Acute Toxicity From Intravenous Use of the Tricyclic Antidepressant Tianeptine.

Tianeptine (7-[([3-chloro-6,11]-dihydro-6-methyldibenzo[c,f][1,2]thiazepin-11-yl) amino] heptanoic acid S, S dioxide) is a tricyclic compound prescribed as an antidepressant in European countries, but is not currently approved for use in the United States. There are few published case reports of tianeptine intoxication. Presented is the first case of acute toxicity associated with the intravenous use of tianeptine. A 36-year-old male intentionally injected tianeptine powder intravenously to "help him see into the future". He became unresponsive and a bystander called emergency medical services. Upon arrival to the Emergency Department, excessive constriction of the pupils, sedation, and a respiratory rate of 6 respirations per minute (rpm) were noted. Blood and urine were collected ~2 h post admission. The patient's serum ethanol concentration was 133 mg/dL. His toxicity was successfully reversed with two doses of naloxone 0.4 mg IV. He was started on a naloxone infusion at 0.2 mg/h and discharged 13 h after admittance awake, alert and oriented. The patient's urine sample screened negative for common drugs of abuse and total tricyclic antidepressants. A high performance liquid chromatography tandem mass spectrometry method was developed and validated to quantify tianeptine in urine. The calibration range was 1-100 ng/mL with linear regression correlation (r2) of 0.9996 or greater. The limit of quantitation was administratively set at 1 ng/mL. The bias of the assay was determined to be within ±20% of the target value for each quality control specimen. The intra-day and inter-day precision did not exceed 15% coefficient of variation for each quality control specimen. Matrix effects, absolute recovery, carryover and specificity were also evaluated. The patient's tianeptine urine concentration was determined to be 2 ng/mL.

Investigation of the in vitro metabolism of the emerging drug candidate S107 for doping-preventive purposes.

The metabolic fate of the emerging drug candidate S107, possessing the potential for misuse as performance-enhancing agent in sports, was investigated by in vitro phase I and II experiments with human microsomal and S9 liver enzymes. The metabolites were identified by liquid chromatography-mass spectrometry with electrospray ionisation in positive mode (LC-ESI-MS/MS). Their collision-induced dissociation behaviour was studied by high-resolution/high accuracy Orbitrap MS(n) analysis, supported by stable isotope labelling, H/D-exchange experiments and density functional theory calculations. Monooxygenation accounted for the main phase I metabolic transformation due to N- and S-oxidation of the 1,4-benzothiazepine core, as substantiated by chemical synthesis, selective reduction methods and characteristic APCI in source fragmentation behaviour of the metabolites. Another dominant metabolic pathway was demethylation, yielding the N- and O-demethylated metabolite, respectively. The latter was further conjugated by glucuronidation as well as sulfonation in subsequent phase II metabolic reactions, whereas the N-demethylated metabolite was not amenable to conjugation. The active drug molecule itself was converted to two glucuronic acid conjugates, which are proposed to consist of two quaternary S107-N(+)-glucuronide isomers. All glucuronides were susceptible to enzymatic hydrolysis with ß-glucuronidase (Escherichia coli). A comprehensive LC-ESI-MS(/MS)-based detection method for urine was developed and its fitness for purpose was assessed. The assay can serve as a potential screening and/or confirmation method for S107 in clinical drug testing and doping control analysis in the future.

Electron ionization mass spectrometry of the ryanodine receptor-based Ca(2+)-channel stabilizer S-107 and its implementation into routine doping control.

