Formulation development and evaluation of nasal in situ gel of promethazine hydrochloride.

OBJECTIVE: The present work aims to develop mucoadhesive thermosensitive nasal in situ gel for Promethazine hydrochloride using quality by design (QbD) approach. It can reduce nasal mucociliary clearance (MCC) and increase residence of the drug on nasal mucosa. This might increase drug absorption to improve bioavailability of the drug as compared to oral dosage form. SIGNIFICANCE: Promethazine hydrochloride is an antiemetic drug administered by oral, parenteral and rectal routes. These routes have poor patient compliance or low bioavailability. Nasal route is a better alternative as it has large surface area, high drug absorption rate and no first pass effect. Its only limitation is short drug retention time due to MCC. By formulating a mucoadhesive in situ gel, the MCC can be reduced, and drug absorption will be prolonged. Thus, improving bioavailability. METHOD: In-situ gel was prepared by cold method having material attributes as concentration of Poloxamer 407 (X1) as gelling agent and hydroxypropyl methyl cellulose K4M (X2) as mucoadhesive agent. Critical Quality Attributes (CQA) were gelation temperature, mucoadhesive force and ex-vivo diffusion. Central composite design (CCD) was adopted for optimization. RESULT: Optimized formulation satisfied all the CQA significant for nasal administration. Moreover, the formulation was found to be stable in accelerated stability studies for 3 months. CONCLUSION: It can be concluded that since the drug can easily permeate through nasal mucosa and can gain access directly in the brain without undergoing first pass metabolism along with increased residence due to mucoadhesion, mucoadhesive in situ gel has potential to increase drug bioavailability.

Promethazine inhibits efflux, enhances antifungal susceptibility and disrupts biofilm structure and functioning in Trichosporon.

Trichosporon spp. are emerging opportunistic fungi associated with invasive infections, especially in patients with haematological malignancies. The present study investigated the in vitro inhibition of efflux pumps by promethazine (PMZ) as a strategy to control T. asahii and T. inkin. Planktonic cells were evaluated for antifungal susceptibility to PMZ, as well as inhibition of efflux. The effect of PMZ was also studied in Trichosporon biofilms. PMZ inhibited T. asahii and T. inkin planktonic cells at concentrations ranging from 32 to 256 µg ml-1. Subinhibitory concentrations of PMZ inhibited efflux activity in Trichosporon. Biofilms were completely eradicated by PMZ. PMZ potentiated the action of antifungals, affected the morphology, changed the amount of carbohydrates and proteins and reduced the amount of persister cells inside biofilms. The results showed indirect evidences of the occurrence of efflux pumps in Trichosporon and opens a perspective for the use of this target in the control of trichosporonosis.

LYTIC COCKTAIL ATTENUATES CATECHOLAMINE SURGE AFTER SEVERE BURNS BY BLOCKING HISTAMINE H1 RECEPTOR/PKA/CREB/TYROSINE HYDROXYLASE SIGNALING IN CHROMAFFIN CELLS.

ABSTRACT: Severe burns develop a catecholamine surge, inducing severe damage to the organism, raising the possibility of multisystem organ failure, and even death. The mechanisms of catecholamine surge have not been fully elucidated, and few strategies are generally acceptable to reduce catecholamine surge postburn. Thus, it is valuable to investigate the underlying mechanisms of catecholamine surge postburn to develop targeted interventions to attenuate it. We have found that the lytic cocktail alleviates the surge of catecholamine and organ injury after severe burn; however, the underlying mechanisms were still unclear. Moreover, the lytic cocktail has side effects, such as significant arterial hypotension and breathing depression, limiting its clinical application. This study aims to investigate the therapeutic mechanism of the lytic cocktail in regulating catecholamine levels postburn. We find that promethazine, a classic histamine H1 receptor blocker and a component of the lytic cocktail, can effectively reduce catecholamine surge and organ injury postburn. Our study confirms that blood histamine levels increase after severe burns. We find that histamine can amplify the catecholamine surge by elevating tyrosine hydroxylase expression and catecholamine synthesis in chromaffin cells through the histamine H1 receptor/Protein Kinase A /cAMP-response element binding protein signaling pathway. In summary, for the first time, we find that histamine plays a vital role in catecholamine surge postburn. We also confirm that the lytic cocktail effectively alleviates catecholamine surge and organ injury postburn through promethazine.

