Inhibition of antipyrine metabolite formation in rats in vivo

1. The effect of four inhibitors of cytochrome P-450-mediated drug oxidations (SKF 52SA, cimetidine, metyrapone and α-naphthoflavone) on the urinary metabolite pattern and ¹⁴CO₂ exhalation rate (CER)-time profile following [N-methyl-¹⁴C]antipyrine administration has been investigated.

2. The CER-time profiles indicated that inhibition of antipyrine metabolism was in the rank order SKF 525A >cimetidine > metyrapone >ANF.

3. The urinary metabolite patterns showed selectivity in action towards particular pathways, 3-hydroxylation being primarily decreased by SKF 525A and cimetidine, and N-demethylation by ANF. The results provide further evidence for involvement of multiple forms of cytochrome P-450 in antipyrine metabolism.     

Effect of cytochrome P450 isozyme induction and glutathione depletion on the metabolism of CS2 to TTCA in rats

 Analysis of 2-thiothiazolidine-4-carboxylic acid (TTCA), a metabolite of carbon disulfide (CS₂), is used in the biological monitoring exposure to CS₂ at work. In order to clarify the metabolic reasons for individual variation in the urinary excretion of TTCA, the latter was studied in rats pretreated with model cytochrome P450 (CYP) enzyme inducers or glutathione (GSH) depletors. Ethanol, phenobarbital (PB) or 3-methylcholanthrene (MC) did not increase 24-h TTCA output following CS₂ inhalation (50 or 500 ppm, 6 h). After oral dosing (10 mg/rat), PB had an inhibiting effect on the excretion rate of TTCA. Tissue GSH depletors phorone, L-buthionine-(RS)-sulfoximine (BSO) and diethylmaleate (DEM) decreased TTCA excretion in rats given an oral dose (10 mg/rat) of CS₂. The initial inhibition by phorone and DEM was reversed after 6 h and from 12 h onward the TTCA in urine exceeded the control level, an effect not seen with BSO. The proportion of CS₂ excreted in urine as TTCA within 24 h was 1.7% in control rats and 1% after BSO treatment, 1.3% after PB, 1.7% after acetone, 1.8% after MC, 2.0% after phorone and 2.5% after DEM treatment. The amount of TTCA in urine increased with the CS₂ dose in a non-linear fashion: 1.6 μmol (50 ppm/6 h) vs. 4.9 μmol (500 ppm/6 h), and 0.2 μmol (1 mg/kg) versus 3.6 μmol (100 mg/kg). It is concluded that induction of different cytochrome P450 isoforms and transient glutathione depletion have only minor effects on the disposition of TTCA in rats following low-level CS₂ exposure persistently low glutathione level as achieved by E.G. BSO, markedly decreased the metabolism of CS₂ to TTCA; these metabolic effectors are unlikely to have a major role in the individual variation of CS₂ metabolism in exposed workers. Received: 14 June 1994/Accepted: 25 August 1994     

Influence of renal failure on cytochrome P450 activity in hypertensive patients using a “cocktail” of antipyrine and nifedipine

Objective. Two substrates were coadministered in a “cocktail” approach to evaluate the contribution of renal failure to drug oxidation. Patients. Nineteen hypertensive patients, nine of them with chronic renal failure (CL_CR 38.9 vs 102.3 ml⋅min⁻¹⋅1.73 m⁻²), were investigated after peroral administration of a combination of antipyrine (500 mg, in capsules) and nifedipine (10 mg, in Oxcord capsules) in the morning after an overnight fast. Results. This “cocktail” approach made it possible to characterize in vivo the activities of different forms of cytochrome P₄₅₀ in a single-study protocol using the total clearance of nifedipine and clearance for production of 3-hydroxymethylantipyrine (HMA), 4-hydroxyantipyrine (OHA) and norantipyrine (NORA). With this “cocktail” approach (antipyrine plus nifedipine), we can suggest a selective effect on the activities of cytochrome P₄₅₀ forms associated with the formation of dehydronifedipine (P₄₅₀ III A₄) and of NORA in patients with mild renal failure under long-term antihypertensive therapy. Received: 12 July 1994 /Accepted in revised form: 7 October 1995     

Lack of multiple dosing effect of sertindole on the pharmacokinetics of alprazolam in healthy volunteers

