RESUMEN
BACKGROUND: Given the importance of achieving optimal therapeutical concentration in patients treated with antidepressants, this study investigates a novel technique for the simultaneous determination of trazodone (TRZ) and doxepin (DOX) in human plasma and serum samples for the first time. RESULTS: To achieve simultaneous determination of two antidepressants, TRZ and DOX, a novel detection system was designed: a non-enzymatic voltammetric biosensor based on boron-reduced graphene oxide/manganese oxide nanoparticles (GCE/B-rGO/MnO NPs). The detection was accomplished after pre-concentration and extraction trace amounts of the analytes using the thin film-solid phase microextraction (TF-SPME) technique, which employed polyvinyl alcohol/polyvinyl acetate/copper oxide nanoparticles (PVA/PVAc/CuO NPs) electrospun nanofibers. The successful preparation of composite nanofibers and modified electrodes was confirmed using the evaluation of field emission-scanning electron microscopy (FE-SEM) and energy-dispersive X-ray spectroscopy (EDX). Also, the composite nanofibers were characterized with attenuated total reflectance-Fourier transform-infrared (ATR-FT-IR) and X-ray diffraction (XRD). In the solution of TRZ and DOX, under optimum experimental conditions, the linear dynamic ranges (LDRs) were 0.1-20.0 µmol L-1 and 0.5-27.0 µmol L-1, respectively. Also, the limit of detection (LOD) values of TRZ and DOX were 0.032 and 0.150 µmol L-1. SIGNIFICANCE: PVAc acts as a cross-linking agent for PVA, and their mixture is effective for sample preparation and pre-concentration of analytes in complex matrices. Also, adding CuO NPs to this polymeric mixture enhanced the adsorption efficiency. Taking advantage of the high surface area of MnO NPs and the high electrical conductivity of B-rGO, and considering the superiority of their simultaneous utilization, the constructed electrochemical biosensor is both cost-effective and rapid. It demonstrates excellent stability, repeatability, and sensitivity for the simultaneous determination of TRZ and DOX under optimal conditions. This biosensor, the first of its kind, is specifically designed for the simultaneous determination of TRZ and DOX in human plasma and serum samples, representing a significant advancement in biosensing technology.
Asunto(s)
Técnicas Biosensibles , Doxepina , Técnicas Electroquímicas , Grafito , Trazodona , Humanos , Doxepina/sangre , Doxepina/aislamiento & purificación , Doxepina/química , Doxepina/análisis , Grafito/química , Técnicas Biosensibles/métodos , Trazodona/sangre , Trazodona/análisis , Trazodona/aislamiento & purificación , Trazodona/química , Óxidos/química , Compuestos de Manganeso/química , Microextracción en Fase Sólida/métodos , Límite de Detección , Cobre/química , Cobre/sangre , AdsorciónRESUMEN
Doxepin is an antihistamine and tricyclic antidepressant that binds to the histamine H1 receptor (H1R) 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 H1R/doxepin complex, the hydroxyl group of Thr1123.37 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 H1R protein complexed with doxepin. The ratio of the E- and Z-isomers bound to wild-type (WT) H1R was 55:45, indicating that the Z-isomer was bound to WT H1R with an approximately 5.2-fold higher affinity than the E-isomer. For the T1123.37V 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 T1123.37V. Furthermore, MD simulations revealed that the hydroxyl group of T1123.37 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 T1123.37 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.
Asunto(s)
Doxepina , Simulación de Dinámica Molecular , Unión Proteica , Receptores Histamínicos H1 , Doxepina/química , Doxepina/metabolismo , Receptores Histamínicos H1/química , Receptores Histamínicos H1/metabolismo , Humanos , Ligandos , Enlace de Hidrógeno , Isomerismo , Sitios de Unión , TermodinámicaRESUMEN
The histamine H1 receptor (H1R) 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 H1 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 H1R 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.
