In vivo identification and characterization of binding sites for selective serotonin reuptake inhibitors in mouse brain

The present study was undertaken to identify and characterize in vivo binding sites of selective serotonin reuptake inhibitors (SSRIs) in the mouse brain by using [3H]paroxetine as radioligand. Relatively higher concentration of [3H]paroxetine was detected in the whole brain (minus cerebellum) than in the plasma of mice after the i.v. injection of the radioligand, and the half-life (t1/2) of elimination was much slower. The in vivo specific [3H]paroxetine binding in the mouse brain after the i.v. injection was defined as the difference of particulate-bound radioactivity between the whole brain and cerebellum, and it was dose-dependently attenuated by oral or intraperitoneal administration of fluoxetine (8.68-116 micromol/kg). Furthermore, oral administration of fluvoxamine, fluoxetine, paroxetine and sertraline at the pharmacologically relevant doses reduced significantly (25-94%) in vivo specific [3H]paroxetine binding in the cerebral cortex, striatum, hippocampus, thalamus and midbrain of mice, and their significant decreases were observed up to at least 8 h (fluvoxamine), 24 h (fluoxetine), and 12 h (paroxetine and sertraline) later. The value of area under the curve (AUC) for decrease in [3H]paroxetine binding vs. time in each brain region was largest for fluoxetine among these SSRIs, due to the relatively longer-lasting occupation of brain serotonin transporter. The AUC value in mouse brain after oral administration of each SSRI was 1.2-3.2 times greater in the thalamus and midbrain than in the cerebral cortex, striatum and hippocampus. Thus, the present study has revealed that [3H]paroxetine may be a suitable radioligand for in vivo characterization of brain binding sites and pharmacological effects of SSRIs.     

Clickable photoaffinity ligands for the human serotonin transporter based on the selective serotonin reuptake inhibitor (S)-citalopram

To date, the development of photoaffinity ligands targeting the human serotonin transporter (hSERT), a key protein involved in disease states such as depression and anxiety, have been radioisotope-based (i.e., ³H or ¹²⁵I). This letter instead highlights three derivatives of the selective serotonin reuptake inhibitor (SSRI) (S)-citalopram that were rationally designed and synthesized to contain a photoreactive benzophenone or an aryl azide for protein target capture via photoaffinity labeling and a terminal alkyne or an aliphatic azide for click chemistry-based proteomics. Specifically, clickable benzophenone-based (S)-citalopram photoprobe 6 (hSERT K_i?=?0.16?nM) displayed 11-fold higher binding affinity at hSERT when compared to (S)-citalopram (hSERT K_i?=?1.77?nM), and was subsequently shown to successfully undergo tandem photoaffinity labeling-biorthogonal conjugation using purified hSERT. Given clickable photoprobes can be used for various applications depending on which reporter is attached by click chemistry subsequent to photoaffinity labeling, photoprobe 6 is expected to find value in structure-function studies and other research applications involving hSERT (e.g., imaging).     

High-performance liquid chromatographic method to screen and quantitate seven selective serotonin reuptake inhibitors in human serum

A high-performance liquid chromatographic screening method (HPLC) is described for the determination of seven selective serotonin reuptake inhibitors (SSRIs) (fluvoxamine, milnacipran, paroxetine, sertraline, fluoxetine, citalopram, venlafaxine) and for three pharmacologically active N-demethylated metabolites (desmethylcitalopram, didesmethylcitalopram and norfluoxetine). A tricyclic antidepressant, clomipramine, was used as an internal standard. The method consists of liquid extraction of serum after alcalinisation at pH 9.50, followed by chromatography on a Beckman C₁₈ reversed-phase column. Compounds were detected at 200.4 nm. The standard curves were linear over a working range of 50–1000 ng/ml for fluvoxamine, 15–1000 ng/ml for fluoxetine, 25–500 ng/ml for norfluoxetine, 50–500 ng/ml for sertraline, 20–500 ng/ml for paroxetine, 25–550 ng/ml for citalopram, 25–750 ng/ml for desmethylcitalopram, 25–800 ng/ml for didesmethylcitalopram, 25–650 ng/ml for milnacipran, and 25–500 ng/ml for venlafaxine. The quantitation limits of the method were 15 ng/ml for fluoxetine, 20 ng/ml for paroxetine, 25 ng/ml for venlafaxine, norfluoxetine and citalopram, and its metabolites, 40 ng/ml for sertraline and 50 ng/ml for fluvoxamine. No interferences were noted with this sensitive and specific method which can be used for therapeutic drug monitoring.     

