Combination of experimental and in silico methods for the assessment of the phototransformation products of the antipsychotic drug/metabolite Mesoridazine.

The lack of studies on the fate and effects of drug metabolites in the environment is of concern. As their parent compounds, metabolites enter the aquatic environment and are subject to biotic and abiotic process. In this regard, photolysis plays an important role. This study combined experimental and in silico quantitative structure-activity relationship (QSAR) methods to assess the fate and effects of Mesoridazine (MESO), a pharmacologically active human drug and metabolite of the antipsychotic agent Thioridazine, and its transformation products (TPs) formed through a Xenon lamp irradiation. After 256min, the photodegradation of MESO⋅besylate (50mgL-1) achieved 90.4% and 6.9% of primary elimination and mineralization, respectively. The photon flux emitted by the lamp (200-600nm) was 169.55Jcm-2. Sixteen TPs were detected by means of liquid chromatography-high resolution mass spectrometry (LC-HRMS), and the structures were proposed based on MSn fragmentation patterns. The main transformation reactions were sulfoxidation, hydroxylation, dehydrogenation, and sulfoxide elimination. A back-transformation of MESO to Thioridazine was evidenced. Aerobic biodegradation tests (OECD 301 D and 301F) were applied to MESO and the mixture of TPs present after 256min of photolysis. Most of TPs were not biodegraded, demonstrating their tendency to persist in aquatic environments. The ecotoxicity towards Vibrio fischeri showed a decrease in toxicity during the photolysis process. The in silico QSAR tools QSARINS and US-EPA PBT profiler were applied for the screening of TPs with character of persistence, bioaccumulation, and toxicity (PBT). They have revealed the carbazole derivatives TP 355 and TP 337 as PBT/vPvB (very persistent and very bioaccumulative) compounds. In silico QSAR predictions for mutagenicity and genotoxicity provided by CASE Ultra and Leadscope® indicated positive alerts for mutagenicity on TP 355 and TP 337. Further studies regarding the carbazole derivative TPs should be considered to confirm their hazardous character.

The conditional stimulation of rat organic cation transporter 2, but not its human ortholog, by mesoridazine: the possibility of the involvement of the high-affinity binding site of the transporter in the stimulation.

OBJECTIVES: To study the functional consequences of the human and rat forms of OCT2 in the presence of phenothiazines. METHODS: MDCK cells expressing human or rat OCT2 were established, and MPP+ transport was determined by uptake assays. Concentration dependency was studied for the stimulatory/inhibitory effects of phenothiazines on MPP+ transport. KEY FINDINGS: Among the 11 phenothiazines examined, the majority were found to have comparable effects on transporter function between the orthologous forms, while three phenothiazines, particularly mesoridazine, had complex impacts on transporter function. For rOCT2, mesoridazine stimulated transport at 0.1 and 1 µmMPP+ with the mesoridazine concentration-uptake curve becoming bell-shaped. This conditional effect became less pronounced at 30 µmMPP+, resulting in an inhibition curve with a typical profile. For hOCT2, mesoridazine behaved as a typical inhibitor of transporter function at all MPP+ concentrations, although the kinetics of inhibition were still affected by the substrate concentration. CONCLUSIONS: The conditional stimulation by mesoridazine in rOCT2, and the lack thereof in hOCT2, may be a manifestation of the interaction of phenothiazine with substrate binding at the high-affinity site of the OCT2. As OCT2 was previously indicated in some drug-drug interactions, the conditional stimulation of OCT2 and its potential species-differences may be of practical relevance.

Topically applied mesoridazine exhibits the strongest cutaneous analgesia and minimized skin disruption among tricyclic antidepressants: The skin absorption assessment.