Asunto(s)
Doxepina , Histamina , Sitios de Unión , Doxepina/química , Doxepina/metabolismo , Histamina/metabolismo , Antagonistas de los Receptores Histamínicos H1 , Humanos , Iones , Receptores Acoplados a Proteínas G/metabolismo , Receptores Histamínicos/metabolismo , Receptores Histamínicos H1/metabolismoRESUMEN
The aim of this study was to establish a high-throughput and sensitive LC-MS/MS method for the determination of doxepin and its major active metabolite nordoxepin in human plasma. It has been designed for bioequivalence study for formulations containing 25 mg of doxepin. Doxepin and nordoxepin were extracted from human plasma samples by protein precipitation with acetonitrile by using protein precipitation 96-well plates. The analyte was separated using a Phenomenex Kinetex Biphenyl column (100 × 2.1 mm, 2.6 µm) using isocratic elution with a mobile phase of 20 mM ammonium formate (30%) and acetonitrile:methanol 3:7 v:v (70%) at a flow rate of 0.5 mL/min and an injection volume of 10 µL. The detection was performed using a triple quadrupole mass spectrometer by multiple reaction monitoring mode to monitor the precursor-to-product ion transitions of m/z 280.4 â 107.0 and 283.4 â 235.0 for doxepin and doxepin-D3, respectively, and 266.3 â 106.9 and 269.3 â 235.0 for nordoxepin and nordoxepin-D3, respectively, in positive electrospray ionization mode. The total run time was 3.5 min. The method was validated over a concentration range of 50-10,000 pg/mL using a Triple Quad 4500 MS System (Sciex) for both analytes. The developed and validated method can be successfully used to study the bioequivalence/pharmacokinetics of doxepin and nordoxepin.
Asunto(s)
Cromatografía Liquida/métodos , Doxepina/análogos & derivados , Doxepina/sangre , Espectrometría de Masas en Tándem/métodos , Precipitación Química , Doxepina/química , Doxepina/aislamiento & purificación , Estabilidad de Medicamentos , Ensayos Analíticos de Alto Rendimiento , Humanos , Modelos Lineales , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Binding of histamine to the G-protein coupled histamine H1 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 Asp1073.32, Tyr1083.33, Thr1945.43, Asn1985.46, Trp4286.48, Tyr4316.51, Phe4326.52, and Phe4356.55, 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 Asp1073.32 for histamine binding.
Asunto(s)
Histamina/química , Simulación de Dinámica Molecular , Dominios Proteicos , Receptores Histamínicos H1/química , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Doxepina/química , Doxepina/metabolismo , Histamina/metabolismo , Antagonistas de los Receptores Histamínicos/química , Antagonistas de los Receptores Histamínicos/metabolismo , Humanos , Ligandos , Estructura Molecular , Mutación , Unión Proteica , Receptores Histamínicos H1/genética , Receptores Histamínicos H1/metabolismoRESUMEN
The reversed-phase chromatographic behaviour of six tricyclic antidepressants (amitryptiline, clomipramine, doxepin, imipramine, nortryptiline and maprotiline) was examined in this work with acetonitrile-water mobile phases, in the absence and presence of the ionic liquids 1-hexyl-3-methylimidazolium chloride and 1-hexyl-3-methylimidazolium tetrafluoroborate, which have interesting features for the separation of basic compounds, in terms of peak shape combined with reduced retention. Tricyclic antidepressants are low polarity drugs that strongly associate to the alkyl chains of conventional stationary phases. They are also positively charged in the usual working pH range (2-8) in reversed-phase liquid chromatography, due to their strong basic character. In consequence, they may interact with the residual ionised silanols present in conventional silica-based stationary phases, which is translated in stronger retention, and tailed and broad peaks. A simple chromatographic procedure for the control of tricyclic antidepressants in pharmaceutical formulations was developed using a C8 column and a mobile phase containing 30% acetonitrile/10 mM 1-hexyl-3-methylimidazolium chloride at pH 3, with UV detection. Intra- and inter-day precisions were usually below +1.0%, and intra- and inter-day bias (trueness) ranged between â2.1% and +2.4%, and between â3.0% and +2.3%, respectively. Sample preparation was simple and only required solubilisation and filtration previous to injection.
