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Doxepin is an antihistamine and tricyclic antidepressant that binds to the histamine H 1 receptor (H 1 R) with high affinity. Doxepin is an 85:15 mixture of the E- and Z-isomers. The Z-isomer is well known to be more effective than the E-isomer, whereas based on the crystal structure of the H 1 R/doxepin complex, the hydroxyl group of Thr112 3.37 is close enough to form a hydrogen bond with the oxygen atom of the E-isomer. The detailed binding characteristics and reasons for the differences remain unclear. In this study, we analyzed doxepin isomers bound to the receptor following extraction from a purified H 1 R protein complexed with doxepin. The ratio of the E- and Z-isomers bound to wild-type (WT) H 1 R was 55:45, indicating that the Z-isomer was bound to WT H 1 R with an approximately 5.2-fold higher affinity than the E-isomer. For the T112 3.37 V mutant, the E/Z ratio was 89:11, indicating that both isomers have similar affinities. Free energy calculations using molecular dynamics (MD) simulations also reproduced the experimental results of the relative binding free energy differences between the isomers for WT and T112 3.37 V. Furthermore, MD simulations revealed that the hydroxyl group of T112 3.37 did not form hydrogen bonds with the E-isomer, but with the adjacent residues in the binding pocket. Analysis of the receptor-bound doxepin and MD simulations suggested that the hydroxyl group of T112 3.37 contributes to the formation of a chemical environment in the binding pocket, which is slightly more favorable for the Z-isomer without hydrogen bonding with doxepin., (© 2024 The Author(s). Journal of Molecular Recognition published by John Wiley & Sons Ltd.)

Conventional formulation of topical doxepin has similar antihistaminic effects as oral doxepin; however, its efficacy is limited due to poor localized effects on the skin. This study was designed to compare the ex vivo permeation and retention of two topical doxepin formulations; liposomal cream and plain cream. Doxepin-containing liposomes were prepared with the thin-film hydration method and assessed for size, size distribution, morphology, entrapment efficiency (EE%) and stability Using rat skin specimens in a Franz diffusion cell. Doxepin concentration in skin and receptor fluid was quantified by a validated HPLC method. The optimized liposomal formulation represented a uniform shape with narrow size distribution and an average diameter of 208.7±5.6nm. EE% of doxepin was 79±1.3 and the liposomes were stable at least for six weeks at 4 ° C. Ex vivo studies showed that while a significantly higher amount of doxepin has passed through the skin and entered the receptor compartment from conventional dosage form (47.06±2.5µg/cm2vs 11.20±0.6µg/cm2 for liposomal formulation), liposomal doxepin favoured accumulation in dermis and epidermis. These results suggest that the liposomal doxepin cream is an effective and easy-to-use formulation and may improve the cutaneous retention of doxepin, thus decreasing its systemic side effects.

The histamine H 1 receptor (H 1 R) is a G protein-coupled receptor (GPCR) and represents a main target in the treatment of allergic reactions as well as inflammatory reactions and depressions. Although the overall effect of antagonists on H 1 function has been extensively investigated, rather little is known about the potential modulatory effect of ions or sequence variants on antagonist binding. We investigated the dynamics of a phosphate ion present in the crystal structure and of a sodium ion, for which we determined the position in the allosteric pocket by metadynamics simulations. Both types of ions exhibit significant dynamics within their binding site; however, some key contacts remain stable over the simulation time, which might be exploited to develop more potent drugs targeting these sites. The dynamics of the ions is almost unaffected by the presence or absence of doxepin, as also reflected in their small effect (less than 1 kcal·mol -1 ) on doxepin binding affinity. We also examined the effect of four H 1 R sequence variants observed in the human population on doxepin binding. These variants cause a reduction in doxepin affinity of up to 2.5 kcal·mol -1 , indicating that personalized medical treatments that take into account individual mutation patterns could increase precision in the dosage of GPCR-targeting drugs.

Although histamine plays a major role in animal models of stress-related disorders, human neuroimaging data are sparse. Histamine H1 receptors in the human brain were first imaged by Professor Kazuhiko Yanai in 1992 by using 11 C-doxepin, a potent ligand of H1 receptors, and positron emission tomography (PET). Subsequent work revealed that H1 receptors are reduced in the prefrontal and anterior cingulate cortices in patients with major depressive disorders. A sex difference in H1 receptor binding in the brain has also been found, with women exhibiting more abundant H1 receptor binding than men. Moreover, female patients with anorexia nervosa show higher H1 receptor binding in the amygdala and lentiform nucleus. These studies also found an inverse correlation of depression scores with H1 receptor binding. Histamine is considered to play a major role in the pathophysiology of irritable bowel syndrome (IBS), a representative disorder of brain-gut interactions. Along these lines, hypnotic suggestion dramatically changes the waveforms of viscerosensory cerebral evoked potentials in response to electrical rectal stimulation and these changes are modified by the administration of H1 antagonist. The direction of the H1 antagonist-induced changes in the viscerosensory cerebral evoked potentials differs between IBS patients and healthy controls. Thus, histamine likely plays an important role in stress-related disorders. Further histamine brain imaging studies of humans are warranted., (© 2021. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Binding of histamine to the G-protein coupled histamine H 1 receptor plays an important role in the context of allergic reactions; however, no crystal structure of the resulting complex is available yet. To deduce the histamine binding site, we performed unbiased molecular dynamics (MD) simulations on a microsecond time scale, which allowed to monitor one binding event, in which particularly the residues of the extracellular loop 2 were involved in the initial recognition process. The final histamine binding pose in the orthosteric pocket is characterized by interactions with Asp107 3.32 , Tyr108 3.33 , Thr194 5.43 , Asn198 5.46 , Trp428 6.48 , Tyr431 6.51 , Phe432 6.52 , and Phe435 6.55 , which is in agreement with existing mutational data. The conformational stability of the obtained complex structure was subsequently confirmed in 2 μs equilibrium MD simulations, and a metadynamics simulation proved that the detected binding site represents an energy minimum. A complementary investigation of a D107A mutant, which has experimentally been shown to abolish ligand binding, revealed that this exchange results in a significantly weaker interaction and enhanced ligand dynamics. This finding underlines the importance of the electrostatic interaction between the histamine ammonium group and the side chain of Asp107 3.32 for histamine binding.

