Elucidation of individual cytochrome P450 enzymes involved in the metabolism of clozapine

The atypical antipsychotic clozapine has been reported to be metabolised mainly to its N-oxide and N-demethylated products. In the present study, individual recombinant cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO) enzymes were used to elucidate which enzymes are responsible for these metabolic con-versions. In vitro metabolism of clozapine was investigated using human CYP1A1, CYP1A2, CYP2C8, CYP2EI, CYP2C9-arg₁₄₄, CYP2C9-cys₁₄₄, CYP2C19, CYP2D6, CYP3A4, CYP3A5 and FMO3 supplemented with an NADPH generating system. Clozapine and its N-oxidation and N-demethylation metabolites were determined by an HPLC method with a Hypersil CN column and a UV detector. Of the enzymes investigated, CYP1A2, CYP3A4, CYP2D6, CYP2C8, CYP2C19 and, to a lesser extent, CYP2C9-cys, CYP2C9-arg and CYP3A5 were apparently involved in N-demethylation, while CYP1A2, CYP3A4, FMO3 and, to a lesser extent, CYP2C8, CYP2C19 and CYP3A5 were found to catalyse the formation of clozapine N-oxide. A bank of 16 human liver microsome preparations was investigated for ability to catalyze the production of clozapine N-oxide and N-desmethylclozapine. Attempts were made to correlate the rates of formation of these metabolites of clozapine to previously determined catalytic activities of CYP1A2, CYP2C19, CYP2D6, CYP2E1 and CYP3A4. At a clozapine concentration of 20 μM, the rate of formation of clozapine N-oxide showed significant correlations with activities of CYP3A4 (P<0.01) and CYP1A2 (P<0.05). The formation of N-desmethylclozapine exhibited significant correlations with CYP1A2 (P<0.01) and CYP3A4 (P<0.01). Similar correlations were observed when the clozapine concentration was increased to 300 μM except that the formation of clozapine N-oxide no longer correlated with CYP1A2 activity. It can be seen from these results that although some recombinant enzymes individually are capable of metabolising clozapine, the activities of several of these enzymes did not correlate with clozapine metabolism when mixtures of the enzymes are used. By combining the results of the current study and those reported in the literature, it is proposed that CYP3A4 and FMO3 are primarily responsible for the production of clozapine N-oxide, and CYP3A4 and CYP1A2 are primarily responsible for the formation of N-desmethylclozapine. The present study demonstrates the importance of the use of multiple techniques for the elucidation of the enzymes involved in the metabolism of certain drugs. Received: 2 December 1997 / Accepted: 15 June 1998     

用2H-标记物和GC-MS分离鉴定联苯双酯在大鼠尿中的一个代谢产物

Dimethyl-4, 4′-dimethoxy-5, 6, 5′, 6′-dimethylenedioxybiphenyl-2, 2′-dicarboxylate (biphenyldimethyldicarboxylate; BDD), a synthetic compound, has been used in the treatment of chronic hepatitis with good results in reducing s-GPT. Previous work in our laboratory studied its metabolites using ³H-labeled compound in combination with TLC and found that its main methabolic pathway is demethylation followed by conjugation with glucuronic acid. This paper reports the isolation and identification of a metabolite of BDD from rat urine using ²H-labeled compound and GC-MS. Rats fasted for 12h were intragastrically given a mixture of ²H-labeled (consisting of monodeutero-and dideutero-BDD in the ratio about 1:1.3)and non-labeled BDD 150mg/kg and placed in metabolism cages for urine collection. The 24h urine was filtered and extracted three times each with 5ml of methylenedichloride. The extracts were pooled and evaporated to dryness under reduced pressure at 35℃. The residue was redissolved in chloroform and subjected to GC-MS analysis. The mass spectrum (m/z: 404, 405, 406; 373, 374, 375; 345, 346, 347; 330, 331, 332; etc)indicates that the molecular ionic and fragment peaks of the metabolite all have 14 amu less than those of BDD. This means that the metabolite isolated is mono-O-demethylated BDD. The result confirmed our findings reported previously.     

