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Background: Entrectinib is an oral, CNS-active, potent inhibitor of tyrosine receptor kinases A/B/C, tyrosine kinase ROS proto-oncogene 1, and anaplastic lymphoma kinase approved for use in patients with solid tumors. We describe 3 clinical studies, including one investigating the single/multiple dose pharmacokinetics of entrectinib in patients and two studies in healthy volunteers investigating the absorption/distribution/metabolism/excretion (ADME) of entrectinib, its relative bioavailability, and effect of food on pharmacokinetics., Methods: The patient study is open-label with dose-escalation and expansion phases. Volunteers received entrectinib (100-400 mg/m 2 , and 600-800 mg) once daily with food in continuous 28-day cycles. In the ADME study, volunteers received a single oral dose of [ 14 C]entrectinib 600 mg. In the third study, volunteers received single doses of entrectinib 600 mg as the research and marketed formulations in the fasted state (Part 1), and the marketed formulation in the fed and fasted states (Part 2). Entrectinib and its major active metabolite M5 were assessed in all studies., Results: Entrectinib was absorbed in a dose-dependent manner with maximum concentrations at ~4 h postdose and an elimination half-life of ~20 h. Entrectinib was cleared mainly through metabolism and both entrectinib and metabolites were eliminated mainly in feces (minimal renal excretion). At steady-state, the M5-to-entrectinib AUC ratio was 0.5 (with 600 mg entrectinib research formulation in patients). The research and marketed formulations were bioequivalent and food had no relevant effect on pharmacokinetics., Conclusions: Entrectinib is well absorbed, with linear PK that is suitable for once-daily dosing, and can be taken with or without food.

Synthetic opioids (SO) associated with the recent alarming increase of deaths and intoxications in United States of America and Europe are not detected by the usual first-line opiates drug screening assays. We developed a liquid chromatography tandem mass spectrometry analytical method for the multiplex detection of 14 fentanyl analogues (2-furanylfentanyl, 4-ANPP, 4-methoxybutyrylfentanyl, acrylfentanyl, alfentanil, carfentanil, despropionyl-2-fluorofentanyl, fentanyl, methoxyacetylfentanyl, norfentanyl, ocfentanil, remifentanil, sufentanil and valerylfentanyl) and U-47700 in whole blood and urine samples. The method was validated according to the requirements of ISO 15189. A simple and fast liquid-liquid extraction (LLE) with De-Tox Tube-A was performed leading to better recovery of molecules in urine than in blood samples. Depending on the compound, the limits of detection (LODs) ranged from 0.01 to 0.10 ng/mL and from 0.02 to 0.05 ng/mL in whole blood and urine, respectively. Calibration curves were linear in the range 0.5-50.0 ng/mL and the limit of quantification (LOQ) ranged from 0.10 to 0.40 ng/mL in blood. Internal quality controls at 1 and 40 ng/mL showed intra-day and between-day precision and accuracy bias below 10% in urine and 15% in blood. The method was applied to the screening of 211 urine samples from patients admitted in emergency or addiction departments. The presence of legal fentanyl analogues in 5 urine samples was justified by their therapeutic use as analgesics. Only one patient was concerned by fentanyl misuse and addiction whereas no illegal SO was detected. This study is not in favor of a huge misuse of SO in the Lorraine region., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2020. Published by Elsevier B.V.)

Being highly potent, New Synthetic Opioids (NSO) have become a public health concern. Little is known though about the metabolism and toxicokinetics (TK) of many of the non fentanyl NSO such as U-47700. Obtaining such data in humans is challenging and so we investigated if pigs were a suitable model species as TK model for U-47700. The metabolic fate of U-47700 was elucidated after intravenous administration to one pig in vivo and results were compared to metabolic patterns formed by different other in vitro systems (human and pig liver microsomes, human liver S9 fraction) and compared to rat and human in vivo data. Furthermore, monooxygenase isozymes responsible for the major metabolic steps were elucidated. In total, 12 phase I and 8 phase II metabolites of U-47700 could be identified. The predominant reactions were N-demethylation, hydroxylation, and combination of them followed by glucuronidation or sulfation. The most predominant monooxygenase catalyzed conversions were N-demethylation, and hydroxylation by CYP3A4 and 2B6, and FMO3 catalyzed N-oxidation. Similar main phase I metabolites were found in vitro as compared to in vivo (pig/human). The metabolic pattern elucidated in the pig was comparable to human in vivo data. Thus, pigs seem to be a suitable animal model for metabolism and further TK of U-47700., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Recently, the number of adverse events, including death, involving novel opioids has continued to increase, providing additional and sustained challenges for forensic and medical communities. Identification of emerging novel opioids can be challenging, compounded by detection windows and unknown metabolic profiles. In this study, human liver microsomes were used for the generation of in vitro metabolic profiles of U-47700 and U-49900. Generated metabolites were analyzed via a SCIEX TripleTOF® 5600+ quadrupole time-of-flight mass spectrometer and resulting data files were processing using MetabolitePilot™. Characterized metabolites were verified in vivo by analysis of authentic human urine specimens collected after analytically confirmed cases of overdose involving U-47700 or U-49900. In total, four metabolites were identified and present in urine specimens for U-47700, and five metabolites for U-49900. N-Desmethyl-U-47700 was determined to be the primary metabolite of U-47700. Parent U-47700 was identified in all urine specimens. N-Desmethyl-U-47700 and N,N-didesmethyl-U-47700 were structurally confirmed for the first time during this study following acquisition of standard reference material. N-Desethyl-U-49900 was determined to be the primary metabolite of U-49900 following microsomal incubations, while N,N-didesethyl-N-desmethyl-U-49900 was the most abundant in a urine specimen. Similarities in metabolic transformation were identified between U-47700 and U-49900, resulting in a common metabolite and isomeric species. These phenomena should be considered in cases involving U-47700 or U-49900. This study is the first to map the metabolic profiles of U-47700 and U-49900 using human liver microsomes, as well as the first to report any literature involving U-49900 and analysis of case specimens., (Copyright © 2017 John Wiley & Sons, Ltd.)