Promethazine Downregulates Wnt/ß-Catenin Signaling and Increases the Biomechanical Forces of the Injured Achilles Tendon in the Early Stage of Healing.

BACKGROUND: Wnt/ß-catenin signaling suppresses the differentiation of cultured tenocytes, but its roles in tendon repair remain mostly elusive. No chemical compounds are currently available to treat tendon injury. HYPOTHESIS: We hypothesized that the inhibition of Wnt/ß-catenin signaling would accelerate tendon healing. STUDY DESIGN: Controlled laboratory study. METHODS: Tendon-derived cells (TDCs) were isolated from rat Achilles tendons. The right Achilles tendon was injured via a dermal punch, while the left tendon was sham operated. A Wnt/ß-catenin inhibitor, IWR-1, and an antihistamine agent, promethazine (PH), were locally and intramuscularly injected, respectively, for 2 weeks after surgery. The healing tendons were histologically and biomechanically evaluated. RESULTS: The amount of ß-catenin protein was increased in the injured tendons from postoperative weeks 0.5 to 2. Inhibition of Wnt/ß-catenin signaling by IWR-1 in healing tendons improved the histological abnormalities and decreased ß-catenin, but it compromised the biomechanical properties. As we previously reported that antihistamine agents suppressed Wnt/ß-catenin signaling in human chondrosarcoma cells, we examined the effects of antihistamines on TDCs. We found that a first-generation antihistamine agent, PH, increased the expression of the tendon marker genes Mkx and Tnmd in TDCs. Intramuscular injection of PH did not improve histological abnormalities, but it decreased ß-catenin in healing tendons and increased the peak force and stiffness of the healing tendons on postoperative week 2. On postoperative week 8, however, the biomechanical properties of vehicle-treated tendons became similar to those of PH-treated tendons. CONCLUSION: IWR-1 and PH suppressed Wnt/ß-catenin signaling and improved the histological abnormalities of healing tendons. IWR-1, however, compromised the biomechanical properties of healing tendons, whereas PH improved them. CLINICAL RELEVANCE: PH is a candidate repositioned drug that potentially accelerates tendon repair.

Biophysical investigation of promethazine hydrochloride binding with micelles of biocompatible gemini surfactants: Combination of spectroscopic and electrochemical analysis.

Knowledge of binding parameters for drug and surfactant complexations is crucially vital in order to design effective drug carrier systems with requisite features. To this end, this work was designed to demonstrate the biophysical characterization of the interaction of a phenothiazine drug promethazine hydrochloride (PMT) with relatively lower cytotoxic and easily degradable biomimetic micellar self-assemblies of oxy-diester functionalized gemini surfactants (Cm-E2O-Cm, m = 12, 14 and 16), possessing different hydrophobic character. The binding propensity of Cm-E2O-Cm increases upon increasing the hydrophobic tail length as manifested through both intrinsic fluorescence and absorption spectral profiles of PMT ̶ Cm-E2O-Cm, showing 1:1 stoichiometry. Ksv values also follow the trend of increasing hydrophobic character (i.e., C12-E2O-C12 < C14-E2O-C14 < C16-E2O-C16). Moreover, the determined thermodynamic parameters, particularly the positive values of ΔHbo and ΔSbo, reveal that the involved complexations are dominated by the hydrophobic interactions. In addition, micropolarity assay was done to deduce the microenvironmental changes upon PMT ̶ Cm-E2O-Cm complexations. Beside this, comparative appraisal of all the three systems helps to underpin a reasonable knowledge of the effect of structural variation of surfactants on their binding ability with drug which, in turn, may also open new avenues for the designing of potential tunable drug carrier systems.

Interaction of promethazine and adiphenine to human hemoglobin: A comparative spectroscopic and computational analysis.