This study describes the development of semisolid formulations containing doxepin (DOX) for pain relief in oral mucositis, frequently related to chemotherapy and/or radiotherapy treatments in patients with head and neck cancer. Chemical permeation enhancers were evaluated and selected according to the results obtained from rheological studies, drug release, and drug permeation and retention through buccal mucosa. Finally, the selected formulation was compared in vivo, with a reference DOX mouthwash, whose clinical efficacy had been previously reported. The obtained findings showed that an orabase® platform loading transcutol® (10%) and menthol (5%) for the buccal vehiculization of DOX exhibited a decreased elastic and viscous behavior improving its application. The main drug release mechanism could be considered as diffusion according to Higuchi model. Obtained DOX permeation rates were considered optimal for an analgesic effect and far below to an antidepressant activity. Similar in vivo plasma concentrations were found for the semisolid formulation and the reference mouthwash. However, DOX amounts retained in the mucosa of animals for the semisolid formulation were higher than the reference, which let us hypostatize even stronger potential local therapeutic effect with additional advantages such as, mucoadhesive properties, absence of alcohol, some degree of freshness, as well as, drug palatability improvement., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The interaction of dothiepin (DOT) and doxepin (DOX) with bovine serum albumin (BSA) and a DNA base (adenine) was studied using UV-visible, fluorescence, attenuated total reflection-infra-red (ATR-IR), cyclic voltammetry and molecular docking methods. Strong fluorescence quenching was observed upon interaction of DOT and DOX with BSA/adenine and the mechanism suggested static quenching. Hydrophobic and hydrogen bonding interactions were the predominant intermolecular forces needed to stabilize the copolymer. Upon addition of the drugs: (i) the tautomeric equilibrium structure of the adenine was changed; and (ii) the oxidation and the reduction peaks of the adenine/BSA interaction shifted towards high and low potentials, respectively. In ATR-IR, the band shift of amides I and II indicated a change in secondary structure of BSA upon binding to DOT and DOX drugs. The reduction in voltammetric current in the presence of BSA/adenine was attributed to slow diffusion of BSA/adenine binding with DOX/DOT. The docking method indicated that the drug moiety interacted with the BSA molecule. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Rationale: Histamine H1 antagonists have hypnotic, appetite-promoting, and sedative side effects. Most second-generation antipsychotics have potent antagonistic effects on histamine H1 receptor (H1R). Positron emission tomography (PET) can measure the H1R occupancy (H1RO) in vivo, although there are no reports regarding antipsychotics., Objectives: We studied the H1RO of olanzapine and quetiapine in vivo with respect to their plasma concentrations and subjective drowsiness by performing human PET imaging studies with [(11)C]doxepin, a potent PET ligand of H1R., Methods: Six healthy Japanese male volunteers were enrolled. Cross-randomized PET imaging was performed after a single oral administration of olanzapine (2.5 mg), quetiapine (25 mg), or placebo. PET data were analyzed by region of interest and voxel-by-voxel analysis. We concurrently measured plasma drug concentrations by liquid chromatography/tandem mass spectrometry and evaluated subjective sleepiness., Results: The binding potential ratios of olanzapine and quetiapine in the cerebral cortex were significantly lower than that of the placebo. The H1RO values of olanzapine and quetiapine in the cortex were approximately 61-80 and 56-81%, respectively. The binding potential ratios of the drugs were significantly lower than that of the placebo in the dorsolateral prefrontal and lateral temporal cortices, and anterior and posterior cingulate gyri. The H1RO values in the cortex were significantly correlated with subjective sleepiness but not plasma drug concentrations., Conclusions: Olanzapine and quetiapine have high H1RO values in the human brain under their clinical minimum doses. This study provides a foundation of the properties by which new-generation antipsychotics block the central histaminergic system in humans.

Histamine H4 receptor (H4R), a member of histamine receptor family, which belongs to class A of G-protein coupled receptors (GPCRs), has been reported to play a critical role in histamine-induced chemotaxis in mast cells and eosinophils. Recently, the crystal structure of human histamine H1 receptor (H1R) was reported, which facilitates structure-based drug discovery of histamine receptor significantly. In the current work, the homology models of H4R and H3R are first constructed based on the crystal structure of H1R. Clobenpropit is then docked into the binding pocket of H4R and two different binding modes can be identified. In order to select a reasonable binding mode, several other ligands including agonists and antagonists are docked into H4R, and the results reveal that all ligands share one preferable binding mode: the protonated NH tightly interacts with Asp(3.32) and the imidazole NH interacts with Glu(5.46). By comparing H3R and H4R, we find that Glu(5.20) and Thr(6.55) in H4R involve in the selectivity of H4R. Then, we perform molecular dynamics (MD) simulations for H4R in complex with its compounds. MD results indicate that the preferable docking mode is more stable. Finally, we dock agonist histamine into H1R and H4R, and then perform 20ns MD simulations for the complexes. H1R or H4R bound with histamine show strong conformational changes from TM5, TM6 and TM7, outward movement of intracellular part of TM6, and conformational change of Tyr(7.53), which is consistent with the recent crystal structures of active GPCRs. Our results reveal the mechanism of selectivity and activation for H4R, which is important for developing selective antagonists and agonists for H4R., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The biogenic amine histamine is an important pharmacological mediator involved in pathophysiological processes such as allergies and inflammations. Histamine H(1) receptor (H(1)R) antagonists are very effective drugs alleviating the symptoms of allergic reactions. Here we show the crystal structure of the H(1)R complex with doxepin, a first-generation H(1)R antagonist. Doxepin sits deep in the ligand-binding pocket and directly interacts with Trp 428(6.48), a highly conserved key residue in G-protein-coupled-receptor activation. This well-conserved pocket with mostly hydrophobic nature contributes to the low selectivity of the first-generation compounds. The pocket is associated with an anion-binding region occupied by a phosphate ion. Docking of various second-generation H(1)R antagonists reveals that the unique carboxyl group present in this class of compounds interacts with Lys 191(5.39) and/or Lys 179(ECL2), both of which form part of the anion-binding region. This region is not conserved in other aminergic receptors, demonstrating how minor differences in receptors lead to pronounced selectivity differences with small molecules. Our study sheds light on the molecular basis of H(1)R antagonist specificity against H(1)R., (©2011 Macmillan Publishers Limited. All rights reserved)

