Metabolism of isovanillin by aldehyde oxidase, xanthine oxidase, aldehyde dehydrogenase and liver slices

Aromatic aldehydes are good substrates of aldehyde dehydrogenase activity but are relatively poor substrates of aldehyde oxidase and xanthine oxidase. However, the oxidation of xenobiotic-derived aromatic aldehydes by the latter enzymes has not been studied to any great extent. The present investigation compares the relative contribution of aldehyde dehydrogenase, aldehyde oxidase and xanthine oxidase activities in the oxidation of isovanillin in separate preparations and also in freshly prepared and cryopreserved liver slices. The oxidation of isovanillin was also examined in the presence of specific inhibitors of each oxidizing enzyme. Minimal transformation of isovanillin to isovanillic acid was observed in partially purified aldehyde oxidase, which is thought to be due to residual xanthine oxidase activity. Isovanillin was rapidly metabolized to isovanillic acid by high amounts of purified xanthine oxidase, but only low amounts are present in guinea pig liver fraction. Thus the contribution of xanthine oxidase to isovanillin oxidation in guinea pig is very low. In contrast, isovanillin was rapidly catalyzed to isovanillic acid by guinea pig liver aldehyde dehydrogenase activity. The inhibitor studies revealed that isovanillin was predominantly metabolized by aldehyde dehydrogenase activity. The oxidation of xenobiotic-derived aromatic aldehydes with freshly prepared or cryopreserved liver slices has not been previously reported. In freshly prepared liver slices, isovanillin was rapidly converted to isovanillic acid, whereas the conversion was very slow in cryopreserved liver slices due to low aldehyde dehydrogenase activity. The formation of isovanillic acid was not altered by allopurinol, but considerably inhibited by disulfiram. It is therefore concluded that isovanillin is predominantly metabolized by aldehyde dehydrogenase activity, with minimal contribution from either aldehyde oxidase or xanthine oxidase.     

Metabolism of cryptolepine and 2-fluorocryptolepine by aldehyde oxidase

Objectives To investigate the metabolism of cryptolepine and some cryptolepine analogues by aldehyde oxidase, and to assess the implications of the results on the potential of cryptolepine analogues as antimalarial agents. Methods The products resulting from the oxidation of cryptolepine and 2‐fluorocryptolepine by a rabbit liver preparation of aldehyde oxidase were isolated and identified using chromatographic and spectroscopic techniques. The antiplasmodial activity of cryptolepine‐11‐one was assessed against Plasmodium falciparum using the parasite lactate dehydrogenase assay. Key findings Cryptolepine was oxidized by aldehyde oxidase give cryptolepine‐11‐one. Although 2‐fluorocryptolepine was found to have less affinity for the enzyme than cryptolepine, it was a better substrate for aldehyde oxidase than the parent compound. In contrast, quindoline, the 11‐chloro‐ , 2,7‐dibromo‐ and 2‐methoxy analogues of cryptolepine were not readily oxidized. Cryptolepine‐11‐one was found to be inactive against P. falciparum in vitro raising the possibility that the effectiveness of cryptolepine as an antimalarial, may be compromised by metabolism to an inactive metabolite by liver aldehyde oxidase. Conclusions Cryptolepine and 2‐fluorocryptolepine are substrates for aldehyde oxidase. This may have implications for the design and development of cryptolepine analogues as antimalarial agents.     

In vivo-in vitro relationship of methotrexate 7-hydroxylation by aldehyde oxidase in four different strain rats

The in vivo metabolism of methotrexate (MTX) to 7-hydroxymethotrexate (7-OH-MTX) was studied using four strains of rats. When MTX was administered to these rats, 7-OH-MTX was detected as the major in vivo metabolite, mainly in bile and feces, and also slightly in the urine. There were marked strain differences in the amounts of 7-OH-MTX excreted in bile, feces and urine. The highest recovery of 7-OH-MTX in bile, feces and urine was observed in Sea:SD rats (6.2%, 4.2% and 0.8% of dose, respectively), followed by Jcl:SD and Crj:SD rats. The lowest recovery (0.02%, 0.2% and 0.003%, respectively) was observed in WKA/Sea rats. The variations of excreted amount of 7-OH-MTX were closely correlated with the strain differences of cytosolic MTX 7-hydroxylase and benzaldehyde oxidase activities. Our results indicate that variation of formation of 7-OH-MTX from MTX in vivo in rats is due primarily to variation of aldehyde oxidase.     