Influence of renal failure,rheumatoid arthritis and old age on the pharmacokinetics of diflunisal

Summary The single-dose plasma kinetics of diflunisal was studied in healthy young and old subjects, in patients with rheumatoid arthritis, and in patients with renal failure. The plasma and urine kinetics of the glucuronidated metabolites of diflunisal were studied in the healthy elderly subjects and in the patients with renal failure. In addition, the multiple-dose plasma kinetics of diflunisal was assessed in healthy volunteers and in patients with rheumatoid arthritis. After a single dose of diflunisal the terminal plasma half-life, mean residence time and apparent volume of distribution were higher in elderly subjects than in young adults. No difference was observed in any pharmacokinetic parameter between age-matched healthy subjects and patients with rheumatoid arthritis. The elimination half-life of unchanged diflunisal was correlated with the creatinine clearance (r=+0.89) and its apparent total body clearance exhibited linear dependence on creatinine clearance (r=+0.78). In patients with renal failure, the terminal plasma half-life and mean residence time of diflunisal were prolonged. The renal and apparent total body clearances were lower, the mean apparent volume of distribution was higher and the mean area under the concentration-time curve extrapolated to infinity (AUC) was greater in the renal failure patients than in controls. The plasma concentration of the glucuronidated metabolites rapidly rose to levels above those of unchanged drug in renal patients, whereas they were lower than those of unchanged diflunisal in controls. The AUC (0–96 h) of diflunisal glucuronides in the patients was four-times that in controls, and the terminal elimination half-life of the glucuronides was prolonged in them. The renal excretion and clearance of diflunisal glucuronides were reduced when renal function was impaired. After multiple dosing, the pre-dose steady-state plasma-concentration increased with decreasing creatinine clearance (r=-0.79). When the plasma concentration exceeded 200 μmol·1⁻¹, the elimination half-life was doubled, due to partial saturation of diflunisal conjugation. This finding suggests that lower doses could be used in long-term treatment. Thus, old age and arthritic disease appear to have little influence on the kinetics of diflunisal in the absence of renal functional impairment. Ordinary doses can be given for short term treatment of elderly patients with or without RA. In patients with renal failure, however, reduced doses of diflunisal are recommended.     

手性毛细管气相色谱法测定人尿中美芬妥英光学异构体含量的方法学研究

A gas chromatographic method equipped with nitrogen-phosphorus detector was developed for the determination of the S-and R-enantiomers of the anticonvulsant, mephenytoin (MP) in human urine. Dichioromethane (4 ml) was added to 1 ml urine, the mixture was shaken and centrifuged. The organic phase was transferred to another tube and blown to dryness under nitrogen on water bath (37℃). The residue was dissolved in 10 μl ethylacetate and 1～2 μl was injected into the GC. Our results showed that direct enantiomeric separation of mephenytoin was obtained by using a chiral capillary column, the retention times for S-and R-mephenytoin were 25. 5 and 26. 2 min respectively, with a detection limit less than 50 ng/ml of mephenytoin. Similar linear and reproducible standard curves were obtained over the concentration range of 53.2 to 2128. 0 ng/ml (for S-MP, r=0. 9914±0. 0070, n=6; and for R-MP, r=0. 9939±0. 0070, n=6), and the mean recoveries of S-and R-MP were 95. 4% and 95. 8% respectively. The within--day relative standard deviations were less than 8. 8% for both S-and R-MP, and that of between--days were less than 14.3%. There was a good reproducibility of the urine S/R mephenytoin determined in China and in Sweden by using similar method in 107 Chinese volunteers after a single oral dose of 100 mg racemic mephenytoin (r=0. 9091, P<0. 001).     