Tricyclic antidepressants (TCAs) are found to have an analgesic action for relieving cutaneous pain associated with neuropathies. The aim of this study was to assess cutaneous absorption and analgesia of topically applied TCAs. Percutaneous delivery was investigated using nude mouse and pig skin models at both infinite and saturated doses. We evaluated the cutaneous analgesia in nude mice using the pinprick scores. Among five antidepressants tested in the in vitro experiment, mesoridazine, promazine and doxepin showed a superior total absorption percentage. The drug with the lowest total absorption percentage was found to be fluphenazine (<7%) either at an infinite dose or at saturated solubility. The follicular pathway was important for mesoridazine and promazine delivery. Mesoridazine showed stronger skin analgesia than the other TCAs although the in vivo skin absorption of mesoridazine (0.34nmol/mg) was less than that of promazine (0.80nmol/mg) and doxepin (0.74nmol/mg). Mesoridazine had a prolonged duration of pain relief (165min) compared to promazine (83min) and doxepin (17min). The skin irritation test demonstrated an evident barrier function deterioration and cutaneous erythema by promazine and doxepin treatment, whereas mesoridazine caused no obvious adverse effect by topical application for up to 7days.

Determination of mesoridazine by liquid chromatography-tandem mass spectrometry and its application to pharmacokinetic study in rats.

The object of the present study was to develop and validate an assay method of mesoridazine in rat plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Plasma samples from rats were prepared by simple protein precipitation and injected onto the LC-MS/MS system for quantification. Mesoridazine and chlorpromazine as an internal standard (IS) were separated by a reversed phase C18 column. A mobile phase was composed of 10mM ammonium formate in water and acetonitrile (ACN) (v/v) by a linear gradient system, increasing the percentage of ACN from 2% at 0.4min to 98% at 2.5min with 4min total run time. The ion transitions monitored in positive-ion mode [M+H](+) of multiple-reaction monitoring (MRM) were m/z 387>126 for mesoridazine and m/z 319>86 for IS. The detector response was specific and linear for mesoridazine at concentrations within the range 0.001-4µg/ml and the correlation coefficient (R(2)) was greater than 0.999 and the signal-to-noise ratios for the samples were ≥10. The intra- and inter-day precision and accuracy of the method were determined to be within the acceptance criteria for assay validation guidelines. The matrix effects were approximately 101 and 99.5% from rat plasma for mesoridazine and chlorpromazine, respectively. Mesoridazine was stable under various processing and/or handling conditions. Mesoridazine concentrations were readily measured in rat plasma samples after intravenous and oral administration. This assay method can be practically useful to the pharmacokinetic and/or toxicokinetic studies of mesoridazine.

Oncologic outcomes of robotic-assisted total mesorectal excision for the treatment of rectal cancer.

OBJECTIVE: To evaluate local recurrence and survival after robotic-assisted total mesorectal excision (RTME) for primary rectal cancer. SUMMARY BACKGROUND DATA: RTME is a novel approach for the treatment of rectal cancer and has been shown to be safe and effective. However, the oncologic results of this approach have not been reported in terms of local recurrence and survival rate. METHODS: Sixty-four consecutive rectal cancer patients with stage I-III disease treated between November 2004 and June 2008 were analyzed prospectively. RESULTS: All patients underwent RTME: 34 had colorectal anastomosis, 18 underwent coloanal anastomosis, and 12 received abdominoperineal resection. Operative mortality rate was 0%. The median operative time was 270 min and median blood loss was 200 mL. The conversion rate was 9.4%. Anastomotic leakage occurred in 4 of 52 (7.7%) patients with anastomosis. Median number of harvested lymph nodes was 14.5. Median distal margin of tumor was 3.4 cm. The circumferential resection margin was negative in all surgical specimens. No port-site recurrence occurred in any patient. Six patients developed recurrence: 2 combined local and distant, and 4 distal alone (mean follow-up of 20.2 months; range, 1.7-52.5). None of the patients developed isolated local recurrence. The mean time to local recurrence was 23 months. The 3-year overall and disease-free survival rates were 96.2% and 73.7%, respectively. CONCLUSIONS: RTME can be carried out safely and effectively in terms of recurrence and survival rates. Further prospective randomized trials are necessary to better define the absolute benefits and limitations of robotic rectal surgery.