Aims: The strength of sedation due to antihistamines can be evaluated using positron emission tomography (PET). The purpose of the present study is to measure histamine H(1) receptor (H(1)R) occupancy following oral administration of cetirizine (10 and 20 mg) in order to examine dose dependency., Methods: Fifteen healthy male volunteers (age range, 20-35 years) were divided into 3 subgroups and were studied following single oral administration of cetirizine at 10 mg (n = 5) and 20 mg (n = 5) or hydroxyzine at 30 mg (n = 5) using PET with 11C-doxepin. Each subject was scanned also following the administration of placebo. Binding potential and H(1)RO values were calculated in the prefrontal and anterior cingulate cortices. Subjective sleepiness was also measured, and the correlation to H(1)RO was examined for each antihistamine., Results: The averaged H(1)ROs of cetirizine 10 mg, 20 mg, and hydroxyzine 30 mg in the prefrontal and cingulate cortices was 12.6%, 25.2%, and 67.6%, respectively. The H(1)RO of hydroxyzine 30 mg correlated well with subjective sleepiness (p < 0.001); however, those of cetirizine 10 and 20 mg showed no correlation with subjective sleepiness., Conclusion: It was demonstrated that the brain penetration of orally administered cetirizine was dose-dependent. Cetirizine 10 mg, with its low H(1)RO and thus minimal sedation, could be more safely used than cetirizine 20 mg for the treatment of various allergic disorders.

Unlabelled: Some antihistamine drugs that are used for rhinitis and pollinosis have a sedative effect as they enter the brain and block the H(1) receptor, potentially causing serious accidents. Receptor occupancy has been measured with PET under single-dose administration in humans to classify antihistamines as more sedating or as less sedating (or nonsedating). In this study, the effect of repeated administration of olopatadine, an antihistamine, on the cerebral H(1) receptor was measured with PET., Methods: A total of 17 young men with rhinitis underwent dynamic brain PET with (11)C-doxepin at baseline, under an initial single dose of 5 mg of olopatadine (acute scan), and under another 5-mg dose after repeated administration of olopatadine at 10 mg/d for 4 wk (chronic scan). The H(1) receptor binding potential was estimated using Logan graphical analysis with cerebellum as reference region input., Results: The acute scan showed a slight decrease in H(1) receptor binding potential across the cerebral cortex (by 15% in the frontal cortex), but the chronic scan showed a marked decrease (by 45% from the acute scan in the frontal cortex). Behavioral data before and after the PET scans did not reveal any sedative effect., Conclusion: The results may be interpreted as either intracerebral accumulation of olopatadine or H(1) receptor downregulation due to repeated administration. The study shows feasibility and potential value for PET in evaluating the pharmacologic effect of a drug not only after a single dose but also after repeated administration.

We introduce a novel experimental method to determine both the extent of ex vivo receptor occupancy of administered compound and its dissociation rate constant (k4). [Here, we reference k4 as the rate of offset of unlabeled ligand in convention with Motulsky and Mahan (1)]. We derived a kinetic rate equation based on the dissociation rate constant for an unlabeled compound competing for the same site as a labeled compound and describe a model to simulate fractional occupancy. To validate our model, we performed in vitro kinetics and ex vivo occupancy experiments in rat cortex with varying concentrations of (R)-dimethindene, a sedating antihistamine. Brain tissue was removed at various times post oral administration, and histamine H1 receptor ligand [3H]-doxepin binding to homogenates from drug-treated or vehicle-treated rats was measured at multiple time points at room temperature. Fractional occupancy and k4 for (R)-dimethindene binding to H1 receptors were calculated by using our proposed model. Rats dosed with 30 and 60 mg/kg (R)-dimethindene showed 42% and 67% occupancy of central H1 receptors, respectively. These results were comparable to occupancy data determined by equilibrium radioligand binding. In addition, drug k4 rate determined by using our ex vivo method was equivalent to k4 determined by in vitro competition kinetics (dissociation half-life t(1/2) approximately 30 min). The outlined method can be used to assess, by simulation and experiment, occupancy for compounds based on dissociation rate constants and contributes to current efforts in drug optimization to profile antagonist efficacy in terms of its kinetic drug-target binding parameters. Data described by the method may be analyzed with commercially available software. Suggested fitting procedures are given in the appendix.

Increasing evidence has shown that the histaminergic neuron system is implicated in the pathophysiology of schizophrenia. The aim of this study was to compare the distribution of histamine H1 receptors between schizophrenics and normal human subjects in vivo using positron emission tomography (PET). H1 receptor binding was measured in 10 normal subjects and 10 medicated schizophrenic patients by PET and [11C] doxepin, a radioligand for the H1 receptor. The binding potential (BP=Bmax/K(D)) of [11C] doxepin for available brain H1 receptors was calculated by a graphical analysis on voxel-by-voxel basis and compared between schizophrenics and normal subjects using the regions of interest (ROIs) and the statistical parametrical mapping (SPM99). BP values for H1 receptors in the frontal and prefrontal cortices and the cingulate gyrus were significantly lower among the schizophrenic patients than among the control subjects. On the contrary, there were no areas of the brain where H1 receptors were significantly higher among the schizophrenic patients than the control subjects. The results of our study suggest that the central histaminergic neuron system could be involved in the pathophysiology of schizophrenia, although further studies are needed to confirm this hypothesis.

The central histaminergic neuron system modulates the wakefulness, sleep-awake cycle, appetite control, learning and memory, and emotion. Previous studies have reported changes in neuronal histamine release and its metabolism under stress conditions in the mammalian brain. In this study, we examined, using positron emission tomography (PET) and [(11)C]-doxepin, whether the histaminergic neuron system is involved in human depression. Cerebral histamine H1 receptor (H(1)R) binding was measured in 10 patients with major depression and in 10 normal age-matched subjects using PET and [(11)C]-doxepin. Data were calculated by a graphical analysis on voxel-by-voxel and ROI (region of interests) basis. Binding potential (BP) values for [(11)C]-doxepin binding in the frontal and prefrontal cortices, and cingulate gyrus were significantly lower in the depressed patients than those in the normal control subjects. There was no area of the brain where [(11)C]-doxepin binding was significantly higher in the depressed patients than in the controls. ROI-based analysis also revealed that BP values for [(11)C]-doxepin binding in the frontal cortex and cingulate gyrus decreased in proportion to self-rating depressive scales scores. The results of this study demonstrate that depressed patients have decreased brain H(1)R binding and that this decrease correlates with the severity of depression symptoms. It is therefore suggested that the histaminergic neuron system plays an important role in the pathophysiology of depression and that its modulation may prove to be useful in the treatment of depression.

