bluuebunny/crossref_metadata_2025 huggingface dataset:
Production of Disulfide‐Bonded Proteins in Escherichia coli
Authors: Ke Na , Berkmen Mehmet
Published: October 2014

Production of recombinant proteins at high yields in Escherichia coli requires extensive optimization of expression conditions. Production is further complicated for proteins that require specific post‐translational modifications for their eventual folding. One common and particularly important post‐translational modification is oxidation of the correct pair of cysteines to form a disulfide bond. This unit describes methods to produce disulfide‐bonded proteins in E. coli in either the naturally oxidizing periplasm or the cytoplasm of appropriately engineered cells. The focus is on variables key to improving the oxidative folding of disulfide‐bonded proteins, with the aim of helping the researcher optimize expression conditions for a protein of interest. Curr. Protoc. Mol. Biol. 108:16.1B.1‐16.1B.21. © 2014 by John Wiley & Sons, Inc.
Overview of Cell‐Free Protein Synthesis: Historic Landmarks, Commercial Systems, and Expanding Applications
Authors: Chong Shaorong
Published: October 2014

During the early days of molecular biology, cell‐free protein synthesis played an essential role in deciphering the genetic code and contributed to our understanding of translation of protein from messenger RNA. Owing to several decades of major and incremental improvements, modern cell‐free systems have achieved higher protein synthesis yields at lower production costs. Commercial cell‐free systems are now available from a variety of material sources, ranging from “traditional” E. coli, rabbit reticulocyte lysate, and wheat germ extracts, to recent insect and human cell extracts, to defined systems reconstituted from purified recombinant components. Although each cell‐free system has certain advantages and disadvantages, the diversity of the cell‐free systems allows in vitro synthesis of a wide range of proteins for a variety of downstream applications. In the post‐genomic era, cell‐free protein synthesis has rapidly become the preferred approach for high‐throughput functional and structural studies of proteins and a versatile tool for in vitro protein evolution and synthetic biology. This unit provides a brief history of cell‐free protein synthesis and describes key advances in modern cell‐free systems, practical differences between widely used commercial cell‐free systems, and applications of this important technology. Curr. Protoc. Mol. Biol. 108:16.30.1‐16.30.11. © 2014 by John Wiley & Sons, Inc.
Analysis of Genetic Linkage Data for Mendelian Traits
Authors: Dueker Nicole D. , Pericak‐Vance Margaret A.
Published: October 2014

This unit describes linkage analysis, an invaluable tool in mapping human disease genes. Linkage analysis is one of several methods used to map genes. Specifically, in linkage analysis, cosegregation of two or more genes (traits) is examined in a family unit to determine if they segregate independently of each other according to Mendel's laws or if they do not segregate independently because of their close physical proximity. The procedures outlined in this unit are not always straightforward, and a number of caveats are presented in the commentary regarding possible complications and confounding factors that may arise. Specific examples are given, but it is not possible to cover all possible scenarios or variables. The intention is to enable the reader to master basic principles used in the linkage approach, and thus be in a position to undertake, with proper consultation, a linkage‐analysis study. This unit deals specifically with lod score analysis in Mendelian disorders. Curr. Protoc. Hum. Genet. 83:1.4.1‐1.4.31. © 2014 by John Wiley & Sons, Inc.
RNA‐seq Data: Challenges in and Recommendations for Experimental Design and Analysis
Authors: Williams Alexander G. , Thomas Sean , Wyman Stacia K. , Holloway Alisha K.
Published: October 2014

RNA‐seq is widely used to determine differential expression of genes or transcripts as well as identify novel transcripts, identify allele‐specific expression, and precisely measure translation of transcripts. Thoughtful experimental design and choice of analysis tools are critical to ensure high‐quality data and interpretable results. Important considerations for experimental design include number of replicates, whether to collect paired‐end or single‐end reads, sequence length, and sequencing depth. Common analysis steps in all RNA‐seq experiments include quality control, read alignment, assigning reads to genes or transcripts, and estimating gene or transcript abundance. Our aims are two‐fold: to make recommendations for common components of experimental design and assess tool capabilities for each of these steps. We also test tools designed to detect differential expression, since this is the most widespread application of RNA‐seq. We hope that these analyses will help guide those who are new to RNA‐seq and will generate discussion about remaining needs for tool improvement and development. Curr. Protoc. Hum. Genet. 83:11.13.1‐11.13.20. © 2014 by John Wiley & Sons, Inc.
High‐Resolution Cytometry for High‐Content Cell Cycle Analysis
Authors: Furia Laura , Pelicci Piergiuseppe , Faretta Mario
Published: October 2014

One of the major limitations of flow cytometry (FCM) is the absence of an intracellular view. Automated microscopy and image analysis, together with technological developments, led to new approaches in cytometry that bypass the above limitation, introducing high resolution, high content, and large statistical sampling. However, few attempts have been made, until now, to translate the wide repertoire of FCM assays into high‐content image screening. This unit describes the implementation of an acquisition and analysis protocol for evaluation of the cell cycle by automated microscopy. The approach grants the possibility to perform simultaneous analysis of a high number of different parameters. A large part of this unit is devoted to the description of hardware features that can optimize the recorded information together with the acquisition and analysis procedures employed to produce good‐quality data.Curr. Protoc. Cytom. 70:7.41.1‐7.41.15. © 2014 by John Wiley & Sons, Inc.
Correlative Fluorescence and Electron Microscopy
Authors: Schirra Randall T. , Zhang Peijun
Published: October 2014