 The effect of sertindole (a new selective antipsychotic compound) on the pharmacokinetic disposition of alprazolam was investigated. Fourteen subjects who completed the study received a single 1 mg dose of alprazolam without or with concomitant sertindole 12 mg daily. Coadministration of sertindole and alprazolam led to a half-hour decrease (P < 0.05) in mean T_max value (alone: 1.2 h, in combination: 0.7 h) and a 1.6-h increase in the mean t_1/2value (12.5 ± 3.2 versus 14.3 ± 3.4 h, P < 0.05) of alprazolam. The mean C_max (18.5 ± 4.9 versus 18.5 ± 4.8 ng/ml) and AUC (266 ± 68 versus 275 ± 57 ng⋅h/ml) values of alprazolam did not change statistically significantly in the presence of sertindole (P > 0.05). These pharmacokinetic changes are minor and not considered to be of clinical significance. Although both sertindole and alprazolam are substrate for CYP3A4 (cytochrome P-450 3A4), the results in this study suggest that sertindole is not an inhibitor of the metabolism of alprazolam. Received: 5 March 1997/Final version: 28 July 1997     

Human cytochromes mediating N-demethylation of fluoxetine in vitro

Biotransformation of the selective serotonin reuptake inhibitor antidepressant, fluoxetine, to its principal metabolite, norfluoxetine, was evaluated in human liver microsomes and in microsomes from transfected cell lines expressing pure human cytochromes. In human liver microsomes, formation of norfluoxetine from R,S-fluoxetine was consistent with Michaelis-Menten kinetics (mean K_m = 33 μM), with evidence of substrate inhibition at high substrate concentrations in a number of cases. The reaction was minimally inhibited by coincubation with chemical probes inhibitory for P450-2D6 (quinidine), -1A2 (furafylline, α-naphthoflavone), and -2E1 (diethyldithiocarbamate). Substantial inhibition was produced by coincubation with sulfaphenazole (K_i = 2.8 μM), an inhibitory probe for P450-2C9, and by ketoconazole (K_i = 2.5 μM) and fluvoxamine (K_i = 5.2 μM). However, ketoconazole, relatively specific for P450-3A isoforms only at low concentrations, reduced norfluoxetine formation by only 20% at 1 μM, and triacetyloleandomycin (≥ 5 μM) reduced the velocity by only 20–25%. Microsomes from cDNA-transfected human lymphoblastoid cells containing human P450-2C9 produced substantial quantities of norfluoxetine when incubated with 100 μM fluoxetine. Smaller amounts of product were produced by P450-2C19 and -2D6, but no product was produced by P450-1A2, -2E1, or 3A4. Cytochrome P450-2C9 appears to be the principal human cytochrome mediating fluoxetine N-demethylation. P450-2C19 and -3A may make a further small contribution, but P450-2D6 is unlikely to make an important contribution. Received: 23 December 1996 / Final version: 4 March 1997     

Radiosynthesis of 13N‐labeled thalidomide using no‐carrier‐added [13N]NH3

Recent studies revealed that thalidomide (1) has unique and broad pharmacological effects on multi‐targets although the application of 1 in therapy is still controversial. In this study, we synthesized nitrogen‐13‐labeled thalidomide ([¹³N]1) as a potential positron emission tomography (PET) probe using no‐carrier‐added [¹³N]NH₃ as a labeling agent. By use of an automated system, [¹³N]1 was prepared by reacting N‐phthaloylglutamic anhydride (2) with [¹³N]NH₃, following by cyclization with carbonyldiimidazole in a radiochemical yield of 56±12% (based on [¹¹N]NH₃, corrected for decay) and specific activity of 49±24 GBq/µmol at the end of synthesis (EOS). At EOS, 570–780 MBq (n=7) of [¹³N]1 was obtained at a beam current of 15 µA after 15 min proton bombardment with a synthesis time of 14 min from the end of bombardment. Using a small animal PET scanner, preliminary biodistribution of [¹³N]1 in mice was examined. Copyright © 2010 John Wiley & Sons, Ltd.     