The binding nature of amphiphilic drugs viz. promethazine hydrochloride (PMT) and adiphenine hydrochloride (ADP), with human hemoglobin (Hb) was unraveled by fluorescence, absorbance, time resolved fluorescence, fluorescence resonance energy transfer (FRET) and circular dichroism (CD) spectral techniques in combination with molecular docking and molecular dynamic simulation methods. The steady state fluorescence spectra indicated that both PMT and ADP quenches the fluorescence of Hb through static quenching mechanism which was further confirmed by time resolved fluorescence spectra. The UV-Vis spectroscopy suggested ground state complex formation. The activation energy (Ea) was observed more in the case of Hb-ADP than Hb-PMT interaction system. The FRET result indicates the high probability of energy transfer from ß Trp37 residue of Hb to the PMT (r=2.02nm) and ADP (r=2.33nm). The thermodynamic data reveal that binding of PMT with Hb are exothermic in nature involving hydrogen bonding and van der Waal interaction whereas in the case of ADP hydrophobic forces play the major role and binding process is endothermic in nature. The CD results show that both PMT and ADP, induced secondary structural changes of Hb and unfold the protein by losing a large helical content while the effect is more pronounced with ADP. Additionally, we also utilized computational approaches for deep insight into the binding of these drugs with Hb and the results are well matched with our experimental results.

In vitro effects of promethazine on cell morphology and structure and mitochondrial activity of azole-resistant Candida tropicalis.

The aim of this study was to evaluate the effect of promethazine on the antifungal minimum inhibitory concentrations against planktonic cells and mature biofilms of Candida tropicalis, as well as investigate its potential mechanisms of cell damage against this yeast species. Three C. tropicalis isolates (two azole-resistant and one azole-susceptible) were evaluated for their planktonic and biofilm susceptibility to promethazine alone and in combination with itraconazole, fluconazole, voriconazole, amphotericin B, and caspofungin. The antifungal activity of promethazine against C. tropicalis was investigated by performing time-kill curve assays and assessing rhodamine 6G efflux, cell size/granularity, membrane integrity, and mitochondrial transmembrane potential, through flow cytometry. Promethazine showed antifungal activity against planktonic cells and biofilms at concentrations of 64 and 128 µg/ml, respectively. The addition of two subinhibitory concentrations of promethazine reduced the antifungal MICs for all tested azole drugs against planktonic growth, reversing the resistance phenotype to all azoles. Promethazine decreased the efflux of rhodamine 6G in an azole-resistant strain. Moreover, promethazine decreased cell size/granularity and caused membrane damage, and mitochondrial membrane depolarization. In conclusion, promethazine presented synergy with azole antifungals against resistant C. tropicalis and exhibited in vitro cytotoxicity against C. tropicalis, altering cell size/granularity, membrane integrity, and mitochondrial function, demonstrating potential mechanisms of cell damage against this yeast species.

Studies on drug metabolism by fungi colonizing decomposing human cadavers. Part II: biotransformation of five model drugs by fungi isolated from post-mortem material.

The present study investigated the in vitro metabolic capacity of 28 fungal strains isolated from post-mortem material towards five model drugs: amitriptyline, metoprolol, mirtazapine, promethazine, and zolpidem. Each fungal strain was incubated at 25 °C for up to 120 h with each of the five models drugs. Cunninghamella elegans was used as positive control. Aliquots of the incubation mixture were centrifuged and 50 µL of the supernatants were diluted and directly analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with product ion scanning. The remaining mixture was analyzed by full scan gas chromatography-mass spectrometry (GC-MS) after liquid-liquid extraction and acetylation. The metabolic activity was evaluated through the total number of detected metabolites (NDM) produced in each model and fungal strains and the percentage of parent drug remaining (%RPD) after up to five days of incubation. All the tested fungal strains were capable of forming mammalian phase I metabolites. Fungi from the normal fungal flora of the human body such as Candida sp., Geotrichum candidum, and Trichosporon asahii) formed up to seven metabolites at %RPD values greater than 52% but no new fungal metabolites (NFM). In contrast, some airborne fungal strains like Bjerkandera adusta, Chaetomium sp, Coriolopsis sp., Fusarium solani and Mucor plumbeus showed NDM values exceeding those of the positive control, complete metabolism of the parent drug in some models and formation of NFM. NFM (numbers in brackets) were detected in four of the five model drugs: amitriptyline (18), metoprolol (4), mirtazapine (8), and zolpidem (2). The latter NFM are potential candidates for marker substances indicating post-mortem fungal metabolism.