The illicit drug market has rapidly evolved from synthetic cannabinoids to cathinone derivatives and now a new emerging threat of synthetic opioids. These compounds were mostly developed by pharmaceutical companies during drug discovery. The new psychoactive substances are not routinely covered in drug screening and may go undetected. Recently fentanyl analogous, AH-7921, MT-45 and now U-47700 have been encountered in clinical and forensic casework. U-47700 is gaining popularity on drug user forms as a legal alternative to heroin. It is a µ-receptor agonist that is part of the trans-1-2-diamine opioid analgesic drug class developed by The Upjohn Company in an attempt to develop a non-addicting analgesic. A LC-MS-MS method was developed and validated to detect and quantify U-47700. Additional analysis was conducted with an LC-QToF to identify the presence of the parent drug and metabolites. A total of four cases have been evaluated by the LC-MS-MS methodology which has an analytical range of 1-1,250 ng/mL and limit of detection of 1 ng/mL. The concentration of U-47700 in urine specimens ranged from below the limit of quantification to 224 ng/mL. The ToF analysis detected the presence of suspected phase I demethylated metabolites that may assist future analysis of this compound. The prevalence of designer opioids in casework highlights the importance of analysis for new psychoactive substances. Traditional opiates/opioids were not detected in the presented cases, but the available case histories revealed an opioid toxidrome. These findings suggest that U-47700 drug may cause significant morbidity and mortality within the United States as an emerging drug threat., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: In the last decade there has been a worldwide surge in the recreational abuse of novel psychoactive substances, particularly amphetamine derivatives and synthetic cannabinoids. Synthetic opioids such as AH-7921, MT-45, and U-47700, with structures distinct from those ever used therapeutically or described recreationally, have also recently emerged., Case Details: We report a patient who suffered respiratory failure and depressed level of consciousness after recreationally using a novel synthetic opioid labeled U-47700. A single dose of naloxone administered by paramedics completely reversed his opioid poisoning. Comprehensive laboratory analysis confirmed the presence of a novel synthetic opioid and excluded other drugs. The drug used appeared to have caused a false positive benzodiazepine result on the initial urine drugs of abuse panel., Conclusion: The case we describe of toxicity from the synthetic opioid labeled U-47700 highlights the emerging trend of novel synthetic opioid abuse.

Background: Novel substances often referred to as "designer drugs" have emerged as drugs of abuse, and recognition of these is difficult as routine blood and urine screening tests do not detect these agents. U-47700 is a synthetic selective μ-opioid agonist that can be bought online for as little as $40 per gram. We report two patients presenting after insufflation of U-47700, with subsequent confirmation of this substance in urine samples., Case Details: A 26-year-old man and 24-year-old woman insufflated a substance they believed to be "synthetic cocaine." The man was found down with cyanosis and agonal respirations. He was intubated and taken to hospital where he recovered well with supportive care. The woman presented with anxiety, tremors and drowsiness and was admitted for observation. Urine samples from both patients were analyzed using GC/MS/MS and LC/QToF, and U-47700 was isolated in both cases. No other opioids were detected., Discussion: These cases are concerning because U-47700 is a relatively new agent that is easy to obtain over the internet and has the potential to cause significant morbidity and mortality.

Context: The opioid epidemic has included use of traditional drugs and recently newer synthetics. It is critically important to recognize and identify these new drugs both clinically and through appropriately designed toxicology testing. There is little available information on a synthetic gaining popularity, U-47700., Case Details: A 23-year-old female presented after using "U4" by nasal insufflation and injection. She was cyanotic with respiratory depression and responded to naloxone in the field. She was found to have non cardiogenic pulmonary edema and hemoptysis which improved with BiPAP. Urine and serum samples were analyzed using mass spectrometry, confirming 3,4-dichloro-N-[(1R,2R)-2-(dimethylamino)cyclohexyl]-N-methylbenzamide or U-47700. The sample was further analyzed elucidating metabolism specifics. Drug and metabolite concentrations were subsequently measured in both serum and urine. The parent compound of U-47700 was detected at 394 ng/mL and 228 ng/mL in serum and urine, respectively. Metabolites detected in appreciable amounts included the desmethyl (1964 ng/mL in urine), bisdesmethyl (618 ng/mL), desmethyl hydroxy (447 ng/mL), and bisdesmethyl hydroxy forms (247 ng/mL) of U-47700., Discussion: U-47700 is a potent μ-opioid receptor agonist and has recently been used recreationally, contributing to hospitalizations and likely deaths in the community. This is a case report describing an exposure to U-47700 with subsequent laboratory analysis. Based upon this one case, parent U-47700 appear to be an appropriate marker of use in a serum sample. However, demethylated metabolites appear dominant as urinary markers of U-47700 use.

Histone deacetylase inhibitors (HDAC inhibitors) are used to treat malignancies such as cutaneous T cell lymphoma and peripheral T cell lymphoma. Only four drugs are approved by the US Food and Drug Administration, namely vorinostat, romidepsin, panobinostat and belinostat, while chidamide has been approved in China. There are a number of bioanalytical methods reported for the measurement of HDAC inhibitors in clinical (human plasma and serum) and preclinical (mouse plasma, rat plasma, urine and tissue homogenates, etc.) studies. This review covers various HDAC inhibitors such as vorinostat, romidepsin, panobinostat, belinostat and chidamide. In addition to providing a comprehensive review of the available methods for the above mentioned HDAC inhibitors, it also provides case studies with perspectives for chosen drugs. Based on the review, it is concluded that the published methodologies using either HPLC or LC-MS/MS are well suited for the quantification of HDAC inhibitors in various biological fluids to delineate pharmacokinetic data., (Copyright © 2016 John Wiley & Sons, Ltd.)

We report here for the first time on the use of Molecularly Imprinted Polymers as modifiers in bulk optodes, Miptode, for the determination of a pharmaceutical compound, itopride hydrochloride as an example in a concentration range of 1×10(-1)-1×10(-4)molL(-1). In comparison to the optode containing the ion exchanger only (Miptode 3), the optode containing the ion exchanger and the MIP particles (Miptode 2) showed improved selectivity over the most lipophilic species, Na(+) and K(+), by more than two orders of magnitude. For instance, the optical selectivity coefficients using Miptode 2, [Formula: see text] , were as follow: NH4(+)˂-6; Na(+)=-4.0, which were greatly enhanced in comparison with that obtained by Miptode 3. This work opens a new avenue for using miptodes for the determination of all the pharmaceutical preparations without the need for the development of new ionophores., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A 30-year old man was found dead in his home after inhaling fumes of a powder burned on aluminum foil. Blood and urine were taken by the medical examiner during the external body examination and submitted to the laboratory for a comprehensive systematic toxicological analysis. A toxic fentanyl level of 10.9μg/L was measured in the subclavian blood. Police investigation revealed that the man searched the internet for information on new psychotropic substances, among others including U-47700. A powder found in the victims' home was transferred to the laboratory for analysis, in which trace amounts of fentanyl (0.0035%, m/m) and U-47700 (0.0012%, m/m) were identified by gas chromatography mass spectrometry. U-47700 is an opioid analgesic drug, considered to have a potency of approximately 7.5 times that of morphine. A target analysis on U-47700 was performed using liquid-liquid extraction and ultra performance liquid chromatography tandem mass spectrometry operating in multiple reaction monitoring mode. The method validation was based on the Scientific Working Group of Forensic Toxicology document 'Standard Practices of Method Validation in Forensic Toxicology'. In blood and urine the U-47700 concentration was 13.8 and 71.0μg/L, respectively. To the author's knowledge, this is the first case report of a fatal intoxication involving U-47700 abused as a new psychotropic substance., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