Correlative fluorescence and electron microscopy (CFEM) is a multimodal technique that combines dynamic and localization information from fluorescence methods with ultrastructural data from electron microscopy, to give new information about how cellular components change relative to the spatiotemporal dynamics within their environment. In this review, we will discuss some of the basic techniques and tools of the trade for utilizing this attractive research method, which is becoming a very powerful tool for biology labs. The information obtained from correlative methods has proven to be invaluable in creating consensus between the two types of microscopy, extending the capability of each, and cutting the time and expense associated with using each method separately for comparative analysis. The realization of the advantages of these methods in cell biology has led to rapid improvement in the protocols and has ushered in a new generation of instruments to reach the next level of correlation—integration. Curr. Protoc. Cytom. 70:12.36.1‐12.36.10. © 2014 by John Wiley & Sons, Inc.
Heating of the sunlit polar cap ionosphere by reflected photoelectrons
Authors: Varney R. H. , Solomon S. C. , Nicolls M. J.
Published: October 2014

Photoelectrons escape from the ionosphere on sunlit polar cap field lines. In order for those field lines to carry zero current without significant heavy ion outflow or cold electron inflow, field‐aligned potential drops must form to reflect a portion of the escaping photoelectron population back to the ionosphere. Using a 1‐D ionosphere‐polar wind model and measurements from the Resolute Bay Incoherent Scatter Radar (RISR‐N), this paper shows that these reflected photoelectrons are a significant source of heat for the sunlit polar cap ionosphere. The model includes a kinetic suprathermal electron transport solver, and it allows energy input from the upper boundary in three different ways: thermal conduction, soft precipitation, and potentials that reflect photoelectrons. The simulations confirm that reflection potentials of several tens of eV are required to prevent cold electron inflow and demonstrate that the flux tube integrated change in electron heating rate (FTICEHR) associated with reflected photoelectrons can reach 109eV cm−2s−1. Soft precipitation can produce FTICEHR of comparable magnitudes, but this extra heating is divided among more electrons as a result of electron impact ionization. Simulations with no reflected photoelectrons and with downward field‐aligned currents (FAC) primarily carried by the escaping photoelectrons have electron temperatures which are ∼250–500 K lower than the RISR‐N measurements in the 300–600 km region; however, simulations with reflected photoelectrons, zero FAC, and no other form of heat flux through the upper boundary can satisfactorily reproduce the RISR‐N data.
Acetaminophen
Authors: Wehr Angela
Published: October 2014

Acetaminophen (APAP) is one of the most commonly used drug molecules. APAP is a cyclooxygenase (COX) inhibitor with anti‐inflammatory properties and is also used as a common analgesic. One adverse effect of concern is that APAP and its metabolites may induce liver, blood cell, and kidney damages. CYP‐mediated oxidation of APAP produces
N
‐acetyl‐
p
‐benzoquinone imine (NAPQI), a highly reactive species that rapidly conjugates with glutathione. Upon glutathione depletion, NAPQI covalently binds with proteins to form protein conjugates in the cell and is responsible for the liver toxicity associated with APAP overdose.
Atenolol
Authors: Yuan Tao
Published: October 2014

Atenolol is a β‐adrenoceptor blocking agent, mainly used for the treatment of angina pectoris, hypertension, cardiac arrhythmia, and myocardial infarction. The in vitro metabolism of [
14
C‐phenyl]‐atenolol was investigated in rat and dog liver microsomes. The in vivo metabolism of [
14
C‐phenyl]‐atenolol was studied in dogs, rats, and mice. The metabolism of [
14
C‐methylen]‐atenolol was studied in humans following oral and intravenous (i.v.) doses. Hydroxylation at the methylene moiety of the carbamoylmethyl group resulted in a hydroxylated metabolite. Atenolol also underwent glucuronidation to form a conjugate metabolite, which was mediated by UGT1A6.
Benzoyl peroxide
Authors: Xu Lin
Published: October 2014

Benzoyl peroxide, an oxidizing agent, degrades via hydrolysis after contact with skin, producing benzoic acid and oxygen.
Bosentan
Authors: Lin Lishan
Published: October 2014

Bosentan is a dual endothelial receptor antagonist in the treatment of pulmonary artery hypertension. Bosentan is metabolized in human liver, mainly mediated by CYP2C9 and 3A4, followed by biliary excretion. Hydroxylation at the
t
‐butyl group yielded
2
, an active metabolite that retained the parent pharmacological activity. The phenol metabolite
3
was formed by oxidative O‐demethylation of the guaiacol ether, catalyzed by 3A4. Metabolite 4 was formed as a minor product from both
2
and
3
. The hepatic uptake of bosentan and 2 were mediated by OATP1B1 and OATP1B3.
Cefepime
Authors: Du Jiangbo
Published: October 2014

Cefepime is a fourth‐generation cephalosporin antibiotic against both Gram‐negative and Gram‐positive organisms. The metabolism and disposition of cefepime were studied in healthy and renally impaired male volunteers after administration of a single intravenous 1000 mg dose of [
14
C]‐cefepime. The parent drug comprised 95 and 76% of the total plasma radioactivity in subjects with normal and impaired renal functions, respectively. Cefepime was primarily excreted unchanged in urine of normal subjects, accounting for 88% of the total recovered radioactivity. Minor metabolites included
N
‐methylpyrrolidine and its
N
‐oxide, and the 7‐epimer of cefepime.
Celecoxib
Authors: Prakash Chandra
Published: October 2014

Celecoxib, a cyclooxygenase‐2 (COX‐2) inhibitor, is a nonsteroidal anti‐inflammatory drug used to relieve pain caused by osteoarthritis and other ailments. It is extensively metabolized in both preclinical species and humans. The primary metabolic pathway of celecoxib was hydroxylation of the methyl group to form a hydroxmethyl metabolite, which was further oxidized to the carboxylic acid metabolite. Conjugation of alcohol and carboxylic acid metabolite of celecoxib yielded the glucuronide conjugates.
Clopidogrel
Authors: Prakash Chandra
Published: October 2014

