT1) and 2 (OCT2).

PURPOSE: The organic cation transporters (OCTs) and multidrug and toxin extrusions (MATEs) together are regarded as an organic cation transport system critical to the disposition and response of many organic cationic drugs. Patient response to the analgesic morphine, a characterized substrate for human OCT1, is highly variable. This study was aimed to examine whether there is any organic cation transporter-mediated drug and drug interaction (DDI) between morphine and commonly co-administrated drugs. METHODS: The uptake of morphine and its inhibition by six drugs which are commonly co-administered with morphine in the clinic were assessed in human embryonic kidney 293 (HEK293) cells stably expressing OCT1, OCT2 and MATE1. The in vivo interaction between morphine and the select irinotecan was determined by comparing the disposition of morphine in the absence versus presence of irinotecan treatment in mice. RESULTS: The uptake of morphine in the stable HEK293 cells expressing human OCT1 and OCT2 was significantly increased by 3.56 and 3.04 fold, respectively, than that in the control cells, with no significant uptake increase in the cells expressing human MATE1. All of the six drugs examined, including amitriptyline, fluoxetine, imipramine, irinotecan, ondansetron, and verapamil, were inhibitors of OCT1/2-mediated morphine uptake. The select irinotecan significantly increased the plasma concentrations and decreased hepatic and renal accumulation of morphine in mice. CONCLUSIONS: Morphine is a substrate of OCT1 and OCT2. Clinician should be aware that the disposition of and thus the response to morphine may be altered by co-administration of an OCT1/2 inhibitor, such as irinotecan.

Differential effects of imipramine and CORT118335 (Glucocorticoid receptor modulator/mineralocorticoid receptor antagonist) on brain-endocrine stress responses and depression-like behavior in female rats.

Depression is commonly associated with hypothalamic-pituitary adrenal (HPA) axis dysfunction that primarily manifests as aberrant glucocorticoid secretion. Glucocorticoids act on Type I mineralocorticoid (MR) and Type II glucocorticoid receptors (GR) to modulate mood and endocrine responses. Successful antidepressant treatment normalizes HPA axis function, in part due to modulatory effects on MR and GR in cortico-limbic structures. Although women are twice as likely to suffer from depression, little is known about how antidepressants modulate brain, endocrine, and behavioral stress responses in females. Here, we assessed the impact of CORT118335 (GR modulator/MR antagonist) and imipramine (tricyclic antidepressant) on neuroendocrine and behavioral responses to restraint or forced swim stress (FST) in female rats (n=10-12/group). Increased immobility CORT118335 in the FST is purported to reflect passive coping or depression-like behavior. CORT118335 dampened adrenocorticotropic hormone (ACTH) and corticosterone responses to the FST, but did not affect immobility. Imipramine suppressed ACTH, but had minimal effects on corticosterone responses to FST. Despite these marginal effects, imipramine decreased immobility, suggesting antidepressant efficacy. In an effort to link brain-endocrine responses with behavior, c-Fos was assessed in HPA axis and mood modulatory regions in response to the FST. CORT118335 upregulated c-Fos expression in the paraventricular nucleus of the hypothalamus. Imipramine decreased c-Fos in the basolateral amygdala and hippocampus (CA1 and CA3), but increased c-Fos in the central amygdala. These data suggest the antidepressant-like (e.g., active coping) properties of imipramine may be due to widespread effects on cortico-limbic circuits that regulate emotional and cognitive processes.

Studies of drug interactions with alpha1-acid glycoprotein by using on-line immunoextraction and high-performance affinity chromatography.