Purpose: This study was conducted to identify the cytochrome P450s (CYPs) responsible for the metabolism of the cis- and trans-isomers of the tricyclic antidepressant doxepin to its pharmacologically active N-desmethylmetabolite by in vitro techniques., Methods: The doxepin N-demethylation was studied by means of pooled human liver microsomes and chemical inhibitors, recombinant human (rh)-CYPs, and geno- and phenotyped human liver microsomes., Results: The N-demethylation of both isomers was inhibited most prominently by tranylcypromine (CYP2C19) to more than 50%. Furafylline (CYP1A2) and sulfaphenazole (CYP2C9) inhibited the N-demethylation to a lesser extent while quinidine (CYP2D6) or troleandomycine (CYP3A4) had no effect. Rh-CYP2C19, -CYP1A2, and -CYP2C9 were able to N-demethylate cis- and trans-doxepin. Only traces of trans-desmethyldoxepin were detectable when CYP3A4 was used. The maximum velocity in the cis- and transdoxepin N-demethylation was significantly (P < 0.05) lower in microsomes with low CYP2C19 activity (345 +/- 44 and 508 +/- 75 pmol/min/ mg protein, respectively) compared to those with high CYP2C19 activity (779 +/- 132 and 1,189 +/- 134 pmollmin/mg)., Conclusion: The present study demonstrates a significant contribution of the polymorphic CYP2C19 to the N-demethylation of doxepin. CYP2C9 and CYP1A2 play a minor role and CYP3A4 does not contribute substantially.

Due to its tranquilizing properties, the tricyclic antidepressant doxepin may be misused as a doping agent in competition horses. Therefore, efficient analytical procedures are required to detect this drug in samples submitted for doping control. To screen for parent doxepin in equine blood and urine, a less specific method has been accepted employing gas chromatography (GC) combined with electron impact (EI) mass spectrometry (MS). The aim of this study was identification of doxepin metabolites providing more specific MS data to verify positives resulting from screening. Thus, after a horse was given doxepin-HCl (1 mg/kg, i.v.), blood and urine were analyzed for free or conjugated metabolites using GC combined with EI- and positive chemical ionization (PCI) MS. In both of the sample materials, cis- and trans-isomers of desmethyldoxepin were detected for up to 48 h after treatment using trifluoracetylation and GC/EI-MS. Following enzymic hydrolysis of urine and propionylation of extracts, each four isomers of hydroxy desmethyldoxepin and hydroxydoxepin were recovered for up to 24 and 48 h, respectively. These compounds were characterized by their EI- and PCI-mass spectra. Although distinct positions of the hydroxyl groups could not be determined, the presence of each two cis/trans-isomeric pairs of differently monohydroxylated metabolites may be assumed. Results reported here suggest, that screening horses for parent doxepin should be completed by analysis of its major isomeric metabolites, desmethyldoxepin and hydroxydoxepin, providing MS data specific enough for confirmatory analysis.

A stereoselective method of analysis of the antidepressant drug doxepin (DOX, an 85:15% mixture of E-Z stereoisomers), its principal metabolites E- and Z-N-desmethyldoxepin (desDOX) and ring-hydroxylated metabolites in microsomal incubation mixtures is described. DOX and its metabolites were extracted from alkalinised incubation mixtures by either: 9:1 hexane-propan-2-ol (method 1) or 1:1 hexane-dichloromethane (method 2), derivatised with trifluoroacetic anhydride and analysed by GC-MS with selected ion monitoring. Both methods were suitable for the analysis of individual desDOX isomers as indicated by correlation coefficients of > or = 0.999 for calibration curves constructed between 50 and 2500 nM, and good within-day precision at 125 nM (C.V. < or = 14%) and 1000 nM (C.V. < or = 8%). Method 1, however, was unsuitable for the analysis of ring-hydroxylated metabolites of DOX, whereas the hydroxylated metabolites of E-DOX and E-desDOX (generated in situ) were extracted by method 2 with a C.V. of ca. 13%. This is the first assay method that permits the simultaneous measurement of desDOX and hydroxylated metabolites of DOX in microsomal mixtures.

Antihistamines are efficacious drugs to be used for the symptomatic relief of allergic diseases. The safety issue of antihistamines is of central importance because of their widespread use in current medical practice. To better understand the pharmacological effects of antihistamines on the central nervous system (CNS), we used two kinds of new methods, positron emission tomography (PET) and gene targeting regarding on histamine H1 receptors. The histamine H1 receptor occupancy was examined in young male volunteers with[11C]-doxepin (a potent H1 antagonist) after the oral or intravenous administration of antihistamines. In other studies, the cognitive performance was also measured tachistoscopically before and after taking antihistamines. The mutant mice lacking H1 receptors were used in the behavioural and neurochemical experiments to re-evaluate the role of H1 receptors. The H1-receptor occupancy in the human frontal cortex caused by antihistamines is significantly correlated with the reported values of incidence of sleepiness in clinical trials, and the occupancy is well proportional to the impaired cognitive performance. The behavioural studies of the H1-receptor knock-out mice confirmed the role of H1 receptors in arousal, the sleep-wake cycle, locomotion, nociception and aggressive behaviour. The pharmacological effects induced by H1 antagonism were re-evaluated by the PET and gene-targetting. Although any serious effects could not be observed in mice by the destruction of the H1-receptor gene, the cognitive performance was impaired in humans after taking first generation antihistamines in recommended doses.

Objective: To report adverse effects in a newborn infant whose mother had been treated with doxepin during pregnancy and while breast-feeding., Case Summary: The nine-day-old white boy was admitted because of poor sucking and swallowing, with muscle hypotonia and vomiting. He was drowsy and had lost 150 g. At the time of admission, he was breast-fed by his mother who was being treated with doxepin 35 mg/d. Samples of plasma and breast milk were taken and analyzed by HPLC and fluorescence polarization immunoassay. The amount of doxepin and N-desmethyldoxepin (DDP) ingested via breast-feeding was approximately 10-20 micrograms/kg/d (i.e., only 2.5% of the weight-adjusted dose of the mother). Doxepin was detectable in small amounts in the infant's plasma (approximately 10 micrograms/L); DDP was below the lower limit of detection of 10 micrograms/L. All adverse effects subsided within 48 hours after breast-feeding was stopped., Discussion: Despite the small doses of doxepin and its active metabolite ingested by breast-fed babies, there is a risk of accumulation and resultant adverse effects. In newborns, the metabolic activity is considerably decreased and may be further reduced by hyperbilirubinemia., Conclusions: Available data suggest that women treated with doxepin should breast-feed their infants with great caution, if at all, although much larger databases are needed to confirm this.