Asunto(s)
Antidepresivos Tricíclicos/química , Boratos/química , Imidazoles/química , Líquidos Iónicos/química , Amitriptilina/análisis , Amitriptilina/química , Antidepresivos Tricíclicos/análisis , Cromatografía de Fase Inversa/métodos , Doxepina/análisis , Doxepina/química , Composición de Medicamentos , Límite de Detección , Nortriptilina/análisis , Nortriptilina/química , Espectrofotometría UltravioletaRESUMEN
The main purpose of this study was to develop a semisolid mucoadhesive formulation for the non-invasive vaginal administration of doxepin (DOX) for relief of pain derived from the scarring process after surgery. An orafix® platform loading DOX was tested for adequate stability, rheology and vaginal mucoadhesion capacity. The formulation exhibited appropriate pH and was microbiologically stable. The rheological studies confirmed its pseudoplastic and thixotropic nature with prevalence of the elastic behavior component over the viscous one. Appropriate syringeability and spreadability results were also confirmed. Different experiments showed adequate mucoadhesion capacity even in the presence of simulated vaginal fluid. Finally, DOX release, permeation and retention in vaginal mucosa studies were also accomplished with promising results. DOX release kinetics followed the modified Higuchi model and the permeation studies did not render such high values as to suggest potential systemic absorption which could lead to undesirable systemic side effects. Therefore, we can hypostatize that the proposed formulation may assist to fill in the therapeutic gap regarding pure pain relief at local level in vagina.
Asunto(s)
Analgésicos/administración & dosificación , Doxepina/administración & dosificación , Sistemas de Liberación de Medicamentos , Adhesividad , Administración Intravaginal , Analgésicos/química , Animales , Doxepina/química , Liberación de Fármacos , Estabilidad de Medicamentos , Femenino , Membrana Mucosa/química , Dolor/tratamiento farmacológico , Reología , Porcinos , Vagina , ViscosidadRESUMEN
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.
Asunto(s)
Doxepina/metabolismo , Neoplasias de Cabeza y Cuello/metabolismo , Mucosa Bucal/metabolismo , Antisépticos Bucales/metabolismo , Dolor/metabolismo , Estomatitis/metabolismo , Animales , Doxepina/administración & dosificación , Doxepina/química , Femenino , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Antagonistas de los Receptores Histamínicos/administración & dosificación , Antagonistas de los Receptores Histamínicos/química , Antagonistas de los Receptores Histamínicos/metabolismo , Masculino , Mucosa Bucal/efectos de los fármacos , Antisépticos Bucales/administración & dosificación , Antisépticos Bucales/química , Técnicas de Cultivo de Órganos , Dolor/tratamiento farmacológico , Estomatitis/tratamiento farmacológico , Porcinos , Resultado del TratamientoRESUMEN
The crystal structure of the human histamine H1 receptor (H1R) has been determined in complex with its inverse agonist doxepin, a first-generation antihistamine. The crystal structure showed that doxepin sits deeply inside the ligand-binding pocket and predominantly interacts with residues highly conserved among other aminergic receptors. This binding mode is considered to result in the low selectivity of the first-generation antihistamines for H1R. The crystal structure also revealed the mechanism of receptor inactivation by the inverse agonist doxepin. On the other hand, the crystal structure elucidated the anion-binding site near the extracellular portion of the receptor. This site consists of residues not conserved among other aminergic receptors, which are specific for H1R. Docking simulation and biochemical experimentation demonstrated that a carboxyl group on the second-generation antihistamines interacts with the anion-binding site. These results imply that the anion-binding site is a key site for the development of highly selective antihistamine drugs.
Asunto(s)
Receptores Histamínicos H1/química , Animales , Sitios de Unión/fisiología , Doxepina/química , Doxepina/farmacología , Histamina/química , Histamina/metabolismo , Antagonistas de los Receptores Histamínicos/química , Antagonistas de los Receptores Histamínicos/farmacología , Antagonistas de los Receptores Histamínicos H1/química , Antagonistas de los Receptores Histamínicos H1/metabolismo , Humanos , Unión Proteica/efectos de los fármacos , Receptores Histamínicos H1/metabolismoRESUMEN
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.
