G protein-coupled receptors (GPCRs) are major therapeutic drug targets and represent more than 30% of the market share of all prescription drugs. The high-resolution three-dimensional structures of the target receptors provide good initial models for structure-based approaches to drug screening and drug design, which are considered to accelerate drug discovery. However, significant bottlenecks at the expression, purification and crystallization stages of structure determination of GPCRs have existed. Here, we review recent techniques for the determination of GPCR structures. In particular, we focus on the protein engineering techniques that have been used to overcome bottlenecks in expression/purification and crystallization, including our development of a platform using budding yeast for the rapid construction and evaluation of GPCR variants for structural studies. We also present our success in determining the crystal structure of the histamine H1 receptor (H1R) in complex with doxepin, an inverse agonist antihistamine. The H1R structure revealed the low selectivity of doxepin to aminergic receptors and provides key information that should aid the development of highly selective antihistamines.

PURPOSE:

The aim of this study was to estimate the prevalence of potentially inappropriate medication (PIM) in the elderly as indicated by Germany's recently published list (PRISCUS) and to assess factors independently associated with PIM prescribing, both overall and separately for therapeutic groups.

METHODS:

Claims data analysis (Health Insurance Sample AOK Hesse/KV Hesse, 18.75% random sample of insurants from AOK Hesse, Germany) is used in the study. The study population is composed of 73 665 insurants >64 years of age continuously insured in the last quarter of 2009 and either continuously insured or deceased in 2010. Prevalence estimates are standardized to the population of Germany (31 December 2010). The variables age, sex, polypharmacy, hospital stay and nursing care are assessed for their independent association with general PIM prescription and among 11 therapeutic subgroups using multivariate logistic regression analysis.

RESULTS:

In 2010, 22.0% of the elderly received at least one PIM prescription (men: 18.3%, women: 24.8%). The highest PIM prevalence was observed for antidepressants (6.5%), antihypertensives (3.8%) and antiarrhythmic drugs (3.5%). Amitriptyline, tetrazepam, doxepin, acetyldigoxin, doxazosin and etoricoxib were the most frequently prescribed PIMs. Multivariate analyses indicate that women (OR 1.39; 95% CI: 1.34-1.44) and persons with extreme polypharmacy (≥10 vs. <5 drugs: OR 5.16; 95% CI: 4.87-5.47) were at higher risk for receiving a PRISCUS-PIM. Risk analysis for therapeutic groups shows divergent associations.

CONCLUSION:

PRISCUS-PIMs are widely used. Educational programs should focus on drugs with high treatment prevalence and call professionals' attention to those elderly patients who are at special risk for inappropriate medication. Copyright © 2013 John Wiley & Sons, Ltd.

INTRODUCTION:

Although a variety of pharmacologic and non-pharmacologic treatments are effective for insomnia in the general population, insomnia in Parkinson's disease differs in important ways and may need different treatments. No studies have conclusively demonstrated effective insomnia treatments in Parkinson's disease.

METHODS:

We conducted a three-arm six-week randomized pilot study assessing non-pharmacologic treatment (cognitive behavioural therapy with bright light therapy) or doxepin (10 mg daily), compared to an inactive placebo in Parkinson's patients with insomnia. Sleep outcomes included insomnia scales, clinical global impression, sleep diaries and actigraphy. Secondary outcomes included motor severity, fatigue, depression and quality of life.

RESULTS:

18 patients were randomized, 6 to each group. Compared to placebo, doxepin improved the Insomnia Severity Index (-9 ± 5.4 vs. -2 ± 3.9, p = 0.03), the SCOPA-night score (-5.2 ± 1.5 vs. -2.3 ± 2.8, p = 0.049), the Pittsburgh Sleep Quality Index-sleep disturbances subscale (-0.5 ± 0.5 vs 0.2 ± 0.4, p = 0.02), and both patient and examiner-rated clinical global impression of change (1.7 ± 0.8 vs. 0.5 ± 0.8, p = 0.03 and 1.4 ± 0.5 vs. 0.3 ± 0.5, p = 0.003). On secondary outcomes doxepin reduced the fatigue severity scale (p = 0.02) and improved scores on the Montreal Cognitive Assessment (p = 0.007). Non-pharmacological treatment reduced the Insomnia Severity Index (-7.8 ± 3.8 vs. -2.0 ± 3.9, p = 0.03), and the examiner-reported clinical global impression of change (p = 0.006), but was associated with decline in Parkinson Disease Questionnaire-39. There were no changes in other primary and secondary outcomes, including actigraphy outcomes. Adverse events were comparable in all groups.