Clopidogrel is an antiplatelet agent used to inhibit blood clots in coronary artery, peripheral vascular, and cerebrovascular diseases. It is extensively metabolized in humans by two main metabolic pathways: one mediated by esterases to form an inactive carboxylic acid derivative and the other mediated by multiple cytochrome P450 enzymes to form the 2‐oxo‐clopidogrel, which is then metabolized to form the pharmacologically active thiol derivative of clopidogrel.
Desloratadine
Authors: Penner Natasha
Published: October 2014

3‐Hydroxy‐desloratadine‐glucuronide was the major circulating metabolite in humans. 3‐Hydroxy‐desloratadine, its glucuronide, and dihydroxy‐desloratadine glucuronides were the major metabolites in excreta.
Dexfenfluramine
Authors: Zhou Xin
Published: October 2014

Dexfenfluramine is a serotoninergic anorectic drug that was once used for weight loss. The drug was withdrawn from the market after reports of heart valve disease and pulmonary hypertension. The disposition of dexfenfluramine was studied in healthy human subjects. Following a 30‐mg dose of dexfenfluramine hydrochloride, metabolites in urine and plasma were analyzed. Primary metabolic reactions included N‐deethylation to form nordexfenfluramine, deamination of nordexfenfluramine yielded 1‐(3‐trifluoromethylphenyl)propan‐2‐ol, its hydroxylation product of a diol metabolite, and decarboxylation reaction yielded
m
‐trifluoromethylbenzoic acid and its glycine conjugate.
Divalproex
Authors: Zhang Zhi‐Yi
Published: October 2014

Divalproex, consisting of sodium valproate and valproic acid (VPA) in a 1 : 1 molar ratio, elicits anticonvulsant activities and is used for the treatment of bipolar disorder, depressive disorder, and epilepsy. In vitro, it is metabolized by mitochondrial β‐oxidation to form the major primary metabolite 2‐ene‐VPA and cytochromes P450‐mediated hydroxylation and dehydration/dehydrogenation at multiple carbon positions, resulting in the formation of numerous oxidative products including the hydroxylated, ketone, acid, and diene metabolites. O‐Glucuronidation of VPA to yield an acyl glucuronide is the only conjugative pathway. Mitochondrial β‐oxidation and the formation of acyl glucuronide have been associated with VPA‐induced hepatotoxicity, potentially because of the intrusion of endogenous fatty acid biotransformation and the attenuation of GSH. Urinary excretion was the major route for elimination. The acyl glucuronide, β‐oxidative and ω‐hydroxylated metabolites appeared to be major in vivo.
Ethionamide
Authors: Chen Meixia
Published: October 2014

Ethionamide is a second‐line antitubercular agent that inhibits mycolic acid synthesis. It underwent extensive hepatic metabolism into several different metabolites in the human study, with <1% of the administered dose excreted unchanged in urine. The pharmacologically active
S
‐oxide and the amide were the major metabolites observed, accounting for 17 and 14% of the administered dose, respectively. Other minor secondary metabolic reactions were also observed. Such reactions included the N‐methylation of the pyridine ring and oxidation of the pyridine ring to yield pyridine‐2‐one metabolites.
Galantamine
Authors: Yu Jinghua
Published: October 2014

Galantamine is a competitive and reversible cholinesterase inhibitor used for the treatment of mild to moderate Alzheimer's disease and various other memory impairments. The metabolism and excretion of [
3
H]‐galantamine in healthy male adult subjects were investigated. After an oral dose of 4 mg, the metabolites detected were galantaminone, epigalantamine, glucuronide of
O
‐desmethyl‐galantamine, glucuronide of
O
‐desmethyl‐epigalantamine, glucuronide of galantamine,
O
‐desmethyl‐galantamine,
N
‐desmethyl‐galantamine,
N
‐oxide of galantamine, sulfate conjugate of
O
‐desmethyl‐galantamine,
N
‐desmethyl‐epigalantamine,
N
‐oxide of epigalantamine, and
O
‐desmethyl‐epigalantamine.
Glipizide
Authors: Tan Bo
Published: October 2014

Glipizide is a sulfonylurea drug used to treat type 2 diabetes. Rapid elimination of the administered radioactive dose of [
14
C‐carbamoyl]‐glipizide in healthy male volunteers after a single oral administration of 5 mg was observed. Urine and feces contained 88 and 11% of the administered dose, respectively, 72‐h post dosing. In urine, two primary metabolites of glipizide were identified. Hydroxylation was the primary metabolic pathway to yield 4‐
trans
‐hydroxy‐cyclohexyl‐glipizide (14%) and 3‐
cis
‐hydroxy‐cyclohexyl‐ glipizide (65%).
N
‐(β‐Acetylaminoethylbenzene‐sulfonyl)‐
N
′
‐cyclohexyl‐urea was detected as a minor metabolite. It is likely generated by the cleavage of the amide linkage and the
N
‐acetylation of the amino group (about 1% of the dose).
Losartan
Authors: Chen Meixia
Published: October 2014

Losartan is a nonpeptide angiotensin II receptor antagonist used for the treatment of hypertension. The in vitro metabolism of losartan was investigated in incubations with liver slice preparations from rats, monkeys, and humans. Incubation of losartan with human liver slices formed six metabolites of similar quantity. The aliphatic hydroxylation at various positions yielded three metabolites. Oxidation of the parent hydroxymethyl moiety yielded the corresponding acid, which was postulated via the aldehyde intermediate. The acid metabolite was found to be more potent than losartan as a nonpeptide angiotensin II receptor antagonist. It formed further an
N
‐glucuronide conjugate.
Mirabegron
Authors: Gao Ruina
Published: October 2014