1. Doxepin is marketed as an irrational mixture of geometric isomers such that the more active Z-isomer comprises only 15% of the total doxepin whereas the less active E-isomer makes up the remaining 85%. 2. The ratio of isomers of the doxepin remains approximately Z:E = 15:85 in the plasma of depressed patients whereas plasma levels of the active Z-N-desmethyl metabolite are similar to those of E-N-desmethyldoxepin. 3. After examination of four animal species (dog, rabbit, guinea pig, rat), rat was closest to human in terms of the Z:E ratio of the geometric isomers of N-desmethyldoxepin excreted in the 0-24-h urine. 4. Changes in the urinary Z:E ratio of the metabolite were observed after oral but not after intravenous or intraperitoneal administration of commercial doxepin to rat. 5. There was no evidence of Z/E interconversion after administration of the pure isomers to rat in vivo, or after incubation of rat or human liver homogenates with pure isomers. 6. In vitro data suggested that the distortion of the Z:E ratio of N-desmethyldoxepin was a consequence of faster metabolism of the E-isomer in comparison with Z-N-desmethyldoxepin rather than 'enrichment' of the Z-isomer at the expense of the E-isomer.

Little information exists on the concentrations of antidepressants and their metabolites in CSF. We measured plasma and CSF levels of trans-doxepin (trans-DOX) and DOX metabolites in 12 depressed patients treated with DOX (250 mg/day) for 6 days. Spinal taps and blood samples were taken on day 7, 10 h after drug administration. Trans-DOX, cis-desmethyldoxepin (cis-DM-DOX), trans-desmethyldoxepin (trans-DM-DOX) and di-desmethyldoxepin (DDM-DOX) were analyzed in CSF and plasma samples by HPLC with column-switching. Although DOX was given as a mixture of 85% trans-DOX and 15% of the pharmacologically more active cis-DOX, we found similar amounts of cis-DM-DOX and trans-DM-DOX in plasma (59.8 +/- 45.1 versus 72.0 +/- 60.0 ng/ml; NS), suggesting that isomerization of DOX had taken place. Trans-DOX and DOX metabolites could be detected in CSF of most patients. Relatively low CSF concentrations of the active metabolite cis-DM-DOX were measured. Clinical efficacy, as assessed by HAMD scores, was not significantly related to plasma or CSF concentrations of trans-DOX or its metabolites. Trans-DOX and DOX metabolites were distributed differently between plasma and CSF. It is concluded that isomerization of DOX is not only relevant for neuronal uptake inhibition, but also for the transport of the metabolites.

From pharmacological studies using histamine antagonists and agonists, it has been demonstrated that histamine modulates many physiological functions of the hypothalamus, such as arousal state, locomotor activity, feeding, and drinking. Three kinds of receptors (H1, H2, and H3) mediate these actions. To define the contribution of the histamine H1 receptors (H1R) to behavior, mutant mice lacking the H1R were generated by homologous recombination. In brains of homozygous mutant mice, no specific binding of [3H]pyrilamine was seen. [3H]Doxepin has two saturable binding sites with higher and lower affinities in brains of wild-type mice, but H1R-deficient mice showed only the weak labeling of [3H]doxepin that corresponds to lower-affinity binding sites. Mutant mice develop normally, but absence of H1R significantly increased the ratio of ambulation during the light period to the total ambulation for 24 hr in an accustomed environment. In addition, mutant mice significantly reduced exploratory behavior of ambulation and rearings in a new environment. These results indicate that through H1R, histamine is involved in circadian rhythm of locomotor activity and exploratory behavior as a neurotransmitter.

1. Histamine H1 receptor occupancy in the human brain was measured in 20 healthy young men by positron emission tomography (PET) using [11C]-doxepin. 2. (+)-Chlorpheniramine, a selective and classical antihistamine, occupied 76.8 +/- 4.2% of the averaged values of available histamine H1 receptors in the frontal cortex after its administration in a single oral dose of 2 mg. Intravenous administration of 5 mg (+)-chlorpheniramine almost completely abolished the binding of [11C]-doxepin to H1 receptors (H1 receptor occupancy: 98.2 +/- 1.2%). 3. Terfenadine, a nonsedative antihistamine, occupied 17.2 +/- 14.2% of the available H1 receptors in the human frontal cortex after its administration in a single oral dose of 60 mg. 4. There was no correlation between H1 receptor occupancy by terfenadine and the plasma concentration of the active acid metabolite of terfenadine in each subject. 5. PET data on human brain were essentially compatible with those on H1 receptor occupancy in guinea-pig brain determined by in vivo binding techniques, although for the same H1 receptor occupancy the dose was less in human subjects than in guinea-pigs. 6. The PET studies demonstrated the usefulness of measuring H1 receptor occupancy with classical and second-generation antihistamines in human brain to estimate their unwanted side effects such as sedation and drowsiness quantitatively.

Commercial preparations of the tricyclic anti-depressant doxepin contain 15% of the more active cis-doxepin and 85% of the trans-isomer. The single dose pharmacokinetics of doxepin and its major metabolite N-desmethyldoxepin were examined in 30 healthy young men. Results for total doxepin showed wide intersubject variation in all pharmacokinetic parameters except tmax and Cmax. Plasma levels of cis-doxepin were extremely low and it was only possible to estimate the stereoselective pharmacokinetics of the parent drug in 3 subjects. The data from those particular subjects resulted in an average ratio of cis- to trans-doxepin isomers in plasma of 15:85. In contrast, the mean plasma levels of cis-N-desmethyldoxepin in 28 subjects exceeded those of the trans-isomer at every time point after 10 h, such that the areas under the plasma concentration versus time curves (AUC) of cis-N-desmethyldoxepin were significantly higher than those of the corresponding trans-isomer. This phenomenon may play an important role in the therapeutic action of doxepin since it has been suggested that cis-N-desmethyldoxepin is pharmacologically active. In 2 subjects, however, the AUC0-inf of trans-N-desmethyldoxepin were respectively 4 and 8 fold higher than those of the cis-isomer.