蝙蝠葛中的新生物碱——N-去甲基蝙蝠葛碱

A new phenolic dauricine-type alkaloid together with the know dauricine, were iso-lated from the rhizoma of Menispermum dauricum DC cultivated in Xianning district, Hubei province.Dauricine was obtained as the major alkaloid and was confirmed by comparison with authentic sample.The new alkaloid is an unstable white powder. Based on spectrometric analysis (UV, IR, FAB--MSand ¹HNMR) and N-methylation which offered clauricine dimethiodide(Ⅴ), the structure was elucidat-ed as RR, N-desmethyldauricine(Ⅱ), which was isolated for the first time from nature.     

抗心肌缺血药3-{4-[(2-羟基-3-烷氨基)丙氧基]苯基(苄基)}-取代4(3H)-喹唑酮的合成

In order to search for effective antimyocardial ischemic agents, fourteen new 3 4-[(3-alkylamino-2-hydroxy)propoxy] phenyl(benzyl)]-substituted 4(3H)-quin zolinones (Ⅱ) were synthesized. Substituted o-aminobenzoic acids used as the starting materials were allowed to react with acetic anhydride and then p-aminophenol (method A), or with N- (4- hydroxyphenyl)formamide (method B), or with thionyl chloride and then N - (4 - hydroxybenzyl) formamide (methode C) to form 3-[(4-hydroxyphenyl(benzyl)]-substituted 4(3H)-quinazolinones (Ⅲ). The intermediate Ⅲ reacted with epichiorohydrin to form the epoxides (Ⅳ). The reaction of Ⅳ with an excess of isopropylamine or tert-butylamine in boiling chloroform gave the desired products. Of all the compounds synthesized, Compounds Ⅱ_3～5 and Ⅱ₁₃ were found to increase the tolerance of mice to hypoxia. Further evaluation is in progress.     

中药秦艽中总生物碱的含量测定

A method for determining the total alkaloids of the Chinese drug Tsingjiu (秦艽) (Gentiana macrophylla) based on titration with silicotungstic acid was found. Weigh out accurately 2 g of pulverized (80 mesh) sample. Macerate it in a glass-stoppered flask with 50 ml of a mixture of chloroform-methanol-concentrated ammonia water (75: 25: 5) for 48 hours with occasional shaking. Take exactly 25 ml of the filtrate, evaporate and dissolve the residue in 0.6 N acetic acid, Impurities in the acetic acid solution are removed with lead acetate and the excess of Pb⁺² is removed by sodium sulphate. The clear filtrate is adjusted to about 50 ml with an acidity at about 0.5 N. The solution is titrated with 0.01 M silicotungstic acid, using malachite green as an outside indicator. Results are calculated on gentianine (C₁0H₉O₂N).     

Antagonism of nitrous oxide-induced anxiolytic-like behavior in the mouse light/dark exploration procedure by pharmacologic disruption of endogenous nitric oxide function

Rationale. Previous studies have shown the anxiolytic-like effects of nitrous oxide (N₂O) to be sensitive to antagonism by non-specific inhibitors of nitric oxide synthase (NOS). Objectives. The present study was conducted to demonstrate further the involvement of nitric oxide (NO) and ascertain whether a specific isoform of NOS is involved in N₂O-induced behavior in mice. Methods. Male NIH Swiss mice were tested in the light/dark exploration test to determine how N₂O-induced behavior was affected by the following pretreatments: the NO scavenger hemoglobin (Hb); the selective nNOS-inhibitor S-methyl-l-thiocitrulline (SMTC); the selective eNOS-inhibitor N(5)-(1-iminoethyl)-l-ornithine (l-NIO); and the selective iNOS-inhibitor 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT). Furthermore, NOS activity was assessed in the whole brain as well as five brain areas of N₂O- versus room air-exposed mice to determine the effects of N₂O on NOS activity. Results. The behavioral effects of N₂O in the light/dark exploration test were significantly attenuated following pretreatment with Hb (2.0 nmol, i.c.v.), SMTC (0.3 μg and 1.0 μg per mouse, i.c.v.) and the higher dose of l-NIO (30 mg/kg, s.c.). However, the N₂O-induced behavioral effect was unaltered by pretreatment with either the lower dose of l-NIO (10 mg/kg, s.c.) or AMT (1.0 mg/kg and 3.0 mg/kg, s.c.). Finally exposure to 50% N₂O for 15 min significantly increased NOS activity in the cerebellum and corpus striatum but not in other brain regions or whole brain. Conclusion. These findings provide further support for the hypothesis that NO is involved in N₂O-induced anxiolytic-like behavior and that this NO is the product of nNOS enzyme activity. Electronic Publication     