Relevance of CYP2D6 -1584C>G polymorphism for thioridazine:mesoridazine plasma concentration ratio in psychiatric patients.

AIMS: The CYP2D6 -1584C>G (rs1080985) polymorphism has been identified as another major factor for CYP2D6 function that is possibly associated with ultrarapid metabolism. The mutant -1584G promoter genotype seems to be consistently related to a higher protein expression than -1584C. However, the impact this SNP in the CYP2D6 promoter region has on plasma levels of patients taking CYP2D6 substrates, such as thioridazine, has not been studied. Previously, we showed the validity of the mesoridazine:thioridazine ratio to assess CYP2D6 activity in clinical settings. Therefore, the aim of this study was to analyze the relationship between the presence of the CYP2D6 -1584C>G polymorphism and the plasma concentrations of thioridazine and its metabolites in a previously studied population of patients in order to evaluate the implications for CYP2D6 hydroxylation capacity. MATERIALS & METHODS: The CYP2D6 -1584C>G polymorphism was determined by using a PCR-RFLP method in 61 Caucasian psychiatric patients receiving thioridazine monotherapy. RESULTS: Among patients with two active CYP2D6 genes, there were significant differences in the thioridazine:mesoridazine plasma concentrations ratio (p < 0.05) among the three CYP2D6 -1584C>G genotype groups. Moreover, in this group of patients the thioridazine:mesoridazine ratio was lower (p < 0.05) in carriers of CYP2D6 -1584G allele than in patients homozygous for CYP2D6 -1584C allele. However, no differences in thioridazine or its metabolite concentrations between homozygous CYP2D6 -1584C allele carriers and carriers of the -1584G allele were found. CONCLUSION: According to the present results the concentration ratio of thioridazine to mesoridazine was related to the CYP2D6 -1584C>G polymorphism. It is likely that individuals who carry CYP2D6 -1584G versus homozygotes for the -1584C allele may present an increased CYP2D6 activity.

Comparison of the effects of thioridazine and mesoridazine on the QT interval in healthy adults after single oral doses.

We compared the effects of single doses of thioridazine and mesoridazine on the heart rate-corrected QT (QTc) interval in healthy adult volunteers. QTc intervals and plasma concentrations of thioridazine, mesoridazine, and metabolites were measured after single oral doses of thioridazine hydrochloride 50 mg, mesoridazine besylate 50 mg, or placebo in a double-blind, crossover study. Mean maximum increases in the QTc interval following thioridazine (37.3+/-4.1 ms, P=0.023) and mesoridazine (46.6+/-7.4 ms, P=0.021) were similar and significantly greater than following placebo (12.9+/-8.1 ms). The area under the effect-time curve over 8 h following drug administration was similar between the two drugs (129.3+/-22.1 vs 148.3+/-43.0 ms h). In conclusion, thioridazine and mesoridazine are associated with similar effects on the QTc interval.

Factors affecting drug concentrations and QT interval during thioridazine therapy.

The objective of this study was to investigate factors affecting steady-state plasma concentrations of thioridazine. A cross-sectional study of patients receiving chronic thioridazine was employed. Common allelic variants of CYP2D6 and CYP2C19, as well as thioridazine and metabolite concentrations and QTc intervals, were determined. In 97 patients, dose-corrected plasma concentrations (C/Ds) of thioridazine and metabolites were correlated with age but not sex or CYP2C19 genotype. Patients with no functional CYP2D6 alleles (n=9) had significantly higher C/D for thioridazine (P=0.017) and the ring sulfoxide metabolite and a significantly higher thioridazine/mesoridazine ratio compared with those with >/=1 functional CYP2D6 allele (n=82). Smokers had significantly lower C/D for thioridazine, mesoridazine, and sulforidazine and significantly lower thioridazine/ring sulfoxide ratios than non-smokers. QTc interval was not significantly affected by CYP2D6 or CYP2C19 genotypes. Plasma concentrations of thioridazine are influenced by age, smoking, and CYP2D6 genotype, but CYP2D6 genotype does not appear to influence on-treatment QTc interval.