The N-demethylation of the individual Z-(cis) and E-(trans) isomers of the tricyclic antidepressant doxepin was studied by examining the 0-8 hr serum concentration-time and the 0-72 hr urinary excretion profiles of parent drug and metabolite in eight healthy males who had received a single oral dose consisting of 25 mg each of Z-[2H0]- and E-[2H4]-labeled drug as the hydrochloride salt. Interconversion of doxepin's isomers was not observed but stereoselective excretion was present. Significant amounts of Z-N-desmethyldoxepin were formed and excreted after dosing with E-doxepin, the urinary Z/E ratio ranging from 0.08 to 3.06. A small amount of nondeuterated E-N-desmethyldoxepin was formed from Z-doxepin in most, but not all, subjects. These findings indicate that isomerization occurs during the N-demethylation of doxepin, possibly involving the formation of an intermediate in which the exocyclic double bond is hydrated and then subseqently dehydrated. This novel biotransformation process accounts for the observation that the Z/E plasma concentration ratio of N-desmethyldoxepin is often greater than that of the administered doxepin in patients receiving the drug therapeutically.

Histamine H1 receptors were visualized in the living dog brain using [11C]pyrilamine or [11C]doxepin by positron emission tomography (PET). The regional distribution of these carbon-11 labeled compounds in the brain corresponded well with that of the histamine H1 receptors separately determined by in vitro binding assay. The radioactivity in the brain was reduced by treatment with triprolidine (1 mg/kg), a histamine H1 antagonist. The results of our study indicate that it is feasible to visualize histamine H1 receptors in human brain using these 11C-labeled compounds and PET.

The effect of doxepin on ventricular arrhythmias, the ECG, and left ventricular function was evaluated in 10 cardiac patients with symptoms with frequent ventricular premature depolarizations in a dose-ranging protocol. Four patients (40%) had greater than or equal to 80% ventricular premature depolarization suppression; four of eight with pairs and four of six with ventricular tachycardia had greater than or equal to 90% suppression. The mean maximal doxepin dose was 115 +/- 41 mg/day; mean nadir total doxepin concentration was 61 +/- 48 ng/ml and mean nadir total desmethyldoxepin concentration was 51 +/- 42 ng/ml. Doxepin increased the heart rate and the PR, QRS, and QTc intervals of the surface ECG (P not significant). There was no significant change in resting mean left ventricular ejection fraction with doxepin: 41% +/- 15% vs. 43% +/- 19% (P not significant). Complaints of sedation (eight patients) limited dose ranging and tolerance to the drug. Although doxepin suppressed ventricular premature depolarizations in four patients, marked sedation limits its usefulness for primary treatment of arrhythmias in this population.

[3H]Doxepin, a tricyclic antidepressant, binds to brain homogenates with two saturable components. The high affinity component, with a dissociation constant (KD) of 0.26 nM, is associated with histamine H1-receptors. This high affinity binding shows stereospecificity in that d-chlorpheniramine is 100 times more potent than the pharmacologically less active l-isomer. Its drug specificity and regional variation closely parallel those exhibited by [3H]mepyramine binding. The drug specificity of the low affinity component is distinct from that of histamine H1-receptors, with no stereospecificity for chlorpheniramine isomers. Furthermore, all the H1-histamine antagonists tested display micromolar potency at the low-affinity doxepin sites but nanomolar potency at the high-affinity doxepin sites associated with a physiological histamine H1-receptor. The drug specificity of the low affinity site does not correspond to that of any known neurotransmitter receptor. Tricyclic antidepressants display IC50 values of 30-600 nM for the inhibition of [3H]doxepin binding to the low-affinity component with most values in the 0.1-0.3 microM affinity range.

The binding of the radioactively labeled tricyclic antidepressant, [3H]doxepin, to rat brain tissue was examined. Scatchard plots of specific [3H]doxepin binding indicated the presence of two distinct binding sites. The equilibrium dissociation constant (KD) of the high-affinity site was 0.020 nM with a maximal binding capacity (Bmax) of 13.7 fmol/mg protein. The corresponding values for the high-affinity site were 3.6 nM and 740 fmol/mg protein, respectively. The high-affinity site was sensitive to competition by pharmacologically relevant concentrations of histamine H1 antagonists such as pyrilamine (KD = 1.0 nM), diphenhydramine (KD = 20 nM), d-chlorpheniramine (KD = 1.7 nM), and 1-chlorpheniramine (KD = 97 nM). The Bmax for [3H]doxepin binding to the high-affinity H1-receptor, however, was approximately 10% of the Bmax obtained using [3H]pyrilamine to label the H1-receptor. Various tricyclic antidepressants were very potent inhibitors at the high-affinity [3H]doxepin site. Their potencies, however, did not correlate with their potencies previously reported for the H1-receptor. The regional distribution of [3H]doxepin high-affinity sites correlated with the known distribution of H1-receptors in the rat brain. These results suggest that [3H]doxepin is binding to a subclass of histamine H1-receptors.

The affinity constant for doxepin obtained from inhibition of histamine-induced contraction of guinea-pig intestinal smooth muscle at 30 degrees C was 2.6 +/- 0.18 X 10(10)M-1. The slope of a Schild plot was not significantly different from unity. The affinity constant of doxepin did not vary markedly with temperature. At 37 degrees C it was 3.75 +/- 0.02 X 10(10)M-1 and at 25 degrees C 2.1 X 10(10)M-1. Doxepin was a competitive inhibitor of [3H]-mepyramine binding to guinea-pig cerebellar homogenates. The affinity constant derived for doxepin at 30 degrees C was 1.12 +/- 0.45 X 10(10)M-1. Hill coefficients for curves of doxepin or mepyramine inhibition of [3H]-mepyramine binding in guinea-pig cerebellum, cerebral cortex and hippocampus did not differ significantly from unity. The mean affinity of mepyramine for histamine H1-receptors in rat brain homogenates at 30 degrees C was 3.5 X 10(8)M-1. Hill coefficients for curves of doxepin or mepyramine inhibition of [3H]-mepyramine binding to homogenates of rat cerebral cortex or rat whole brain were near unity. These studies provide no evidence that doxepin binds preferentially to a sub-class of histamine H1-receptors in rat brain.