CONCLUSION:

Doxepin and non-pharmacologic treatment substantially improved insomnia in Parkinson's disease. These potential benefits must be replicated in a full confirmatory randomized controlled trial.

Polymorphisms in CYP2D6 and CYP2C19 affect the efficacy and safety of tricyclics, with some drugs being affected by CYP2D6 only, and others by both polymorphic enzymes. Amitriptyline, clomipramine, doxepin, imipramine, and trimipramine are demethylated by CYP2C19 to pharmacologically active metabolites. These drugs and their metabolites, along with desipramine and nortriptyline, undergo hydroxylation by CYP2D6 to less active metabolites. Evidence from published literature is presented for CYP2D6 and CYP2C19 genotype-directed dosing of tricyclic antidepressants.

We developed a sensitive and robust electrogenerated chemiluminescence (ECL) flow sensor based on Ru(bpy)(3) (2+) immobilized with a Nepem-211 perfluorinated ion exchange conductance membrane, which has robustness and stability under a wide range of chemical and physical conditions, good electrical conductivity, isotropy and a high exchange capacity for immobilization of Ru(bpy)(3) (2+) . The flow sensor has been used as a post-column detector in high-performance liquid chromatography for determination of erythromycin and clarithromycin in honey and pork, and tricyclic antidepressant drugs in human urine. Under optimal conditions, the linear ranges were 0.03-26 ng/μL and 0.01-1 ng/μL for macrolides and tricyclic antidepressant drugs, respectively. The detection limits were 0.02, 0.01, 0.01, 0.06 and 0.003 ng/μL for erythromycin, clarithromycin, doxepin, amitriptyline and clomipramine, respectively. There is no post-column reagent addition. In addition to the conservation expensive reagents, the experimental setup was simplified. The flow sensor was used for 2 years with high sensitivity and stability. Copyright © 2013 John Wiley & Sons, Ltd.

In the present work, electromembrane surrounded solid phase microextraction (EM-SPME) is introduced for the first time. The organic liquid membrane, which consists of 2-nitrophenyl octyl ether (NPOE), was immobilized in the pores of a hollow fiber (HF) and the basic analytes migrated in an electrical field from aqueous sample solution through the liquid membrane and into aqueous acceptor phase and then they were adsorbed on the solid sorbent, which acts as the cathode. Effective parameters such as composition of organic liquid membrane, pH of donor and acceptor phases, applied voltage and extraction time were optimized for extraction of amitriptyline (AMI) and doxepin (DOX) as model analytes and figures of merit of the method were investigated in pure water, human plasma, and urine samples. To extract the model analytes from 24 mL neutral sample solution across organic liquid membrane and into aqueous acceptor phase, 120 V electrical potential was applied for 20 min and finally the drugs were adsorbed on a carbonaceous cathode. Regardless of high sample cleanup, which make the proposed method suitable for the analysis of drugs from complicated matrices, extraction efficiencies in the range of 3.1-11.5% and good detection limits (less than 5 ngmL(-1)) with admissible repeatability and reproducibility (intra- and inter-assay precisions ranged between 4.0-8.5% and 7.5-12.2%, respectively) were obtained from different extraction media. Linearity of the method was studied in the range of 2.0-500.0 ngmL(-1) and 5.0-500.0 ngmL(-1) for AMI and DOX, respectively and coefficient of determination higher than 0.9947 were achieved. Finally, the proposed method was applied for the analysis of AMI and DOX in real samples.