New insights into the biochemistry of cardiac arrhythmia and skeletal muscle fatigue have yielded new drug candidates to counteract these phenomena. Major biological targets have become ryanodine receptor (RyR)-based Ca(2+)-release channels, which tend to 'leak' under various circumstances including strenuous exercise and, thus, cause aberrant calcium sparks that entail impaired muscle function. Therapeutics, which are referred to as rycals, are currently being developed to treat cardiac arrhythmia via enhancement of calstabin-ryanodine affinities that causes a stabilization of the RyR. These therapeutics possess potential for misuse in sports, and an early implementation of target analytes such as the benzothiazepine derivatives S-107 and JTV-519 or putative metabolites into doping control screening procedures is recommended. Reference compounds, deuterated analogues, and a putative metabolic product were synthesized, and electron ionization mass spectra of these products were studied and dissociation pathways elucidated by means of tandem mass spectrometry (MS/MS) and accurate mass measurements. The characterized analytes were incorporated into existing sports drug testing assays based on liquid-liquid extraction and subsequent gas chromatography/mass spectrometry (GC/MS) analysis, and specificity, lower limit of detection (4-6 ng/mL), intraday and interday precision (1.5-17.2%), as well as recovery (63-66%) were determined. The established procedure proved suitable for routine doping control analysis to detect a potential misuse of the drug candidate S-107 in elite sport.

Screening for the calstabin-ryanodine receptor complex stabilizers JTV-519 and S-107 in doping control analysis.

Recent studies outlined the influence of exercise on the stability of the skeletal muscle calstabin1-ryanodine receptor1-complex, which represents a major Ca(2+) release channel. The progressive modification of the type-1 skeletal muscle ryanodine receptor (RyR1) combined with reduced levels of calstabin1 and phosphodiesterase PDE4D3 resulted in a Ca(2+) leak that has been a suggested cause of muscle damage and impaired exercise capacity. The use of 1,4-benzothiazepine derivatives such as the drug candidates JTV-519 and S-107 enhanced rebinding of calstabin1 to RyR1 and resulted in significantly improved skeletal muscle function and exercise performance in rodents. Due to the fact that the mechanism of RyR1 remodelling under exercise conditions were proven to be similar in mice and humans, a comparable effect of JTV-519 and S-107 on trained athletes is expected, making the compounds relevant for doping controls. After synthesis of JTV-519, S-107, and a putative desmethylated metabolite of S-107, target compounds were characterized using nuclear magnetic resonance spectroscopy and electrospray ionization (ESI)-high-resolution/high-accuracy Orbitrap mass spectrometry. Collision-induced dissociation pathways were suggested based on the determination of elemental compositions of product ions and H/D-exchange experiments. The most diagnostic product ion of JTV-519 was found at m/z 188 (representing the 4-benzyl-1-methyl piperidine residue), and S-107 as well as its desmethylated analog yielded characteristic fragments at m/z 153 and 138 (accounting for 1-methoxy-4-methylsulfanyl-benzene and 4-methoxy-benzenethiol residues, respectively). The analytes were implemented in existing doping control screening procedures based on liquid chromatography, multiple reaction monitoring and simultaneous precursor ion scanning modes using a triple quadrupole mass spectrometer. Validation items such as specificity, recovery (68-92%), lower limit of detection (0.1-0.2 ng/mL), intraday (5.2-18.5%) and interday (8.7-18.8%) precision as well as ion suppression/enhancement effects were determined.

Effects of organic anion transporting polypeptide 1B1 haplotype on pharmacokinetics of pravastatin, valsartan, and temocapril.

