Metabolism of chlorpromazine by pulmonary microsomal enzymes in the rat and rabbit

Pulmonary metabolism of chlorpromazine (CPZ) was compared using isolated microsomes of rat and rabbit lungs. CPZ-metabolizing activity of the rat lung was found to be 10-fold higher than that of the rabbit lung. The principal metabolic pathways were N-oxidation in the rat lung and N-demethylation in the rabbit lung. Kinetic analyses revealed that, although the values for apparent K_m were roughly similar for both pathways, V_max for N-oxidation by the rat lung was approximately ten times greater than that for N-demethylation by the rabbit lung. N-Oxidation by the rat lung had a broad range of pH optimum of 7–8, whereas N-demethylation by the rabbit lung had a pH optimum 8–9. SKF525-A, piperonyl butoxide, n-octylamine and CO did not inhibit N-oxidation by the rat lung, but inhibited N-demethylation by the rabbit lung. SKF525-A and n-octylamine stimulated the CPZ-N-oxidation by the rat lung. Hg²⁺ and Mg²⁺ inhibited N-oxidation by the rat lung. These results indicate that pulmonary metabolism of CPZ in the rat is catalyzed by a microsomal flavoprotein monooxygenase, while pulmonary metabolism in the rabbit is catalyzed by a cytochrome P-450 monooxygenase system, and that a marked species variation exists with respect to pulmonary metabolism of CPZ.     

N-oxidation of N,N-dimethylaniline in the rabbit and rat lung

We have reported previously that chlorpromazine (CPZ) and imipramine (IMP) are metabolized via N-oxidation by the rat lung, while they are not appreciably metabolized by the rabbit lung. Indeed, marked species differences exist in the pulmonary N-oxidation of these pneumophilic drugs. In the present studies, the isolated, ventilated and perfused lung (IPL) preparations as well as in vitro preparations of the rabbit and rat lungs were used to examine the pulmonary disposition of [¹⁴C]-N,N-dimethylaniline (DMA) which has been used frequently as a substrate for N-oxidation. Although the IPLs of both species were active in DMA N-oxidation, the rabbit lung was more active in DMA N-oxidation than the rat lung on the basis of per g lung. The gradual decline in radiolabel concentration in the perfusate was more marked in the rat than in the rabbit IPL. This decline was not due to the drug accumulation in the lung, but to its volatility. There was no dose dependency in the tissue/medium DMA concentration ratios (approximately 1.60), indicating uptake by simple diffusion and low affinity for the lung tissue. In vitro lung preparations showed higher DMA N-oxidase activity in the rabbit than in the rat, regardless of whether whole homogenate, post-mitochondrial supernatant fraction or microsomal fraction was used, or how the activities were expressed (per mg protein or per g tissue). These results suggest that, although DMA is not highly concentrated in the lung, it is N-oxidized by the lung and that DMA N-oxidase is different from CPZ or IMP N-oxidase reported previously.     

Radical formation during autoxidation of 4-dimethylaminophenol and some properties of the reaction products