Asunto(s)
ADN/metabolismo , Dotiepina/química , Dotiepina/metabolismo , Doxepina/química , Doxepina/metabolismo , Albúmina Sérica Bovina/metabolismo , Animales , ADN/química , Electroquímica , Simulación del Acoplamiento Molecular , Oxidación-Reducción , Espectrometría de FluorescenciaRESUMEN
BACKGROUND: Excipients in various formulations of active drugs occasionally include known contact allergens. Their ingestion may trigger dermatitis or cause it to become widespread or refractory to therapy. OBJECTIVE: The aim of this study was to investigate the prevalence of common contact allergens among the excipients of oral antihistamines available in this country. METHODS: We gathered the complete ingredient lists of 2119 different preparations of 12 oral antihistamines from the National Library of Medicine data bank and entered them into an electronic database for analysis. RESULTS: More than half the formulations (55.0%) contained at least 1 member of the 10 allergen families assessed. Most brompheniramine and doxepin preparations included potentially allergenic excipients, whereas fexofenadine was most often free of them. Sorbitan group members, azo dyes, and propylene glycol were the allergens found most frequently in the antihistamines, each present in over 25% of the products. Elixirs, liquids, solutions, suspensions, and syrups were more likely than nonchewable caplets, capsules, and tablets to contain the allergens tabulated (100% vs 39.3%, respectively). Chewable pills frequently contained azo dyes. CONCLUSIONS: Ingestion of antihistamines could precipitate a systemic contact dermatitis in a patient sensitized to an allergen present as an excipient in the medicine.
Asunto(s)
Alérgenos/análisis , Excipientes/análisis , Antagonistas de los Receptores Histamínicos/química , Administración Oral , Compuestos Azo/análisis , Bromofeniramina/química , Química Farmacéutica , Colorantes/análisis , Bases de Datos Farmacéuticas , Dermatitis Alérgica por Contacto/etiología , Formas de Dosificación , Doxepina/química , Antagonistas de los Receptores Histamínicos/administración & dosificación , Medicamentos sin Prescripción/química , Polisorbatos/análisis , Medicamentos bajo Prescripción/química , Propilenglicol/análisis , Terfenadina/análogos & derivados , Terfenadina/químicaRESUMEN
Examination of the stability of clonazepam, diazepam, alprazolam, haloperidol, and doxepin in basic solutions was performed, together with an assessment of the kinetic (k, t0.1i t0.5) and thermodynamic (Ea, ΔH(++)i ΔS(++)) stability-indicating parameters, which were compared with the lipophilicity (logP) of the studied drugs. It was observed that the calculated values of Ea, ΔH(++) and ΔS(++) for the studied drugs increased from 41.04 kJ/mol to 125.50 kJ/mol, from 37.82 kJ/mol to 122.24 kJ/mol and from -167.09 J/Kmol to 53.02 J/Kmol, respectively, along with an increase of lipophilicity (logP) from 2.12 to 4.30 for the most hydrophilic alprazolam to the most lipophilic haloperidol. The degradation products were identified using UPLC/MS/MS method.
Asunto(s)
Alprazolam/química , Clonazepam/química , Diazepam/química , Doxepina/química , Haloperidol/química , Estabilidad de Medicamentos , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , TermodinámicaRESUMEN
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.
Asunto(s)
Cromatografía de Gases/métodos , Membranas Artificiales , Modelos Químicos , Microextracción en Fase Sólida/métodos , Amitriptilina/sangre , Amitriptilina/química , Amitriptilina/aislamiento & purificación , Amitriptilina/orina , Doxepina/sangre , Doxepina/química , Doxepina/aislamiento & purificación , Doxepina/orina , Éteres/química , Humanos , Concentración de Iones de Hidrógeno , Límite de Detección , Modelos Lineales , Reproducibilidad de los ResultadosRESUMEN
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.