Doxepin (DOX) and desmethyldoxepin (DMD) kinetics were examined in seven depressed patients receiving single daily doses of 150 mg DOX for 1 to 3 wk. Blood samples were collected at 0, 4, 12, 15, 18, and 24 hr after the first dose, at bedtime before doses 7, 14, and 21, and at 4, 12, 15, 18, and 24 hr after the last dose. Plasma concentrations of DOX and DMD were analyzed by high-pressure liquid chromatography. Clinical response to DOX treatment was evaluated by the Zung self-rating depression scale and the Hamilton rating scale for depression. Mean DOX t1/2 after the first dose was 17.7 hr, and it rose to 21.8 hr after the last dose. Mean DMD t1/2 was not significantly affected by multiple dosing (34.2 hr after first dose and 37.1 hr after last dose). Mean values for plasma clearance, volume of distribution, and first-pass metabolism were 0.87 l/hr/kg, 23.8 l/kg, and 69.5%. In depressed patients kinetics were in the normal range. Steady-state concentrations of DOX and DMD were reached within 2 wk of beginning DOX dosing. The concentration-response curve indicated strong correlation between total DOX concentration (DOX + DMD) and antidepressant effect (r2 = 0.76).

Doxepin binds to intact astrocytes in primary cultures. The binding is competitively displaceable by excess cold doxepin and it is pronounced (Bmax = 27 nmol/mg protein) but the affinity is rather low (KD = 30 microM). The binding is inhibited by other antidepressants (amitriptyline, desipramine, tranylcypromine, iprindole) and propranolol but not by isoproterenol. Nevertheless, doxepin counteracts effectively the increase in the production of cyclic AMP evoked by isoproterenol.

The pharmacokinetics of two marketed formulations of doxepin HCl administered as a single 100-mg oral dose were compared. Sixteen healthy volunteers between the ages of 21 and 50 years participated in this crossover study. A one-week washout period intervened between doses. Blood samples were drawn before drug administration and at various times up to 48 hours after dosing. The concentrations of doxepin (DOX) and desmethyldoxepin (DDOX), the major active metabolite, were determined. Bioequivalence was determined by statistical comparisons of the area under the curve and maximum concentrations of DOX and DDOX. Statistical comparisons indicated no difference between the two formulations with respect to any of the parameters. The results of this study demonstrate that the two formulations of doxepin HCl are bioequivalent and would be expected to have similar clinical efficacy.

The pharmacological effects of three tricyclic antidepressant agents (desipramine, protriptyline and doxepin) are evaluated in rat isolated atria in relation to their accumulation and efflux kinetics. The pharmacological effects studed are: inhibition of 1-3H-noradrenaline uptake, potentiation of 1-noradrenaline chronotropic response, and changes in spontaneous atrial rate. All drugs inhibit noradrenaline uptake and potentiate noradrenaline chronotropic response (desipramine congruent to protriptyline greater than doxepin). Desipramine and protriptyline, at concentrations of 10(-7) -- 10(-6)M stimulate the spontaneous rate; higher concentrations (greater than 10(-6)M) depress it. Doxepin has only a negative chronotropic effect. When the drugs are removed from the incubation medium, the depressing effect starts to disappear immediately for doxepin and desipramine and after 20 min for protriptyline. On the contrary the stimulating effect persists after repeatedly washing the preparations. Desipramine, protriptyline and doxepin extensively accumulate in the myocardial tissue (desipramine larger than or equal to protriptyline greater than doxepin). In the efflux studies doxepin is washed out more rapidly than desipramine and protriptyline. Although the kinetics of uptake and efflux of the three compounds are not sufficient to interpret their different pharmacological activities in isolated atria, they give useful information on the persistance of the sympathomimetic effect and the rapid disappearing of the negative chronotropic effect after washing.

Doxepin, a tricyclic antidepressant, is one of the most potent histamine H1 antagonists. Therefore, the binding of [3H]doxepin to human brain membranes was examined. Scatchard analysis revealed two distinct binding sites. The high-affinity binding site with a dissociation constant (KD +/- S.E.M.) of 3.1 +/- 0.3 X 10(-10) M was pharmacologically identified as histamine H1 receptors. Dissociation curves at low concentrations of [3H]doxepin were biphasic, suggesting several possibilities about the interaction between [3H]doxepin and histamine H1 receptors. Tetracyclic antidepressants, mianserin and maprotiline, were very potent, with KDs of 3.6 +/- 0.7 X 10(-10) M and 7.9 +/- 0.5 X 10(-10) M, respectively. Mequitazine, a new antihistamine with a weak sedative effect, had a KD of 5.8 +/- 0.8 X 10(-9), making it ten times as potent as the classic antihistamine diphenhydramine. The highest binding of [3H]doxepin to histamine H1 receptors was found in cerebral neocortex and the limbic system. The distribution of histamine H1 receptors in human central nervous system did not correlate with the previously reported distributions in rat brain and guinea pig brain determined by [3H]doxepin binding.

Using radioligand binding techniques, we determined the equilibrium dissociation constants (KDS) for a series of antidepressants at the histamine H1, muscarinic acetylcholine, alpha-1 and alpha-2 adrenergic and dopamine (D-2) receptors of normal human brain tissue obtained at autopsy. Twenty-five different antidepressants were studied at all but the D-2 receptor at which the number was 13 (including a metabolite of an antidepressant). Of all the receptor interactions studied, that at the histamine H1 receptor was in general the most potent interaction of this class of compounds, corresponding to results from studies using animal tissue as the source of receptors. At the human brain histamine H1 receptor, antidepressants remained among the most potent histamine H1 antagonists known, although their affinities for human receptors were lower than those for animal receptors. The most potent and the least potent compounds at the receptors were doxepin (KD = 0.24 nM) and fluvoxamine (KD = 109 microM) at the histamine H1 receptor, amitriptyline (KD = 18 nM) and trazodone (KD = 324 microM) at the muscarinic receptor, doxepin (KD = 24 nM) and viloxazine (KD = 14 microM) at the alpha-1 receptor, mianserin (KD = 73 nM) and bupropion (KD = 81 microM) at the alpha-2 receptor and amoxapine (KD = 160 nM) and trazodone (KD = 3800 nM) at the D-2 receptor, respectively. In general, compared to older drugs, the newer compounds tended to have lower affinities for these receptors.