Indium selectively increases the cytochrome P-450 dependent O-dealkylation of coumarin derivatives in male mice

Summary Indium pretreatment of rats and mice has been reported to decrease the concentration of cytochrome P-450, thereby reducing the activity of some cytochrome P-450 dependent enzymatic reactions. The present study reveals that pretreatment of C57B1/6JHan mice of both sexes with one s. c. dose of 120 mg of In₂(SO₄)₃ · 5 H₂O per kg of body weight decreases the concentration of cytochrome P-450 to about 65% of control levels.Neither cytochrome b₅ nor NADPH-cytochrome P-450 reductase is affected. Hepatic microsomal ethoxyresorufin O-deethylase activity declines to about 75% of control values. In contrast, with coumarin substrates, a sex dependence in the direction of change is observed: in female mice indium decreases the activity to about 75%, whereas in males it enhances the activity to 140%. Moreover, with 7-(methoxy-¹⁴C)coumarin as substrate, indium-pretreated male mice exhale about 180% and females about 65% of ¹⁴C0₂ compared to the corresponding controls. A close correlation between the in vivo and in vitro effects of indium on the metabolism of the coumarin derivatives is suggested. After isolation and purification of cytochrome P-450, SDS-PAGE indicates in indium-pretreated male mice an intensification of a 48.5 kDa protein band which is decreased in females. Immunological studies using antibodies raised against control female cytochrome P-450 show cross reactivity among all microsomes used in these experiments. High percentages of inhibition occur in microsomes with high molecular activity towards coumarin derivatives. The in vitro kinetics of antibody-inhibited O-deethylation of 7-ethoxycoumarin seems to obey a non- or partial-competitive type of inhibition. Indium pretreatment of mice produces sex-dependent effects on the metabolism of coumarin derivatives.Abbreviations 7-EtOC 7-ethoxycoumarin - CER ¹⁴C0₂ exhalation rate Send offprint requests to K. J. Netter at the above address     

Syntheses of [2H3, 15N], [14C]Nexavar™ and its labeled metabolites

Nexavar?, Sorafenib tosylate (BAY 43‐9006 tosylate) is a potent small molecule Raf kinase inhibitor for the treatment of hyperproliferative disorders such as cancer. Both radiolabeled and stable isotope labeled compounds were required for drug absorption, distribution, metabolism and excretion (ADME) and quantitative mass spectrometry bio‐analytical studies. Nexavar? labeled with carbon‐14 in the carboxamide group was prepared in two steps in an overall radiochemical yield of 42% starting from 4‐chloro‐N‐methyl‐2‐pyridine‐[¹⁴C]carboxamide. The [²H₃,¹⁵N] version of Nexavar? was prepared in 75% yield based on 4‐chloro‐N‐[²H₃]methyl‐2‐pyridine‐[¹⁵N]carboxamide. The pyridine N‐oxide metabolite labeled with carbon‐14 as well as with deuterium and nitrogen‐15 and was synthesized by oxidation in yields of 59% and 87%, respectively. Starting from [²H₂, ¹³C]formaldehyde the N‐hydroxymethyl metabolite was labeled with carbon‐13 and deuterium in one step in a 45% overall yield. Copyright © 2006 John Wiley & Sons, Ltd.     

Assessment of the cytochrome P-448 dependent liver enzyme system by a caffeine breath test

Summary [1-Methyl-¹⁴C], [3-Methyl-¹⁴C] and [7-Methyl-¹⁴C] caffeine were used to investigate demethylation in control rats, and in rats pretreated with phenobarbital or 3-methylcholanthrene, by a¹⁴CO₂-exhalation test. Compared to controls, pretreatment with phenobarbital did not enhance demethylation of any of the labelled caffeines. In contrast, induction by 3-methylcholanthrene, presumably of cytochrome P-448, resulted in highly significant increases in peak¹⁴CO₂ exhalation rates,¹⁴CO₂ disappearance constants and areas under the exhalation rate — time curves. Based on these results, [7-methyl-¹⁴C] and [3-methyl-¹⁴C] caffeine were chosen for assessing the feasibility of a caffeine breath test in man, using 5 normal volunteers and 2 patients with compensated liver cirrhosis.¹⁴CO₂ exhalation curves in cirrhotics were clearly different from those in normal volunteers, being characterised by a slower rise and a lower specific activity of exhaled¹⁴CO₂. Since the variability of the levels of the specific activity in subjects with normal livers suggested the influence of extraneous factors, a second group of normal volunteers, smokers and nonsmokers, was investigated. With either labels, the average¹⁴CO₂ exhalation rate was doubled in smokers. From these studies in rats and preliminary results in man it is concluded that specifically labelled caffeine is a suitable and promising substrate for studying demethylation by breath analysis. Presumably, caffeine represents a safe and sensitive indicator of the activity of the cytochrome P-448 system.Supported by the Swiss National Science FoundationH. Wietholtz was a visiting fellow supported by the Deutsche Forschungsgemeinschaft     

