- Synthesis and preliminary binding studies of 4,4-ditritio(-)nicotine of high specific activity
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4,4-Ditritio-(-)nicotine (5) of high specific activity (4.7 Ci/mmol) has been synthesized from (-) nicotine via the readily prepared 4,4-dibromocotinine. Scatchard analysis of the binding of 5 to the crude mitochondrial fraction of whole rat brain revealed a K(a) of 4.7 x 106 M-1 and 13 fmol of binding sites/mg of protein.
- Vincek,Martin,Aceto,Bowman
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- Effect of CYP2A6 genetic polymorphism on the metabolic conversion of tegafur to 5-fluorouracil and its enantioselectivity
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Tegafur (FT), a prodrug of 5-fluorouracil, is a chiral molecule, a racemate of R- and S-isomers, and CYP2A6 plays an important role in the enantioselective metabolism of FT in human liver microsomes (R-FT ? S-FT). This study examined the enantioselective metabolism of FT by microsomes prepared from Sf9 cells expressing wild-type CYP2A6 and its variants (CYP2A6*7,*8, *10, and*11) that are highly prevalent in the Asian population. We also investigated the metabolism of coumarin and nicotine, both CYP2A6 probe drugs, in these variants. Enzyme kinetic analyses showed that CYP2A6.7 (I471T) and CYP2A6.10 (I471T and R485L) had markedly lower Vmax values for both enantiomers than wild-type enzyme (CYP2A6.1) and other variant enzymes, whereas Km values were higher in most of the variant enzymes for both enantiomers than CYP2A6.1. The ratios of Vmax and Km values for R-FT to corresponding values for S-FT (R/S ratio) were similar among enzymes, indicating little difference in enantioselectivity among the wild-type and variant enzymes. Similarly, both CYP2A6.7 and CYP2A6.10 had markedly lower Vmax values for coumarin 7-hydroxylase and nicotine C-oxidase activities than CYP2A6.1 and other variant enzymes, whereas Km values were higher in most of the variant enzymes for both activities than CYP2A6.1. In conclusion, the amino acid substitutions in CYP2A6 variants generally resulted in lower affinity for substrates, while Vmax values were selectively reduced in CYP2A6.7 and CYP2A6.10. Consistent R/S ratios among CYP2A6.1 and variant enzymes indicated that the amino acid substitutions had little effect on enantioselectivity in the metabolism of FT. Copyright
- Yamamiya, Ikuo,Yoshisue, Kunihiro,Ishii, Yuji,Yamada, Hideyuki,Chiba, Masato
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- Nicotine metabolism and urinary elimination in mouse: In vitro and in vivo
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This study aimed at elucidating the in vivo metabolism of nicotine both with and without inhibitors of nicotine metabolism. Second, the role of mouse CYP2A5 in nicotine oxidation in vitro was studied as such information is needed to assess whether the mouse is a suitable model for studying chemical inhibitors of the human CYP2A6. The oxidation of nicotine to cotinine was measured and the ability of various inhibitors to modify this reaction was determined. Nicotine and various inhibitors were co-administered to CD2F1 mice, and nicotine and urinary levels of nicotine and four metabolites were determined. In mouse liver microsomes anti-CYP2A5 antibody and known chemical inhibitors of the CYP2A5 enzyme blocked cotinine formation by 85-100%, depending on the pre-treatment of the mice. The amount of trans-3-hydroxycotine was five times higher than cotinine N-oxide, and ten times higher than nicotine N-1-oxide and cotinine. Methoxsalen, an irreversible inhibitor of CYP2A5, significantly reduced the metabolic elimination of nicotine in vivo, but the reversible inhibitors had no effect. It is concluded that the metabolism of nicotine in mouse is very similar to that in man and, therefore, that the mouse is a suitable model for testing novel chemical inhibitors of human CYP2A6.
- Raunio,Pokela,Puhakainen,Rahnasto,Mauriala,Auriola,Juvonen
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- Functional characterization of 34 CYP2A6 allelic variants by assessment of nicotine C-oxidation and coumarin 7-hydroxylation activities
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CYP2A6, amember of the cytochrome P450 (P450) family, is one of the enzymes responsible for the metabolism of therapeutic drugs and such tobacco components as nicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and N-nitrosodiethylamine. Genetic polymorphisms in CYP2A6 are associated with individual variation in smoking behavior, drug toxicities, and the risk of developing several cancers. In this study, we conducted an in vitro analysis of 34 allelic variants of CYP2A6 using nicotine and coumarin as representative CYP2A6 substrates. These variant CYP2A6 proteins were heterologously expressed in 293FT cells, and their enzymatic activitieswere assessed on the basis of nicotine C-oxidation and coumarin 7-hydroxylation activities. Among the 34 CYP2A6 variants, CYP2A6.2, CYP2A6.5, CYP2A6.6, CYP2A6.10, CYP2A6.26, CYP2A6.36, and CYP2A6.37 exhibited no enzymatic activity, whereas 14 other variants exhibited markedly reduced activity toward both nicotine and coumarin. These comprehensive in vitro findings may provide useful insight into individual differences in smoking behavior, drug efficacy, and cancer susceptibility.