4-Dimethylaminophenol (DMAP), after intravenous injection, rapidly forms ferrihaemoglobin and has been successfully used in the treatment of cyanide poisoning. Since DMAP produces many equivalents of ferrihaemoglobin, it was of interest to obtain further insight into this catalytic process. DMAP autoxidizes readily at pH regions above neutrality, a process which is markedly accelerated by oxyhaemoglobin. The resulting red-coloured product was identified as the N-N,-dimethylamino) phenoxyl radical by EPR spectroscopy. The same radical was also produced by pulse radiolysis and oxidation with ferricyanide. The 4-(N-N-dimethylamino)phenoxyl radical is quite unstable and decays in a pseudo-first order reaction (k = 0.4 sec^?1 at pH 8.5,22°) with the formation of p-benzoquinone and dimethylamine. This observed decay rate is identical with the rate of hydrolysis of N,N-dimethylquinonimine. When a solution containing the phenoxyl radical was extracted with ether, half the stoichiometric amount of DMAP was recovered. Hence it is apparent that the phenoxyl radical decays by disproportionation yielding DMAP and N,N-dimethylquinomine. The latter product then quickly hydrolyses. The equilibrium of this disproportionation reaction is far towards the radical side, and the pseudo-first order hydrolysis controls the radical decay rate. p-Benzoquinone rapidly reacts with DMAP (k₂ = 2 × 10⁴M^?1sec^?1) with the formation of the 4-N,N-dimethylamino)phenoxyl and the semiquinone radicals. This reaction explains the autocatalytic phenoxyl radical formation during the autoxidation of DMAP. DMAP is not oxidized by H₂O₂ or O₁₂^? but the 4-(N-N-dimethylamino)phenoxyl radical is very rapidly reduced by O₂^? (k₂ = 2 × 10⁸M^?1 sec^?1. In addition, the phenoxyl radical is quickly reduced by NAD(P)H or GSH with the formation of NAD(P)⁺ or GSSG. Since DMAP is also able to reduce two equivalents of ferrihaemoglobin (provided that the ferrohaemoglobin produced is trapped by carbon monoxide), electrophilic addition reactions of the phenoxyl radical seem unimportant in contrast to N,N-dimethylquinonimine. Hence, during the catalytic ferrihaemoglobin formation, DMAP is oxidized by oxygen which is activated by haemoglobin, and the phenoxyl radical oxidizes ferrohaemoglobin. This catalytic process is terminated by covalent binding of N,N-dimethylquinonimine to SH groups of haemoglobin (and GSH in red cells).     

Mouse liver microsomal hexose-6-phosphate dehydrogenase: NADPH generation and utilization in monooxygenation reactions

Hexose-6-phosphate dehydrogenase (H6PD) activity in washed hepatic microsomes from male ICR mice, when assayed with NADP⁺ and deoxyglucose-6-phosphate, was partially latent. Brief sonication or detergents activated H6PD causing an approximately 4- and 8.5-fold increase in NADPH generation respectively. The sonicated microsomes exhibited H6PD-linked N-demethylase activity toward aminopyrine. This activity was best sustained in the presence of deoxyglucose-6-phosphate, while galactose-6-phosphate, glucose-6-phosphate, and glucose were less effective. Reaction media containing sonicated microsomes, NADP⁺ and deoxyglucose-6-phosphate also catalyzed N-demethylation of p-chloro-N-methylaniline, N, N-dimethylaniline and nicotine, O-demethylation of p-nitroanisole, p-hydroxylation of aniline, ring hydroxylation of biphenyl at the 2- and 4-positions, dearylation of parathion, and the N-oxidation of N,N-dimethylaniline. In general, the hexose-6-phosphate dehydrogenase-linked monooxygenation rates were 60% or more of those observed in the presence of exogenous NADPH.     