Altering extracellular potassium concentration does not modulate drug block of human ether-a-go-go-related gene (hERG) channels.

1. Drug-induced block of the rapidly activating delayed rectifier K+ current (I(Kr)), encoded by human ether-a-go-go-related gene (hERG), has been linked to acquired long QT syndrome (aLQTS). Hypokalaemia is a recognized risk factor in aLQTS. To further understand why hypokalaemia is a risk factor in aLQTS, we examined the effect of [K+]o on drug block of the hERG potassium channel stably expressed in human embryonic kidney (HEK-293) cells using whole-cell voltage-clamp techniques. 2. The effects of selected [K+]o (1-20 mmol/L) on hERG block with four structurally diverse compounds (dofetilide, mesoridazine, quinidine and terfenadine) from different therapeutic classes were evaluated. Reducing [K+]o from 20 to 1 mmol/L had little effect on IC50 values for hERG current block for all four compounds. For example, evaluating quinidine in external potassium concentrations of 20, 10, 5 and 1 mmol/L resulted in IC50 values of 1.82 +/- 0.33, 2.04 +/- 0.28, 1.57 +/- 0.52 and 1.14 +/- 0.21 mmol/L, respectively. No statistically significant difference (P > 0.35, anova) was observed between drug block of hERG in different external potassium concentrations. These data are in contrast with previously reported results examining hERG channel modulation expressed in AT-1 cells under similar experimental conditions. 3. These results demonstrate that [K+]o does not directly modulate drug block of hERG channels expressed in an HEK-293 cell line. The enhanced risk of Torsades de Pointes associated with hypokalaemia in aLQTS may be due to reduction of other (non-hERG) potassium currents, further reducing the repolarization reserve, and not due to direct modulation of hERG block by [K+]o.

Synthesis, receptor binding and functional studies of mesoridazine stereoisomers.

The four stereoisomers of mesoridazine were synthesized and evaluated in D2, 5-HT1A, 5-HT2A, 5-HT2C, D1, and D3 receptor binding and functional assays. Two isomers demonstrated potent D2 receptor binding (Ki < 3 nM) and functional antagonism (IC50 < or = 10 nM) activities. These two isomers also showed moderate affinity for the 5-HT2A and D3 receptors. A third isomer was devoid of significant D2 receptor binding, but did have moderate affinity for the 5-HT2A and D3 receptors. The fourth isomer demonstrated poor affinity for all the receptors tested. Most significantly, the stereochemistry of the sulfoxide moiety played a dominant role in the observed structure-activity relationship (SAR).

Diastereotopic analysis of mesoridazine besylate (Serentil).

NMR studies were conducted with the aim of determining the diastereoisomeric ratio of a commercially supplied sample of mesoridazine (MES) and to compare the results with a freshly synthesised sample of MES. The results indicated that the commercially supplied MES consisted almost entirely of one diastereoisomeric pair, which was in agreement with previous findings reported by Eap et al. The synthesised sample of MES was analysed by NMR in two stages: 1) as the initial product isolated as the free base from the direct synthesis, and 2) as the free base isolated from the crystallised besylate salt of the synthetic product. The NMR results show that the initial synthetic product consisted of two equal pairs of diastereoisomers. The diastereoisomeric pairs were further separated by the addition of the chiral shift reagent (R)-(-)-N-(3,5 dinitrobenzoyl)-alpha-benzylamine to reveal equal quantities of all four enantiomers, clearly observed at the methyl sulfoxide proton peak of the NMR scan. The sample obtained from the crystallisation of MES besylate, however, indicated a significant difference, with a diastereoisomeric ratio of 75:25. The results suggest that MES besylate undergoes preferential crystallisation of one pair of diastereoisomers, with the other pair remaining in solution.

Mesoridazine: an open-channel blocker of human ether-a-go-go-related gene K+ channel.