Expeditious syntheses of stable and radioactive isotope‐labeled anticonvulsant agent,JNJ‐26990990, and its metabolites

Syntheses of stable and radioactive isotope‐labeled anticonvulsant agent, JNJ‐26990990, that is, N‐(benzo[b]thien‐3‐ylmethyl)‐sulfamide and its metabolites are described. [¹³C¹⁵N]Benzo[b]thiophene‐3‐carbonitrile was first prepared by coupling of 3‐bromo‐benzo[b]thiophene with [¹³C¹⁵N]‐copper cyanide. The resultant [¹³C¹⁵N]benzo[b]thiophene‐3‐carbonitrile was reduced with lithium aluminum deuteride to give [¹³CD₂¹⁵N]benzo[b]thiophen‐3‐yl‐methylamine; which was then coupled with sulfamide to afford [¹³CD₂¹⁵N]‐N‐(benzo[b]thien‐3‐ylmethyl)‐sulfamide, the stable isotope‐labeled compound with four stable isotope atoms. Direct oxidation of [¹³CD₂¹⁵N]‐N‐(benzo[b]thien‐3‐ylmethyl)‐sulfamide with hydrogen peroxide and peracetic acid gave the stable isotope‐labeled sulfoxide and sulfone metabolites. On the other hand, radioactive ¹⁴C‐labeled N‐(benzo[b]thien‐3‐ylmethyl)‐sulfamide was prepared conveniently by sequential coupling of 3‐bromo‐benzo[b]thiophene with [¹⁴C]‐copper cyanide, reduction of the carbonitrile to carboxaldehyde, and reductive amination with sulfamide.     

Heterogeneous response of hepatic mixed function oxidases to chronic phenobarbital administration.

Effects of chronic phenobarbital (PB) administration (75mg/kg, p.o. for 59 days) on certain hepatic mixed function oxidases (HMFO) were investigated in male Sprague-Dawley rats to determine whether maximal induction is maintained throughout this period or whether further alterations occur in the inductive effect. As demonstrated by measurements of ¹⁴CO₂ excretion rates in breath after a singl i.p. injection of [dimethylamine-¹⁴C]aminopyrine ([¹⁴C]aminopyrine), PB in the above dose for 7 days accelerated hepatic aminopyrine demethylation 92 per cent above control values. However, after 59 days of PB administration, ¹⁴CO₂ excretion was only 5 per cent above control values; thus, initial PB-induced enhancement of aminopyrine N-demethylation declined with time. Phenobarbital administration did not affect total (unlabeled) CO₂ output, indicating that decreased ¹⁴CO₂ excretion did not result from potential PB-induced reduction of CO₂ output. In contrast, total cytochrome P450 content and aniline hydroxylase activity remained elevated throughout the 59 days of PB treatment. Ethylmorphine N-demethylase activity and aminopyrine N-demethylase activity (using a 3.0 mM substrate concentration) remained elevated until 28 days of PB administration, after which time these activities decreased significantly (P < 0.05), finally reaching 71 and 83 per cent respectively, of peak activity. Kinetic analyses of aminopyrine N-demethylation by hepatic microsomes revealed the K_m values were unchanged from 4 to 59 days of PB administration, but that V_max increased to a maximum of 15.5 on day 28, after which it decreased to 6.60 on day 59. This pattern of change for V_max of aminopyrine N-demethylase was similar to that for enzyme activity obtained using a constant 3.0 mM concentration of aminopyrine. Although aminopyrine N-demethylase activity (using a 3.0 mM substrate concentration) peaked after 2 days of PB treatment, 7 days of PB treatment reduced this activity to 14 per cent below peak values. After 59 days of PB treatment, aminopyrine N-demethylase activity (at a 0.3 mM substrate concentration) declined to 61 per cent of the peak value attained on day 2. Another chronic PB study was performed and different time points selected to determine the reproducibility of these differential alterations of MFO activity. Results in the second study were similar to those obtained in the first study. Appreciable loss with time of the inductive effects produced by PB on aminopyrine metabolism both in vitro and in vivo suggests a heterogeneous response of hepatic MFO to PB; metabolism of aniline remained at peak inductive values throughout the 59 days of PB administration, whereas ethylmorphine metabolism declined only slightly.     