Characterization of moclobemide N-oxidation in human liver microsomes

1. Moclobemide undergoes morpholine ring N-oxidation to form a major metabolite in plasma, Ro12-5637. 2. The kinetics of moclobemide N-oxidation in human liver microsomes (HLM) (n = 6) have been investigated and the mixed-function oxidase enzymes catalysing this reaction have been identified using inhibition, enzyme correlation, altered pH and heat pretreatment experiments. 3. N-oxidation followed single enzyme Michealis-Menten kinetics (0.02-4.0 mM). K_{m app} and V_max ranged from 0.48 to 1.35 mM (mean ± SD 0:77 ± 0:34 mM) and 0.22 to 2.15 nmol mg^?1 min^?1 (1:39 ± 0:80 nmol mg^?1 min^?1), respectively. 4. The N-oxidation of moclobemide strongly correlated with benzydamine N-oxidation, a probe reaction for flavin-containing monooxygenase (FMO) activity, (0.1 mM moclobemide, r_S = 0:81; p < 0:005; 4 mM moclobemide, r_S = 0:94; p = 0:0001). Correlations were observed between moclobemide N-oxidation and specific cytochrome P450 (CYP) activities at both moclobemide concentrations (0.1 mM moclobemide, CYP2C19 r_S = 0:66; p < 0:05; 4 mM moclobemide, CYP2E1 r_S = 0:56; p < 0:05). 5. The general P450 inhibitor, N-benzylimidazole, did not affect the rate of Ro12-5637 formation (0% inhibition versus control) at 1.3 mM moclobemide. Furthermore, the rate of Ro12-5637 formation in HLM was unaffected by inhibitors or substrates of specific P450s (<10% inhibition versus control). 6. Heat pretreatment of HLM in the absence of NADPH (inactivating FMOs) resulted in 97% inhibition of Ro12-5637 formation. N-oxidation activity was greatest when incubated at pH 8.5. These results are consistent with the reaction being FMO mediated. 7. In conclusion, moclobemide N-oxidation activity has been observed in HLM in vitro and the reaction is predominantly catalysed by FMOs with a potentially small contribution from cytochrome P450 isoforms.     

Halidohydrolytic conversion of N,N'-bis-(dichloroacetyl)-N,N'-diethyl-1,4-xylylenediamine in rat liver

N,N′-bis-(dichloroacetyl)-N,N′-diethyl-1,4-xylylenediamine (WIN 13099) was synthesized with tritium on the xylylene carbon which did not exchange with body water. This compound was metabolized by 140,000 g supernatant liquid and the mitochondrial fraction of rat liver. Deproteinized 140,000 g supernatant liquid was required for the mitochondrial activity. Although WIN 13099 effectively inhibits drug metabolism by rat liver microsomes in vitro and in vivo, it is not metabolized by rat liver microsomes in vitro. The major metabolite in vitro of WIN 13099, N-dichloroacetyl-N′-hydroxyacetyl-N,N′-diethyl-1,4-xylylenediamine, was isolated and characterized and found not as effective as the parent compound in inhibiting electron transport by sub-mitochondrial particles or drug metabolism by rat liver microsomes.     

Comparison of the drug metabolising ability of rat intestinal mucosal microsomes with that of liver.

A study of the drug metabolizing capabilities of rat intestinal microsomes was undertaken and a direct comparison made with analogous activity in hepatic microsomes. The cofactor requirements and K_m values of the cytochrome P-450 mediated 7-ethoxycoumarin O-de-ethylation in the epithelial cell microsomes was similar to that in hepatic microsomes, whereas the V_max values were 5–6 fold lower in the intestinal preparation. Greater differences were seen in other cytochrome P-450 dependent reactions. The K_m value for biphenyl-4-hydroxylation was an order of magnitude lower in the intestinal microsomes, whereas the 4-hydroxylation of aniline and acetanilide and the N-demethylation of ethylmorphine and aminopyrine, although very active in hepatic microsomes, were barely detectable in intestinal microsomes. The O-de-ethylation inhibition characteristics of metyrapone and 7,8-benzoflavone but not SKF 525 A, were similar in the liver and intestinal preparations.These findings and the alterations in drug oxidation activity after PB and 3MC pretreatment suggest that there are both quantitative and qualitative differences in the cytochrome P-450's between liver and intestine. This suggestion is further supported by the difference in the safrole adduct binding spectrum with NADPH or with cumene hydroperoxide between the two tissues from control, phenobarbitone and 3-MC pretreated rats.O-Deacetylation of 4-nitrophenyl acetate and indoxyl acetate in intestinal and liver preparations was very comparable while acetanilide N-deacetylation was three times more active in the liver. Glucuronidation of 1-naphthol by the intestinal microsomes showed a similar K_m but a lower V_max than the liver microsomes. No change was observed in any of these enzymes following pretreatment of rats with PB or with 3-MC.     