AH-7921 (3,4-dichloro-N-[(1-dimethylamino)cyclohexylmethyl]benzamide) is a new synthetic opioid and has led to multiple non-fatal and fatal intoxications. To comprehensively study AH-7921 metabolism, we assessed human liver microsome (HLM) metabolic stability, determined AH-7921's metabolic profile after human hepatocytes incubation, confirmed our findings in a urine case specimen, and compared results to in silico predictions. For metabolic stability, 1 µmol/L AH-7921 was incubated with HLM for up to 1 h; for metabolite profiling, 10 µmol/L was incubated with pooled human hepatocytes for up to 3 h. Hepatocyte samples were analyzed by liquid chromatography quadrupole/time-of-flight high-resolution mass spectrometry (MS). High-resolution full scan MS and information-dependent acquisition MS/MS data were analyzed with MetabolitePilot™ (SCIEX) using multiple data processing algorithms. The presence of AH-7921 and metabolites was confirmed in the urine case specimen. In silico prediction of metabolite structures was performed with MetaSite™ (Molecular Discovery). AH-7921 in vitro half-life was 13.5 ± 0.4 min. We identified 12 AH-7921 metabolites after hepatocyte incubation, predominantly generated by demethylation, less dominantly by hydroxylation, and combinations of different biotransformations. Eleven of 12 metabolites identified in hepatocytes were found in the urine case specimen. One metabolite, proposed to be di-demethylated, N-hydroxylated and glucuronidated, eluted after AH-7921 and was the most abundant metabolite in non-hydrolyzed urine. MetaSite™ correctly predicted the two most abundant metabolites and the majority of observed biotransformations. The two most dominant metabolites after hepatocyte incubation (also identified in the urine case specimen) were desmethyl and di-desmethyl AH-7921. Together with the glucuronidated metabolites, these are likely suitable analytical targets for documenting AH-7921 intake. Copyright © 2015 John Wiley & Sons, Ltd., (Copyright © 2015 John Wiley & Sons, Ltd.)

Background: YM758 monophosphate is a novel If channel inhibitor that has an inhibitory action for If current and shows a strong and specific activity, selectively lowering the heart rate and decreasing oxygen consumption by heart muscle., Objectives: The objectives of the current study were to investigate the in vivo metabolic profiles of YM758 in mice, rats, rabbits, dogs, and monkeys and to elucidate the structures of YM758 metabolites., Methods: Biological samples were analyzed by liquid chromatography hyphenated with a radiometric detection system and liquid chromatography coupled with a mass spectrometer to clarify their metabolic patterns. To elucidate their structures, metabolites were isolated and analyzed by mass spectrometry and nuclear magnetic resonance spectroscopy., Results: Our results from in vivo metabolic profiling in humans and animals indicated there is no significant species difference in the metabolism of YM758, and the metabolic pathways of YM758 are considered to be oxidation, hydration, and demethylation followed by sulfate or glucuronide conjugation.

Detection times and screening limits (SL) are methods used to ensure that the performance of horses in equestrian sports is not altered by drugs. Drug concentration-response relationship and knowledge of concentration-time profiles in both plasma and urine are required. In this study, dexamethasone plasma and urine concentration-time profiles were investigated. Endogenous hydrocortisone plasma concentrations and their relationship to dexamethasone plasma concentrations were also explored. A single dose of dexamethasone-21-isonicotinate suspension (0.03 mg/kg) was administered intramuscularly to six horses. Plasma was analysed for dexamethasone and hydrocortisone and urine for dexamethasone, using UPLC-MS/MS. Dexamethasone was quantifiable in plasma for 8.3 ± 2.9 days (LLOQ: 0.025 μg/L) and in urine for 9.8 ± 3.1 days (LLOQ: 0.15 μg/L). Maximum observed dexamethasone concentration in plasma was 0.61 ± 0.12 μg/L and in urine 4.2 ± 0.9 μg/L. Terminal plasma half-life was 38.7 ± 19 h. Hydrocortisone was significantly suppressed for 140 h. The plasma half-life of hydrocortisone was 2.7 ± 1.3 h. Dexamethasone potency, efficacy and sigmoidicity factor for hydrocortisone suppression were 0.06 ± 0.04 μg/L, 0.95 ± 0.04 and 6.2 ± 4.6, respectively. Hydrocortisone suppression relates to the plasma concentration of dexamethasone. Thus, determination of irrelevant plasma concentrations and SL is possible. Future research will determine whether hydrocortisone suppression can be used as a biomarker of the clinical effect of dexamethasone., (© 2014 John Wiley & Sons Ltd.)

1.  Mosapride citrate (mosapride) is a potent gastroprokinetic agent. The only previous study on mosapride metabolism in human reported one phase I oxidative metabolite, des-p-fluorobenzyl mosapride, in human plasma and urine using HPLC method. Our aim was to identify mosapride phase I and phase II metabolites in human urine, feces and plasma using UPLC-ESI-MS/MS. 2.  A total of 16 metabolites were detected. To the best of our knowledge, 15 metabolites have not been reported previously in human. 3.  Two new metabolites, morpholine ring-opened mosapride (M15) and mosapride N-oxide (M16), alone with one known major metabolite, des-p-fluorobenzyl mosapride (M3), were identified by comparison with the reference standards prepared by our group. The chemical structures of seven phase I and six phase II metabolites of mosapride were elucidated based on UPLC-MS/MS analyses. 4.  There were two major phase I reactions, dealkylation and morpholine ring cleavage. Phase II reactions included glucuronide, glucose and sulfate conjugation. The comprehensive metabolic pathway of mosapride in human was proposed for the first time. 5.  The metabolites in humans were compared with those in rats reported previously. In addition to M10, the other 15 metabolites in humans were also found in rats. This result suggested that there was little qualitative species difference in the metabolism of mosapride between rats and humans. 6.  In all, 16 mosapride metabolites including 15 new metabolites were reported. These results allow a better understanding of mosapride disposition in human.

A case is presented of a 19-year-old white male who was found dead in bed by a friend. While no anatomic cause of death was observed at autopsy, toxicological analysis of his blood identified AH-7921, a synthetic opioid. AH-7921 was isolated by liquid-liquid extraction into n-butyl chloride from alkalinized samples. Extracts were analyzed and quantified by gas chromatography mass spectrometry in selected ion monitoring mode. The heart blood had an AH-7921 concentration of 3.9 mg/L and the peripheral blood concentration was 9.1 mg/L. In addition to the blood, all submitted postmortem specimens including urine, liver, kidney, spleen, heart, lung, brain, bile and stomach content were quantified. The following concentrations of AH-7921 were reported: 6.0 mg/L in urine, 26 mg/kg in liver, 7.2 mg/kg in kidney, 8.0 mg/kg in spleen, 5.1 mg/kg in heart, 21 mg/kg in lung, 7.7 mg/kg in brain, 17 mg/L in bile and 120 mg/125 mL in the stomach content. The medical examiner reported that the cause of death was opioid intoxication and the manner of death was accident.