海岛轮环藤碱-N-氧化物的制备和结构

Oxidation of insularine (Ⅰ) with m-chloroperbenzoic acid yielded four insularine-N-oxides. Two of them are identical in every respect with our insularine-2β-N-oxide (Ⅱ) and insularine-2'β-N-oxide (Ⅲ), two rare examples of naturally occurring head-to-tail bisbenzyliso-quinoline N-oxides newly isolated from Cyclea sutchnenensis, which confirmed the structures of the two novel natural N-oxides. The other two are new. compounds and their structures have been established as insularine-2'α-N-oxide (Ⅳ) and insularine-2β-2'β-N, N-dioxide (Ⅴ), on the basis of spectral data (UV, IR. ¹HNMR, NOEDS AND MS) analysis.     

CYP2D6在印度卡纳塔克邦和安得拉邦人群中的遗传多态性(英文)

AIM: To study the prevalence of cytochrome P-450 2D6 (CYP2D6) polymorphism in Kamataka ( KA) and Andhra Pradesh (AP) population. METHODS: Two hundred and eleven healthy human volunteers participated in the study (100 from KA and 111 from AP). At bed time, after voiding their bladder, the volunteers ingested 30 mg of dextromethorphan hydrobromide (DM). Urine samples were collected for 8 h. DM and its metabolite dextrorphan ( DT) were estimated in the urine using HPLC. The metabolic ratio (DM/DT) was used for phenotyping. RESULTS: The prevalence of poor metabolisers (PM) in KA is 4 % and AP is 1.8 %. CONCLUSION: The frequency of PM phenotype in South Indian population is in between the Western and Oriental population.     

Identification of etazene (etodesnitazene) metabolites in human urine by LC-HRMS

Etazene (or etodesnitazene) is a novel and highly active synthetic opioid belonging to the rapidly evolving and emerging group of “nitazenes.” Etazene metabolites were identified through analysis of a human urine sample. The sample was obtained from a 25-year-old man who attempted suicide by taking a new psychoactive substances (NPS) cocktail purchased online and was analyzed by ultrahigh performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). Etazene metabolites were predicted with BioTransformer 3.0, and the exact masses were added to the inclusion list. Eight possible metabolites were identified in the urine sample. N- and O-deethylation were identified as the predominant metabolism routes, resulting in M1 (O-deethylated etazene; most abundant metabolite based on the peak area), M2 (N-deethylated etazene), and M3 (N,O-dideethylated etazene) metabolites. Less abundant hydroxylated products of these deethylated metabolites and etazene were also found. Additionally, in the analysis without β-glucuronidase treatment, M1- and M3-glucuronide phase II metabolites were found. As N- and O-deethylated products seem to be the predominant urinary metabolites, the detection of these metabolites in urine can be useful to demonstrate etazene exposure.     