The influence of concurrent cimetidine administration on the disposition of doxepin was evaluated in 10 healthy volunteers. Each subject ingested 100 mg of doxepin on two different occasions, once while otherwise drug free and once while receiving cimetidine, 300 mg every 6 hours. Doxepin absorptive parameters--time to peak doxepin plasma concentration (2.3, control, vs. 2.4 hours during cimetidine co-administration) and peak concentration achieved (43.3. vs. 55.5 ng/ml)--were not changed during cimetidine administration. Likewise, doxepin elimination half-life was similar in the control state (12.5 hours) and during cimetidine administration (13.2 hours). However, doxepin area under the plasma concentration-time curve (AUC) was increased during concurrent cimetidine administration (533 vs. 695 ng/ml . hour; p less than 0.05), resulting in a trend toward decreased doxepin oral clearance (4404 vs. 3278 ml/min; 0.05 less than p less than 0.1). Relative bioavailability during concurrent cimetidine treatment was 123% of that during the control trial. Desmethyldoxepin AUC was no different between trials (478, control, vs. 433 ng/ml . hour during cimetidine ingestion). Plasma protein binding of doxepin was similar between trials (percent unbound; 10.5, control, vs. 11.2%) and therefore did not influence calculated AUC. These data indicate that doxepin relative bioavailability is increased during concurrent cimetidine administration and suggest that doxepin hepatic extraction is impaired by cimetidine after oral administration. During chronic doxepin therapy, addition of cimetidine to a therapeutic regimen may result in increased doxepin plasma concentration.

[3H]Doxepin, a tricyclic antidepressant, binds with high affinity to guinea pig brain membranes with a drug specificity indicating an association with H1 histamine receptors. The [3H]doxepin binding site has been solubilized, with digitonin being the only detergent able to maintain specific binding after solubilization. After solubilization, the kinetics and drug specificity of binding are virtually identical with those obtained in the intact membranes, indicating a conservation of the transmitter binding site after removal of the receptor from its lipid environment. The regulation of agonist affinity for the histamine H1 receptor by cations is maintained after solubilization. Sodium and to a similar extent lithium, but not potassium, rubidium, or cesium, decrease the affinity of agonists for the receptor. The divalent cations manganese and magnesium maintain their ability to increase agonist affinity after solubilization. Guanine nucleotides, however, lose their ability to decrease agonist affinity for the histamine H1 receptor after solubilization. Histamine receptors in rat brain differ from guinea pig brain receptors in the potency of several antihistamines. The difference is maintained in the solubilized receptors. Sucrose gradient and gel filtration experiments indicated Mr approximately 430,000 for the receptor-digitonin complex. The isoelectric point of the receptor is 4.8. None of these physical techniques distinguishes between guinea pig and rat brain receptors.

Doxepin is closely related in structure and general pharmacological properties to other tricyclic antidepressant drugs such as amitriptyline and imipramine. It combines antidepressant activity with a sedative effect and in this respect resembles amitriptyline, with which it shares a similar profile of clinical action. The mood elevating effect of doxepin appears to be similar to that of amitriptyline but is probably less marked than that of imipramine and in some studies has been slower to take effect than imipramine. At dosages which have achieved a similar overall response rate, doxepin tends to cause fewer or less troublesome side-effects than imipramine, amitriptyline or amitriptyline-prephenazine. The more marked sedative properties of doxepin make it more useful than imipramine in depressed patients with sleep distrubances and in depression associated with anxiety. The benzodiazepines remain the drugs of choice in anxiety states. but when anxiety is accompained by significant depression, doxepin is more effective than chlordiazepoxide or diazepam. Doxepin is usually well tolerated, and in particular by the elderly and those with cardiovascular disease. Side-effects are similar in nature to those of other tricyclic antidepressants, with dry mouth, drowsiness and constipation being the most common. Postural hypotension is uncommon. Although doxepin appears to cause fewer cardiovascular side-effects in usual therapeutic doses, it has an intrinsic cardiotoxicity on overdosage similar to other tricyclics.

A method for the identification of antidepressants and their metabolites in urine after acid hydrolysis is described. The acetylated extract is analysed by computerized gas chromatography--mass spectrometry. An on-line computer allows rapid detection using mass fragmentography with the masses 58, 84, 86, 100, 191, 193, 194, 205. The identity of positive signals in the reconstructed mass fragmentogram is established by a comparison of the entire mass spectra with those of standards. The mass fragmentogram, the underlying mass spectra and the gas chromatographic retention indices (OV-101) are documented.

Doxepin and N-desmethyldoxepin concentrations in plasma and breast milk from a lactating mother and in plasma from her infant were measured during an 8 week period using high performance liquid chromatography. Plasma concentrations ranged from 35-68 microgram l-1 for doxepin and 65-131 microgram l-1 for N-desmethyldoxepin. Mean pre-feed milk/plasma ratios were 1.08 and 1.02 for doxepin and N-desmethyldoxepin respectively and increased to 1.66 and 1.53 at the post-feed samples. Only N-desmethyldoxepin (15 microgram l-1) was detectable in the infant's plasma sampled after 43 days of maternal therapy.

The protein binding of doxepin (DOX) and desmethyldoxepin (DDOX) were studied in serum and plasma samples from healthy volunteers and psychiatric patients. Binding was measured by equilibrium dialysis (16 hrs at 37 degrees) and drug concentrations by radioimmunoassay. In addition, albumin and alpha 1-acid glycoprotein concentrations of the samples were measured by radial immunodiffusion. The mean +/- SEM percentages of unbound DOX were: 20.4 +/- 1.2 and 15.9 +/- 1.2 in healthy subjects (n = 16) and patients (n = 15) respectively, and those of DDOX: 21.4 +/- 0.9 and 19.0 +/- 1.4 for healthy subjects and patients, respectively. There was a significant negative correlation between serum alpha 1-acid glycoprotein concentration and free fraction of DOX in both groups. In healthy subjects a significant negative correlation was also found between albumin concentrations and free fraction of both DOX and DDOX. Binding experiments with isolated protein fractions revealed that all of the total binding in plasma could be explained by binding to albumin and alpha 1-acid glycoprotein. The observed 2--4-fold interindividual variability in the free fractions of these drugs is probably less important than the much larger variability in the total serum concentrations.