Asunto(s)
Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Receptores Acoplados a Proteínas G/química , Receptores Histamínicos H1/química , Receptores Histamínicos/química , Doxepina/química , Doxepina/metabolismo , Histamina/química , Histamina/metabolismo , Humanos , Enlace de Hidrógeno , Ligandos , Unión Proteica , Conformación Proteica , Receptores Acoplados a Proteínas G/metabolismo , Receptores Histamínicos/metabolismo , Receptores Histamínicos H1/metabolismo , Receptores Histamínicos H4RESUMEN
The capillary isotachophoresis (cITP) separation of the isomers of the tricyclic antidepressant doxepin using ß-cyclodextrin (ß-CD) as a buffer additive is investigated by online microcoil NMR detection. Capillary electrophoresis (CE) is also used to determine the binding constant between the doxepin E and Z geometric isomers and ß-CD. Although the doxepin isomers could be easily baseline resolved by CE, their separation by cITP was more challenging due in part to the high concentration of doxepin after cITP-focusing. The use of online (1)H NMR detection allows observation of changes in doxepin dynamics due to formation of the ß-CD inclusion complex, changes in the fraction complexed and the intracapillary pH. It also provides novel experimental evidence that a weak complex between ß-CD and acetate contributes to its active transport from the leading electrolyte through the sample band to the trailing electrolyte in this cationic cITP separation. The results of these cITP-NMR experiments provide new mechanistic details about the interactions of the buffer counterion acetate with various components of the separation system and have important implications for other analyses based on formation of cyclodextrin inclusion complexes.
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Acetatos/química , Doxepina/química , Doxepina/aislamiento & purificación , Electroforesis Capilar/métodos , Isotacoforesis/métodos , beta-Ciclodextrinas/química , Antidepresivos/química , Antidepresivos/aislamiento & purificación , Tampones (Química) , Espectroscopía de Resonancia Magnética , Movimiento (Física) , EstereoisomerismoRESUMEN
OBJECTIVES: (11)C-Doxepin is an established positron emission tomography (PET) probe for imaging the histamine H1 receptor, which is associated with various neurological disorders and allergic diseases. A fully automated current Good Manufacturing Practices (cGMP)-compliant radiosynthesis is therefore desirable in order to facilitate clinical PET studies. We report here a fully automated production method for (11)C-doxepin using a multipurpose PET module for clinical use. METHODS: (11)C-Doxepin was radiosynthesized by N-[(11)C]methylation of nordoxepin using [(11)C]methyl iodide in DMF solvent, and then purified by HPLC, and finally reformulated with solid phase extraction (SPE) using a cGMP-compliant automated multipurpose PET module developed in house. The final product was analyzed and subjected to quality control according to current US Pharmacopeia requirements. RESULTS: The radiochemical yield (decay corrected) of (11)C-doxepin for clinical use was 47.0 ± 5.2% (n = 12) based on [(11)C]methyl iodide, moreover the radiochemical purity of (11)C-doxepin was more than 97.5% with 1,200 ± 500 Ci/mmol specific activity(end of production). The total production time of (11)C-doxepin was 37 min from end of bombardment (EOB) with the final product passing all tests under cGMP requirements for clinical use. CONCLUSIONS: A simplified and reliable fully automated production of (11) C-doxepin for clinical use was developed, allowing the synthesis of the tracer with high yield using a cGMP-compliant module and procedure. The success of this approach could make the PET tracer (11) C-doxepin more accessible for clinical studies.
Asunto(s)
Doxepina/síntesis química , Tomografía de Emisión de Positrones/métodos , Receptores Histamínicos H1/metabolismo , Automatización , Radioisótopos de Carbono , Cromatografía Líquida de Alta Presión , Doxepina/química , Humanos , Control de CalidadRESUMEN
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.
