Chronic administration of clomipramine inhibits morphine hot plate analgesia

Clomipramine, chronically administered in mice, for 3 days, inhibits partially but significantly morphine analgesia in the hot plate test, when used at dose of 10 mg/kg/day, i.p.; 2.5 and 5 mg/kg/day were ineffective. Neither higher doses (20 and 40 mg/kg/day) nor longer duration of pretreatment (8 and 16 days) modified the intensity of this inhibition. Reduction in morphine analgesia was obtained after a 24h delay between the last injection of clomipramine and that of morphine (30 min before testing), while clomipramine did not induce any antinociceptive effect and clomipramine and desmethylclomipramine plasma and brain levels were low or undetectable. These results provide new evidence for the interaction between clomipramine and the endogenous opiate system. A pharmacokinetic interaction between clomipramine and morphine was excluded; involvement of change in opiate and 5 HT2 receptors by chronic administration of clomipramine is discussed.     

Determination of clomipramine by flow‐injection analysis with acidic potassium permanganate–formic acid chemiluminescence detection

A sensitive and simple chemiluminescent (CL) method for the determination of clomipramine has been developed by combining the flow‐injection analysis (FIA) technique, which is based on the CL intensity generated from the redox reaction of potassium permanganate (KMnO₄)–formic acid in sulphuric acid (H₂SO₄) medium. Under the optimum conditions, the linear range for the determination of clomipramine was 0.04–4 µg/mL, with a correlation coefficient of 0.9988 (n = 10) and a detection limit of 0.008 µg/mL (3σ), and the relative standard deviation (RSD) for 2.0 µg/mL clomipramine (n = 11) is 1.26%. The proposed method has been successfully applied to the determination of the studied clomipramine in pharmaceutical preparations. The possible reaction mechanism is discussed. Copyright © 2011 John Wiley & Sons, Ltd.     

Quantification of antidepressants and antipsychotics in human serum by precipitation and ultra high pressure liquid chromatography-tandem mass spectrometry

The present article describes the quantification of mirtazapine, O-desmethylvenlafaxine, quetiapine, venlafaxine, and ziprasidone (group 1), and amitriptyline, citalopram, clomipramine, clozapine, desmethylclomipramine, desipramine, imipramine, and nortriptyline (group 2) in human serum for therapeutic drug monitoring. The method was developed to replace old techniques which applied solid phase extraction and ultra-violet detection. The old methods had reached their limit of capacity regarding the number of samples and co-medicated drugs interfering with the detection. Serum samples were precipitated with zinc sulphate and methanol containing a stable isotope labelled analog for each analyte. Quantitative analysis was performed by ultra high pressure liquid chromatography combined with a tandem mass spectrometer using a Zorbax SB-C8 column (2.0 × 50 mm; 1.8 μm) with a mobile phase consisting of 0.1% formic acid in water and methanol, respectively. The total run time of the chromatography was 4 min. Precision and trueness varied from 2.6% to 14.9% and 87.6% to 103.5%, respectively. At the lower limit of quantification, precision was up to 17.9% and trueness varied from 89.5% to 111.5%. A five point standard curve covering the clinically relevant ranges with a power function fit was applied for calibration. Ion suppression from matrix effects and internal standards were thoroughly investigated and are discussed. Process efficiency rates varied from 42% to 99%. The method has shortened the response time, reduced interference from other drugs, avoided acetonitrile usage, and reduced the amount of serum needed for analysis 50-fold.     

Developing imidazole analogues as potential inhibitor for Leishmania donovani trypanothione reductase: virtual screening,molecular docking,dynamics and ADMET approach

Visceral leishmaniasis (VL) affects Indian subcontinent, African and South American continent, and it covers 70 countries worldwide. Visceral form of leishmaniasis is caused by Leishmania donovani in Indian subcontinent which is lethal if left untreated. Extensive resistance to antileishmanial drugs such as sodium stibogluconate, pentamidine and miltefosine and their decreased efficacy has been reported in the endemic region. Amphotericin B drug has shown good antileishmanial activity with significant toxicity, but its cost of treatment has limited the outreach of this treatment to affected people living in endemic zone. So, there is an urgent need to identify new antileishmanial drugs with excellent activity and minimal toxicity issues. Trypanothione reductase, a component of antioxidant system, is necessary for parasite growth and survival to raise infection. To develop potential inhibitor, we docked nine hundred and eighty-four 5-nitroimidazole analogues along with clomipramine which is a well-known inhibitor for TR. Total one hundred and forty-seven 5-nitroimidazole analogues with better docking score than clomipramine were chosen for ADMET and QikProp studies. Among these imidazole analogues, total twenty-four imidazole analogues and clomipramine were chosen on the basis of their ADMET, QikProp, and prime MM-GBSA study. Later on, two analogues with best MM-GBSA dG bind were undergone molecular dynamic simulation to ensure protein–ligand interactions. Using above approach, we confirm that ethyl 2-acetyl-5-[4-butyl-2-(3-hydroxypentyl)-5-nitro-1H-imidazol-1-yl]pent-2-enoate can be a drug candidate against L. donovani for the treatment of VL in the Indian subcontinent.     