Maintenance of monooxygenase activities and detection of cytochrome P-450-mediated cytotoxicity in Mongolian gerbil hepatocyte cultures

1. Hepatocytes were isolated from untreated and phenobarbitone (PB)-treated Mongolian gerbils by lobe perfusion. Yields were approx. 20 ± 10⁶ cells/g liver and viability was 95 ± 1%.

2. PB treatment significantly increased the total cytochrome P-450 content, and the 7-ethoxycoumarin O-deethylase, p-nitrophenol hydroxylase and coumarin 7-hydroxylase activities, relative to those of untreated gerbils, measured in homogenates of freshly isolated hepatocytes.

3. After 24?h in culture the cytochrome P-450 content of hepatocyte homogenates from both untreated and PB-treated gerbils was 40–45% that of the corresponding values of freshly isolated hepatocytes. This decrease was accompanied by selective losses of cytochrome P-450-dependent enzyme activities.

4. Erythromycin and benzphetamine N-demethylase, and p-nitrophenol hydroxylase, activities were well maintained over 24?h in culture, whilst 7-ethoxycoumarin O-deethylase and coumarin 7-hydroxylase activities were poorly maintained. In general, the stability of the monooxygenase activities measured was improved by PB treatment of gerbils.

5. The toxicity of coumarin, precocene I and precocene II to gerbil hepatocyte cultures was dose-dependent. Precocene II was significantly more toxic to hepatocytes cultured from PB-treated, compared with untreated, gerbils.

6. Gerbil hepatocyte cultures would seem to be appropriate for investigating species differences in metabolism-mediated cytotoxicity.     

Gepirone and 1-(2-pyrimidinyl)-piperazine in vitro: human cytochromes mediating transformation and cytochrome inhibitory effects

Biotransformation of gepirone to its principal metabolite, 1-(2-pyrimidinyl)-piperazine (1-PP), was studied in human liver microsomes and in microsomes from cDNA-transfected human lymphoblastoid cells. Formation of 1-PP from gepirone in liver microsomes proceeded with a mean apparent K _m ranging from 335 to 677 μM. Coincubation with 1 μM ketoconazole reduced reaction velocity to less than 5% of control values at a gepirone concentration of 250 μM. Three other metabolites, presumed to be hydroxylated products, were also formed from gepirone. Formation of all three products was reduced to approximately 20% of control values by 1 μM ketoconazole; quinidine at 1 μM produced a small reduction in formation (91–94% of control) of two of the metabolites. 1-PP was formed from gepirone exclusively by pure P450-3A4 with a K _m of 849 μM; K _m values for the other metabolites were 245, 240, and 415 μM. Two of the products were also formed by P450-2D6. The results indicate that 3A4 is the principal cytochrome mediating 1-PP formation, as well as formation of the other metabolites. The properties of gepirone and 1-PP themselves as cytochrome inhibitors were tested in human liver microsomes using index reactions representing activitiy of P450-1A2, -2C9, -2C19, -2D6, -2E1 and -3A. Gepirone and 1-PP produced negligible inhibition of all these reactions. Thus gepirone at therapeutic doses in humans has a low likelihood of inhibiting P450-mediated drug metabolism involving these cytochromes. Received: 26 November 1997/Final version: 6 February 1998     

Non-response to citalopram in depressive patients: pharmacokinetic and clinical consequences of a fluvoxamine augmentation