Asunto(s)
Doxepina/metabolismo , Antagonistas de los Receptores Histamínicos/metabolismo , Receptores Histamínicos H1/química , Receptores Histamínicos H1/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Doxepina/química , Antagonistas de los Receptores Histamínicos/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Isomerismo , Ligandos , Modelos Moleculares , Fosfatos/química , Fosfatos/metabolismo , Unión Proteica , Conformación Proteica , Receptores Adrenérgicos beta 2/química , Receptores de Dopamina D3/química , Especificidad por SustratoRESUMEN
Using an isocratic high-performance liquid chromatography (HPLC) system and UV detection, a simple and precise analytical procedure was developed to quantify levels of the CB(1) receptor antagonist rimonabant in the plasma of rhesus monkeys. Rimonabant was extracted from plasma samples into 5% isopropanol in hexane. After separation, the isopropanol-hexane fractions were dried to residue, redissolved in mobile phase, and then injected into the HPLC. The HPLC system included an acetonitrile-phosphate buffer (62:38, v/v) mobile phase (pH 6.7), flow rate of 1.5 mL/min, C(18) column (4.6 mm i.d. x 150 mm length, 5 microm), and UV detection at 280 nm. Retention times for rimonabant and doxepin (internal standard) were 9.9 and 2.4 min, respectively. The regression of the spiked calibrator curve was linear from 60 to 4000 ng/mL (r(2) = 0.996). The lower limit of quantification was 60 ng/mL, and recovery was 83.6%. Rimonabant was stable in stock solutions and monkey plasma across a range of temperatures and concentrations. To demonstrate utility, plasma rimonabant was measured in six rhesus monkeys at 60 and 240 min after intramuscular administration of 1 mg/kg rimonabant. Rimonabant levels ranged from 175 to 1290 ng/mL. The analytical assay described here provides a simple and accurate procedure for multiple within-subject measurements of the CB(1) antagonist rimonabant.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Macaca mulatta/sangre , Piperidinas/sangre , Pirazoles/sangre , Espectrofotometría Ultravioleta/métodos , Animales , Doxepina/análisis , Doxepina/química , Femenino , Modelos Lineales , Masculino , Piperidinas/química , Pirazoles/química , Reproducibilidad de los Resultados , Rimonabant , Sensibilidad y EspecificidadRESUMEN
Perhexiline is a drug that is used for treatment of moderate to severe angina pectoris that has not responded to other treatment. It has a low therapeutic index, and saturable metabolism that is also subject to genetic polymorphism (CYP2D6). Concentration monitoring of the parent drug and its major metabolite is considered necessary to optimise efficacy and reduce the risk of hepatotoxicity and neuropathy. A rapid, simple and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) assay was developed for the determination of perhexiline and its metabolite cis-hydroxyperhexiline in human plasma. After proteins were precipitated with acetonitrile, perhexiline, the major metabolite cis-hydroxyperhexiline and nordoxepin as the internal standard were resolved on a phenyl-hexyl column using gradient elution of 0.05% formic acid and methanol. The three compounds were detected using electrospray ionisation in the positive mode. Standard curves were linear over the concentration range 10-2000microg/L (r>0.999), bias was Asunto(s)
Cromatografía Liquida/métodos
, Perhexilina/análogos & derivados
, Perhexilina/sangre
, Espectrometría de Masas en Tándem/métodos
, Doxepina/análogos & derivados
, Doxepina/química
, Humanos
, Modelos Lineales
, Reproducibilidad de los Resultados
, Sensibilidad y Especificidad
RESUMEN
BACKGROUND: Doxepin is a tricyclic compound that has been used extensively for the treatment of depressive and anxiety disorders for approximately thirty years. It was noted early to have sedative effects and assist with the improvement of disrupted sleep patterns, but in higher antidepressant doses it was also noted to have significant anticholinergic and antinoradrenergic properties. These properties led to significant dose-limiting side effects, which at times precluded its effective use. Recently, doxepin has seen renewed interest in low doses as an H1 specific antagonist in sleep disorders. OBJECTIVE: The review seeks systematically to examine currently published data on the use of doxepin for the treatment of insomnia, and its pharmacological basis. METHODS: Medline articles showing from a search of 'doxepin and insomnia' were included in the review. RESULTS/CONCLUSION: Currently available data support the use of low-dose doxepin as preferential H1 antagonist for the treatment of primary insomnia. There are likely preferential effects upon sleep maintenance insomnia compared with sleep initiation given the role of histamine in the sleep-wake cycle.