Mirabegron is a potent and selective agonist for the human β
3
‐adrenoceptor. It is used to control overactive bladder. After a single oral dose of 160 mg of [
14
C]‐mirabegron to healthy male subjects, plasma, urine, and fecal samples were collected for metabolite identification. In plasma, in addition to the parent drug, metabolites identified included the amide hydrolysis metabolite and its
N
‐acetylated metabolite,
N
‐dealkylated metabolite,
O
‐glucuronide,
N
‐
O
‐glucuronide,
N
‐glucuronide, and carbamoyl glucuronides. In urine, further two conjugated metabolites were identified. In fecal samples, no metabolites were observed.
Moricizine
Authors: Yuan Tao
Published: October 2014

Moricizine is a phenothiazine derivative with class I antiarrhythmic properties. The metabolism of [
14
C‐phenyl]‐moricizine was studied in human plasma and urine. The biotransformation of moricizine was extensive, with >99% of the parent drug metabolized. More than 35 radiolabeled chromatographic peaks in plasma and more than 51 peaks in urine were observed. Eight metabolites were structurally identified. S‐Oxidation, hydrolysis of the amide bond from the morpholino side chain and the ester bond from the carbamate side chain, N‐dealkylation of the morpholine ring, and glucuronide conjugation were the primary metabolic reactions.
Ramelteon
Authors: Zhong Kan
Published: October 2014

Ramelteon is a selective melatonin agonist used for the treatment of insomnia. The in vivo metabolism of [
14
C]‐ramelteon in healthy male subjects was studied after a single oral dose of 16 mg. CYP1A2 was the primary enzyme involved in the metabolism of ramelteon. The principal metabolite in human serum was the side chain C‐2 hydroxylation metabolite. Systemic exposure of this metabolite (major active metabolite) was 20‐ to 30‐fold greater than that of ramelteon. Other metabolic pathway included hydroxylation at aliphatic and aromatic rings, further oxidation from alcohol to keto form, and opening of the furan ring. Phase II metabolism generated several glucuronide conjugates. Approximately, 84% of the administered radioactivity was recovered in urine and 4% in feces. Negligible urinary excretion of the parent compound (<1%) and low serum concentration indicated that ramelteon underwent extensive first‐pass metabolism.
Zolpidem
Authors: Lin Lishan
Published: October 2014

Zolpidem is used for the short‐term treatment of insomnia, sleeping disorder, and brain disorders. Human metabolism of zolpidem was investigated in young volunteers. After a single oral administration of [
14
C]‐zolpidem, plasma, urine, and fecal samples were collected and analyzed. The primary metabolic reactions observed were methyl and benzyl hydroxylations/oxidations, which yield alcoholic metabolites and their carboxylic acid metabolites. Two carboxylic acid metabolites and the hydroxylated imidazopyridine metabolite were detected accounting for, respectively, 52, 12, and 10% of the administered dose. The carboxylic acid from benzyl oxidation was the only circulating metabolite detected.
Donepezil
Authors: Zhong Kan
Published: October 2014

Donepezil is a reversible acetylcholinesterase inhibitor used for the treatment of moderate‐to‐severe Alzheimer's disease. The in vivo metabolism of donepezil was investigated in eight healthy male subjects after a single 5‐mg oral dose of [
14
C]‐donepezil. O‐Demethylation followed by glucuronide conjugation, N‐dealkylation, and N‐oxidation were identified as the major metabolic reactions.
Irinotecan
Authors: Gao Ruina
Published: October 2014

Irinotecan is a topoisomerase I inhibitor used to treat several types of tumors. It is a prodrug of SN‐38. It underwent reversible and pH‐dependent interconversion between the lactone (closed ring) and carboxylate (open ring) forms. The major in vivo human metabolic pathway of irinotecan included the formation of the active metabolite SN‐38 lactone and the conjugation of SN‐38 to its glucuronide conjugate, and opening of the piperidine ring by N‐monodealkylation and further N,N‐didealkylation. Minor metabolic reactions included decarboxylation of the ring‐opening form of irinotecan, further dehydration yielded the ring‐closure product, hydroxylation of the piperidine ring, and combinations of reactions (hydroxylation, oxidation, N‐dealkylation, and hydrolysis of the lactone) yielded metabolites.
Simvastatin
Authors: Wehr Angela
Published: October 2014

Simvastatin (brand name: Zocor) is a 3‐hydroxy‐3‐methyl‐glutaryl‐CoA (HMGCoA) reductase inhibitor. It is used to lower blood cholesterol and triglyceride levels. The reversible conversion of simvastatin lactone to the active hydroxy acid form is NADPH independent. While simvastatin acid is not a substrate for microsomal metabolism, the lactone form is converted to the major metabolites, 3″‐hydroxy simvastatin, 3′,5′‐dihydrodiol simvastatin, 6′‐exomethylene simvastatin, and 6′‐hydroxysimvastatin, and in an NADPH dependent fashion. Active metabolites 6′‐hydroxysimvastatin and 3″‐hydroxy simvastatin inhibit HMG‐CoA reductase activity in vitro.
Ximelagatran
Authors: Yu Jinghua
Published: October 2014

Ximelagatran is an orally active anticoagulant. It was withdrawn from the market after reports of hepatotoxicity. Ximelagatran is a prodrug, and can be converted to the active agent melagatran. The metabolism of [
3
H]‐ximelagatran was studied in healthy male volunteers following either a single oral or intravenous dosing. The reduction of amidoximes and hydrolysis of the ethyl ester were the major metabolic reactions observed, leading to the formation of
N
‐hydroxy‐melagatran, ethyl‐melagatran, and melagatran, which were detected in human urine and feces.
Ambroxol
Authors: Liu Jia
Published: October 2014