Mesoridazine, a phenothiazine antipsychotic agent, prolongs the QT interval of the cardiac electrocardiogram and is associated with Torsade de pointes-type arrhythmias. In this study, we examined the effects of mesoridazine on human ether-a-go-go-related gene (HERG) K+ currents. HERG channels were stably expressed in human embryonic kidney 293 cells and studied using standard whole-cell patch-clamp technique (37 degrees C). Mesoridazine blocked HERG currents in a concentration-dependent manner (IC50 550 nM at 0 mV); block increased significantly over the voltage range where HERG activates and saturated at voltages eliciting maximal HERG channel activation. Tonic block of HERG current by mesoridazine (1.8 microM) was minimal (< 2-4%). The rate of the onset of HERG channel block was rapid and dose dependent (tau = 54 +/- 7 ms at 0 mV and 1.8 microM mesoridazine), but not significantly affected by test potentials ranging from -30 to +30 mV. The V1/2 for steady-state activation was shifted from -31.2 +/- 1.0 to -39.2 +/- 0.5 mV (P < 0.01). The apparent rate of HERG channel deactivation was significantly reduced (fast tau = 153 +/- 8 vs. 102 +/- 6 ms at -50 mV, P < 0.01; slow tau = 1113 +/- 63 vs. 508 +/- 27 ms, P < 0.01). The inactivation kinetics and voltage dependence of steady-state inactivation of the HERG channel were not significantly altered by mesoridazine. These findings demonstrate that mesoridazine is a potent and rapid open-channel blocker of HERG channels. This block would explain the QT prolongation seen clinically at therapeutic concentrations (0.3-3.6 microM).

Cinqüenta anos de medicamentos antipsicóticos em psiquiatria: III. fenotiazinas piperidínicas / Fifty years of antipsychotic drugs in psychiatry: III. piperidine phenothiazines

Na iminência de uma nova era na terapêutica psiquiátrica com a redescoberta da clozapina e a introduçäo dos novos antipsicóticos atípicos, é tempo de um inventário das substâncias desenvolvidas nos últimos cinqüenta anos. É feito um breve histórico dos antecedentes dos antipsicóticos tradicionais na era que se encerra. As substâncias introduzidas até o presente poderiam ser reunidas nos grupos tradicionais - fenotiazinas (alifáticas, piperazínicas e piperidínicas), tioxantenos, butirofenonas, difenilbutilpiperidinas, benzaminas, indóis, dibenzoxazepinas - e nos grupos químicos mais recentes - diidroindolonas, dbenzodiazepinas, benzisoxazólicos -, além de compostos ainda em desenvolvimento. Neste artigo, o terceiro de uma série concebida com esta finalidade, säo examinados os derivados fenotiazínicos com cadeia lateral piperidínica que tenham demonstrado utilidade na prática clínica e ou guardem importância histórica: mepazina, mesoridazina, nortioridazina, piperacetazina, propericiazina, sulforidazina e toridazina. Com base em bibliografia básica específica, säo discutidos aspectos técnicos e revisado o conhecimento científico acumulado através da experimentaçäo e utilizaçäo clínica destes compostos, desde seu lançamento até a presente data, com informaçöes sistemáticas sobre fórmula estrutural, fórmula molecular, nomes químicos, nomes de fantasia e códigos de cada composto, além de dados sobre eventual comercializaçäo no país

Use of the mesoridazine/thioridazine ratio as a marker for CYP2D6 enzyme activity.