The effect of comedication with fluvoxamine on the plasma concentrations of the enantiomers of citalopram and its metabolites in dextromethorphan/mephenytoin phenotyped patients pretreated with citalopram (CIT) was studied: seven female patients (45.1 ± 13.9 years) suffering from a major depressive episode [ICD-10: F _32.2 (n = 3 patients), F _33.2 (n = 2), F _32.10 (n = 1) or F _32.11 (n = 1)] , who were non-responders to a 3-week treatment with 40 mg/day CIT (From day-21 to day 0) (day 0: MADRS score ≥12), were comedicated for another 3 weeks with fluvoxamine (50 mg/day from day 1–7, 100 mg/day from day 14–21). All patients were extensive metabolizers of mephenytoin (CYP2C19) and dextromethorphan (CYP2D6), except one patient, who had a genetic deficiency of CYP2D6. There was a significant increase of the plasma concentrations of S- and R-citalopram from day 0 (27 ± 14 μg/l and 55 ± 23 μg/l, respectively) to day 21 (83 ± 38 μg/l and 98 ± 44 μg/l, respectively), after addition of fluvoxamine (P < 0.02, for each comparison), and the mean ratio S/R-citalopram increased from 0.48 to 0.84. S-Citalopram inhibits more potently 5-HT uptake than R-citalopram: therefore, fluvoxamine increases the pharmacologically more active S-citalopram with some stereoselectivity. According to a previous in vitro study, this pharmacokinetic interaction occurs on the level of CYP2C19, but also of CYP2D6 and CYP3A4 which, in contrast to CYP1A2, contribute to the N-demethylation of citalopram and which are stereoselectively inhibited by fluvoxamine. All but one patient showed clinical improvement by a decrease of the MADRS score by at least 50% and a final score ≤13 (mean ± SD: day 0:30.6 ± 9.2; day 21:11.0 ± 6.5). Some patients showed minor symptoms, such as nausea and tremor, but the combined treatment was generally well tolerated. Received: 29 May 1996 /Final version: 2 September 1996     

Studies of the metabolism of N-methyl containing anti-tumour agents: 14CO2 breath analysis after administration of 14C-labelled N-methyl drugs, formaldehyde and formate in mice

The ¹⁴CO₂; content of the breath was analysed after administration of the following N-¹⁴ CH₃ labelled drugs to mice: aminopyrine, hexamethylmelamine (HMM), pentamethylmelamine (PMM), procarbazine and caffeine. Except for aminopyrine, the ¹⁴CO₂; exhalation rate plots declined monophasically with half lives of 91 min ([¹⁴C]-HMM), 97 min ([¹⁴C]-PMM), 68 min ([¹⁴C]procarbazine) and 92 min ([¹⁴C]caffeine). The ¹⁴CO₂ exhalation rate peaked rapidly after aminopyrine administration and declined bi-phasically with an initial $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 15 min and a terminal $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 126 min. The ¹⁴CO₂; plots after both [¹⁴C]-HMM and [¹⁴C]aminopyrine were influenced by pre-treatment of mice with proadifen. Pretreatment with phenobarbitone shortened the $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of the ¹⁴CO₂ appearance rate after [¹⁴C]HMM by 24% but did not change the ¹⁴CO₂ curve after administration of [¹⁴C] aminopyrine. The ¹⁴CO₂ exhalation rate plots after administration of H¹⁴CHO and H¹⁴COOH were virtually identical with that obtained after [¹⁴C]aminopyrine and not influenced by either proadifen or phenobarbitone pretreatment. The ¹⁴CO₂ exhalation rate profile obtained on metabolism of [¹⁴C]aminopyrine in mice thus appears to be determined by the rate of the oxidation of formaldehyde or formate to CO₂. Only 24% of the label injected with the N-methyl moieties of [¹⁴C]HMM and 21% of the label in [¹⁴C]procarbazine were exhaled as ¹⁴CO₂, whereas 49% of the N-¹⁴CH₃ in [¹⁴C]aminopyrine were metabolized to ¹⁴CO₂. It remains to be determined whether this difference and the difference in the shapes of the ¹⁴CO₂ exhalation profiles obtained with the cytotoxic N-¹⁴CH₃ drugs as compared to [¹⁴C]aminopyrine, are related to the biochemical processes mediating their antineoplastic activity.     

Synthesis of highly potent lymphocyte function‐associated antigen‐1 antagonists labeled with carbon‐14 and with stable isotopes,part 3

The drug candidates ( 2 ) and ( 3 ) are highly potent LFA‐1 inhibitors. They were efficiently prepared labeled with carbon‐14 using a palladium‐catalyzed carboxylation of an iodo‐precursor ( 5 ) and sodium formate‐¹⁴C to afford acid [¹⁴C]‐( 6 ), which was coupled via an amide bond to chiral amines ( 7 ) and ( 8 ) in 52% and 48% overall yield, respectively, and with specific activities higher than 56 mCi/mmol and radiochemical purities of 99%. For stable isotopes synthesis, the amine [²H₈]‐( 7 ) was synthesized in three steps from 2‐cyanopyridine‐²H₄ using Kulinkovich‐Szymonik aminocyclopropanation, followed by coupling to L ‐alanine‐2,3,3,3‐²H₄‐N‐t‐BOC, and then removal of the BOC‐protecting group. Amide bond formation with acid ( 6 ) gave [²H₈]‐( 2 ) in 36% overall yield. The amine [¹³C₄,¹⁵N]‐( 8 ) was obtained in two steps using L‐threonine‐¹⁴C₄,¹⁵N and then coupled to acid [¹³C]‐( 6 ) to give [¹³C₅,¹⁵N]‐( 3 ) in 56% overall yield.     