Development and characterization of a small electromembrane extraction probe coupled with mass spectrometry for real-time and online monitoring of in vitro drug metabolism.

A small and very simple electromembrane extraction probe (EME-probe) was developed and coupled directly to electrospray ionization mass spectrometry (ESI-MS), and this system was used to monitor in real time in vitro metabolism by rat liver microsomes of drug substances from a small reaction (incubation) chamber (37 °C). The drug-related substances were continuously extracted from the 1.0 mL metabolic reaction mixture and into the EME-probe by an electrical potential of 2.5 V. The extraction probe consisted of a 1-mm long and 350-µm ID thin supported liquid membrane (SLM) of 2-nitrophenyl octyl ether. The drugs and formed metabolites where extracted through the SLM and directly into a 3 µL min(-1) flow of 60 mM HCOOH inside the probe serving as the acceptor solution. The acceptor solution was directed into the ESI-MS-system, and the MS continuously monitored the drug-related substances extracted by the EME-probe. The extraction efficiency of the EME-probe was dependant on the applied electrical potential and the length of the SLM, and these parameters as well as the volume of the reaction chamber were set to the values mentioned above to avoid serious depletion from the reaction chamber (soft extraction). Soft extraction was mandatory in order not to affect the reaction kinetics by sample composition changes induced by the EME-probe. The EME-probe/MS-system was used to establish kinetic profiles for the in vitro metabolism of promethazine, amitriptyline and imipramine as model substances.

Interactions of phenothiazine drugs with bile salts: micellization and binding studies.

An evaluation of the interactions of phenothiazine tranquilizer drugs (promazine hydrochloride; PMZ and promethazine hydrochloride; PMT) with bile salts viz., sodium cholate (NaC) and sodium deoxycholate (NaDC) in aqueous medium, investigated through different physicochemical measurements is presented in this work. The mixed micellization behavior and surface properties of the phenothiazine-bile salt systems have been analyzed by conductivity and surface tension measurements. Application of different theoretical approaches to all the phenothiazine-bile salt mixtures shows a non-ideal behavior. Further, the spectroscopic techniques such as UV-visible and steady state fluorescence have been employed to study the binding of phenothiazines with bile salts. The stoichiometric ratios, binding constants (K), and free energy change (ΔG) for the phenothiazine-bile salt complexes were estimated from the Benesi-Hildebrand (B-H) double reciprocal plots obtained by using the changes in spectral intensities of phenothiazines on addition of bile salts. The results are discussed in the light of use of bile salts as promising drug delivery agents for phenothiazines and hence improve their bioavailabilty.

Studies on the metabolism of five model drugs by fungi colonizing cadavers using LC-ESI-MS/MS and GC-MS analysis.

It is well-known that cadavers may be colonized by microorganisms, but there is limited information if or to what extent these microbes are capable of metabolizing drugs or poisons, changing the concentrations and metabolic pattern of such compounds in postmortem samples. The aim of the present study was to develop a fungal biotransformation system as an in vitro model to investigate potential postmortem metabolism by fungi. Five model drugs (amitriptyline, metoprolol, mirtazapine, promethazine, and zolpidem) were each incubated with five model fungi known to colonize cadavers (Absidia repens, Aspergillus repens, Aspergillus terreus, Gliocladium viride, and Mortierella polycephala) and with Cunninghamella elegans (positive control). Incubations were performed in Sabouraud medium at 25 °C for 5 days. After centrifugation, a part of the supernatants was analyzed by liquid chromatography-tandem mass spectrometry with product ion scanning. Another part was analyzed by full scan gas chromatography-mass spectrometry after extraction and derivatization. All model drugs were metabolized by the control fungus resulting in two (metoprolol) to ten (amitriptyline) metabolites. Of the model fungi, only Abs. repens and M. polycephala metabolized the model drugs: amitriptyline was metabolized to six and five, metoprolol to two and two, mirtazapine to five and three, promethazine to six and nine, and zolpidem to three and four metabolites, respectively. The main metabolic reactions were demethylation, oxidation, and hydroxylation. The presented in vitro model is applicable to studying drug metabolism by fungi colonizing cadavers.