Effect of N6-methyladenosine on fat-cell glucose metabolism: Evidence for two modes of action

N⁶-Methyladenosine strongly stimulates [1-¹⁴C]glucose oxidation in rat adipocytes [J.E. Souness and V. Chagoya de Sanchez, Fedn Eur. Biochem. Soc. Lett.125, 249 (1981)]. The effect of the adenosine analogue is largely independent of its action as an R-site agonist. Removal of endogenous adenosine was a prerequisite for the manifestation of the effect of N⁶-methyladenosine. Nucleoside uptake inhibitors, dipridamole and nitrobenzylthioinosine, did not block the action of N⁶-methyladenosine on [1-¹⁴C]glucose oxidation. The effect of the adenosine analogue was not greatly influenced by N⁶-phenylisopropyladenosine, nicotinic acid or theophylline. Although N⁶-methyladenosine stimulated 3-O-methylglucose uptake into fat cells, it is uncertain whether this was its only effect on glucose metabolism, in view of the comparable enhancement of hexose transport elicited by N⁶-phenylisopropyladenosine, a much weaker stimulator of glucose oxidation. That hexose transport is not the sole site of action of N⁶-methyladenosine was supported by the finding that, under conditions which have been proposed to make glucose transport rate limiting, the adenosine analogue only weakly enhanced [1-¹⁴C]glucose oxidation. The conversion of glucose carbon 1 to ¹⁴CO₂ was enhanced by N⁶-methyladenosine to a greater degree than that of carbon 6, suggesting an increase in pentose phosphate shunt activity. Mechanisms by which this could arise are discussed. Although similarities exist between the effects of insulin and N⁶-methyladenosine on adipocyte glucose metabolism, the mechanisms by which they stimulate glucose oxidation appear to be distinct, in view of the additivity of their actions on [1-¹⁴C]glucose conversion to ¹⁴CO₂. The results indicate that N⁶-methyladenosine affects fat-cell glucose metabolism via two different mechanisms: (1) a weak R-site-dependent mode of action related to stimulation of glucose transport and inhibition of lipolysis, and (2) a strong R-site-independent effect of unknown mechanism.     

The reversibility of N-oxidation in vivo: The fate of 14C-N-hydroxychlorphentermine and 14C-nitrochlorphentermine in the rat

The interrelationships between primary amines and their in vivoN-oxidized metabolites are unclear. We have therefore synthesized ¹⁴C-N-hydroxychlorphentermine and ¹⁴C-nitrochlorphentermine and examined their metabolism and excretion in the rat. ¹⁴C-N-hydroxychlorphentermine was excreted slowly in the urine (66 per cent of dose in 6 days) with a further 8 per cent in the faeces (3 per cent) and as ¹⁴CO₂ (5 per cent), and the only urinary metabolites were the unchanged hydroxylamine and its glucuronic acid conjugate. ¹⁴C-Nitrochlorphentermine was eliminated more rapidly (92 per cent of dose in 4 days), with 41 per cent in the urine, 1 per cent in the faeces and 50 per cent as ¹⁴CO₂. The only urinary metabolites were the reduction product N-hydroxychlorphentermine and its glucuronide but the large amount of ¹⁴CO₂ found indicated that side chain oxidation was a major metabolic route. The results are discussed with reference to the possible reversibility of N-oxidation in vivo and putative mechanisms for the oxidation of the side chain.     

Inhibition of NADPH oxidation and related drug oxidation in liver microsomes by zinc.