Thioridazine is metabolized in humans by CYP2D6 to mesoridazine, which is an active metabolite. Two or more CYP2D6 substrates are seldom given simultaneously to elderly patients because potentially dangerous metabolic interactions may occur. It may be valuable to know the CYP2D6 metabolic capacity of such patients to avoid drug interactions, which depend on the metabolic phenotype. The goal of this study was to evaluate the use of the mesoridazine/thioridazine ratio for the estimation of CYP2D6 enzyme capacity. A sensitive and reliable method has been developed for the determination of thioridazine and its metabolites, mesoridazine and sulforidazine. Commonly used central nervous system (CNS) comedications do not interfere with the method. A group of 27 chronic patients with mental illness receiving monotherapy with thioridazine were studied. There were 23 men and 4 women between 37 and 80 years old (mean +/- SD: 61.2 +/- 10.2). The thioridazine/mesoridazine ratio correlated with the debrisoquine metabolic ratio (r = 0.74, p < 0.001). Therefore, the authors suggest that the measurement of thioridazine and its metabolite might be a useful tool to assess CYP2D6 activity during treatment.

Pharmacokinetic interaction of fluvoxamine and thioridazine in schizophrenic patients.

This study investigated to what extent fluvoxamine affects the pharmacokinetics of thioridazine (THD) in schizophrenic patients under steady-state conditions. Concentrations of THD, mesoridazine, and sulforidazine were measured in plasma samples obtained from 10 male inpatients, aged 36 to 78 years, at three different time points: A, during habitual monotherapy with THD at 88 +/-54 mg/day; B, after addition of a low dosage of fluvoxamine (25 mg twice a day) for 1 week; and C, 2 weeks after fluvoxamine discontinuation. After the addition of fluvoxamine, THD concentrations relative to time point A significantly increased approximately threefold from 0.40 to 1.21 micromol/L (225%) (p < 0.002), mesoridazine concentrations increased from 0.65 to 2.0 micromol/L (219%) (p < 0.004), and sulforidazine levels increased from 0.21 to 0.56 micromol/L (258%) (p < 0.004). The THD-mesoridazine and THD-sulforidazine ratios remained unchanged during the study. Mean plasma THD, mesoridazine, and sulforidazine levels decreased at time point C, but despite fluvoxamine discontinuation for 2 weeks, three patients continued to exhibit elevated concentrations of THD and its metabolites. In conclusion, fluvoxamine markedly interferes with the metabolism of THD, probably at the CYP2C19 and/or CYP1A2 enzyme level. Therefore, clinicians should be aware of the potential for a clinical drug interaction between both compounds, and careful monitoring of THD levels is valuable to prevent the accumulation of the drug and resulting toxicity.

Pharmacokinetics of thioridazine and its metabolites in blood plasma and the brain of rats after acute and chronic treatment.

This study was aimed at investigation of the pharmacokinetics of thioridazine and its metabolites after a single and repeated administrations. Male Wistar rats received thioridazine as a single dose (10 mg/kg i.p.) or they were treated chronically with the neuroleptic (10 mg/kg i.p., twice a day for two weeks). Plasma and brain concentrations of thioridazine and its metabolites (N-desmethylthioridazine, mesoridazine, sulforidazine, and the ring sulfoxide) were determined using the HPLC method. The obtained data showed that sulfoxidation in position 2 of the thiomethyl substituent and in the thiazine ring are main metabolic pathways of thioridazine, and showed that, in contrast to humans, in the rat N-desmethylthioridazine is formed in appreciable amount. The biotransformation of thioridazine was rather fast yielding plasma peak concentrations of metabolites lower than that of the parent compound. The maximum concentrations of thioridazine and its metabolites in the brain appeared later than in plasma. The peak concentrations and AUC values of thioridazine and its metabolites were higher in the brain than in plasma and this corresponded well with their longer half-lives in the brain as compared to plasma. The drug was not taken up by the brain as efficiently as other phenothiazines. Chronic treatment with thioridazine produced significant increases (with the exception of thioridazine ring sulfoxide) in the plasma concentrations of the parent compound and its metabolites which was accompanied with the prolongation of their plasma half-lives. The observed plasma levels of thioridazine were within 'therapeutic range' while the concentrations of its metabolites were relatively lower as compared to those observed in psychiatric patients. The increased plasma concentrations of thioridazine and its metabolites observed in plasma after chronic treatment were not followed by parallel changes in the brain.