Synthesis and activity of a new class of dual acting norepinephrine and serotonin reuptake inhibitors: 3-(1H-indol-1-yl)-3-arylpropan-1-amines

Compounds with a combination of norepinephrine and serotonin reuptake inhibition have been approved in the US and Europe for a number of indications, including major depressive disorder and pain disorders such as diabetic neuropathy and fibromyalgia. Efforts to design selective norepinephrine reuptake inhibitors based on SAR from the aryloxypropanamine series of monoamine reuptake inhibitors have led to the identification of a potent new class of dual acting norepinephrine and serotonin reuptake inhibitors, namely the 3-(1H-indol-1-yl)-3-arylpropan-1-amines.     

Designing rapid onset selective serotonin re-uptake inhibitors. 2: structure-activity relationships of substituted (aryl)benzylamines

A series of substituted benzylamines 2–48 were prepared as part of a strategy to identify structurally differentiated and synthetically more accessible selective serotonin reuptake inhibitors, relative to clinical candidate 1. In particular, 44 and 48; demonstrated low nanomolar potency and good selectivity, in a structurally simplified template and, in vivo, very low Vdu, significantly lower than l, and a more rapid T_max, consistent with our clinical objectives.     

Comparison of the effects of serotonin selective,norepinephrine selective,and dual serotonin and norepinephrine reuptake inhibitors on lower urinary tract function in cats

Previous studies showed that the dual serotonin (5-hydroxytryptamine, 5-HT) and norepinephrine (NE) reuptake inhibitor, duloxetine, increases bladder capacity and urethral sphincter electromyographic (EMG) activity in a cat model of acetic acid-induced bladder irritation. The present study aimed to determine the relative importance of 5-HT versus NE reuptake inhibition for mediating these effects by examining drugs that are selective for either the 5-HT or NE system or both. Similar to duloxetine, venlafaxine (0.1 to 10 mg/kg), also a dual serotonin and norepinephrine reuptake inhibitor, produced marked increases in bladder capacity and EMG activity that were reversed by methiothepin (0.3 mg/kg). S-norfluoxetine (0.01 to 10 mg/kg), a serotonin selective reuptake inhibitor, produced small but significant increases in bladder capacity and EMG activity at doses of 3 and 10 mg/kg. Thionisoxetine (0.01 to 3.0 mg/kg), a NE selective reuptake inhibitor, produced no effects on bladder capacity or sphincter EMG activity. Surprisingly, co-administration of thionisoxetine and s-norfluoxetine up to doses of 1 mg/kg of each compound produced no effect on lower urinary tract function. These doses were the maximum that could be administered in combination due to drug-induced emergence of skeletal muscle activity in chloralose-anesthetized animals. These results indicate that there are unexplained pharmacological differences between the effects of single compounds that exhibit dual NE and 5-HT reuptake inhibition and a combination of compounds that exhibit selective NE and 5-HT reuptake inhibition on lower urinary tract function.     