- Hosono, Hiroki,Kumondai, Masaki,Maekawa, Masamitsu,Yamaguchi, Hiroaki,Mano, Nariyasu,Oda, Akifumi,Hirasawa, Noriyasu,Hiratsuka, Masahiro
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- Roles of CYP2A6 and CYP2B6 in nicotine C-oxidation by human liver microsomes
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Nicotine C-oxidation by recombinant human cytochrome P450 (P450 or CYP) enzymes and by human liver microsomes was investigated using a convenient high-performance liquid chromatographic method. Experiments with recombinant human P450 enzymes in baculovirus systems, which co-express human nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH)-P450 reductase, revealed that CYP2A6 had the highest nicotine C-oxidation activities followed by CYP2B6 and CYP2D6; the K(m) values by these three P450 enzymes were determined to be 11.0, 105, and 132 μM, respectively, and the V(max) values to be 11.0, 8.2, and 8.6 nmol/min per nmol P450, respectively. CYP2E1, 2C19, 1A2, 2C8, 3A4, 2C9, and 1A1 catalysed nicotine C-oxidation only at high (500 μM) substrate concentration. CyP1B1, 2C18, 3A5, and 4A11 had no measurable activities even at 500 μM nicotine. In liver microsomes of 16 human samples, nicotine C-oxidation activities were correlated with CYP2A6 contents at 10 μM substrate concentration, whereas such correlation coefficients were decreased when the substrate concentration was increased to 500 μM. Contribution of CYP2B6 (as well as CYP2A6) was demonstrated by experiments with the effects of orphenadrine (and also coumarin and anti- CYP2A6) on the nicotine C-oxidation activities by human liver microsomes at 500 μM nicotine. CYP2D6 was found to have minor roles since quinidine did not inhibit microsomal nicotine C-oxidation at both 10 and 500 μM substrate concentrations. These results support the view that CYP2A6 has major roles for nicotine C-oxidation at lower substrate concentration and both CYP2A6 and 2B6 play roles at higher substrate concentrations in human liver microsomes.
- Yamazaki, Hiroshi,Inoue, Kiyoshi,Hashimoto, Masafumi,Shimada, Tsutomu
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- Interconversion of nicotine enantiomers during heating and implications for smoke from combustible cigarettes, heated tobacco products, and electronic cigarettes
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Physiological properties of (R)-nicotine have differences compared with (S)-nicotine, and the subject of (S)- and (R)-nicotine ratio in smoking or vaping related items is of considerable interest. A Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS) method for the analysis of (S)- and (R)-nicotine has been developed and applied to samples of nicotine from different sources, nicotine pyrolyzates, several types of tobacco, smoke from combustible cigarettes, smoke from heated tobacco products, e-liquids, and particulate matter obtained from e-cigarettes aerosol. The separation was achieved on a Chiracel OJ-3 column, 250 × 4.6 mm with 3-μm particles using a nonaqueous mobile phase. The detection was performed using atmospheric pressure chemical ionization (APCI) in positive mode. The only transition measured for the analysis of nicotine was 163.1 → 84.0. The method has been summarily validated. For the analysis, the samples of tobacco and smoke from combustible cigarettes were subject to a cleanup procedure using solid phase extraction (SPE). It was demonstrated that nicotine upon heating above 450°C for several minutes starts decomposing, and some formation of (R)-enantiomer from a sample of 99% (S)-nicotine is observed. An analogous process takes place when a 99% (R)-nicotine is heated and forms low levels of (S)-nicotine. This interconversion has the effect of slightly increasing the content of (R)-nicotine in smoke compared with the level in tobacco for combustible cigarettes and for heated tobacco products. The (S)/(R) ratio of nicotine enantiomers in e-liquids was identical with the ratio for the particulate phase of aerosols generated by e-cigarette vaping.
- Moldoveanu, Serban C.