Ambroxol is an expectorant, which increases the production of serous mucus in the respiratory tract. After oral administration of [
14
C‐benzyl]‐ambroxol to human subjects, about 90 and 10% of the administered radioactivity were recovered from urine and feces, respectively. Metabolites observed included 6,8‐dibromo‐3‐(
trans
‐4‐hydroxycylcohexyl)‐1,2,3,4‐tatrahydroquinazoline, which could proceed nonenzymatically, and dibromoanthranilic acid via N‐dealkylation and oxidative deamination, which was catalyzed by CYP3A4 predominantly. Glucuronides of the parent drug and the two phase I metabolites were also detected.
Aranidipine
Authors: Gao Chunying
Published: October 2014

Aranidipine is a 1,4‐dihydropyridine Ca
2+
antagonist used for the treatment of hypertension. In human plasma, it was metabolized by the reduction of the keto moiety of the oxopropyl group to the corresponding secondary alcohol. Two stereoisomers of the resulting alcohol were isolated. Both metabolites exhibited similar antihypertensive activity as the parent compound.
Apremilast
Authors: Zhou Xin
Published: October 2014

Apremilast is currently under phase III trials as an anti‐inflammatory drug. The metabolism and disposition of [
14
C]‐apremilast were investigated in healthy male volunteers following a single oral dose of 20 mg. About 39 and 58% of the administered radioactivity were recovered in feces and urine, respectively. Major metabolite was the glucuronide conjugate of
O
‐desmethyl apremilast, which accounted for 39% of the circulating radioactivity and 34% of the recovered radioactivity. Minor metabolites detected in excreta were
O
‐demethylated apremilast and its hydrolysis product. More than 20 metabolites from O‐demethylation, O‐deethylation, N‐deacetylation, hydroxylation, hydrolysis, and conjugation were found in plasma, urine, and feces. None of these metabolites were pharmacologically active.
Bisoprolol
Authors: Chen Meixia
Published: October 2014

Bisoprolol is a β
1
‐selective adrenergic blocking agent used in the treatment of hypertension and angina pectoris. The metabolism of racemic bisoprolol was studied in healthy male volunteers after oral dosing of 20 mg. The amount of unchanged bisoprolol excreted in urine was 50–60% of the administered dose. The major metabolite was the acid metabolite generated by
O
‐dealkylation and further oxidation, which accounted for 15–20% of the dose. Other metabolites were generated by the combination of the following reactions: hydroxylation to yield alcohols and further oxidation to carboxylic acids,
O
‐dealkylation/oxidation, and
N
‐dealkylation/deamination/oxidation to yield acid metabolites. Phase II reaction yielded
O
‐glucuronide conjugate of bisoprolol.
Lapatinib
Authors: Lin Lishan
Published: October 2014

Lapatinib is an orally active dual tyrosine kinase inhibitor. It is used in combination with other drugs to treat patients with progressive HER2‐overexpressing metastatic breast cancer. Metabolism and excretion of [
14
C]‐lapatinib were studied in healthy volunteers after a single oral 250‐mg dose. An average of 92% of the dose was excreted in feces and 1% in urine. Intact lapatinib was the predominant component in plasma. Other circulating metabolites were
N
‐hydroxylamine lapatinib, oxime derivatives, nitrone derivatives, and oxidative deamination products.
O
‐Dealkylated lapatinib, its quinazoline moiety hydroxylated derivative, intramolecular cyclized hydroxypyridine derivative, the pyridinium salt derivative, N‐dealkylated metabolite, and oxidative deamination product were found in feces.
Valsartan
Authors: Chen Meixia
Published: October 2014

Valsartan is an angiotensin II receptor antagonist acting on the AT
1
subtype. It is used to treat high blood pressure and congestive heart failure. The disposition and metabolism of [
14
C]‐valsartan were investigated in healthy male volunteers. After a single oral administration of 80 mg, approximately 51% of the dose was absorbed, and valsartan was the predominant radioactive compound in plasma. A minor, pharmacologically inactive metabolite valeryl‐4‐hydroxy‐valsartan was detected in plasma. The hydroxylation was catalyzed only by CYP2C9. Valsartan was mainly excreted unchanged (81% of the dose in the excreta).
Pefloxacin
Authors: Jin Jing
Published: October 2014

Pefloxacin is a broad‐spectrum antibiotic used to treat severe and life‐threatening bacterial infections. The metabolism and excretion of pefloxacin were investigated in healthy male volunteers. Primary metabolic reactions were N‐demethylation of piperazine ring, N‐oxidation of the
N
‐methyl moiety, hydroxylation/oxidation at the piperazine ring, and glucuronide conjugation at the carboxyl group. Metabolites norfloxacin and pefloxacin
N
‐oxide were detected in human plasma. Major products in human urine were the parent, norfloxacin, pefloxacin
N
‐oxide, and oxonorfloxacin.
Triamcinolone Acetonide
Authors: Lin Lishan
Published: October 2014

Triamcinolone acetonide is a synthetic corticosteroid used to treat various skin conditions and chronic bronchial asthma. The metabolism and excretion of [
14
C]‐triamcinolone acetonide were investigated in healthy male volunteers after a single oral 0.8‐mg dose. The parent drug was extensively metabolized and accounted for 24% of the total plasma radioactivity. Approximately, 40 and 54% of the total radioactivity were recovered in urine and feces, respectively. Hydroxylation (6β‐OH) and oxidation (21‐carboxylic acid) metabolites were detected in plasma, urine, and feces. None of the major metabolites showed anti‐inflammatory effects.
Olopatadine
Authors: Yang Yong
Published: October 2014

Olopatadine is a histamine receptor antagonist used to reduce tear levels in eye due to allergic inflammation. The metabolism of [
14
C]‐olopatadine was investigated in rats and dogs. Major metabolites included
N
‐mono‐demethylolopatadine, olopatadine
N
‐oxide, C
8
‐hydroxyl‐olopatadine and its sulfate conjugate, and
N
‐di‐demethylolopatadine and its C
8
‐hydroxylated metabolite. After an oral 80‐mg dose to healthy volunteers, the parent drug and
N
‐mono‐ and
N
‐di‐demethylolopatadines, and olopatadine
N
‐oxide were detected in human plasma.
Paroxetine
Authors: Deng Pan
Published: October 2014