Colorimetric determination of hydroxyurea in human serum using high-performance liquid chromatography

A high-performance liquid chromatography assay for hydroxyurea in human serum was developed based on a commercial colorimetric assay kit for urea (Sigma Diagnostics). Serum (0.5 ml), spiked with methylurea as an internal standard, was treated with 70% perchloric acid. Supernatant (0.2 ml) was combined with 0.7 ml of BUN acid reagent and 0.6 ml of BUN color reagent. The resulting colored reactant (100 μl) was analyzed on a 300×3.9 mm Bondclone 10 C₁₈ column coupled with a UV–Vis detector, at 449 nm. The mobile phase was 13% acetonitrile in water. Retention times of colored derivatives of hydroxyurea and methylurea were 6.5 and 12.2 min, respectively. The log–log calibration curve was linear from 0.0065 to 1.31 mM. Average accuracy was 99.9±4.0% and the intra- and inter-day error of assay did not exceed 11%.     

Enantioselective determination of isradipine in human serum using chiral stationary phase liquid chromatography and gas chromatography with nitrogen selective detection

rac-Isradipine is a dihydropyridine type calcium antagonist. Its calcium entry blocking effect is due primarily to the (+)-(S)-enantiomer. This study describes a sensitive enantioselective method for the determination of isradipine in human serum. Following alkaline extraction into hexane, the enantiomers of isradipine are separated quantitatively by high-performance liquid chromatography on a Chiralcel OJ column at 39°C. The collected fractions were evaporated and assayed using capillary gas chromatography on a HP 50+ column with nitrogen selective detection. Using 2.0 ml of serum, 0.7 nmol/1 (0.26 ng/ml) of each enantiomer could be determined with acceptable precision. The method has successfully been used to measure (+)-(S)- and (−)-(R)-isradipine concentrations in samples from volunteers after intravenous and oral administration of isradipine. Chirality 10:808–812, 1998. © 1998 Wiley-Liss, Inc.     

Simple and selective determination of the cyclophosphamide metabolite phosphoramide mustard in human plasma using high-performance liquid chromatography

A simple and selective assay for the determination of the alkylating cyclophosphamide metabolite phosphoramide mustard (PM) in plasma was developed and validated. PM was determined after derivatisation by high-performance liquid chromatography (HPLC) with ultraviolet detection at 276 nm. Sample pre-treatment consisted of derivatisation of PM with diethyldithiocarbamate (DDTC) at 70°C for 10 min, followed by extraction with acetonitrile in the presence of 0.7 M sodium chloride. Phase separation occurred due to the high salt content of the aqueous phase. The HPLC system consisted of a C₈ column with acetonitrile–0.025 M potassium phosphate buffer, pH 8.0, (32:68, v/v) as the mobile phase. The entire sample handling procedure, from collection at the clinical ward until analysis in the laboratory, was optimised and validated. Calibration curves were linear from 50 to 10 000 ng/ml. The lower limit of quantification and the limit of detection (using a signal-to-noise ratio of 3) were 50 and 40 ng/ml, respectively, using 500 μl of plasma. Within-day and between-day precisions were below 11% over the entire concentration range and the accuracies were between 100 and 106%. PM was found to be stable at −30°C for at least 10 weeks both in plasma and as a DDTC-derivative in a dry sample. A pharmacokinetic pilot study in two patients receiving 1000 mg/m² CP in a 1-h infusion demonstrated the applicability of the assay.     

Enantioselective determination of thiamylal in human serum by high-performance liquid chromatography

Thiamylal, a widely used anesthetic drug, has two enantiomers. We developed a novel and simple method for measuring thiamylal enantiomers in human serum using reversed-phase high-performance liquid chromatography, R(+)- and S(−)-Thiamylal were separated using a chiral mobile phase containing β-cyclodextrin, and detected at the range of 50 ng/ml-25 μg/ml in serum. The relative standard deviations R(+)- and S(−)-thiamylal were 3.4–8.7% and 2.8–8.7% for the intra-day assay, and 2.8–12.0% and 2.8–13.0% for the inter-day assay. This method may be applied to enantioselective pharmacokinetic studies of thiamylal.     