A capillary electrophoretic (CE) method for screening of six tricyclic antidepressants: amitriptyline, nortriptyline, imipramine, desipramine, doxepin and nordoxepin, in human serum, has been developed. The drugs were separated in a bare fused silica capillary (50 microm i.d.) using the background electrolyte: 50 mM CAPSO (pH 9.54) in methanol/water with KCI addition. For increasing the method sensitivity, the sample concentration in the capillary (sample stacking) using pressure and electrokinetic injection has been applied. The standard addition method was used for calibration of the developed analytical procedure. The precision of the identification parameter (the relative migration time) and the quantitative parameter (the relative peak area) was within the range of 0.05-1.65 and 0.73-6.7 (RSD %), respectively. The detection limits were found to be 30 ng/mL for desipramine, 62.5 ng/mL for nortriptyline, and 50 ng/mL for remaining analytes.

Antidepressants have been considered by their analgesic activity in numerous studies, and specifically tricyclic antidepressants to possess the greatest efficacy. Imipramine and doxepin have been reported to exhibit local anaesthetic properties. In order to investigate their cutaneous analgesic effect after topical application a nanoemulsion vehicle was developed. This nanoemulsion is composed of propilenglicol, Transcutol, water, Labrasol, Plurol Oleique, isostearyl isostearate, oleic acid, and d-limonene. The final concentration of imipramine or doxepin in the nanoemulsion system was 3% (w/w). The nanoemulsions were characterized by pH, viscosity, droplet size, polydispersity index and finally, a morphological and structural examination was carried out by using transmission electron microscopy. Furthermore, the present work also reports stability studies on the nanoemulsion formulations to evaluate the integrity of the formulation; these indicate that formulations are stable for a period of three months. Moreover ex vivo studies were performed to evaluate permeation behaviour through human skin and predict plasma concentrations concluding that topically applied imipramine and doxepin loaded nanoemulsions were safe for a local effect. Similarly, the in vivo analgesic and anti-allodynic activity in rats was evaluated being stronger for the doxepin loaded nanoemulsion. This study demonstrated that nanoemulsion containing doxepin could be promising as an alternative analgesic therapy with a potential clinical application.

Pruritus and psyche are intricately and reciprocally related, with psychophysiological evidence and psychopathological explanations helping us to understand their complex association. Their interaction may be conceptualized and classified into 3 groups: pruritic diseases with psychiatric sequelae, pruritic diseases aggravated by psychosocial factors, and psychiatric disorders causing pruritus. Management of chronic pruritus is directed at treating the underlying causes and adopting a multidisciplinary approach to address the dermatologic, somatosensory, cognitive, and emotional aspects. Pharmcotherapeutic agents that are useful for chronic pruritus with comorbid depression and/or anxiety comprise selective serotonin reuptake inhibitors, mirtazapine, tricyclic antidepressants (amitriptyline and doxepin), and anticonvulsants (gabapentin, pregabalin); the role of neurokinin receptor-1 antagonists awaits verification. Antipsychotics are required for treating itch and formication associated with schizophrenia and delusion of parasitosis (including Morgellons disease).

Doxepin is a tricyclic antidepressant from the group of dibenzoxepines. Apart from the antidepressant effect, it has also the sedative and anxiolytic effect, so it is used in the treatment of anxiety disorder in the course of psychosis, organic diseases and alcoholism. Doxepin increases concentration of norepinephrine and serotonin in the brain by preventing inactivation and blocking their reuptake. In addition, the drug has an antagonistic effect on receptors in CNS (muscarinic M1, histamine H1, alpha-1adrenergic, serotonergic 5-HT2) and also blocks sodium and potassium channels in cardiomyocytes. In this paper we present a case of severe poisoning by various drugs, including doxepin. The dominating symptom was prolonged toxic coma. Slow return of consciousness was observed from the fifth day after intoxication, logical verbal contact with the patient was possible on the seventh day. It seems that this symptom results from the pharmacokinetic properties of doxepin as well as the high drug dose and a synergistic effect of lorazepam and phenobarbital. Transient anisokoria was an unusual symptom that appeared in the course of intoxication. The patient was examined by a neurologist and underwent CT of the head twice - no organic causes were revealed. This phenomenon can be explained by physiological anisokoria, caused by asymmetry in the sympathetic innervation of pupil rectractor muscle, exacerbated by a complete parasympathetic conduction block as a result of anticholinergic action of doxepin. Tricyclic antidepressants are often used by patients for the purpose of suicide. Because of the serious cardiological and neurological symptoms the course of doxepin intoxication may be severe.