Disposition and metabolism of N,N-dimethylacetoacetamide in male F344 and Wistar-Han rats and female B6C3F1 mice

1. N,N-dimethylacetoacetamide (DMAAm) is a β-dicarbonyl compound used as an industrial intermediate. This study investigated the disposition and metabolism of [¹⁴C]DMAAm in male rats and female mice.

2. A single oral dose of [¹⁴C]DMAAm (target dose of 10 or 130?mg/kg) was administered to male F344 and Wistar-Han rats. [¹⁴C]DMAAm was almost completely absorbed and excreted in urine, with ca. 80?90% of the dose recovered within 24?h for both rat strains. Fecal excretion and CO₂ exhalation were minimal (1 and 2%, respectively). Less than 3% of the dose remained in tissues at 24?h. There was no apparent dose- or strain-related difference in the disposition of [¹⁴C]DMAAm in rats.

3. In female B6C3F1 mice administered 8?mg/kg [¹⁴C]DMAAm, 80% of the administered radioactivity was recovered in urine and cage rinse in 24?h.

4. Urinary metabolites were isolated and characterized by liquid chromatography /mass spectrometry following oral administration of 435?mg/kg [¹⁴C]DMAAm in male F344 rats. Metabolism occurred via reduction of the 3-keto group and oxidation of the N-methyl groups, to give N,N-dimethyl-3-hydroxybutanamide, N-methyl-N-hydroxymethyl-3-hydroxybutanamide, and N-hydroxymethyl-3-hydroxybutanamide, and N-demethylation to give N-monomethylacetoacetamide (MMAAm).

     

The role of cytochrome P-450 in the dual pathways of N-demethylation of N,N'-dimethylaniline by hepatic microsomes

1. The role of cytochrome P-450 in the demethylation of N, N'-dimethyl aniline (DMA) has been investigated by studying carbon monoxide (CO) inhibition and its reversal by light.

2. Two distinct pathways of N-demethylation are present in hepatic microsomes from phenobarbital-(PB) and 3-methyIcholanthrene-(3-MC) induced rats.

3. Pathway A, the demethylation of DMA, is catalysed by a microsomal cytochrome P-450 system similar to the one we have reported for the dealkylation of benzphetamine and 3-ethylmorphine, in that there is a maximal, although weak, light reversal of CO inhibition by wavelengths of light around 445 nm.

4. The second pathway consists of two enzyme steps (pathways B + C).

5. Pathway B is the step in which the N-oxide of DMA, N, N'-dimethylaniline N-oxide (DMAO) is formed and this step is catalysed by the flavin amine mixed-function oxidase described by Ziegler and his co-workers.

6. Pathway C, the demethylation of DMAO, requires a cytochrome P-450 with rather unusual properties.

7. This reaction is inhibited by SKF 525A and CO, but is not significantly affected by the removal of oxygen even though CO strongly inhibits in this anaerobic atmosphere.

8. Higher CO/O₂ ratios (2 :1 to 4:1) are required for inhibition equal to that of pathway A and light reversal of this inhibition is almost complete and maximally produced by wavelengths of light around 455 nm.     

Modulation of aflatoxin B1 biotransformation by β-naphthoflavone in isolated rabbit lung cells