Determination of the cell lytic properties of amphiphilic inhibitors of the cytosolic phospholipase A2 against human platelets by measuring the liberation of serotonin with high-performance liquid chromatography and fluorescence detection

A procedure for the determination of the influence of detergents on the cell integrity of human platelets by measuring the release of serotonin with high-performance liquid chromatography and fluorescence detection is presented. After exposure of the platelets to the test compounds the cells and cell fragments, respectively, are centrifuged off. In the supernatants the liberated serotonin is determined directly without a further sample clean up. The amphiphilic inhibitors of cytosolic phospholipase A2 (cPLA2), arachidonyltrifluoromethyl ketone (AACOCF3), palmityltrifluoromethyl ketone (PACOCF3) and methyl arachidonylfluorophosphonate (MAFP), and the polyoxyethylene detergent Brij 58 were investigated for their cell lytic properties with this method. All compounds lysed the platelets liberating serotonin at a concentration of 33 microM. AACOCF, and Brij 58 even caused cell lysis at lower concentrations.     

N-Benzyl-N-(pyrrolidin-3-yl)carboxamides as a new class of selective dual serotonin/noradrenaline reuptake inhibitors

The structure–activity relationship and the synthesis of novel N-benzyl-N-(pyrrolidin-3-yl)carboxamides as dual serotonin (5-HT) and noradrenaline (NA) monoamine reuptake inhibitors are described. Compounds such as 18 exhibited dual 5-HT and NA reuptake inhibition, good selectivity over dopamine (DA) reuptake inhibition and drug-like physicochemical properties consistent with CNS target space. Compound 18 was selected for further preclinical evaluation.     

Simple high-performance liquid chromatography determination of ampicillin in human serum using solid-phase extraction disk cartridges

A simple and reproducible method for the analysis of ampicillin in human serum was developed. Serum samples were extracted using solid-phase extraction disk cartridges containing a sorbent of styrene divinyl/benzene. Extracts were separated by reversed-phase C₁₈ high-performance liquid chromatography with UV detection at 220 nm. The mobile phase consisted of acetonitrile–10 mM NaH₂PO₄ (6.5:93.5, v/v). Using this extraction procedure, recovery from serum was 98.4±5.6%. The quantitation limit was 0.19 μg/ml using 0.5 ml of serum. The calibration curves from 0.19 to 9.41 μg/ml were linear with correlation coefficients of 0.999. This method is suitable for therapeutic drug monitoring of ampicillin (ABPC) after oral administration of lenampicillin hydrochloride.     

Synthesis and biological evaluation of novel 3,4-diaryl lactam derivatives as triple reuptake inhibitors

A series of 3,4-diarylpyrrolidin-2-one was designed, prepared and evaluated as triple reuptake inhibitors for antidepressant. Most compounds exhibited comparable in vitro efficacy as norepinephrine and dopamine transporter reuptake inhibitors. Especially, 2i showed better potency than GBR-12909 (IC₅₀ = 14 nM) which was used as reference compound for dopamine transporter. In addition, 2a and 2b showed inhibition (5.17 μM–85.6 nM) for three transporters.     

Simultaneous determination of catecholamines in rat brain tissue by high-performance liquid chromatography

A novel and highly sensitive method has been developed for the determination of catecholamines [noradrenaline (NA), dopamine (DA), serotonin (5-HT) and their metabolites 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA)] in brain tissue. The method uses isocratic reversed-phase HPLC with amperometric end-point detection. The calibration curve was linear over the range 10–150 pg on-column. The assay limits of detection for NA, DA, 5-HT, 5-HIAA and HVA were 3.8, 3.8, 6.8, 5 and 7.5 pg on-column, respectively. The mean inter- and intra-assay relative standard deviations (RSDs) over the range of the standard curve were less than 5%. The absolute recoveries averaged 99.1%, 99.5%, 97.7%, 99.5% and 98.8% for NA, DA, 5-HT, 5-HIAA and HVA, respectively.