Simultaneous determination of sulphamonomethoxine and its N4-acetyl metabolite in blood serum by high-performance liquid chromatography with direct injection

A high-performance liquid chromatographic method with direct injection has been developed for the simultaneous determination of sulphamonomethoxine and its N⁴-acetyl metabolite in serum of animals and fish. A HISEP shielded hydrophobic-phase column (15 cm × 4.6 mm I.D.), a mobile phase of 0.05 M citric acid–0.2 M disodium hydrogenphosphate-acetonitrile (70:15:15, v/v), and ultraviolet detection at 265 nm were used. The standard calibration curves in serum of chicken, pig, cattle, rainbow trout and yellowtail were linear over the range 0.5–20 μg/ml. The recoveries of sulphamonomethoxine and its N⁴-acetyl metabolite from all serum samples determined at different concentrations (0.5, 2.0 and 10.0 μg/ml) were 93–103% and 90–103%, respectively. The lowest measurable sulphamonomethoxine and N²-acetyl metabolite concentrations were 0.04 and 0.1 μg/ml, respectively, for all serum samples.     

Simultaneous determination of a new anticancer agent (NB-506) and its active metabolite in human plasma and urine by high-performance liquid chromatography with ultraviolet detection

A high-performance liquid chromatographic method with ultraviolet detection has been developed to quantify NB-506 and its active metabolite in human plasma and urine. This method is based on solid-phase extraction, thereby allowing the simultaneous measurement of the drug and metabolite with the limit of quantification of 0.01 μg/ml in plasma and 0.1 μg/ml in urine. Standard curves for the compounds were linear in the concentration ranges investigated. The range for the drug in plasma was 0.01–2.5 μg/ml, and for the metabolite 0.01–1 μg/ml. In urine, the range for both compounds was 0.1–10 μg/ml. The method was validated and applied to the assay of plasma and urinary samples from phase I studies.     

Determination of a major metabolite of tipredane in rat urine by high-performance liquid chromatography with column switching

An automated method, based on column-switching reversed-phase high-performance liquid chromatography, has been developed for the determination of a major metabolite of tipredane in rat urine. Samples are injected directly onto a cyanopropyl extraction column. The portion of eluate containing the metabolite is switched, via an injection loop, onto an octadecylsilane analytical column. The limit of quantification of the method was 25 ng/ml for a 20 μl injection volume of urine. The intra-assay precision (0.7–4.8%) and accuracy (94–105%), and the inter-assay precision (2.7–12.6%) and accuracy (94–105%), were acceptable. The analyte was found to be stable in rat urine when stored at room temperature for six days, in a freezer at or below −20°C for twelve weeks, and when the samples were subjected to two freeze–thaw cycles. No significant interference was observed from tipredane and its major human metabolites, or urine constituents in male and female rats. The method was successfully used to analyse samples from a long-term toxicology study.     

Rapid microsample analysis of imipramine and desipramine by reversed-phase high-performance liquid chromatography with ultraviolet detection

A rapid and highly sensitive HPLC assay method was developed to measure small amounts of imipramine and its major metabolite, desipramine. The assay involved simple extraction procedures using clomipramine as the internal standard. The mobile phase consisted of acetonitrile (60%) and 0.01 M triethylamine in distilled water (40%) with the pH adjusted to 3.0. Separations were achieved on a C₁₈ column and the effluent measured for UV absorption at 260 nm. The chromatographic separation was excellent, with no interference from endogenous serum constituents. This assay was suitable for measuring drug concentrations in the range of 10–1000 ng/ml using a 0.1-ml serum sample. The method was applied to a drug disposition study in transgenic mice with increased plasma α₁-acid glycoprotein.     