[Simultaneous determination of chlorpromazine and promethazine and their main metabolites by capillary electrophoresis with electrochemiluminescence].

Based on the phenomenon that each of chlorpromazine (CPZ), promethazine (PMZ), chlorpromazine sulfoxide (CPZSO) and promethazine sulfoxide (PMZSO) could enhance the electrochemiluminescence (ECL) intensity of tris(2,2'-bipyridyl) ruthenium, a novel and sensitive method was proposed for the simultaneous determination of CPZ, PMZ and their main metabolites using capillary electrophoresis (CE) coupled with ECL detection. The influences of several experimental parameters were explored. The optimum experimental conditions were as follows: detection potential of 1. 20 V (Ag/AgCl), 40 mmol/L of phosphate buffer solution (pH 6.5) containing 5 mmol/L tris(2,2'-bipyridyl) ruthenium in ECL detection cell, running buffer solution of 18 mmol/L (pH 4.8), sample injection of 8 s at 11 kV, and separation voltage of 13.5 kV. The detection limits (3sigma) of this method were 8.3 x 10(-7) g/L for CPZ, 7.2 x 10(-6) g/L for PMZ, 1.9 x 10(-5) g/L for CPZSO and 3.7 x 10(-6) g/L for PMZSO. The linear ranges of ECL intensity versus mass concentration of medicaments were 7. 1 x 10(-6) - 6. 3 x 10(-3) g/L for CPZ, 7.5 x 10(-5) - 4.6 x 10(-3) g/L for PMZ, 9.7 x 10(-5) - 3.6 x 10(-3) g/L for CPZSO and 8.1 x 10(-5) - 7.7 x 10(-3) g/L for PMZSO. The relative standard deviations (RSDs) of the four target compounds were not more than 3% for ECL intensity and 1% for migration time. This method has the merits of simplicity, speediness, sensitivity, small sample injection, and free from interference. This method was successfully utilized to directly and simultaneously detect CPZ, PMZ, CPZSO and PMZSO in urine samples of pet dogs.

Spectroscopic approach of the interaction study of amphiphilic drugs with the serum albumins.

The interaction of the amphiphilic drugs, i.e., amitriptyline hydrochloride (AMT) and promethazine hydrochloride (PMT), with serum albumins (i.e., human serum albumin (HSA) and bovine serum albumin (BSA)), has been examined by the various spectroscopic techniques, like fluorescence, UV-vis, and circular dichroism (CD). Fluorescence results indicate that in case of HSA-drug complexes the quenching of fluorescence intensity at 280 nm is less effective as compared to at 295 nm while in case of BSA-drug complexes both have almost same effect and for most of drug-serum albumin complexes there is only one independent class of binding. For all drug-serum albumin complexes the quenching rate constant (K(q)) values suggest the static quenching procedure. The UV-vis results show that the change in protein conformation of PMT-serum albumin complexes was more prominent as compared to AMT-serum albumin complexes. The CD results also explain the conformational changes in the serum albumins on binding with drugs. The increase in α-helical structure for AMT-serum albumin complexes is found to be more as compared to PMT-serum albumin complexes. Hence, the various spectroscopic techniques provide a quantitative understanding of the binding of amphiphilic drugs with serum albumins.

Simultaneous determination of cyclodol and diprazin by thin layer chromatography and high performance liquid chromatography.