Rationale: The recent discovery of resveratrol's capability to inhibit cAMP-specific phosphodiesterases (PDEs) and, as a consequence, to enhance particularly the activity of Sirt1 in animal models has reinforced the interest of preventive doping research organizations, especially in PDE4 inhibitors. Among these, the archetypical PDE4-inhibitor rolipram significantly increased the number of mitochondria in laboratory rodents, which further demonstrated a performance increase in a treadmill-test (time-to-exhaustion) of approximately 40%. Besides rolipram, a variety of new PDE4-inhibiting substances including cilomilast, roflumilast, and numerous additional new drug entities were described, with roflumilast being the first-in-class having received clinical approval for the treatment of chronic obstructive pulmonary disease (COPD). Due to the availability of these substances, and the fact that a misuse of such compounds in sport cannot be excluded, it deems relevant to probe for the prevalence of these compounds in sports drug testing programs., Methods: Known urinary phase-I metabolites of rolipram, roflumilast, and cilomilast were generated by in vitro incubations employing human liver microsomal preparations. The metabolites obtained were studied by liquid chromatography with high-resolution/high-accuracy tandem mass spectrometry (LC/MS/MS) and the reference product ion mass spectra of established and most relevant metabolites were utilized to provide the information necessary for comprehensive doping controls. The analytical procedure was based on conventional routine doping control assays employing enzymatic hydrolysis followed by liquid-liquid extraction and subsequent LC/MS/MS measurement., Results: Structures of diagnostic product ions and dissociation pathways of target analytes were elucidated, providing the information required for implementation into an existing test method for routine sports drug testing. The established method allowed for detection limits for the intact drugs of 1-5 ng/mL, and further assay characteristics (intraday precision 1.5-13.7%, interday precision 7.3-18.6%, recovery 20-100%, ion suppression/enhancement, and specificity) were determined. In addition, proof-of-concept analyses concerning roflumilast were conducted with a urine sample obtained from a COPD patient under roflumilast treatment., (Copyright © 2013 John Wiley & Sons, Ltd.)

The article presents the data concerning the application of technique of thin-layer chromatography in the conditions of laboratory diagnostics to identify benzamide derivative neuroleptics (amisupride, sulpiride, tiapride) in derivate from human biologic fluids and tissues with the possibility to separate the analyzed tissues from soextractive tissues.

The exposure of a drug candidate and its metabolites in humans and preclinical species during drug development needs to be determined to ensure that the safety of drug-related components in humans is adequately assessed in the standard toxicology studies. The in vivo radiolabeled studies in preclinical species and human volunteers provide the total fate of the drug-derived radioactivity including the relative abundance of metabolites. Here, we describe how the single-dose radiolabeled human studies could provide the exposure of circulating metabolites at steady state using a case study of an extensively metabolized drug, lixivaptan. After an oral dose of [(14)C]lixivaptan to humans, a total of nine metabolites were detected in the systemic circulation; eight of them exceeded 10% of the parent exposure (2-41% of total radioactivity). The plasma samples were profiled for all subjects at each time point by high-performance liquid chromatography, and metabolites were quantified using a radioactive detector. On the basis of single-dose area under the concentration-time curve (AUC) values, exposure of six human metabolites was greater at least in one preclinical species used in toxicology evaluation. On the basis of the t(1/2) of lixivaptan and two major metabolites from a single dose in humans, their AUC and C(max) values were simulated at the steady state. The simulated exposure (C(max) and AUC) values of parent drug and the two most abundant metabolites were similar to those from a 7-day clinical study obtained using a validated liquid chromatography-mass spectrometry assay, suggesting that a well designed single-dose radiolabeled human study can help in addressing the metabolites in safety testing-related issues.

S-nitrosoglutathione reductase is the primary enzyme responsible for the metabolism of S-nitrosoglutathione (GSNO), the body's main source of bioavailable nitric oxide. Through its catabolic activity, GSNO reductase (GSNOR) plays a central role in regulating endogenous S-nitrosothiol levels and protein S-nitrosation-based signaling. By inhibiting GSNOR, we aim to increase pulmonary GSNO and induce bronchodilation while reducing inflammation in lung diseases such as asthma. To support the clinical development of N6022, a first-in-class GSNOR inhibitor, a 14-day toxicology study was conducted. Sprague-Dawley rats were given 2, 10 or 50 mg/kg/day N6022 via IV administration. N6022 was well tolerated at all doses and no biologically significant adverse findings were noted in the study up to 10 mg/kg/day. N6022-related study findings were limited to the high dose group. One male rat had mild hepatocellular necrosis with accompanying increases in ALT and AST and several male animals had histological lung assessments with a slight increase in foreign body granulomas. Systemic exposure was greater in males than females and saturation of plasma clearance was observed in both sexes in the high dose group. Liver was identified as the major organ of elimination. Mechanistic studies showed dose-dependent effects on the integrity of a rat hepatoma cell line., (Copyright © 2011 Elsevier Inc. All rights reserved.)

4-(4-Methyl-piperazin-1-ylmethyl)-N-[6-methyl-5-(4-pyridin-3-yl-pyrimidin-2-ylamino)-pyridin-3-yl]-3-trifluoromethyl-benzamide (flumatinib, HH-GV678), an antineoplastic tyrosine kinase inhibitor, is currently in Phase I clinical trials in China for the treatment of chronic myelogenous leukemia (CML). The purpose of this study was to identify the metabolites of flumatinib in CML patients, with the aim of determining the main metabolic pathways of flumatinib in humans after oral administration. Ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry revealed 34 metabolites; 7 primary metabolites were confirmed by comparison with synthetic reference standards. The results show that the parent drug flumatinib was the main form recovered in human plasma, urine, and feces. The main metabolites of flumatinib in humans were the products of N-demethylation, N-oxidation, hydroxylation, and amide hydrolysis. In addition to these phase I metabolites, several phase II glucuronidation and acetylation products were detected in plasma, urine, and feces. The observed circulating metabolites included an N-demethylated metabolite (M1), two hydrolytic metabolites (M3, M4), oxidation metabolites (M2-1, M2-4, M2-7, M2-9, and M14), a glucuronide conjugate (M16-2), and several multiple metabolic products. Flumatinib was predominantly metabolized by amide bond cleavage to yield two corresponding hydrolytic products. By comparison with the related drug 4-(4-methyl-piperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-benzamide (imatinib), we concluded that the electron-withdrawing groups of trifluoromethyl and pyridine facilitated the amide bond cleavage and led to the in vivo formation of a carboxylic acid and an amine.