OBJECTIVE: Recent reports have shown that genetic polymorphisms in organic anion transporting polypeptide (OATP) 1B1 have an effect on the pharmacokinetics of drugs. However, the impact of OATP1B1*1b alleles, the frequency of which is high in all ethnicities, on the pharmacokinetics of substrate drugs is not known after complete separation of subjects with OATP1B1*1a and *1b. Furthermore, the correlation between the clearances of OATP1B1 substrate drugs in individuals has not been characterized. We investigated the effect of genetic polymorphism of OATP1B1, particularly the *1b allele, on the pharmacokinetics of 3 anionic drugs, pravastatin, valsartan, and temocapril, in Japanese subjects. METHODS: Twenty-three healthy Japanese volunteers were enrolled in a 3-period crossover study. In each period, after a single oral administration of pravastatin, valsartan, or temocapril, plasma and urine were collected for up to 24 hours. RESULTS: The area under the plasma concentration-time curve (AUC) of pravastatin in *1b/*1b carriers (47.4 +/- 19.9 ng.h/mL) was 65% of that in *1a/*1a carriers (73.2 +/- 23.5 ng.h/mL) (P = .049). Carriers of *1b/*15 (38.2 +/- 15.9 ng.h/mL) exhibited a 45% lower AUC than *1a/*15 carriers (69.2 +/- 23.4 ng.h/mL) (P = .024). In the case of valsartan we observed a similar trend as with pravastatin, although the difference was not statistically significant (9.01 +/- 3.33 microg.h/mL for *1b/*1b carriers versus 12.3 +/- 4.6 microg.h/mL for *1a/*1a carriers [P = .171] and 6.31 +/- 3.64 microg.h/mL for *1b/*15 carriers versus 9.40 +/- 4.34 microg.h/mL for *1a/*15 carriers [P = .213]). The AUC of temocapril also showed a similar trend (12.4 +/- 4.1 ng.h/mL for *1b/*1b carriers versus 18.5 +/- 7.7 ng.h/mL for *1a/*1a carriers [P = .061] and 16.4 +/- 5.0 ng.h/mL for *1b/*15 carriers versus 19.0 +/- 4.1 ng.h/mL for *1a/*15 carriers [P = .425]), whereas that of temocaprilat (active form of temocapril) was not significantly affected by the haplotype of OATP1B1. Interestingly, the AUC of valsartan and temocapril in each subject was significantly correlated with that of pravastatin (R = 0.630 and 0.602, P < .01). The renal clearance remained unchanged for each haplotype for all drugs. CONCLUSION: The major clearance mechanism of pravastatin, valsartan, and temocapril appears to be similar, and OATP1B1*1b is one of the determinant factors governing the interindividual variability in the pharmacokinetics of pravastatin and, possibly, valsartan and temocapril.

Comparison of urinary excretion of pravastatin and temocapril in bile duct-ligated rats and Eisai hyperbilirubinemic rats (EHBR).

BACKGROUND/PURPOSE: In patients with complete bile duct obstruction, the only pathway for the elimination of cholephilic compounds is through the urine. Although changes in various transporters in the liver and kidney in cholestasis have been elucidated, little is known about how effectively the elimination of these compounds is compensated for by urinary excretion. METHODS: In the present study, the urinary excretion of pravastatin and temocapril was studied in bile-duct-ligated rats (BDLR) for 3 days and in Eisai hyperbilirubinemic rats (EHBR). After urinary bladder cannulation, radiolabeled pravastatin and temocapril were injected intravenously. Urine samples were collected every 1 h for 4 h, and the radioactivity was counted. RESULTS: Urinary excretion of pravastatin was markedly increased in BDLR (85.9% of the dose after 4 h) and moderately increased in EHBR (35.9% of the dose after 4 h) compared with that in control rats (5.5% of the dose after 4 h). Similar but less prominent differences were observed with temocapril after it was administered (50.7%, 38.2%, and 22.0% of the dose after 4 h in BDLR, EHBR, and the controls, respectively). CONCLUSIONS: The absence of biliary excretion of anionic drugs was compensated for by urinary excretion in BDLR and EHBR, and the compensation was more efficient with pravastatin than with temocapril. In patients with complete bile duct obstruction, the only pathway for the elimination of cholephilic compounds is through the urine. Although changes in various transporters in the liver and kidney in cholestasis have been elucidated, little is known about how effectively the elimination of these compounds is compensated for by urinary excretion.

Metabolism of [(14)C]omapatrilat, a sulfhydryl-containing vasopeptidase inhibitor in humans.