A method that combined on-line immunoextraction with high-performance affinity chromatography was developed to examine the binding of drugs with α1-acid glycoprotein (AGP). Affinity microcolumns containing immobilized polyclonal anti-AGP antibodies were developed that had a capture efficiency of up to 98.4% for AGP and a binding capacity of 0.72nmol AGP when using a 20mm×2.1mm i.d. microcolumn. These microcolumns were employed in various formats to examine the binding of drugs to normal AGP and AGP that had been adsorbed from serum samples for patients with systemic lupus erythematosus (SLE). Drugs that were screened in zonal elution experiments for their overall binding to these types of AGP included chlorpromazine, disopyramide, imipramine, propranolol, and warfarin. Most of these drugs showed an increase in their binding to the AGP from SLE serum when compared to normal AGP (i.e., an increase of 13-76%); however, disopyramide gave a 21-25% decrease in retention when the same AGP samples were compared. Frontal analysis was used to further evaluate the binding of disopyramide and imipramine to these forms of AGP. Both drugs gave a good fit to a model that involved a combination of saturable and non-saturable interactions with AGP. Changes in the non-saturable interactions accounted for most of variations seen in the binding of disopyramide and imipramine with the AGP samples. The methods used in this study could be adapted for use in personalized medicine and the study of other proteins or drugs using aqueous mixtures or clinical samples.

An Investigation into the Prediction of in Vivo Clearance for a Range of Flavin-containing Monooxygenase Substrates.

Flavin-containing monooxygenases (FMO) are metabolic enzymes mediating the oxygenation of nucleophilic atoms such as nitrogen, sulfur, phosphorus, and selenium. These enzymes share similar properties to the cytochrome P450 system but can be differentiated through heat inactivation and selective substrate inhibition by methimazole. This study investigated 10 compounds with varying degrees of FMO involvement to determine the nature of the correlation between human in vitro and in vivo unbound intrinsic clearance. To confirm and quantify the extent of FMO involvement six of the compounds were investigated in human liver microsomal (HLM) in vitro assays using heat inactivation and methimazole substrate inhibition. Under these conditions FMO contribution varied from 21% (imipramine) to 96% (itopride). Human hepatocyte and HLM intrinsic clearance (CLint) data were scaled using standard methods to determine the predicted unbound intrinsic clearance (predicted CLint u) for each compound. This was compared with observed unbound intrinsic clearance (observed CLint u) values back calculated from human pharmacokinetic studies. A good correlation was observed between the predicted and observed CLint u using hepatocytes (R2 = 0.69), with 8 of the 10 compounds investigated within or close to a factor of 2. For HLM the in vitro-in vivo correlation was maintained (R2 = 0.84) but the accuracy was reduced with only 3 out of 10 compounds falling within, or close to, twofold. This study demonstrates that human hepatocytes and HLM can be used with standard scaling approaches to predict the human in vivo clearance for FMO substrates.

Chromatographic studies of drug interactions with alpha1-acid glycoprotein by ultrafast affinity extraction and peak profiling.

Interactions with serum proteins such as alpha1-acid glycoprotein (AGP) can have a significant effect on the behavior and pharmacokinetics of drugs. Ultrafast affinity extraction and peak profiling were used with AGP microcolumns to examine these processes for several model drugs (i.e., chlorpromazine, disopyramide, imipramine, lidocaine, propranolol and verapamil). The association equilibrium constants measured for these drugs with soluble AGP by ultrafast affinity extraction were in the general range of 104-106M-1 at pH 7.4 and 37°C and gave good agreement with literature values. Some of these values were dependent on the relative drug and protein concentrations that were present when using a single-site binding model; these results suggested a more complex mixed-mode interaction was actually present, which was also then used to analyze the data. The apparent dissociation rate constants that were obtained by ultrafast affinity extraction when using a single-site model varied from 0.14 to 7.0s-1 and were dependent on the relative drug and protein concentrations. Lower apparent dissociation rate constants were obtained by this approach as the relative amount of drug versus protein was decreased, with the results approaching those measured by peak profiling at low drug concentrations. This information should be useful in better understanding how these and other drugs interact with AGP in the circulation. In addition, the chromatographic approaches that were optimized and used in this report to examine these systems can be adapted for the analysis of other solute-protein interactions of biomedical interest.