Identification of YH439 and its metabolites in rat urine by gas chromatography–mass spectrometry and liquid chromatography–mass spectrometry

YH439 is a potential drug candidate for the treatment of various hepatic disorders. YH439 and its three metabolites have been identified in rat urine by liquid chromatography–mass spectrometry (LC–MS) and by gas chromatography (GC)–MS. Identification of YH439 and its metabolites was established by comparing their GC retention times and mass spectra with those of the synthesized authentic standards. Both electron impact- and positive chemical ionization MS have been evaluated. The metabolism study was performed in the rat using oral administration of the drug. A major metabolite (YH438) was identified as the N-dealkylation product of YH439. Other identified metabolites were caused by the loss of the methyl thiazolyl amine group (metabolite II) from YH439, the isopropyl hydrogen malonate group (metabolite IV) and the decarboxylated product (metabolite III) of metabolite II.     

Improved high-performance liquid chromatography determination of methotrexate and its major metabolite in plasma using a poly(styrene-divinylbenzene) column

A sensitive high-performance liquid chromatographic assay has been developed for measuring plasma concentrations of methotrexate and its major metabolite, 7-hydroxymethotrexate. Methotrexate and metabolite were extracted from plasma using solid-phase extraction. An internal standard, aminopterin was used. Chromatographic separation was achieved using a 15-cm poly(styrene-divinylbenzene) (PRP-1^®) column. This column is more robust than a silica-based stationary phase. Post column, the eluent was irradiated with UV light, producing fluorescent photolytic degradation products of methotrexate and the metabolite. The excitation and emission wavelengths of fluorescence detection were at 350 and 435 nm, respectively. The mobile phase consisted of 0.1 M phosphate buffer (pH 6.5), with 6% N,N-dimethylformamide and 0.2% of 30% hydrogen peroxide. The absolute recoveries for methotrexate and 7-hydroxymethotrexate were greater than 86%. Precision, expressed as a coefficient of variation (n=6), was <10% at each of five methotrexate concentrations in the range 2.5–50 ng/ml. The limits of quantitation of methotrexate were 1 and 2.5 ng/ml for methotrexate and 7-hydroxymethotrexate, respectively (using 1 ml plasma). A robust HPLC method has been developed for the reproducible quantitation of methotrexate in plasma of patients taking a weekly dose of methotrexate for rheumatoid arthritis.     

Simultaneous determination of tolmetin and its metabolite in biological fluids by high-performance liquid chromatography

A highly sensitive and selective high-performance liquid chromatographic assay has been developed for the separation and quantitation of tolmetin and its major metabolite in human biological fluids, viz. plasma, urine and synovial fluid. Analysis of plasma and synovial fluid required only 0.5 ml of the sample. The sample was washed with diethyl ether and extracted with diethyl ether—chloroform (2:1). The extracted compounds were injected onto a reversed-phase column (RP-2) and absorbance was measured at 313 nm. The standard curves in plasma were found to be linear for both tolmetin and the metabolite at concentrations from 0.04 to 10.0 μg/ml. Urine samples (0.5 ml) were diluted (1:1) with methanol containing the internal standard and were directly injected onto the reversed-phase (RP-2) column. Standard curves of tolmetin and metabolite in urine were linear in the range 5–300 μg/ml. Serum and synovial fluid concentrations of tolmetin and its metabolite in patients receiving multiple doses of tolmetin sodium were determined using the assay procedure.     

Estrogen metabolism in nonhuman primates I. In vitro biosynthesis of estrogen glucosiduronates in rhesus monkey liver

Estrone glucosiduronate, 17β-estradiol-3-glucoslduronate, 17β-estradiol-17-glucosiduronate and estriol-16α-glucoslduronate have been biosynthesized in substantial yield by incubation of radioactive estrone, 17β-estradiol, estriol and uridlne diphosphoglucosiduronic acid with rhesus monkey liver homogenates. The metabolites were characterized by chromatography on Celite and DEAE-Sephadex, enzyme hydrolysis, derivative formation and crystallization to constant specific activity. The percent conversion to 17β-estradiol-17-glucosiduronate from 17β-estradlol ranged between 56–71%; from estrone, the conversion was 49–54%. Other metabolites formed from estradiol were estrone glucosiduronate(12–21%) and 17β-estradiol-3-glucosiduronate(5–12%). The same metabolites derived from estrone accounted for 18–28% and 10–14% respectively. After estriol incubation, more than 90% of the steroid was converted to estriol-16α-glucosiduronate with no detectable estriol-3-glucosiduronate. This report represents the first time that 17β-estradiol-17-glucosiduronate has been reported as a metabolite in the rhesus monkey and this is the only known species which forms 17β-estradiol-17-glucosiduronate as the predominant metabolite of either estrone or estradiol $\text{in vitro}$.Rhesus monkey liver is similar to the human and baboon in that it metabolizes estriol exclusively to estriol-16-glucosiduronate.     