Only a few COX-1-selective inhibitors are currently available, and the research on COX-1 selective inhibitors is not fully developed. The authors have produced several COX-1 selective inhibitors including N-(5-amino-2-pyridinyl)-4-trifluoromethylbenzamide: TFAP (3). Although 3 shows potent analgesic effect without gastric damage, the urine after administration of 3 becomes red-purple. Since the colored-urine should be avoided for clinical use, in this research we examined the cause of the colored-urine. UV-vis spectra and LC-MS/MS analyses of urine samples and metabolite candidates of 3 were performed to afford information that the main reason of the colored urine is a diaminopyridine (4), produced by metabolization of 3. This information is useful to design new COX-1 selective inhibitors without colored urine based on the chemical structure of 3., (Copyright 2010 Elsevier Ltd. All rights reserved.)

(-)-N-{2-[(R)-3-(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)piperidino]ethyl}-4-fluorobenzamide (YM758) is a novel inhibitor of the "funny" If current channel (If channel) that is expressed in the sinus node of heart and is being developed as a treatment for stable angina and atrial fibrillation. Its metabolites were identified in human urine, plasma, and feces by radio-high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry analyses after oral administration of [(14)C]YM758. 6,7-Dimethoxy-2-[(3R)-piperidin-3-ylcarbonyl]-1,2,3,4-tetrahydroisoquinoline (YM-252124), (5R)-5-[(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)carbonyl]piperidin-2-one (YM-385459), 2-{[(3R)-1-{2-[(4-fluorobenzoyl)amino]ethyl}piperidin-3-yl]carbonyl}-7-methoxy-1,2,3,4-tetrahydroisonolin-6-yl beta-D-glucopyranosiduronic acid (AS2036329), and the unchanged drug were detected as major constituents in both urine and plasma, whereas N-(4-fluorobenzoyl)glycine (YM-385461) was detected in plasma, but not in urine. The renal and hepatic uptake transporters for these metabolites were investigated by assessing their inhibitory effect on uptake activity in human (h) organic cation transporter (OCT) 1-3/rat (r) Oct1-3, human organic anion transporter (OAT) 1/rOat1, hOAT3/rOat3, and organic anion-transporting protein 1B1/1B3-expressing HEK293 cells. IC(50) values of YM-252124 for 1-methyl-4-phenylpyridinium uptake via hOCT2 and rOct2 were 93.9 and 1700 microM, respectively, suggesting that this metabolite is secreted into urine via hOCT2/rOct2 and that the large difference in the inhibitory potentials between hOCT2 and rOct2 explains the species difference in the urinary excretion ratio of the radioactivity. The renal secretion of YM-385461, one derivative of p-aminohippuric acid, via hOAT1/rOat1, and hepatic uptake of YM-252124 via hOCT1/rOct1 was also expected. This kind of study was useful in investigating the relationship between the urinary/hepatic elimination and the transport activity for metabolites.

This study was initiated to characterize the metabolism and pharmacokinetics of SNC80 in rats and to evaluate the impact of Freund's complete adjuvant (FCA)-induced inflammation on its body disposition. In vitro, the disappearance and intrinsic clearance (CL(int)) of SNC80 were measured following incubations in recombinant rat CYPs and in phenotyped liver microsomes from naive and 24-h FCA-treated rats. The unbound fraction (f(u)) was assessed by ultrafiltration. Based on the Clint values, in vivo blood clearance of 3.35 and 2.48 L/h/kg were predicted in naive and FCA-treated rats. In vivo, SNC80 was administered to naive and 24-h FCA-treated rats at 10 micromol/kg i.v. and 50 micromol/kg p.o. The naive animals showed high plasma clearance (3.1 L/h/kg), low renal clearance (<0.02 L/h/kg) and poor bioavailability (<4%). Following i.v. administration, plasma clearance was lower (22%) in FCA-treated vs. untreated rats. Despite the decreases in f(u) (approximately 30%) and CL(int) (approximately 40%) in vitro, in vivo the apparent bioavailability and oral clearance were not significantly different between FCA-treated and naive rats. Hepatic and possibly intestinal losses contribute to the low bioavailability of SNC80. Non-hepatic mechanisms may compensate for the decrease in plasma clearance found in FCA-treated rats, preventing an increase in the oral bioavailability of SNC80.

Gentisamide (GAM, 2,5-dihydroxybenzamide), a minor first-pass metabolite of salicylamide (SAM, 2-hydroxybenzamide), was studied using FT-IR, 1D and 2D homo- and heteronuclear 1H and 13C NMR spectroscopy. GAM was isolated from human urine eight hours after oral administration of SAM. FT-IR, 1H and 13C NMR spectra unequivocally confirmed the chemical structure of GAM through chemical and substituent shifts, coupling constants and connectivities in COSY, NOESY, HETCOR and HBMC spectra. From NOESY spectra of GAM in DMSO-d6, it was concluded that the amide protons are oriented toward the ortho-proton at C-6. Obtained results indicate that the presence of the additional phenol group at C-5 in GAM favours the formation of intramolecular hydrogen bonding of the O...HO type between C2-OH proton and oxygen atom of the amide group.

Melanoma is a neoplasia of dramatically increasing incidence that has a propensity to spread rapidly. Early detection is fundamental and patient management requires reliable, sensitive and reproducible staging methods, such as a single examination by planar scintigraphy or single-photon emission tomography (SPET) using a radiopharmaceutical with selectivity for melanoma tissue. Among iodobenzamides reported to possess an affinity for melanoma, a new compound, N-(2-diethylaminoethyl)-2-iodobenzamide (BZA(2)), was selected for a clinical trial in view of its pharmacokinetic experimental profile in melanoma-bearing mice. Planar whole-body scintigraphy using (123)I-BZA(2) was performed in 25 patients with histologically proven cutaneous melanoma. Performance was evaluated in two groups of patients with one or more documented secondary lesions ( n=13) or with no known secondary lesions ( n=12), and results were compared with those of conventional investigation techniques. No adverse clinical or biological events were recorded. Lesions were imaged by increased tracer uptake, and good quality images were obtained 4 h after administration. After a follow-up of more than 1 year, the overall results of (123)I-BZA(2) scintigraphy on a per patient basis showed a sensitivity of 100%, a specificity of 95%, a positive predictive value of 86% and a negative predictive value of 100%. The proven secondary lesions were imaged with a sensitivity of 100% and a specificity of 91%. In seven patients with suspected metastases, the absence of (123)I-BZA(2) uptake was confirmed as true negative, and in one patient without suspected metastases, (123)I-BZA(2) scintigraphy revealed a gastric lesion. Hence eight diagnoses would have been modified by (123)I-BZA(2) scintigraphy data. (123)I-BZA(2) allowed discrimination between benign and malignant lesions and, in the case of malignancies, between those of melanomatous origin and others. This compound, which is selective for melanoma tissue, appears promising for the staging and restaging of melanoma.