Paroxetine is a selective 5‐HT uptake inhibitor used to treat depression. The metabolic fate of [
14
C]‐paroxetine was examined in humans after a single oral dose of 30 mg. Metabolism of paroxetine initially involved the opening of the methylenedioxyphenyl moiety to the catechol intermediate, followed by O‐methylation at positions 3 and 4, or O‐dealkylation. These hydroxylated metabolites underwent rapid and extensive conjugation to form glucuronide and sulfate conjugates. The glucuronide and sulfate conjugates of the
O
‐methyl metabolite were the major plasma and urinary metabolites.
Fosinopril
Authors: Lin Lishan
Published: October 2014

Fosinopril is a prodrug form of fosinoprilat. It is an ACE inhibitor used to treat hypertension and congestive heart failure. In human study, slow absorption of fosinopril after oral administration of [
14
C]‐fosinopril was observed. The absolute absorption of fosinopril was about 36% of the administered oral dose. The hydrolysis product fosinoprilat was extensively bound to plasma proteins. After the intravenous (i.v.) dose of fosinoprilat, the recovery of the administered radioactivity in the 0–96‐h samples was 44 and 46% in urine and feces, respectively. About 75% of the radioactivity in plasma and urine was identified as the parent, fosinoprilat, 15–20% as the glucuronide conjugate of fosinoprilat, and 5% as the
p
‐hydroxy metabolite of fosinoprilat. The glucuronide conjugate of fosinoprilat was the predominant urinary metabolite; however, it was not detected in feces. Only intact fosinoprilat was detected in plasma, urine, and feces following the i.v. dosing.
Pindolol
Authors: Diao Xingxing
Published: October 2014

Pindolol is a nonselective β‐blocker with partial β‐adrenergic receptor agonist activity. It is used to treat high blood pressure. Metabolism and excretion of pindolol were examined in rhesus monkeys and human patients with renal failure. The excreted amount (0–72 h) ranged from 84 to 90% of the applied [
14
C]‐pindolol radioactive dose. Direct glucuronidation of pindolol was the predominant metabolite in urine, which accounted for 66% of the dosed radioactivity. Two sulfate conjugates were also found of the 3‐hydroxy‐ and 5‐hydroxy pindolols.
20(
S
)‐Protopanaxadiol
Authors: Ma Minkang , Diao Xingxing
Published: October 2014

20(
S
)‐Protopanaxadiol is a natural product found in ginseng and notoginseng. It exhibited a wide range of pharmacological properties, including antiestrogenic, anti‐inflammatory, antitumor, vasodilator, and antinociceptive. The in vitro metabolism of 20(
S
)‐protopanaxadiol was studied in human liver microsomes and human hepatocytes. The initial and primary metabolic pathway was epoxidation of the 24,25‐double bond to generate 24,25‐epoxides (
2
and
3
), followed by hydration reaction to yield the corresponding 24,25‐vicinal diol derivatives (
4
and
5
) and hydration/ring closure to yield 20,24‐oxide (
6
and
7
). Metabolites
6
and
7
underwent dehydrogenation to form ketones, and hydroxylation and conjugation reactions to yield numerous secondary metabolites. In addition, two glucuronide conjugates were detected in human hepatocytes incubation.
Rivaroxaban
Authors: Lin Lishan
Published: October 2014

Rivaroxaban is an oral anticoagulant. In human liver microsomes and hepatocytes, two primary metabolic reactions were observed. The first reaction involved the hydroxylation at the morpholinone moiety to generate three monohydroxylated metabolites. The second primary reaction involves either oxidative N‐ or O‐dealkylation reaction or hydrolytic opening of the lactam bond to generate ring‐opening products. Hydroxylation at the oxazolidinone moiety was a minor metabolic pathway. A morpholinone ring‐opened product was the major metabolite in human hepatocyte incubations. Minor pathways observed included amide hydrolysis of chlorothiophene amide moiety, conjugation with glycine, and oxidative deamination. The in vivo metabolic profile of rivaroxaban in healthy human males after a single oral dose of [
14
C]‐rivaroxaban (10 mg) was similar to that observed in animals and in vitro. No major or pharmacologically active circulating metabolites were detected.
Nicardipine
Authors: Li Liang
Published: October 2014

Nicardipine is a calcium channel blocker. It acts as a cerebral vasodilator and it is used to treat high blood pressure and hypertension. Metabolism of [
14
C]‐nicardipine in healthy male human volunteers was conducted after of a single dose of 30 mg. Plasma, urine, and feces were collected until 168‐h post dosing. Metabolism preceded under two parallel pathways (products with the dihydropyridine moiety and products with the pyridine moiety). Several secondary metabolites were identified where sequential metabolism of carboxylic ethyl ester portion of the molecule occurred.
Phenolphthalein
Authors: Yang Yong
Published: October 2014

Phenolphthalein is a color indicator used in acid/base titration. It has also been used as a laxative, but its over‐the‐counter uses had been withdrawn because of toxicological concerns over carcinogenicity. The metabolism of [
14
C]‐phenolphthalein was investigated in F344 rats and B6C3F1 mice following a single oral dose. At the 800 mg kg‐1 dose level, recovery of the administered radioactivity was nearly 100% in females and about 75% in males, within 72‐h post dosing. Radioactivity was primarily recovered in the feces in rats (>90%), whereas mice excreted 30–40% of administered radioactivity in urine. Phenolphthalein glucuronide was the major metabolite detected. Minor metabolites included sulfate conjugate, hydroxylated metabolite, and a diglucuronide conjugate. Biliary elimination was extensive in rats (35% of dose within 6 h). The only product detected in bile was phenolphthalein glucuronide.
Ciclesonide
Authors: Yu Jinghua
Published: October 2014