Rat liver microsomes were incubated in the presence of zinc and the rate of NADPH oxidation and related metabolism of aniline and ethylmorphine by appropriate oxidases were studied. A competitive mechanism of the inhibition of NADPH oxidation by zinc was found, with V_max = 10.3 nmoles NADP/min/mg of protein and K_i amounting to 7.22 μM zinc. In microsomes dialyzed against EDTA, addition of Mn²⁺ but not of Mg²⁺ enhanced the rate of NADPH oxidation. A complex relation of Zn²⁺ and Mn²⁺ in liver microsomes was found, the data not obeying the rigorous treatment for enzyme kinetics. The activity of aniline hydroxylase and ethylmorphine-N-demethylase was inhibited by zinc; 50 per cent inhibition was reached at 60 and 55 μM Zn²⁺ respectively. Another microsomal enzyme, glucose 6-phosphatase, independent of NADPH, was not affected by zinc. The content and spectral characteristics of cytochrome P-450 were not affected by zinc. It is concluded that Zn²⁺ inhibits oxidation of NADPH and prevents this pyridine nucleotide from functioning in the microsomal electron transport system. The possibility that Zn²⁺ may interfere with other ions or enzymes involved in microsomal electron transport cannot be excluded.     

Activation of the O-glucuronide of the carcinogen N-hydroxy-N-2-fluorenylacetamide by enzymatic deacetylation in vitro: formation of fluorenylamine-tRNA adducts.

The O-glucuronide of N-hydroxy-N-2-fluorenylacetamide (N-GIO-FAA) was deacetylated by guinea pig liver. tRNA reacted with the product of this deacetylation, the O-glucuronide of N-2-fluorenylhydroxylamine (N-GIO-FA), to give fluorenylamine-substituted nucleic acid adducts. The quantity of adduct formation was used to determine deacetylase activity.Of the various guinea pig tissues assayed, only the liver contained enzyme activity, and this activity was confined to the microsomal fraction of the cell. Guinea pig liver microsomes were about four times as active as rabbit liver microsomes and about fourteen times as active as rat liver microsomes in promoting fluorenylamine-tRNA adduct formation. Adduct formation induced by guinea pig microsomes was about seven times greater at pH 8.5 than at pH 7.0.The aglycone of the O-glucuronide, N-hydroxy-N-2-fluorenylacetamide (N-hydroxy-FAA) also yielded flourenylamine-substituted nucleic acid adducts following deacetylation at pH 8.5 by guinea pig liver microsomes in the presence of tRNA. In contrast to resuls obtained with N-GIO-FAA, adduct formation with N-hydroxy-FAA was not as efficient, and it was independent of pH over the range 7.0–8.5. Rabbit and rat liver microsomes were more active in promoting adduct formation of tRNA with N-hydroxy-FAA than with N-GIO-FAA.The differential inhibition of the microsome-induced formation of adducts of N-GIO-FAA and N-hydroxy-FAA with tRNA affirms that the first step in the binding mechanism of N-GIO-FAA with tRNA is enzymatic deacetylation and not hydrolysis to the aglycone N-hydroxy-FAA.     

Biotransformation of benzydamine by microsomes and precision-cut slices prepared from cattle liver

1. Benzydamine (BZ), a non-steroidal anti-inflammatory drug used in human and veterinary medicine, is not licensed for use in food-producing species. Biotransformation of BZ in cattle has not been reported previously and is investigated here using liver microsomes and precision-cut liver slices. 2. BZ was metabolized by cattle liver microsomes to benzydamine N-oxide (BZ-NO) and monodesmethyl-BZ (Nor-BZ). Both reactions followed Michaelis-Menten kinetics (K_m = 76.4 ± 16.0 and 58.9 ± 6.4 µM, V_max = 6.5 ± 0.8 and 7.4 ± 0.5 nmol mg^?1 min^?1, respectively); sensitivity to heat and pH suggested that the N-oxidation is catalysed by the flavin-containing monooxygenases. 3. BZ-NO and Nor-BZ were the most abundant products derived from liver slice incubations, and nine other BZ metabolites were found and tentatively identified by LCMS. Desbenzylated and hydroxylated BZ-NO analogues and a hydroxylated product of BZ were detected, which have been reported in other species. Product ion mass spectra of other metabolites, which are described here for the first time, indicated the formation of a BZ N⁺-glucuronide and five hydroxylated and N⁺-glucuronidated derivatives of BZ, BZNO and Nor-BZ. 4. The results indicate that BZ is extensively metabolized in cattle. Clearly, differences in metabolism compared with, for example, rat and human, will need to be considered in the event of submission for marketing authorization for use in food animals.     