Several radioiodinated N-(dialkylaminoalkyl)benzamides have been used for planar scintigraphy and single-photon emission computed tomography (SPECT) of melanoma metastases. In a quest for improved melanoma uptake and tissue selectivity, structure-activity studies for N-(2-diethylaminoethyl)benzamides with variation of phenyl substituents were performed using C57Bl/6 mice bearing B16 melanoma. Compounds 2 (4-amino-5-bromo-N-(2-diethylaminoethyl)-3-[(131)I]iodo-2-methoxybenz amide) and 6 (4-acetamido-N-(2-diethylaminoethyl)-5-[(131)I]iodo-2-methoxybenzamid e) showed at 6 h post iv injection, for example, melanoma uptake of 16.6 and 23.2% ID/g, respectively (mean values, n = 3). Uptake was 3-5 times higher (P < 0.01) than observed with benzamides known from the literature and was probably facilitated by the relatively slow urinary excretion of 2 or 6. In contrast, analogues lacking either the MeO, Ac, AcNH, or Br substituents exhibited reduced tumor uptake and high urinary excretion of radioactivity in various benzamide metabolites. Uptake of radioiodinated benzamides in B16 melanoma is not mediated by a specific mechanism such as sigma-receptor binding. 2 and 6 exhibited similar melanoma uptake values but quite different sigma(1)-receptor affinities of K(i) = 0.278 +/- 0.018 and 5.19 +/- 0.40 microM, respectively. Uptake studies with IMBA (N-(2-diethylaminoethyl)-3-[(131)I]iodo-4-methoxybenzamide) or BZA (N-(2-diethylaminoethyl)-4-[(131)I]iodobenzamide) showed that with increasing dose of unlabeled compound the measured uptake of label was unchanged (IMBA) or even enhanced (BZA) while receptor binding of label decreased. Differential and equilibrium density-gradient centrifugation revealed that most of the radioactivity from labeled IMBA was associated with fractions containing melanin granules. Thus, structure-activity studies indicate that blood clearance rates and metabolic stability are the main determinants for benzamide uptake in melanoma. The high uptake and slow clearance of 6 offer considerable potential for melanoma imaging in patients, and this compound may also prove to be useful for radionuclide therapy.

Colostomized chickens given oral doses of 3,5-dinitrobenzamide (nitromide) cleared nitromide predominantly through the urine (58% of dose) and feces (21% of dose). Rats cleared 52% of nitromide via urinary excretion and 44% via feces. Major urinary metabolites for both chickens and rats include: 3-amino-5-nitrobenzamide, 3-acetamido-5-nitrobenzamide, 3-acetamide-5-aminobenzamide, and 3,5-diacetamidobenzamide. The major fecal metabolite in chickens was 3-acetamido-5-nitrobenzamide (67% of fecal 14C) and 3-acetamido-5-aminobenzamide in rats (approximately 50%).

Iofratol is currently under evaluation as a potential X-ray contrast medium for angiography and myelography. An HPLC method for assaying iofratol in rat and human plasma and urine samples is described. The analysis is based on the reversed-phase chromatographic separation of iofratol and the internal standard (iopamidol) from the endogenous components of biological fluids, and detection by UV absorption at 242 nm. The selectivity of the method was satisfactory. The mean absolute recovery was greater than 90%. The precision and accuracy of the analytical methods were in the range 0.8-7.4 and -7.8 to +9.7%, respectively. The detection limits in plasma (0.1 ml) and urine (0.5 ml) were 0.1 and 0.4 microg (iofratol)/ml, respectively. The analyte was stable in the different biological matrices when stored at room temperature (20 degrees C) for at least 1 day, 4 degrees C for 1 month and -20 degrees C for 1 year.

1. The metabolic fate of the insecticide teflubenzuron, orally dosed to the male Wistar rat, was investigated. Particular attention was paid to the metabolic fate of the benzoyl and aniline moiety after hydrolysis of the urea bridge. 2. The 0-48-h urinary and faecal metabolic patterns and recoveries showed that for a dose range of 4-53 mumol (1.5-20 mg) teflubenzuron, 90% of the dose was excreted in the faeces mainly in unmodified form, approximately 4.6% was absorbed from the lumen and excreted in the urine, and 5.4% was retained in the body. Metabolites excreted in the urine could be identified as benzoate and aniline derivatives originating from the two aromatic rings of teflubenzuron liberated from the parent molecule by hydrolysis of the urea bridge. 3. The amount of urinary benzoate-type metabolites was about eight times the amount of aniline-type metabolites, indicating significant differences in efficiency of urinary excretion of the benzoate moiety as compared with the aniline ring. 4. To investigate further the possible reason underlying this difference in urinary excretion efficiency between the two aromatic derivatives formed from teflubenzuron, dose-recovery studies of these aniline- and benzoate-type metabolites were performed. These studies confirmed the discrepancy observed between the urinary recovery of the benzoyl and the aniline moiety of teflubenzuron. 5. Additional results of the present study indicate that the above discrepancy can be explained by the fact that the benzoate derivative is excreted mainly in its unmetabolized form, whereas the aniline derivative needs additional phase I and II modifications before it can be excreted from the body, the former being a relatively slow reaction. Furthermore, conversion of the halogenated aniline derivative in phase I metabolism might result in a reactive benzoquinone-type or N-oxidized primary metabolite, which can be retained in the body due to reaction with cellular macromolecules.

The fluorescence properties of five substituted benzamides, including alizapride, metoclopramide, sulpiride, sultopride and tiapride, were investigated at several pH values and in various solvents (dimethyl sulfoxide, ethanol, ethylene glycol, methanol, propan-2-ol, tetrahydrofuran and water). Except for alizapride, the fluorescence intensities were found to be higher at acidic (1-6) than at alkaline (8-12) pH values. Using the optimum solvent (aqueous solutions) and appropriate pH conditions, linear spectrofluorimetric calibration curves were established over a concentration range of about two orders of magnitude, with correlation coefficients larger than 0.996. Limits of detection were between 1 and 13 ng ml-1, depending on the compound. The method was applied to the determination of benzamides in pharmaceutical preparations and in human urine, with recoveries ranging from 94 to 108% and from 93 to 104%, respectively.