 The cytotoxic and carcinogenic mycotoxin aflatoxin (AF) B₁ (AFB₁) is biotransformed by the cytochrome P450 monooxygenases (CYP) to a number of relatively nontoxic metabolites, as well as to the ultimate toxic metabolite, AFB₁–8,9-epoxide. In a number of tissues and species, AFB₁ hydroxylation to the relatively nontoxic metabolite, AFM₁, is induced by β-naphthoflavone (BNF) treatment. Although the liver is the principal target organ for AFB₁ toxicity, the mycotoxin is also toxic and carcinogenic to respiratory tissues. To determine if BNF treatment alters the extent of pulmonary AFB₁ bioactivation by enhancing detoxification and thereby decreasing epoxidation, the effects of BNF on pulmonary AFB₁ metabolism were examined. Rabbit lung cells, isolated by protease digestion and centrifugal elutriation, were incubated with [³H]AFB₁. In nonciliated bronchiolar epithelial (Clara) cell-enriched (45–50%) fractions, [³H]AFM₁ production (pmol/mg DNA per 2 h) was increased by prior treatment of rabbits with BNF (80 mg/kg per day, 3 and 2 days before cell isolation) as follows: with 1.0 μM [³H]AFB₁; control, 10.6±2.3; BNF, 30.0±6.4; with 0.10 μM [³H]AFB₁; control, 9.4±4.7; BNF, 20.6±5.9. With 1.0 μM [³H]AFB₁, prior treatment of animals with BNF abolished formation of [³H]aflatoxicol (AFL) but not [³H]AFQ₁. The activation (epoxidation) of [³H]AFB₁ was measured indirectly as covalent binding to endogenous DNA. With 1.0 μM [³H]AFB₁, treatment of rabbits with BNF did not alter DNA binding (pmol/mg DNA per 2 h) in the Clara cell-enriched fraction: control, 103±41; BNF, 114±49. However, with 0.10 μM [³H]AFB₁, DNA binding in the same fraction was 47% lower in cells from BNF treated animals: control, 17.4±4.2; BNF, 9.3±3.9. Formation of 8,9-dihydro-8,9-dihydroxy- AFB₁, and the glutathione conjugate of the aflatoxin epoxide (AFB₁-GSH) were not detectable at the AFB₁ concentrations and time point studied, in cells from either BNF-treated or control rabbits. Incubation of isolated, unseparated lung cells from untreated rabbits with 5.0 to 50 μM BNF decreased [³H]AFB₁-DNA binding in the presence of 0.1 μM [³H]AFB₁ by 35 to 77%, while lower BNF concentrations did not alter DNA binding. In lung cells isolated from BNF treated rabbits, BNF was not detectable (i.e.<0.5 μM detection limit). Therefore, the amount of BNF present in isolated rabbit lung cells following in vivo treatment with BNF was below that required to directly inhibit AFB₁-DNA adduct formation. The decrease in AFB₁-DNA binding from rabbits treated with BNF is apparently due to the selective induction of CYP isozymes and related increases in AFM₁ formation, and not to direct inhibition of epoxidation or enhanced conjugation of AFB₁-8,9-epoxide with glutathione. Received: 5 March 1996/Accepted: 10 May 1996     

Immunological and enzymatic comparison of hepatic cytochrome P-450 fractions from phenobarbital-, 3-methylcholanthrene-, β-naphtoflavone- and 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rats

A comparison of the cytochrome P-450 forms induced in rat liver microsomes by phenobarbital on the one hand, and 3-methylcholanthrene, β-naphtoflavone and 2,3,7,8-tetrachlorodibenzo-p-dioxin on the other hand, was performed using specific antibodies: anti-P-450 B₂ PB IG (against the phenobarbital-induced cytochrome P-450) and anti-P-450 B₂ BNF IG (against the β-naphtoflavone-induced cytochrome P-450). On DEAE-cellulose chromatography, four cytochrome P-450 fractions were separated, called P-450 A (non-adsorbed), P-450 Ba, P-450 Bb and P-450 Bc, from control, phenobarbital-, 3-methylcholanthrene, /gb-naphtoflavone- and 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rats. Cytochrome P-450 A fractions appeared to be unmodified by the inducers, whereas the specifically induced cytochrome P-450 forms were always recovered in Bb fractions. The P-450 Bb fractions induced by 3-methylcholanthrene, β-naphtoflavone and 2,3,7,8-tetrachlorodibenzo-p-dioxin exhibited common antigenic determinants, comparable catalytic activities (benzphetamine, N-demethylase, benzo[a]pyrene hydroxylase) and similar mol. wts. Moreover, the inhibition patterns by the two antibodies of benzphetamine N-demethylase and benzo[a]pyrene hydroxylase activities catalysed by 3-methylcholanthrene, β-naphtoflavone and 2,3,7,8-tetrachlorodibenzo-p-dioxin microsomes or by the corresponding P-450 Bb fractions in a reconstituted system were quite identical. By these different criteria, β-naphtoflavone, 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin seem to induce a common cytochrome P-450 species in rat liver.