Study of the in vitro and in vivo metabolism of the tryptamine 5‐MeO‐MiPT using human liver microsomes and real case samples

The synthetic tryptamine 5‐methoxy‐N‐methyl‐N‐isopropyltryptamine (5‐MeO‐MiPT) has recently been abused as a hallucinogenic drug in Germany and Switzerland. This study presents a case of 5‐MeO‐MiPT intoxication and the structural elucidation of metabolites in pooled human liver microsomes (pHLM), blood, and urine. Microsomal incubation experiments were performed using pHLM to detect and identify in vitro metabolites. In August 2016, the police encountered a naked man, agitated and with aggressive behavior on the street. Blood and urine samples were taken at the hospital and his premises were searched. The obtained blood and urine samples were analyzed for in vivo metabolites of 5‐MeO‐MiPT using liquid chromatography–high resolution tandem mass spectrometry (LC–HRMS/MS). The confiscated pills and powder samples were qualitatively analyzed using Fourier transform infrared (FTIR), gas chromatography–mass spectrometry (GC–MS), LC‐HRMS/MS, and nuclear magnetic resonance (NMR). 5‐MeO‐MiPT was identified in 2 of the seized powder samples. General unknown screening detected cocaine, cocaethylene, methylphenidate, ritalinic acid, and 5‐MeO‐MiPT in urine. Seven different in vitro phase I metabolites of 5‐MeO‐MiPT were identified. In the forensic case samples, 4 phase I metabolites could be identified in blood and 7 in urine. The 5 most abundant metabolites were formed by demethylation and hydroxylation of the parent compound. 5‐MeO‐MiPT concentrations in the blood and urine sample were found to be 160 ng/mL and 3380 ng/mL, respectively. Based on the results of this study we recommend metabolites 5‐methoxy‐N‐isopropyltryptamine (5‐MeO‐NiPT), 5‐hydroxy‐N‐methyl‐N‐isopropyltryptamine (5‐OH‐MiPT), 5‐methoxy‐N‐methyl‐N‐isopropyltryptamine‐N‐oxide (5‐MeO‐MiPT‐N‐oxide), and hydroxy‐5‐methoxy‐N‐methyl‐N‐isopropyltryptamine (OH‐5‐MeO‐MiPT) as biomarkers for the development of new methods for the detection of 5‐MeO‐MiPT consumption, as they have been present in both blood and urine samples.     

The metabolites of chlorpromazine N-oxide in rat bile

1. The metabolism of chlorpromazine N-oxide was studied in female rats after a 20?mg/kg single i.p. dose.

2. Metabolites identified in urine and faeces were chlorpromazine, 7-hydroxy-chlorpromazine, chlorpromazine sulphoxide, N-desmethylchlorpromazine and N-desmethylchlorpromazine sulphoxide. As these same five metabolites were previously shown to be present after oral administration this indicates that reduction of chlorpromazine N-oxide occurs not only in the gastrointestinal tract but also at other sites.

3. The metabolism of chlorpromazine N-oxide was studied following its administration by either i.p., i.v. or oral routes to female rats in which the bile duct was cannulated.

4. There were no qualitative differences between the three routes of administration with respect to the metabolites identified. With the exception of the absence of N-desmethylchlorpromazine and N-desmethylchlorpromazine sulphoxide, all metabolites previously identified in urine and faeces were also present in bile.

5. Additionally there were three compounds present in rat bile which were not identified in urine or faeces. These were chlorpromazine N-oxide, chlorpromazine N, S-dioxide and 7-hydroxychlorpromazine O-glucuronide. This is the first unequivocal evidence for the identification of intact 7-hydroxychlorpromazine O-glucuronide in any species.

6. The inability to detect chlorpromazine N-oxide and chlorpromazine N, S-dioxide in the faeces of rats is likely to be due to the reduction of the N-oxide group on the passage of these biliary metabolites down the intestinal tract.     

Evaluation of Dextromethorphan N‐Demethylation Activity as a Biomarker for Cytochrome P450 3A Activity in Man

Abstract: The present study evaluates the usefulness of dextromethorphan N‐demethylation activity indices to reflect cytochrome P450 (CYP) 3A activity in man. Indices of dextromethorphan N‐demethylation activity were categorized as N₁=3‐methoxymorphinan/dextromethorphan, N₂=3‐hydroxymorphinan/dextrorphan, N₃=(3‐methoxymorphinan + 3‐hydroxymorphinan)/(dextromethorphan + dextrorphan). Two mg of midazolam were administered orally to 22 Japanese male volunteers, and midazolam clearance determined. Thirty mg of dextromethorphan were also orally administered to these volunteers and N₁, N₂ and N₃ indices determined by 12 hr urine collection. Results showed N₂ and N₃ were highly correlated (r>0.99, P<0.001), and significantly correlated to oral midazolam clearance (r=0.45, P<0.05); suggesting that N₂ and N₃ are more suitable than N₁ when using dextromethorphan as an index of individual CYP3A activity.     