Hepatic microsomal metabolism of N-nitrosodimethylamine to its methylating agent

Incubation of N-nitrosodi[¹⁴C]methylamine with calf thymus DNA and an isolated rat liver microsomal fraction resulted in a transfer of ¹⁴C-label from N-nitrosodi[¹⁴C]methylamine to biological macromolecules present in the in vitro assay system. This transfer of ¹⁴C-label from N-nitrosodi[¹⁴C]methylamine to biological macromolecules, believed to represent a methylation process, was dependent on the integrity and concentration of rat liver microsomes added to the in vitro assay system, as well as on the time of incubation. A requirement for NADPH was also observed. A study of the kinetics of this transfer of ¹⁴C-label from N-nitrosodi[¹⁴C]methylamine to biological macromolecules in vitro yielded values for the apparent K_m and V_max of 0.18 mm and 2.56 pmol methyl groups transferred per milligram of nucleic acid per milligram of microsomal protein per minute, respectively. The transfer reaction was inhibited by exposure of microsomes to carbon monoxide or pretreatment of animals with phenobarbital, 3-methylcholanthrene, or 2,3,7,8-tetrachlorodibenzo-p-dioxin. The capability of other rat liver subcellular fractions to mediate the reaction was examined.     

Hydroperoxide-dependent activation of p-phenetidine catalyzed by prostaglandin synthase and other peroxidases

p-Phenetidine is metabolized by ram seminal vesicle (RSV) microsomes, horseradish peroxidase oxidase (HRP) and rat liver microsomes to protein-binding products. These reactions are very rapid and depend on the presence of arachidonic acid (AA) or various hydroperoxidases. The RSV- and HRP-mediated binding was inhibited more than 80% by the addition of reduced glutathione (1 mM) or the antioxidant butylated hydroxyanisole (0.5 mM). Indomethacin (100 μM) and acetylsalicylic acid (1 mM) reduced the AA-dependent reaction in RSV microsomes to less than 5% of control values. When hydrogen peroxide replaced AA, the RSV/H₂O₂-supported binding in the presence of 50 μM p-phenetidine proceeded at rates similar to that observed with RSV/AA. Unlike the AA-dependent reaction, the H₂O₂-supported reaction showed no inhibition of protein binding at higher p-phenetidine concns. The data in this report are consistent with a peroxidase activation of p-phenetidine possibly involving an amine radical catalyzed by prostaglandin synthase (PGS) present in RSV microsomes as well as by other peroxidases. The mechanism for this activation and physiological implications are discussed.     

Purification and properties of cytochrome P-450 and NADPH-cytochrome c (P-450) reductase from human liver microsomes.

Cytochrome P-450 and NADPH-cytochrome c (P-450) reductase were purified to 10.6 nmoles per mg of protein and 19.9 units per mg of protein, respectively, from human liver microsomes. The purified cytochrome was assumed to be in a low spin state as judged by the absolute spectrum. n-Octylamine and aniline produced type II difference spectra and SKF 525-A and benzphetamine type I spectra when bound to the purified cytochrome P-450. The purified human cytochrome P-450 catalyzed laurate oxidation as determined by NADPH oxidation but not aniline hydroxylation, benzphetamine N-demethylation and 7-ethoxycoumarin O-deethylation when reconstituted with the reductases purified from human and rat liver microsomes. The human cytochrome P-450, however, catalyzed drug oxidations when cumene hydroperoxide was used as the oxygen source. The purified human NADPH-cytochrome c (P-450) reductase contained FAD and FMN at a ratio of 1:0.76. The reductase was capable of supporting 7-ethoxycoumarin O-deethylation activity of cytochrome P-448 purified from 3-methylcholanthrene-treated rat liver microsomes.     