Omapatrilat, a potent vasopeptidase inhibitor, is currently under development for the treatment of hypertension and congestive heart failure. This study describes the plasma profile along with isolation and identification of urinary metabolites of omapatrilat from subjects dosed orally with 50 mg of [(14)C]omapatrilat. Only a portion of the radioactivity in plasma was unextractable (40-43%). Prominent metabolites identified in plasma were S-methyl omapatrilat, acyl glucuronide of S-methyl omapatrilat, and S-methyl (S)-2-thio-3-phenylpropionic acid. Omapatrilat accounted for less than 3% of the radioactivity. However, after dithiothreitol reduction all of the radioactivity was extractable and was characterized to be omapatrilat and its hydrolysis product (S)-2-thio-3-phenylpropionic acid, both apparently bound to proteins via reversible disulfide bonds. Urinary profile of radioactivity showed no parent compound but the presence of several metabolites that can be grouped into three categories. 1) Three metabolites, accounting for 56% of the urinary radioactivity, resulted from the hydrolysis of the exocyclic amide bond of omapatrilat. Two metabolites were diastereomers of S-methyl sulfoxide of (S)-2-thio-3-phenylpropionic acid, and the third was the acyl glucuronide of S-methyl (S)-2-thio-3-phenylpropionic acid. 2) One disulfide, identified as the L-cysteine mixed disulfide of omapatrilat, accounted for 8% of the radioactivity in the urine. 3) Five metabolites, derived from omapatrilat, accounted for 30% of the radioactivity in the urine. Two of these metabolites were mixtures of diastereomers of S-methyl sulfoxide of omapatrilat and the third was the S-methyl omapatrilat ring sulfoxide. The other two metabolites were S-methyl omapatrilat and its acyl glucuronide. These results indicate that omapatrilat undergoes extensive metabolism in humans.

A novel octahydropyridobenzothiazepine metabolite in human urine: biomimetic formation from the melanogen 5-S-cysteinyldopa and formaldehyde via a peculiar sulfur-controlled double Pictet-Spengler condensation.

HPLC evidence is reported demonstrating the occurrence in some human urine samples of a novel catecholic metabolite, (3R,7S)-3, 7-dicarboxy-10,11-dihydroxy-2,3,4,5,6,7,8,9-octahydropyrido[ 4,3-g][1, 4]benzothiazepine (2). The compound was shown to arise by a double Pictet-Spengler condensation of the urinary melanogen 5-S-cysteinyldopa (1) with formaldehyde, in which regioselective formation of the six-membered ring ortho to the activating hydroxyl group lends assistance to the subsequent closure of the seven-membered 1,4-thiazepine moiety. Under physiologically relevant conditions, i.e., in 0.1 M phosphate buffer pH 7.4 and at 37 degrees C, the 7,8-tetrahydroisoquinoline 5 was the sole detectable intermediate in the formation of 2. N-Acetylcysteinyldopa (4) reacted likewise with formaldehyde to give the 7, 8-dihydroxytetrahydroisoquinoline 6. The anomalous regiochemistry underlying formation of 5 and 6 was rationalized with the aid of AM1/PM3 calculations on the model alkylthiocatechol 10, predicting a higher HOMO-controlled reactivity on the position ortho rather than para to the activating hydroxyl group. The potential of the reported chemistry as a convenient synthetic access to the 2,3,4, 5-tetrahydro[1,4]benzothiazepine ring system is suggested by the efficient conversion of the cysteinylcatechol 3 to 8 in the presence of formaldehyde.

Determination of cystathionine and perhydro-1,4-thiazepine-3,5-dicarboxylic acid in the urine of a patient with cystathioninuria using column liquid chromatography-mass spectrometry.