Ciclesonide is a glucocorticoid used to treat obstructive airway diseases such as asthma and hay fever. Major metabolites detected in the 4‐h human hepatocyte incubation mixtures were active metabolite desisobutyrylciclesonide (
2
) via ester hydrolysis, and pharmacologically inactive metabolites via hydroxylations of
2
. Hippuric acid was a unique metabolite identified. It was formed by the cleavage of the cyclohexylmethyl moiety of ciclesonide, followed by aromatization of the cyclohexane ring through multiple steps of hydroxylation, dehydration, and conjugation with glycine. The in vivo metabolism of ciclesonide was studied in patients who were undergoing lung surgery for the treatment of malignant pulmonary lesions. After inhaled a single dose of 1.28 mg ciclesonide at various time‐points between
2
and 24 h before lung tissue resection, metabolite 2 and its oleate and palmitate conjugates were detected in the central and peripheral lung tissues. A substantial portion of ciclesonide was activated rapidly to
2
within the first time point of tissue analysis.
Nifedipine
Authors: Liu Jia
Published: October 2014

Nifedipine is a dihydropyridine drug. It is a calcium channel blocker and is used to treat hypertension. In humans, 65% of the administered drug was excreted in urine and 35% was excreted in bile. In humans, its initial reaction was dehydrogenation, converting the dihydropyridine moiety to the corresponding pyridine. The parent drug and its corresponding pyridine metabolite, a further ester hydrolysis product, a methyl hydroxylation and glucuronide congjugation metabolite, and a lactone closure metabolite were found in urine.
Paliperidone
Authors: Liu Ying
Published: October 2014

Paliperidone is the primary active metabolite of the older antipsychotic risperidone, and is used to treat mania, bipolar disorder, and schizophrenia. The human metabolism of [
14
C]‐paliperidone was examined in five healthy male subjects after an oral administration of 1 mg paliperidone solution. Unchanged parent was the only component detected in the plasma. Approximately 80% and 11% of the radioactivity was excreted in urine and in feces. About 59% of the dose was excreted as the parent drug in urine, and no parent drug was excreted in feces. Major metabolic reactions were N‐dealkylation, monohydroxylation of the alicyclic ring, and alcohol dehydrogenation. Benzisoxazole ring scission and monohydroxylation metabolites were also identified in feces. Phase II reaction was also important, yielding glucuronide conjugates of the hydroxylated metabolites.
Ruxolitinib
Authors: Li Xiuli
Published: October 2014

Ruxolitinib is a potent selective inhibitor of Janus tyrosine kinase. It is used to treat intermediate/high risk myelofibrosis. The metabolism of [
14
C]‐ruxolitinib was investigated in healthy volunteers following a single oral dose of 25 mg. Absorption was rapid. The amount of administered radioactivity recovered in urine and feces was 74 and 22%, respectively. Unchanged drug was the major component in plasma. The primary metabolic reactions were mono‐ and dihydoxylations at various positions, which yielded numerous metabolic products (and isomers). 2‐Hydroxy‐ and 3‐hydroxycyclopentyl metabolites were the major metabolites in plasma, representing 17 and 8% of the AUC of ruxolitinib, respectively. Oxidation of the –OH moiety yielded the corresponding 3‐oxocyclopentyl metabolite, and hydration of the pyrrolopyrimiding moiety yielded a keto/alcohol‐related metabolite. Several mono‐ and dihydroxylated metabolites and their glucuronide conjugates were also detected.
Vemurafenib
Authors: Du Jiangbo
Published: October 2014

Vemurafenib is a B‐Raf enzyme inhibitor for the treatment of late‐stage melanoma. After oral administration of [
14
C]‐vemurafenib to humans, approximately 94% of the total radioactivity was recovered in feces and <1% was recovered in urine. Unchanged parent was the major circulating component detected. Circulating metabolites detected were a hydroxylated metabolite, a glucuronide metabolite, and a glucoside metabolite. These metabolites combined to represent <5% of the total chromatographic radioactivity. They were also present in urine and feces. During the initial 96‐h postdosing, each metabolite accounted for <0.5% of the administered dose in urine and <6% in feces.
Alprazolam
Authors: Ding Juefang
Published: October 2014

Alprazolam is a triazolobenzodiazepine used to treat generalized anxiety disorder, panic disorder, and depressive symptoms. The in vivo metabolism of [
14
C]‐alprazolam was studied in humans after a single oral dose of 2 mg. Urine samples were examined after hydrolysis with β‐glucuronidase. Alprazolam and its metabolites were excreted primarily in urine, as 94% of the dose was excreted in urine in 72 h. Among the recovered drug and metabolites in urine, 20% was alprazolam, 17% was hydroxymethyl alprazolam, and 17% was a benzophenone metabolite (by opening of the benzodiazepine ring). Other minor metabolites identified were 4‐hydroxyalprazolam, α,4‐dihydroxyalprazolam, and 1‐demethylated alprazolam. Alprazolam and its metabolites were excreted in urine mostly as glucuronide conjugates.
Methadone
Authors: Yu Jinghua
Published: October 2014

Methadone is an analgesic drug used to reduce severe chronic pain. It is also used to treat drug addiction. The metabolism of methadone was investigated in three male human subjects who had long history of multidrug abuse following a daily 80–100 mg of methadone dose. N‐Demethylation, hydroxylation, and glucuronide conjugation were the major metabolic pathway observed. Major metabolites detected in urine were 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine and its isomer, whereas glucuronide conjugates, 1,5‐dimethyl‐3,3‐diphenyl‐2‐pyrrolidone, 4‐dimethylamino‐2,2‐diphenylvaleriacid, and normethadol were identified as minor metabolites.
Tamoxifen
Authors: Meng Jian
Published: October 2014