Inhibition of dextromethorphan metabolism by moclobemide

 This pilot study was conducted to evaluate the potential of the new antidepressant moclobemide to inhibit the cytochrome enzyme P4502D6 (CYP2D6) using the cough suppressant dextromethorphan as a substrate in four extensive metabolizers (EM) of debrisoquine. The subjects received seven oral doses of 20 mg dextromethorphan at 4-h intervals over 2 days (1 and 2) and subsequently moclobemide (300 mg b.i.d.) for 9 days. On days 10 and 11, they received seven doses of 20 mg dextromethorphan in addition to moclobemide. During monotreatment and combined treatment, blood was collected on days 2 and 11, respectively, for determination of dextromethorphan and its demethylated metabolites using automated high- performance liquid chromatography with column switching. Concurrent administration of moclobemide markedly reduced the O-demethylation of dextromethorphan, whereas the N-demethylation of dextrorphan to hydroxymorphinan was not affected. The findings indicate that moclobemide can affect the pharmacokinetics of drugs that are mainly metabolized by CYP2D6. Received: 18 November 1996 / Final version: 10 March 1997     

The fate of 4-cyano-N,N-dimethylaniline in rats; a novel involvement of glutathione in the metabolism of anilines

1. 4-Cyano-N, N-dimethylaniline (CDA), when administered to rats as a single oral dose (18.5 mg/kg), was rapidly absorbed and eliminated as a mixture of metabolites in the urine (86% dose after 24 h).

2. Residues in tissues after 48 h, expressed as μg equiv. of CDA, were: liver, 0.35; kidney, 0.28; testes, 0-08; fat, 006; bone marrow, 0.15 and blood, 0.32.

3. Absorption, metabolism and elimination following four consecutive daily doses of CDA (65 mg/kg) were similar to those at the lower dose.

4. The major metabolite was 2-amino-5-cyanophenyl sulphate with its mono- and di-N-methyl analogues as minor metabolites. 4-Cyanoaniline, its N-acetyl derivative and an unstable derivative were also found as metabolites.

5. Three sulphur-containing metabolites with methylsulphinyl-, methylsulphonyl-, and N-acetylcysteinyl-groups attached to the C2 atom of an N-acetyl group were identified, the latter accounting for 10.3% and 41% of the low and high doses, respectively.

6. The sulphur-containing metabolites indicate the involvement of glutathione in metabolism at the N-acetyl group. This implies the generation of an electrophilic intermediate, possibly the sulphate conjugate of an N-glycolylaniline, in the metabolism of CDA.     

The effect of perazine on the CYP2D6 and CYP2C19 phenotypes as measured by the dextromethorphan and mephenytoin tests in psychiatric patients

There is evidence that the antipsychotic drug perazine is an inhibitor of CYP2D6. This study aimed at evaluating its effect on CYP2D6 and CYP2C19 activities in submitting psychiatric patients to phenotyping with dextromethorphan and mephenytoin, respectively, substrates of these enzymes, before and during a treatment with perazine. A total of 31 patients were phenotyped with dextromethorphan (CYP2D6) and mephenytoin (CYP2C19) before and after a 2‐week treatment with 450 ± 51 mg/day (mean ± sd) perazine. At baseline, five patients appeared to be poor metabolizers (PM) of dextromethorphan and two patients of mephenytoin. The metabolic ratio (MR) of dextromethorphan/dextrorphan as determined in collected urine increased significantly (Wilcoxon; P < .0001) from baseline (0.39 ± 1.38 [mean ± sd]) till day 14 (1.46 ± 2.22). In 19 out of 26 extensive metabolizers (EM) of dextromethorphan, the phenotype changed from EM to PM. This suggests an almost complete inhibition of CYP2D6 by perazine and/or its metabolites. On the other hand, perazine (or some of its metabolites) did seemingly not inhibit CYP2C19. In conclusion, this study suggests that in patients treated with perazine and co‐medicated with CYP2D6 substrates, there could be an increased risk of adverse effects as a consequence of a pharmacokinetic interaction.     