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p. 667 - 677
(2022/02/02)
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- Method for preparing bioactive (S)-(-)-nicotine
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The invention relates to the field of organic synthesis, and discloses a method for preparing bioactive (S)-(-)-nicotine. The method comprises the steps of carrying out first reaction on methyl nicotinate and tert-butyl succinic acid diester, and then carrying out second reaction; and carrying out contact reaction on the system after the second reaction and an acidic material to obtain 4-oxo-4-(3-pyridyl) butyric acid; carrying out asymmetric reduction reaction on 4-oxo-4-(3-pyridyl) butyric acid and (R)-(+)-2-methyl-CBS-oxazoborane to obtain 5-(3-pyridyl) dihydrofuran-2 (3H)-ketone; carrying out third reaction on the 5-(3-pyridyl) dihydrofuran-2 (3H)-ketone and methylamine hydrobromide to obtain 1-methyl-5-(3-pyridyl)-2-pyrrolidone; and carrying out fourth reaction on the 1-methyl-5-(3-pyridyl)-2-pyrrolidone and a reducing agent to obtain the bioactive (S)-(-)-nicotine. According to the method, the bioactive body (S)-(-)-nicotine can be obtained with high yield and high purity.
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- Preparation method of artificially synthesized nicotine
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The invention discloses a preparation method of artificially synthesized nicotine, and belongs to the technical field of chemical synthesis. According to the synthesis method of a racemate (+/-)-(R,S)-nicotine and a natural optical active enantiomer (-)-(S)-nicotine, nicotinate and diester of succinic acid (or N-alkyl succinimide) are taken as the primary raw materials; and defects of a conventional nicotine synthesis technology such as difficulty for massive production, high cost, and the like, are overcome. Specifically, the provided synthesis method has the advantages that the primary raw materials are easily available, the preparation technology is simple, the cost is low, the prepared nicotine does not contain any other harmful tobacco compound, and the preparation method is suitable for industrial large-scale production.
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- Evaluation of the Edman degradation product of vancomycin bonded to core-shell particles as a new HPLC chiral stationary phase
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A modified macrocyclic glycopeptide-based chiral stationary phase (CSP), prepared via Edman degradation of vancomycin, was evaluated as a chiral selector for the first time. Its applicability was compared with other macrocyclic glycopeptide-based CSPs: TeicoShell and VancoShell. In addition, another modified macrocyclic glycopeptide-based CSP, NicoShell, was further examined. Initial evaluation was focused on the complementary behavior with these glycopeptides. A screening procedure was used based on previous work for the enantiomeric separation of 50 chiral compounds including amino acids, pesticides, stimulants, and a variety of pharmaceuticals. Fast and efficient chiral separations resulted by using superficially porous (core-shell) particle supports. Overall, the vancomycin Edman degradation product (EDP) resembled TeicoShell with high enantioselectivity for acidic compounds in the polar ionic mode. The simultaneous enantiomeric separation of 5 racemic profens using liquid chromatography-mass spectrometry with EDP was performed in approximately 3?minutes. Other highlights include simultaneous liquid chromatography separations of rac-amphetamine and rac-methamphetamine with VancoShell, rac-pseudoephedrine and rac-ephedrine with NicoShell, and rac-dichlorprop and rac-haloxyfop with TeicoShell.
- Hellinghausen, Garrett,Lopez, Diego A.,Lee, Jauh T.,Wang, Yadi,Weatherly, Choyce A.,Portillo, Abiud E.,Berthod, Alain,Armstrong, Daniel W.
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p. 1067 - 1078
(2018/08/01)
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- The novel assay method for nicotine metabolism to cotinine using high performance liquid chromatography
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Nicotine is the primary psychoactive component in tobacco. It is taken into the body by tobacco smoking, and mainly metabolized to cotinine in the hepatic cytochrme P450 (CYP) 2A6. The objective of this study was to develop a sensitive method for the determination of nicotine metabolism to cotinine using HPLC. The internal standard, trans-4′-carboxycotinine methyl ester was synthesized with a simple method. The nicotine and cotinine were separated completely and detected by C18 5- μm analytical column (L-column Octa decyl silyl (ODS), 150mmx4.6 mm i.d.) equipped with a C18 5- μm guard column (L-column ODS, 10mmx4.6 mm i.d.) and ultraviolet detection at 260 nm. The detection limit of the assay was 0.05 μM for cotinine (n=5, R.S.D) and 0.1 μM for nicotine. Thus the present results provided a sensitive and useful method for the determination of nicotine metabolism catalyzed by CYP2A6.