Tamoxifen is an antagonist of the estrogen receptor in breast tissue via its active metabolite, hydroxytamoxifen. It is used in endocrine therapy for hormone receptor‐positive breast cancer in premenopausal women. The metabolism of [
14
C]‐tamoxifen was investigated in four women after oral drug administration. Most of the radioactivity was slowly excreted in feces, only a small amount was detected in urine. The majority of the tamoxifen‐related materials in feces were conjugates. Unchanged drug and hydroxylated metabolites accounted for <30% of the fecal radioactivity. Tamoxifen was extensively metabolized. Primary metabolic reactions were N‐demethylation, oxidative deamination, O‐dealkylation, N‐oxidation, hydroxylation, and O‐glucuronidation of phase I metabolites.
Hyperglycemia‐ and neuropathy‐induced changes in mitochondria within sensory nerves
Authors: Hamid Hussein S. , Mervak Colin M. , Münch Alexandra E. , Robell Nicholas J. , Hayes John M. , Porzio Michael T. , Singleton J. Robinson , Smith A. Gordon , Feldman Eva L. , Lentz Stephen I.
Published: October 2014

Objective

This study focused on altered mitochondrial dynamics as a potential mechanism for diabetic peripheral neuropathy (DPN). We employed both an in vitro sensory neuron model and an in situ analysis of human intraepidermal nerve fibers (IENFs) from cutaneous biopsies to measure alterations in the size distribution of mitochondria as a result of hyperglycemia and diabetes, respectively.

Methods

Neurite‐ and nerve‐specific mitochondrial signals within cultured rodent sensory neurons and human IENFs were measured by employing a three‐dimensional visualization and quantification technique. Skin biopsies from distal thigh (DT) and distal leg (DL) were analyzed from three groups of patients; patients with diabetes and no DPN, patients with diabetes and confirmed DPN, and healthy controls.

Results

This analysis demonstrated an increase in mitochondria distributed within the neurites of cultured sensory neurons exposed to hyperglycemic conditions. Similar changes were observed within IENFs of the DT in DPN patients compared to controls. This change was represented by a significant shift in the size frequency distribution of mitochondria toward larger mitochondria volumes within DT nerves of DPN patients. There was a length‐dependent difference in mitochondria within IENFs. Distal leg IENFs from control patients had a significant shift toward larger volumes of mitochondrial signal compared to DT IENFs.

Interpretation

The results of this study support the hypothesis that altered mitochondrial dynamics may contribute to DPN pathogenesis. Future studies will examine the potential mechanisms that are responsible for mitochondrial changes within IENFs and its effect on DPN pathogenesis.
Determination of β‐d‐glucan and endotoxin levels in Kampo extracts
Authors: Nakae Hajime , Takahashi Gaku , Sato Ryo , Shibata Shigehiro , Endo Shigeatsu
Published: October 2014

Aims

Kampo medicine is based on the use of established formulations combining natural extracts with no “brand‐name” products or corresponding “generic” formulation. Due to differences in manufacturing practices, products of different pharmaceutical companies may contain different concentrations of β‐d‐glucan and endotoxins. The aim of this study was to compare the concentrations of β‐d‐glucan and endotoxins in five Kampo extracts from four pharmaceutical companies.

Methods

Packages of Kampo extracts were dissolved in distilled water. β‐d‐Glucan and endotoxin concentrations were measured using high‐sensitivity kinetic turbidimetric Limulus assay.

Results

All Kampo extracts examined in this study were found to contain detectable concentrations of β‐d‐glucan and endotoxins. Significant differences in the concentration of β‐d‐glucan and endotoxins (P = 0.0024 and P = 0.0013, respectively) were observed between products of different pharmaceutical companies.

Conclusions

High β‐d‐glucan and endotoxin contents were detected in Kampo extracts, with a large variability in the concentrations of both β‐d‐glucan and endotoxins among extracts from different pharmaceutical companies.
Preservation of electroencephalographic organization in patients with impaired consciousness and imaging‐based evidence of command‐following
Authors: Forgacs Peter B. , Conte Mary M. , Fridman Esteban A. , Voss Henning U. , Victor Jonathan D. , Schiff Nicholas D.
Published: October 2014

Objective

Standard clinical characterization of patients with disorders of consciousness (DOC) relies on observation of motor output and may therefore lead to the misdiagnosis of vegetative state or minimally conscious state in patients with preserved cognition. We used conventional electroencephalographic (EEG) measures to assess a cohort of DOC patients with and without functional magnetic resonance imaging (fMRI)‐based evidence of command‐following, and correlated the findings with standard clinical behavioral evaluation and brain metabolic activity.

Methods

We enrolled 44 patients with severe brain injury. Behavioral diagnosis was established using standardized clinical assessments. Long‐term EEG recordings were analyzed to determine wakeful background organization and presence of elements of sleep architecture. A subset of patients had fMRI testing of command‐following using motor imagery paradigms (26 patients) and resting brain metabolism measurement using 18fluorodeoxyglucose positron emission tomography (31 patients).

Results

All 4 patients with fMRI evidence of covert command‐following consistently demonstrated well‐organized EEG background during wakefulness, spindling activity during sleep, and relative preservation of cortical metabolic activity. In the entire cohort, EEG organization and overall brain metabolism showed no significant association with bedside behavioral testing, except in a few cases when EEG was severely abnormal.

Interpretation

These findings suggest that conventional EEG is a simple strategy that complements behavioral and imaging characterization of DOC patients. Preservation of specific EEG features may be used to assess the likelihood of unrecognized cognitive abilities in severely brain‐injured patients with very limited or no motor responses. Ann Neurol 2014;76:869–879