Differential metabolism of O-ethyl O-4-nitrophenyl phenylphosphonothioate by rat and chicken hepatic microsomes

The in vitro hepatic metabolism of O-ethyl O-4-nitrophenyl phenylphosphonothioate (EPN) was investigated in the hen (a species that is sensitive to EPN delayed neurotoxicity) and the rat (an insensitive species). EPN, which produced a Type I binding spectrum on incubation with cytochrome P-450, was converted by liver microsomes from both species to its oxygen analog, O-ethyl O-4-nitrophenyl phenylphosphonate (EPNO), and to p-nitrophenol (PNP). The formation of EPNO and PNP was dependent on the presence of NADPH in the reaction mixture and could be inhibited by either SKF-525A or by anaerobic conditions. The rates of EPNO and PNP formation by rat liver microsomes were, however, 3- and 20-fold higher, respectively, than the rates of formation by chicken liver microsomes. There was also a 4-fold difference in the cytochrome P-450 contents of the liver microsomes. The EPNO-hydrolyzing activity of rat liver microsomes was much greater than that of chicken liver microsomes. EPNO metabolism, in contrast to EPN metabolism, did not require NAPDH nor was it inhibited by SKF-525A or by anaerobic conditions. Prior exposure of rats to phenobarbital (PB) or Arochlor 1254 resulted in an increase in hepatic microsomal EPN metabolism and cytochrome P-450 content. On the other hand, 3-methylcholanthrene (3-MC) treatment elevated microsomal cytochrome P-450 but did not increase EPNO or PNP formation. Pretreatment with EPN did not alter either microsomal EPN metabolism or cytochrome P-450 levels. In chickens, prior exposure to PB, 3-MC or 100 mg/kg EPN increased EPNO and PNP formation by liver microsomes as well as cytochrome P-450 levels; prior exposure of chickens to 15 mg/kg EPN did not alter these variables. The λ_max Soret bands of the reduced hepatic cytochrome P-450 complexes from these animals differed as follows (rat then chicken): untreated, 450 vs 452 nm; PB-treated, 450 vs 451 nm; and 3-MC-treated, 448 vs 449 nm. None of the above treatments had an effect on EPNO metabolism by liver microsomes.     

Drug oxygenation activities mediated by liver microsomal flavin-containing monooxygenases 1 and 3 in humans,monkeys, rats,and minipigs

Liver microsomal flavin-containing monooxygenases (FMO, EC 1.14.13.8) 1 and 3 were functionally characterized in terms of expression levels and molecular catalytic capacities in human, cynomolgus monkey, rat, and minipig livers. Liver microsomal FMO3 in humans and monkeys and FMO1 and FMO3 in rats and minipigs could be determined immunochemically with commercially available anti-human FMO3 peptide antibodies or rat FMO1 peptide antibodies. With respect to FMO-dependent N-oxygenation of benzydamine and tozasertib and S-oxygenation of methimazole and sulindac sulfide activities, rat and minipig liver microsomes had high maximum velocity values (V_max) and high catalytic efficiency (V_max/K_m, Michaelis constant) compared with those for human or monkey liver microsomes. Apparent K_m values for recombinantly expressed rat FMO3-mediated N- and S-oxygenations were approximately 10–100-fold those of rat FMO1, although these enzymes had similar V_max values. The mean catalytic efficiencies (V_max/K_m, 1.4 and 0.4 min⁻¹ μM⁻¹, respectively) of recombinant human and monkey FMO3 were higher than those of FMO1, whereas V_max/K_m values for rat and minipig FMO3 were low compared with those of FMO1. Minipig liver microsomal FMO1 efficiently catalyzed N- and S-oxygenation reactions; in addition, the minipig liver microsomal FMO1 concentration was higher than the levels in rats, humans, and monkeys. These results suggest that liver microsomal FMO1 could contribute to the relatively high FMO-mediated drug N- and S-oxygenation activities in rat and minipig liver microsomes and that lower expression of FMO1 in human and monkey livers could be a determinant factor for species differences in liver drug N- and S-oxygenation activities between experimental animals and humans.     