Determination of the enantiomers of metoprolol and its major acidic metabolite in human urine by high-performance liquid chromatography with fluorescence detection

Enantiomers of metoprolol and its acidic metabolite H 117/04 were determined in human urine by high-performance liquid chromatography (HPLC) with fluorometric detection after chiral derivatization. The carboxyl functional group of the major metabolite was blocked by esterification after solid-phase extraction, which helped to quantitate this compound from interfering substances. The assay method was validated. The recovery of (−)- and (+)-metoprolol from urine was 86.3–90.5%; and the recovery of the (−)- and (+)-acidic metabolite H 117/04 from urine was 74.4–83.9% at different concentrations.     

Ethanol utilization for metabolite production by Candida utilis strains in liquid medium

Ethanol has been reported to be a gaseous pollutant, originating from the agricultural industry. Interest in its biodegradation has increased over the last two decades. Most of the current studies have focused on its elimination by mixed cultures. This study is part of a broader project intended to utilize Candida utilis strains for gaseous ethanol elimination and to eventually bioconvert them into biomass and/or volatile metabolites. We present here the study of six strains (one from the ATCC and five from the ICIDCA collection) cultivated in a liquid medium, with initial ethanol concentrations of 16 g/l and 32 g/l. At 16 g/l, a maximum ethanol elimination rate of 0.13 g/l × h was obtained in four of the six strains (ATCC 9950, L/375–1, L/375–5 and L/375–10). This rate increased to 0.21 g/l × h with an initial ethanol concentration of 32 g/l. The L/375–5 strain was the best biomass producer (3.3 g/l) at 32 g/l, while the highest ethyl acetate production (0.80 g/l) was obtained with the L/375–1 strain. The L/375–25 and L/375–26 strains which showed very low ethyl acetate production were, by way of contrast, efficient acetaldehyde producers, with 0.54 g/l and 0.66 g/l measured in the broth. While biomass production reached its maximum after two days of culture, the production of acetic acid and ethyl acetate continued during the third day. The results for biomass and metabolite production obtained with the ICIDCA collection strains (L/375–1, L/375–5 and L/375–10) were better than those obtained with the ATCC 9950 strain, although the latter often has been reported to be particularly suitable for metabolite production.     

Metabolic profiling of human saliva before and after induced physiological stress by ultra-high performance liquid chromatography–ion mobility–mass spectrometry

A method has been developed for metabolite profiling of the salivary metabolome based on protein precipitation and ultra-high performance liquid chromatography coupled with ion mobility-mass spectrometry (UHPLC–IM–MS). The developed method requires 0.5 mL of human saliva, which is easily obtainable by passive drool. Standard protocols have been established for the collection, storage and pre-treatment of saliva. The use of UHPLC allows rapid global metabolic profiling for biomarker discovery with a cycle time of 15 min. Mass spectrometry imparts the ability to analyse a diverse number of species reproducibly over a wide dynamic range, which is essential for profiling of biofluids. The combination of UHPLC with IM–MS provides an added dimension enabling complex metabolic samples to be separated on the basis of retention time, ion mobility and mass-to-charge ratio in a single chromatographic run. The developed method has been applied to targeted metabolite identification and untargeted metabolite profiling of saliva samples collected before and after exercise-induced physiological stress. δ-Valerolactam has been identified as a potential biomarker on the basis of retention time, MS/MS spectrum and ion mobility drift time.     

Gas chromatographic–mass spectrometric assay for rocuronium with potential for quantifying its metabolite, 17-desacetylrocuronium, in human plasma

A rapid, sensitive and selective method has been developed for the quantification of plasma concentrations of neuromuscular blocking drug, rocuronium, using gas chromatography with mass spectrometric detection. 3-Desacetylvecuronium served as the internal standard. The method involved iodide ion pair formation and a single-step liquid–liquid extraction with dicholoromethane. This method also permits simultaneous determination of its putative metabolite, 17-desacetylrocuronium, although the high detection limit for the metabolite limits the practical application of this method in pharmacokinetic study of the metabolite. The extraction efficiency was 75% for rocuronium and 50% for 17-desacetylrocuronium. The limit of quantification was 26 ng/ml for rocuronium and 870 ng/ml for its metabolite. The assay was used successfully in a patient undergoing liver transplantation and receiving rocuronium as a constant rate infusion and in a patient undergoing general elective surgery receiving the drug as an intravenous bolus. This assay is a time-saving alternative to published gas or liquid chromatographic methods for assaying rocuronium.     