- Miyazawa, Mitsuo,Kawauchi, Yumi,Okuno, Yoshiharu,Oda, Yoshimitsu
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experimental part
p. 295 - 297
(2011/05/02)
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- Enamine trapping with the oxidation of nicotine and analogues
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Various N-tertiary pyrrolidine derivatives 1 and 6-9 bearing voluminous substituents in 2-position react with Hg(II)-EDTA under double dehydrogenation and an atypical direction to the secondary C-5 with formation of the corresponding lactams 2 and 12-15. The primary products of a two electron withdrawal cannot be isolated. By addition of substituted benzaldehydes b-d as trapping reagents to the Hg(II)-EDTA solution additionally are received condensation products of the type of benzylidene lactams 1b-d, 6b,c and 7b-9b. Here the total yield of isolated products increases, while simultaneously the yield of unsubstituted lactams of the type 2 and 12-15 decreases. Therefore the atypical direction of dehydrogenation with the trapping aldehydes is the same as with the lactam forming pyrrolidines. Additionally the detection of the enamine species in the first stage of dehydrogenation with following reaction to the condensation product is secured. The (E)-configuration of 1b is confirmed by the chemical shift of the proton a-H and the 19F/13C-coupling constant of C-4 by direct comparison with its (Z)-isomer 27. The similar signal dates of the enone protons in the other prepared benzylidene lactams 1c,d, as well as 6b,c and 7b-9b verify also the (E)-configuration.
- Moehrle,Berlitz
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scheme or table
p. 219 - 226
(2010/08/05)
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- Enantioselective syntheses of 2-substituted pyrrolidines from allylamines by domino hydroformylation-condensation: Short syntheses of (S)-nicotine and the alkaloid 225C
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Short routes to chiral 2-substituted pyrrolidines based on rhodium-catalyzed hydroformylations of allylamines and their N-alkyl and N-acyl derivatives, which were prepared by asymmetric allylic substitutions, are described. The outcome of the hydroformyla
- Dübon, Pierre,Farwick, Andreas,Helmchen, Günter
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scheme or table
p. 1413 - 1416
(2009/10/23)
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- Hapten-carrier conjugates for use in drug-abuse therapy and methods for preparation of same
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Hapten-carrier conjugates capable of eliciting anti-hapten antibodies in vivo are disclosed. Methods of preparing the hapten-carrier conjugates and therapeutic compositions are also disclosed. Where the hapten is a drug of abuse, a therapeutic composition containing the hapten-carrier conjugate is particularly useful in the treatment of drug addiction, more particularly, cocaine addiction. Passive immunization using antibodies raised against conjugates of the instant invention is also disclosed. The therapeutic composition is suitable for co-therapy with other conventional drugs.
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- The biosynthesis of [5'-14C]cotinine and other radiolabeled nicotine metabolites
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The present study describes the biosynthesis and isolation of the major radiolabeled nicotine metabolites formed using phenobarbitone (PB)-induced rabbit hepatic homogenates (10,000 g fraction). The optimal incubation and extraction methods for cotinine formation from non-labeled nicotine were established. The biosynthesis and isolation of [5'-14C]cotinine and other radiolabeled metabolites such as [2'-14C]nornicotine and [4-14C]-(3-pyridyl)-4-oxobutyric acid, from commercially available [2'-14C]nicotine, were carried out using the developed methods. Cotinine was isolated using preparative silica gel TLC, whereas the other metabolites were obtained using a cation-exchange HPLC method. This study showed that in addition to the two major metabolites (i.e. cotinine and nornicotine), 4-(3-pyridyl)-4-oxo-butyric acid, 3-hydroxycotinine, norcotinine, nicotine-1'-N-oxide and cotinine-1-N-oxide were also formed when PB-induced rabbit hepatic homogenates were used. Two further metabolites of unknown structure were detected. However, the isolation and further purification were only carried out on cotinine, nornicotine and 4-(3-pyridyl)-4-oxo-butyric acid.
- Tsai, Mui-Chiung,Sai, Yang,Li, Yan,Aislaitner, George,Gorrod, John W.
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p. 387 - 407
(2007/10/03)
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- Kinetic resolution in the oxidation of iminium ion to lactam catalysed by aldehyde oxidase.
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A rabbit liver enzyme preparation oxidised racemic iminium ions 1 and 2 to optically active lactams 3 and 4 with enantiomer ratios ER/S = 5.5 and 0.14, respectively.
- Bielawski,Brand?nge,Rodriguez
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p. 198 - 201
(2007/10/02)
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