1. The structures of major biliary and urinary metabolites of ecabapide in rat were identified by comparison with authentic standards using lc-ms and 1H-nmr spectrometry. 2. A major metabolite was found in the bile obtained from rat after an oral dose of 14C-ecabapide and identified as the amidaldehyde derivative. In the urine, two polar metabolites were characterized as the phenolic sulphates. Further, two lipophilic metabolites were identified as alcohol derivatives, and two others as oxamic acids. 3. From these results, it was estimated that the first step in the metabolism of ecabapide in rat was oxidative N-dealkylation to produce the amidaldehyde. This amidaldehyde was further metabolized by two routes, one by reduction of the amidaldehyde into the corresponding alcohol followed by mono-demethylation and subsequent aromatic O-sulphation, the second by oxidation of the amidaldehyde into the oxamic acid followed by mono-demethylation and subsequent aromatic O-sulphation.

In rats treated ip with N-ethylbenzamide (EB) at doses of 250-500 mg/kg, this amide was metabolized by hydrolysis to ethylamine and benzoic acid, the latter detected in the urine as hippuric acid. Proposed metabolic pathways were investigated by treatment of male Wistar rats with EB and N-ethyl-alpha-13C-benzamide followed by examination of urine samples by high-resolution NMR spectroscopy. Ethylamine was detected by 1H and 13C NMR techniques. Estimation of metabolite levels in 24-hr posttreatment urine by 1H NMR spectroscopy with an internal standard of 3-(trimethylsilyl)-1-propanesulfonic acid and correction for background levels of hippuric acid in control urine showed that the percentage of the dose excreted as hippuric acid was 52-76%. The corresponding range for the excretion of ethylamine (detected as an ethylammonium cation) was 27-41%. Signals for EB, benzamide, and benzoic acid were not above the background in urine of treated animals. A similar NMR study with unlabeled p-chloro-N-ethylbenzamide (p-Cl EB) resulted in the detection of p-chlorohippuric acid and ethylamine. Treatment of rats with N,N-diethylbenzamide and p-chloro-N,N-diethylbenzamide gave the same detectable metabolites in the urine as those found in experiments with EB and p-Cl EB. These observations could be rationalized with a metabolic pathway involving an initial oxidative mono-N-deethylation reaction followed by enzymatic hydrolysis of the secondary amides to ethylamine and benzoic acids and conjugation of the latter with glycine. N-Methylbenzamide was also found to be eliminated in the urine as hippuric acid.

1. 14C-Ecabapide, 3-[[[2-(3,4-dimethoxyphenyl)ethyl]carbamoyl]methyl]amino-N- methyl[14C]benzamide, was dosed orally to rat (100 mg/kg). Within 48h after dosing, 36.7 +/- 5.4 and 55.7 +/- 11.8% of the administered radioactivity was recovered from urine and faeces respectively. 2. The unchanged drug was the major compound excreted in the urine and accounted for 37% of the urinary radioactivity. Seven urinary metabolites were purified by preparative hplc and their structures were elucidated by mass and 1H-nmr spectrometry. 3. The major metabolic pathway of ecabapide was found to be the formation of 3-amino-N-methylbenzamide produced by N-dealkylation of the secondary amine at the 3-position of the benzamide moiety followed by acetylation. 4. Further metabolic pathways of the N-methylbenzamide moiety were N-demethylation via the carbinolamine derivatives, and/or aromatic hydroxylation followed by glucuronidation.

A highly sensitive and specific assay was developed for routine analysis of zacopride at the femtomole level in human plasma and urine. Zacopride and the deuterated internal standard [(2H3)zacopride] were measured by gas chromatography/negative ion chemical ionization mass spectrometry with methane as the reagent gas. A multiple-step liquid-liquid extraction procedure was used to isolate the two compounds of interest from complex biological matrices. Zacopride was converted to the fluorinated derivative with pentafluoropropionic anhydride. The mass spectrometer was tuned to monitor the very intense [M-HF]- ion at m/z 435 which was generated into the ion source by a dissociative capture process. This assay was performed with 1 ml of plasma or 0.2 ml of urine, and the quantification limit of the method was calculated as 10 pg ml-1 using a suitable statistical test. The very low relative standard deviation and mean percentage error values calculated during the within-day or between-day repeatability assays have clearly demonstrated the ruggedness of the technique for the routine quantitative measurement of zacopride in plasma and urine. Some preliminary results on the pharmacokinetics of this potent drug are presented to illustrate the applicability of this new powerful gas chromatographic/mass spectrometric method.

The metabolism of mosapride citrate ((+-)-4-amino-5-chloro-2-ethoxy-N-[[4-(4-fluorobenzyl)-2-morpho li nyl] methyl]benzamide citrate, AS-4370, CAS 112885-42-4) was investigated in rats, dogs and monkeys after a single oral administration. Four metabolites, M-1 to M-4, were isolated from rat urine. Two of them were identified as des-p-fluorobenzyl (M-1) and 5'-oxo-des-p-fluorobenzyl mosapride (M-2) and the other two were considered to be as 3-hydroxy des-p-fluorobenzyl (M-3) and 3-hydroxy 5'-oxo-des-p-fluorobenzyl mosapride (M-4) by mass spectrometry and/or nuclear magnetic resonance spectrometry. Metabolites in biological specimens such as plasma and urine after oral administration of [14C]mosapride in rats, dogs and monkeys were analyzed by thin layer chromatography (TLC). Unchanged mosapride, M-1, M-2 and some other metabolites were observed in the plasma of male rats. On the other hand, female rat plasma contained mosapride as the major radioactive component with a small amount of M-1 and little other metabolite(s). In rat urine, irrespective of sex, M-1, M-2, M-3, M-4 and a few other metabolites were observed but the unchanged drug was hardly detected. These facts suggested that the metabolic rate of mosapride was different between sexes in rats. In male rat bile, mosapride, M-1 and polar metabolite(s) were observed. In dogs, mosapride and M-1 were observed as major radioactive components in plasma, and M-1 was seen also as the major metabolite in urine but unchanged drug was hardly detected. Similar results were obtained in monkeys.(ABSTRACT TRUNCATED AT 250 WORDS)