Ciklodol (trihexyphenidil)--the central and peripheral m-cholinoblocker is currently used with other antipsychotic drugs such as phenotiazines and tricycle antidepressants. For the purpose of simultaneous determination of ciklodol and diprazine, were selected two methods of analysis: Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC). During development of TLC method was studied the 10 visualizing system and 24 mobile systems. For individual or simultaneous determination of ciklodol and diprazine were recommended the following solvents' systems: 1. Toluene-acetone-ethanole-25%NH(4)OH (45:45: 7.5:2.5), 2. Hexane-ethyl acetate (15:5), 3. Chloroform-heptene-25%NH(4)OH (16:3:3), 4. Ethylacetate-hexane (10:10), 5. Acetonitrile-metanol (10:10) and 6.Heptene-chloroform-ethanol-25% NH(4)OH (5:10:3:1). As visualizing systems were chosen: Iodine vapors, blacklight (UV254) and reagent of FNP. Reagent of FNP gives colored spot just with diprazine and it is also could be used for separation of both objects in simultaneous analysis. Developed HPLC method of simultaneous determination of ciklodol and diprazine: like mobile phase is recommended: Acetonitril- 0.05M KH(2)PO4 (55:45) (v/v) +H(3)PO(4) (pH3.5), column EC250 x 4.6mm, with solid phase Nucleosil, flow rate 1ml/min, sample volume 40 microl. In given conditions, the retention time of ciklodol is 6.005min and diprazine 7.227min. Developed method of simultaneous determination and separation of ciklodol and diprazine in respective mixtures could be successfully applied as in the pharmaceutical, as well in the chemical-toxicological laboratories.

Thermodynamic studies on the interaction of dioxopromethazine to beta-cyclodextrin and bovine serum albumin.

The interaction of dioxopromethazine (DOPM) with beta-cyclodextrin (beta-CD) and bovine serum albumin (BSA) were investigated by fluorescence quenching method. It was shown that DOPM has quite a strong ability to quench the fluorescence launching from BSA by reacting with it and forming a certain kind of new compound. The quenching and the energy transfer mechanisms were discussed, respectively. The binding constants and thermodynamic parameters at four different temperatures, the binding locality, and the binding power were obtained. The conformation of BSA was discussed by synchronous and three-dimensional fluorescence techniques. The inclusion reaction between beta-CD and DOPM was explored by both Lineweaver-Burk equation and Benesi-Hildebrand equation. The inclusion constants and the thermodynamic parameters at 297 and 307 K were figured out, respectively. The mechanism of inclusion reaction was speculated and the slow release characteristics of beta-CD to DOPM was attempted to explain at molecule level.

Hydroxyzine, promethazine and thioridazine interaction with phospholipid monomolecular layers at the air-water interface.

In this work the interaction of Hydroxyzine, Promethazine and Thioridazine with Langmuir films of dipalmitoylphosphatidylcholine (dpPC) and dipalmitoylphosphatidic acid (dpPA), is studied. Temporal variations in lateral surface pressure (pi) were measured at different initial pi (pi(i)), subphase pH and drug-concentration. Drugs with the smallest (PRO) and largest (HYD) molecular size exhibited the lowest adsorption (k(a)) and the highest desorption (k(d)) rate constant values, respectively. The affinity binding constants (K(b)) obtained in monolayers followed the same profile (K(b,PRO) < K(b,HYD) < K(b,THI)) of the egg-PC/water partition coefficients (P) determined in bilayers. The drug concentration required to reach the half-maximal Deltapi at pi(i) = 14 mN/m (K(0.5)), was very sensitive to pH. The maximal increment in pi upon drug incorporation into the monolayer (deltapi(max)) will depend on the phospholipid collapse pressure (pi(c)), the monolayers's compressibility and drug's size, shape and charge. The higher pi(c) of dpPC lead to higher pi(cut-off) values (maximal pi allowing drug penetration), if compared with dpPA. In dpPC and dpPA pi(cut-off) decreased as a function of the molecular size of the uncharged drugs. In dpPA, protonated drugs became electrostatically trapped at the monolayer surface hence drug penetration, monolayer deformation and pi increase were impaired and the correlation between pi(cut-off) and drug molecular size was lost.