Liquid chromatographic analysis and preliminary pharmacokinetics of methotrexate in cancer patients co-treated with docetaxel

A new HPLC method has been developed for the quantitative determination of methotrexate (MTX) and its 7-hydroxyl metabolite in human plasma. Samples were purified by protein precipitation with acetone and methanol, and a sample clean-up with a mixture of n-butanol and diethyl ether. The analytes were separated on an RP Inertsil ODS-80A column and eluted in a solvent system containing 5% (v/v) tetrahydrofuran in water (pH 2.0). UV absorption measurement was performed at 313 nm, and the detector response was linear in a concentration range of 10–10 000 ng/ml. The lower limit of quantitation of MTX was 10 ng/ml using 1 ml sample aliquots. Values for accuracy and (within-run and between-run) precision were between 95.5–111% and 3.69–11.0%, respectively, at four concentrations analyzed in quintuplicate on four separate occasions. The assay was applied to study the effects of docetaxel co-administration on the pharmacokinetics and metabolism of MTX in cancer patients.     

Rapid determination of the anti-cancer drug chlorambucil (Leukeran™) and its phenyl acetic acid mustard metabolite in human serum and plasma by automated solid-phase extraction and liquid chromatography–tandem mass spectrometry

A bioanalytical method for the determination of the anticancer drug chlorambucil (Leukeran™) and its phenyl acetic acid mustard metabolite in human serum and plasma is described. Automated solid-phase extraction of the analytes is carried out with C₁₈ sorbent packed in a 96 well format microtitre plate using a robotic sample processor. The extracts are analysed by isocratic reversed-phase liquid chromatography using pneumatically and thermally assisted electrospray ionisation (TurboIonspray) with selected reaction monitoring. The method is specific and sensitive, with a range of 4–800 ng/ml in human serum and plasma for both parent drug and metabolite (sample volume 200 μl). The method is accurate and precise with intra-assay and inter-assay precision (C.V.) of <15% and bias <15% for both analytes. The automated extraction procedure is significantly faster than manual sample pre-treatment methods, a batch of 96 samples is extracted in 50 min allowing for faster sample turnaround. The method has been used to provide pharmacokinetic support to biocomparability studies of Leukeran™ following single doses of oral tablet formulations.     

Quantification of naltrexone and 6,β-naltrexol in plasma and milk using gas chromatography–mass spectrometry: Application to studies in the lactating sheep

A selective gas chromatography–mass spectrometry method using solid-phase extraction has been developed for the detection and quantification of naltrexone and its metabolite, 6,β-naltrexol in plasma and milk from humans and sheep at pharmacologically relevant concentrations. Di- or tri-acetyl derivatives were formed and quantified by selected-ion monitoring. Recoveries of naltrexone (30 μg/l) and 6,β-naltrexol (250 μg/l) from both human plasma and milk were greater than 70%. Intra-assay and inter-day precision ranged from 3 to 21% for naltrexone and 2–18% for 6,β-naltrexol for all matrices investigated, with an overall mean accuracy of 104% for naltrexone, and 99% for 6,β-naltrexol. Human samples containing these analytes were stable for at least 3 weeks at −20°C or 6 weeks at −80°C. Analysis of the plasma and milk from the lactating sheep showed mean milk-to-plasma ratios of 55 for naltrexone and 3 for 6,β-naltrexol.     

The inhibitors of the 5HT-transporter fluoxetine and clomipramine attenuate serotonin-induced constriction of the aorta and the calcium signal in smooth muscle cells of the rat

We found that the inhibitors of the serotonin (5HT) transporter fluoxetine and clomipramine significantly inhibit 5HT-induced constriction of isolated rings of the aorta. The most prominent inhibitory effect was observed for clomipramine, which at a concentration of 2 μM, prevented aorta constriction in response to low and moderate doses of 5HT and multiply attenuated it in response to high doses (10 μM). The inhibitors of the 5HT-transporter attenuated the strength of norepinephrine-induced aorta constriction by 40–60% and eliminated long-term tonic constriction. Application of clomipramine or fluoxetine on the vessels preliminarily constricted by norepinephrine resulted in 100% relaxation, which was maintained in the presence of the NO-synthase inhibitor L-NAME. The inhibitors of the 5HT-transporter decreased but did not prevent 5HT-induced [Ca²⁺]_cyt increase in smooth muscle cells (SMCs) of the aorta even at high concentrations. Clomipramine and fluoxetine did not affect the vasopressin-induced [Ca²⁺]_cyt increase in SMCs and the strength of constriction of isolated aorta rings. We found that the sensitivity of the rat aorta to the vasoconstrictor effect of 5HT and the role of the 5HT-transporter in regulation of the vascular tone increased with aging.