A comprehensive understanding of thioTEPA metabolism in the mouse using UPLC-ESI-QTOFMS-based metabolomics

ThioTEPA, an alkylating agent with anti-tumor activity, has been used as an effective anticancer drug since the 1950s. However, a complete understanding of how its alkylating activity relates to clinical efficacy has not been achieved, the total urinary excretion of thioTEPA and its metabolites is not resolved, and the mechanism of formation of the potentially toxic metabolites S-carboxymethylcysteine (SCMC) and thiodiglycolic acid (TDGA) remains unclear. In this study, the metabolism of thioTEPA in a mouse model was comprehensively investigated using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC–ESI-QTOFMS) based-metabolomics. The nine metabolites identified in mouse urine suggest that thioTEPA underwent ring-opening, N-dechloroethylation, and conjugation reactions in vivo. SCMC and TDGA, two downstream thioTEPA metabolites, were produced from thioTEPA from two novel metabolites 1,2,3-trichloroTEPA (VII) and dechloroethyltrichloroTEPA (VIII). SCMC and TDGA excretion were increased about 4-fold and 2-fold, respectively, in urine following the thioTEPA treatment. The main mouse metabolites of thioTEPA in vivo were TEPA (II), monochloroTEPA (III) and thioTEPA-mercapturate (IV). In addition, five thioTEPA metabolites were detected in serum and all shared similar disposition. Although thioTEPA has a unique chemical structure which is not maintained in the majority of its metabolites, metabolomic analysis of its biotransformation greatly contributed to the investigation of thioTEPA metabolism in vivo, and provides useful information to understand comprehensively the pharmacological activity and potential toxicity of thioTEPA in the clinic.     

The use of 13C-nmr spectroscopy for the detection and identification of metabolites of carbon-13 labelled amitriptyline

The antidepressant drug amitriptyline and two of its metabolites, nortriptyline and desmethylnortriptyline, each containing two ¹³C atoms, have been used to determine the sensitivity and selectivity of ¹³C-nmr spectroscopy for the detection of unchanged amitriptyline and N-desmethyl metabolites in the urine of animals dosed orally with the labelled drug. The resonance signals from the ¹³C atoms detected in the ¹³C-nmr spectrum of the entire extract from a control 12 h rat urine sample to which 1 mg of each labelled compound had been added were easily detected, using an instrument accumulation time of 1 h. The ¹³C-nmr spectrum of an extract of hydrolysed urine from a dog that had received an oral dose of [¹³Q]amitriptyline (30 mg) exhibited signals that could be assigned to metabolites resulting from N-dealkylation and N-oxidation, as well as those bearing the intact amitriptyline side-chain. These assignments were confirmed by analysis of the same extract by g.c.-ms and h.p.l.c.     

The metabolism of (±)-methylephedrine in rat and man

1. Urinary metabolites of methylephedrine and their excretion after oral administration to rat and human volunteers have been studied.

2. The unchanged drug, ephedrine, norephedrine, their aromatic hydroxylated compounds and methylephedrine N-oxide were found in rat urine. The same metabolites, except the p-hydroxylated metabolites, were detected in human urine. The most abundant metabolite in rat urine was methylephedrine N-oxide, and in human urine was the unchanged drug.

3. Metabolites excreted in three days after administration of the drug to rat amounted to about 54% of the dose and those after administration to man, 70-72%.