A method for the measurement of cystathionine and perhydro-1,4-thiazepine-3,5-dicarboxylic acid in the urine of a patient with cystathioninuria has been developed, using column liquid chromatography-mass spectrometry. Cystathionine and perhydro-1,4-thiazepine-3,5-dicarboxylic acid were determined by scanning the [M + H]+ ions of each compound. The recoveries were 80-92.4% for cystathionine and 80-100% for perhydro-1,4-thiazepine-3,5-dicarboxylic acid after ion-exchange treatment. The results agreed well with those obtained using an amino acid analyser. The concentrations found for cystathionine and perhydro-1,4-thiazepine-3,5-dicarboxylic acid were 1.289 +/- 0.099 mg/ml and 0.310 +/- 0.0067 mg/ml, respectively.

Determination of a new angiotensin-converting enzyme inhibitor (CS-622) and its active metabolite in plasma and urine by gas chromatography-mass spectrometry using negative ion chemical ionization.

A sensitive and specific gas chromatographic-mass spectrometric method for the simultaneous determination of angiotensin-converting enzyme inhibitor (I, CS-622) and its active desethyl metabolite (II, RS-5139) in plasma and urine was developed. Compound D5-RS-5139 was used as an internal standard and measurements were made by electron-capture negative ion chemical ionization. Extraction from plasma and urine was carried out using Sep-Pak C18 and silica cartridges. The extract of plasma or urine was treated with diazomethane followed by trifluoroacetic anhydride to convert I and II into their methyl ester trifluoroacetyl derivatives. The detection limit of I and II was 0.5 ng/ml in plasma and 5 ng/ml in urine. The proposed method was satisfactory for the determination of I and II in plasma and urine with respect to accuracy and precision. Thus it is suitable for measurement of bioavailability and pharmacokinetics of I and II in body fluids.

Hexahydro-1,4-thiazepine-3,5-dicarboxylic acid and thiomorpholine-3,5-dicarboxylic acid are present in normal human urine.

Hexahydro-1,4-thiazepine-3,5-dicarboxylic acid and thiomorpholine-3,5-dicarboxylic acid, simply referred to as cyclothionine and TMDA, respectively, are two cyclic sulfur-containing imino acids detected in bovine brain. Human urine has been investigated to establish the occurrence of these imino acids as common constituents under normal conditions. The morning urine of healthy subjects has been analyzed for enrichment of these compounds by using an ion-exchange procedure. Gas/liquid chromatography of the final extracts revealed the presence of peaks coeluting with authentic cyclothionine and TMDA. The latter compound eluted very close to an unknown sulfur-containing compound. A resolved peak of TMDA has been obtained by high-performance liquid chromatography of the final extracts derivatized with phenylisothiocyanate. Selected ion monitoring with multiple-ion detection applied to the compounds separated by gas chromatography revealed the presence of the respective molecular ions and of the decarboxylated fragments, thus confirming the identification of cyclothionine and TMDA in human urine.

Ion-pair extraction and high-performance liquid chromatographic determination of tianeptine and its metabolites in human plasma, urine and tissues.

An isocratic reversed-phase ion-pair liquid chromatographic method for the determination of tianeptine and its two main metabolites in plasma, urine and tissues, using an internal standard, is reported. The influence of two stationary phases on the retention of the drugs was studied. The drugs were extracted as ion pairs, using a heptane-octanol-tetraheptylammonium bromide mixture (98:2:0.5, v/v/w) as extraction solvent. This extraction procedure yielded plasma drug recoveries of greater than 60% and allowed UV detection at 220 nm without interference from endogenous components of plasma, urine or tissues. Linear standard curves up to 1.00 micrograms/ml and drug determination down to 0.01 microgram/ml were observed. This method has been successfully applied to the analysis of human plasma and urine samples and of encephales from tianeptine-dosed rats.

Isolation of two new sulfur-containing amino acids from the urine of a cystathioninuric patient.

Two new sulfur-containing amino acids are isolated from the urine of a cystathioninuric patient. They are presumed to be cystathionine sulfoxide and perhydro-1,4-thiazepine-3,5-dicarboxylic acid.