Promethazine oxidation by redox mediation in peroxidase reactions.

The effect of promethazine on peroxidase-catalyzed oxidation of 3,3', 5,5'-tetramethylbenzidine was investigated at pH 5.4. Promethazine dose dependently introduced a lag in the appearance of tetramethylbenzidine charge-transfer complex monitored at 652 nm. Increasing concentrations of tetramethylbenzidine however decreased the lag period proportional to the tetramethylbenzidine concentration. Addition of promethazine to preformed charge transfer complex caused rapid bleaching of the blue-colored complex. Titration of promethazine with the yellow-colored diimine gave rise to the blue charge-transfer complex and the complete reduction of the species to the colorless parent amine compound. The available evidence suggests that promethazine is oxidized via redox mediation by tetramethylbenzidine peroxidase-oxidized products.

Fluoxetine-related death in a child with cytochrome P-450 2D6 genetic deficiency.

The clinical course of a 9-year-old diagnosed with attention-deficit hyperactivity disorder, obsessive-compulsive disorder, and Tourette's disorder and treated with a combination of methylphenidate, clonidine, and fluoxetine is described. The patient experienced over a 10-month period, signs and symptoms suggestive of metabolic toxicity marked by bouts of gastrointestinal distress, low-grade fever, incoordination, and disorientation. Generalized seizures were observed, and the patient lapsed into status epilepticus followed by cardiac arrest and subsequently expired. At autopsy, blood, brain, and other tissue concentrations of fluoxetine and norfluoxetine were several-fold higher than expected based on literature reports for overdose situations. The medical examiner's report indicated death caused by fluoxetine toxicity. As the child's adoptive parents controlled medication access, they were investigated by social welfare agencies. Further genetic testing of autopsy tissue revealed the presence of a gene defect at the cytochrome P450 CYP2D locus, which results in poor metabolism of fluoxetine. As a result of this and other evidence, the investigation of the adoptive parents was terminated. This is the first report of a fluoxetine-related death in a child with a confirmed genetic polymorphism of the CYP2D6 gene that results in impaired drug metabolism. Issues relevant to child and adolescent psychopharmacology arising from this case are discussed.

Tardive dyskinesia: probing abnormal metabolism with promethazine.

Ten in-patients with tardive dyskinesia (TD) (mean AIMS score 16.7) and 8 controls were recruited to the study, and 3 h after oral administration of promethazine a blood sample was taken for assay of levels of promethazine and its immediate metabolites by high-performance liquid chromatography (HPLC). The TD group had a variety of indicators of impaired or slow metabolism compared to the controls. There was a significant difference in the ratio of promethazine to promethazine sulphoxide (P < 0.05) between patients with TD and the control group. The TD group but not the controls showed increasing metabolic impairment with age. This small study confirms the previous reports of impaired neuroleptic metabolism in TD, particularly in the elderly.

Usefulness of the hydrogen--deuterium exchange method in the study of drug metabolism using liquid chromatography-tandem mass spectrometry.

The usefulness of the hydrogen-deuterium (H-D) exchange method in the study of drug metabolism was investigated. Metabolite samples of denopamine and promethazine prepared in vitro were introduced to a triple stage quadrupole tandem mass spectrometer via a high performance liquid chromatography (HPLC) system using a deuterated mobile phase. Mass spectra by various ionization modes and collisionally induced dissociation (CID) mass spectra were obtained. A metabolite of denopamine was observed to have a shift of 7 mass units by the H-D exchange method, and this shift proved to be a glucuronidated metabolite. Discrimination between N- or S-oxide formation and hydroxylation observed in the metabolism of promethazine was also easily accomplished by this method. In this manner, the structures of the metabolites were elucidated unequivocally by determining the number of labile hydrogen atoms by the use of the H-D exchange method, since various reactions in drug metabolism are accompanied by an increase or a decrease in the number of labile hydrogen atoms. Additional information on the structures was obtained by analysis of the CID spectra of the molecular ion species. Thus, the combination of the H-D exchange method and tandem mass spectrometry was found to be very useful for the study of drug metabolism.