The pharmacokinetics and dose proportionality of mosapride citrate ((+-)-4-amino-5-chloro-2-ethoxy-N-[[4- (4-fluorobenzyl)-2-morpholinyl]methyl]benzamide citrate dihydrate, AS-4370, CAS 112885-42-4) were investigated in healthy male volunteers. The subjects were given a single oral dose (5, 10, 20 and 40 mg, each of 5 subjects) and a multiple oral dose (20 mg t.i.d. for one day, and 10 and 20 mg t.i.d. for 8 days, each of 5 subjects). Food effect on the pharmacokinetics of mosapride was also evaluated after a single oral 10 mg dose by an open, two-way crossover method. Mean plasma levels of mosapride reached a peak 0.5-1 h after single dosing of 5, 10, 20 and 40 mg. The peaks were dose-related with values of 25.1, 51.2, 157.8 and 280.6 ng/ml, respectively, and were followed by a first-order decrease with apparent half-lives of 1.4-2.0 h. The Cmax and AUC increased in proportion to the dose, indicating linear pharmacokinetics of mosapride up to 40 mg. The Cmax of M-1, a des-4-fluorobenzyl metabolite, was 1/6 of that of the unchanged drug. Urinary excretion of the unchanged mosapride and M-1 during 48 h after single dosing accounted for 0.1-0.4% and 7.0-11.0% of the dose, respectively. There were no significant changes in the plasma concentration-time profiles and urinary excretions between single and multiple doses, indicating that the pharmacokinetics of mosapride in man was not altered by its multiple administration. Plasma levels of mosapride reached steady state on day 2 of multiple administration.(ABSTRACT TRUNCATED AT 250 WORDS)

The method for the analysis of anileridine, levorphanol, nalbuphine and ethamivan in urine by means of GC/NPD and GC/MSD is described. TFAA and MSTFA-MBTFA have been used in this procedure for TFA and TMS derivatization. The parent forms and the metabolites of the four drugs can be found by GC/NPD screening and GC/MSD confirmation. The method is reliable, fast and sensitive.

A reversed-phase column liquid chromatographic method for the determination of remoxipride, a novel antipsychotic drug, in biological fluids is described. A simple one-step extraction is used followed by liquid chromatography on a 3-microns octadecylsilica column and ultraviolet absorbance detection. The method is accurate and precise for clinical remoxipride levels in both plasma and urine. For situations where a higher sensitivity is necessary a two-step extraction and a modified mobile phase are used. With this modification plasma concentrations down to 2 nM can be determined with acceptable precision.

Bromadoline and its two N-demethylated metabolites were extracted into ether:butyl chloride after the addition of internal standard and basification of the various biological fluids (blood, plasma, serum, and urine). These compounds were then extracted into dilute phosphoric acid from the organic phase and separated on a reversed-phase chromatographic system using a mobile phase containing acetonitrile and a buffer of 1,4-dimethylpiperazine and perchloric acid. The overall absolute extraction recoveries of these compounds were approximately 50-80%. The background interferences from the biological fluids were negligible and allowed quantitative determination of bromadoline and the metabolites at levels as low as 2-5 ng/mL. At mobile phase flow rate of 1 mL/min, the sample components and the internal standard were eluted at the retention times within approximately 7-12 min. The drug- and metabolite-to-internal standard peak height ratios showed excellent linear relationships with their corresponding concentrations. The analytical method showed satisfactory within- and between-run assay precision and accuracy, and has been utilized in the simultaneous determination of bromadoline and its two N-demethylated metabolites in biological fluids collected from humans and from dogs after administration of bromadoline maleate.

Disulfiram is a drug used in the treatment of chronic alcoholism in man. Accurate assessment of patient compliance is important in this treatment. This paper describes a method for the detection and quantitative analysis of diethylamine, a metabolite of disulfiram, in urine. The method involves conversion of the water-soluble diethylamine in the urine to a derivative, N,N-diethyl-3,5-dinitrobenzamide, that is soluble in an organic solvent. This derivative is extracted from urine with diethyl ether and then subjected to thin-layer chromatography. A spectrophotometric procedure is used for quantification. This method provides a means of determining whether or not a patient is taking his prescribed disulfiram.

A method for on-line radiometric detection in liquid chromatography (LC) is described that permits the detection of low levels of radioactivity in LC effluents by using solvent segmentation and storage of the segmented effluent in a capillary storage loop. The object of the method is to make the flow-rate in the on-line radioactivity monitor (and hence the mean residence time in the monitor) independent of the separation process. Therefore, after storage of the complete chromatogram, the segmented effluent is led through the monitor at flow-rates that can be chosen according to the residence time desired for accurate and precise radioactivity determination. In this system, it is possible to use a flow-cell volume small enough to preserve the chromatographic integrity, while maintaining the possibility of increasing the counting time. Tests have been performed on the reproducibility of 14C detection and the influence of the flow-rate through the monitor on the standard deviation in 14C peak area and, thus, on the detection limit, using 14C-labelled remoxipride. As an application, analyses of urine samples for [14C]remoxipride and one of its potential metabolites are reported.

This paper describes the synthesis of 5-[(3-bromo-2,6-dimethoxybenzamido)-methyl]-5-hydroxy-2-pyrrolidon e (3) and its 1-ethyl analogue 2, two urinary metabolites of the dopamine D-2 antagonist remoxipride [1, (S)-3-bromo-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2, 6-dimethoxybenzamide]. Two synthetic schemes leading to a common intermediate, 5-benzamido-4-oxopentanoic acid 4, were developed. This key intermediate permits conversion into either metabolite. Reaction of 4 with isobutyl chloroformate furnished a mixed carbonic anhydride, which upon treatment with ethylamine or ammonia gave the 4-oxopentanamides 5 and 6, respectively. Ring-closure afforded the corresponding 5-hydroxy-2-pyrrolidones 2 and 3.

Clebopride is extensively metabolized in the rat, rabbit, dog, and man. By use of chromatographic methods, up to 25 metabolites in hydrolyzed and nonhydrolyzed urine have been detected. All four species produced the same main metabolites, as indicated by thin-layer chromatography. These, isolated from urine of the three animal species, were identified as N-(4'-piperidyl)-2-methoxy-4-amino-5-chlorobenzamide, N-(4'-piperidyl-2'-one)-2-methoxy-4-amino-5-chlorobenzamide, and N-(1'-alpha-hydroxybenzyl-4'-piperidyl)-2-methoxy-4-amino-5-chlorobenzamide (tentative structure of a carbinolamine more stable than expected). In the dog, 2-methoxy-4-amino-5-chlorobenzoic acid was also detected. N4-glucuronidation of clebopride and some of its metabolites has been shown to occur in the three animal species. The rabbit produced large amounts of these conjugates. Clebopride N4-sulfonate was not present in the urine of any of the species investigated.

2-Proparglyoxy-5-amino-N-(n-butyl)-benzamide(parsalmide, My 41-6) given s.c. to the cat and excreted in the urine, was present partly as unchanged parsalmide (25-62% of total excreted compounds), partly as unstably conjugated compounds (17-36%, mainly N-glucuronilparsalmide), and partly as stably conjugated compounds (11-36%, mainly N-acetylparsalmide). Urinary excretion of parsalmide and its metabolites continued for some days, indicating a slow excretion from the body, contrasting with other species, man included, previously examined. Summarizing, there are some 5-6 metabolites of parsalmide in the urine of treated cats, apart from unchanged parsalmide.