Regioselective Glucuronidation of Andrographolide and Its Major Derivatives: Metabolite Identification,Isozyme Contribution,and Species Differences

Andrographolide (AND) and two of its derivatives, deoxyandrographolide (DEO) and dehydroandrographolide (DEH), are widely used in clinical practice as anti-inflammatory agents. However, UDP-glucuronosyltransferase (UGT)-mediated phase II metabolism of these compounds is not fully understood. In this study, glucuronidation of AND, DEO, and DEH was characterized using liver microsomes and recombinant UGT enzymes. We isolated six glucuronides and identified them using 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. We also systematically analyzed various kinetic parameters (K_m, V_max, and CL_int) for glucuronidation of AND, DEO, and DEH. Among 12 commercially available UGT enzymes, UGT1A3, 1A4, 2B4, and 2B7 exhibited metabolic activities toward AND, DEO, and DEH. Further, UGT2B7 made the greatest contribution to glucuronidation of all three anti-inflammatory agents. Regioselective glucuronidation showed considerable species differences. 19-O-Glucuronides were present in liver microsomes from all species except rats. 3-O-Glucuronides were produced by pig and cynomolgus monkey liver microsomes for all compounds, and 3-O-glucuronide of DEH was detected in mouse and rat liver microsomes (RLM). Variations in K_m values were 48.6-fold (1.93–93.6 μM) and 49.5-fold (2.01–99.1 μM) for 19-O-glucuronide and 3-O-glucuronide formation, respectively. Total intrinsic clearances (CL_int) for 3-O- and 19-O-glucuronidation varied 4.8-fold (22.7–110 μL min⁻¹ mg⁻¹), 10.6-fold (94.2–991 μL min⁻¹ mg⁻¹), and 8.3-fold (122–1,010 μL min⁻¹ mg⁻¹), for AND, DEH, and DEO, respectively. Our results indicate that UGT2B7 is the major UGT enzyme involved in the metabolism of AND, DEO, and DEH. Metabolic pathways in the glucuronidation of AND, DEO, and DEH showed considerable species differences.

Electronic supplementary material

The online version of this article (doi:10.1208/s12248-014-9658-8) contains supplementary material, which is available to authorized users.KEY WORDS: andrographolide, anti-inflammatory activity, glucuronidation, species difference, UGT2B7     

Synthesis of N-oxide derivatives of metyrapone and their detection as in vitro metabolites†

A variety of possible N-oxidation products of 2-methyl-1, 2-bis(3-pyridyl)propan-1-one (metyrapone) have been synthesized by peracid oxidation, and characterized using various spectroscopic techniques. Specific and sensitive chromatographic techniques have been developed for the separation and identification of its in vitro metabolites. Incubation of metyrapone with rat or mouse hepatic microsomes, in the presence of a NADPH-regenerating system, leads to the formation of metyrapol (keto-reduction), and two mono-N-oxides.     

Influence of substituants on microsomal dealkylation of aromatic N-, O-, and S-alkyl compounds

Dealkylation of aromatic N-, O- an S-alkyl compounds by rat liver microsomes was investigated in relation to the physical and chemical properties of the substrates. The relative rate of demethylation of some of the aniline derivatives tested was only dependent on their lipid solubility and basicity. In other derivatives tested steric effects also influenced the reaction. Generally the highest demethylation rate was found at the O-alkyl or N-alkyl group when a second substituent was in 2-position. N-Demethylation was the primary reaction. The results are discussed in relation to the binding and orientation of the substrates in the microsomal lipid membrane.