Metabolism of zaleplon by human liver: evidence for involvement of aldehyde oxidase

1. The metabolism of Zaleplon (CL-284,846; ZAL) has been studied in precision-cut human liver slices and liver cytosol preparations. 2. Human liver slices metabolized ZAL to a number of products including 5-oxo-ZAL (M2), N-desethyl-5-oxo-ZAL (M1) and N-desethyl-ZAL (DZAL), the latter metabolite being known to be formed by CYP3A forms. 3. Human liver cytosol preparations catalysed the metabolism of ZAL to M2. Kinetic analysis of three cytosol preparations revealed mean (± SEM) K_m and V_max of 93 ± 18 mm and 317 ± 241 pmol/min/mg protein, respectively. 4. Using 16 individual human liver cytosol preparations a 33-fold variability in the metabolism of 80µM ZAL to M2 was observed. Correlations were observed between M2 formation and the metabolism of the aldehyde oxidase substrates phenanthridine (r² = 0.774) and phthalazine (r² = 0.460). 5. The metabolism of 80µM ZAL to M2 in liver cytosol preparations was markedly inhibited by the aldehyde oxidase inhibitors chlorpromazine, promethazine, hydralazine and menadione. Additional kinetic analysis suggested that chlorpromazine and promethazine were non-competitive inhibitors of M2 formation with K_i of 2.3 and 1.9 µM, respectively. ZAL metabolism to M2 was also inhibited by cimetidine. 6. Incubations conducted with human liver cytosol and H₂¹⁸O demonstrated that the oxygen atom incorporated into ZAL and DZAL to form M2 and M1, respectively, was derived from water and not from molecular oxygen. 7. In summary, by correlation analysis, chemical inhibition and H₂¹⁸O incorporation studies, ZAL metabolism to M2 in human liver appears to be catalysed by aldehyde oxidase. With human liver slices, ZAL was metabolized to products dependent on both aldehyde oxidase and CYP3A forms.     

Metabolism of zaleplon by human liver: evidence for involvement of aldehyde oxidase

1. The metabolism of Zaleplon (CL-284,846; ZAL) has been studied in precision-cut human liver slices and liver cytosol preparations. 2. Human liver slices metabolized ZAL to a number of products including 5-oxo-ZAL (M2), N-desethyl-5-oxo-ZAL (M1) and N-desethyl-ZAL (DZAL), the latter metabolite being known to be formed by CYP3A forms. 3. Human liver cytosol preparations catalysed the metabolism of ZAL to M2. Kinetic analysis of three cytosol preparations revealed mean (+/- SEM) K(m) and V(max) of 93 +/- 18 mm and 317 +/- 241 pmol/min/mg protein, respectively. 4. Using 16 individual human liver cytosol preparations a 33-fold variability in the metabolism of 80 micro M ZAL to M2 was observed. Correlations were observed between M2 formation and the metabolism of the aldehyde oxidase substrates phenanthridine (r(2) = 0.774) and phthalazine (r(2) = 0.460). 5. The metabolism of 80 micro M ZAL to M2 in liver cytosol preparations was markedly inhibited by the aldehyde oxidase inhibitors chlorpromazine, promethazine, hydralazine and menadione. Additional kinetic analysis suggested that chlorpromazine and promethazine were non-competitive inhibitors of M2 formation with K(i) of 2.3 and 1.9 micro M, respectively. ZAL metabolism to M2 was also inhibited by cimetidine. 6. Incubations conducted with human liver cytosol and H(2)(18)O demonstrated that the oxygen atom incorporated into ZAL and DZAL to form M2 and M1, respectively, was derived from water and not from molecular oxygen. 7. In summary, by correlation analysis, chemical inhibition and H(2)(18)O incorporation studies, ZAL metabolism to M2 in human liver appears to be catalysed by aldehyde oxidase. With human liver slices, ZAL was metabolized to products dependent on both aldehyde oxidase and CYP3A forms.     

Species variation in hepatic aldehyde oxidase activity

The activity of hepatic aldehyde oxidase from rabbit, guinea pig, rat, marmoset, dog, baboon and man was investigated in vitro with charged and uncharged N-heterocyclic substrates: Km and Vmax values were determined for phthalazine, 6,7-dimethoxy-1-[-4-(ethylcarbamoyloxy)piperidino]phthalazine (carbazeran), quinine and quinidine. The oxidation of N-phenylquinolinium chloride to N-phenyl-2-quinolone and N-phenyl-4-quinolone was followed spectrophotometrically. Rat or dog liver showed low and negligible enzyme activity respectively, whereas baboon liver contained a highly active aldehyde oxidase. Enzyme from marmoset and guinea pig liver had the closest spectrum of activity to human liver aldehyde oxidase. Unlike that from man, rabbit hepatic aldehyde oxidase was refractory towards carbazeran and converted N-phenylquinolinium chloride predominantly to the 2-quinolone. N-Phenyl-4-quinolone was the major oxidation product with enzyme from guinea pig, marmoset, baboon and man.     

Metabolism of azoxy derivatives of procarbazine by aldehyde dehydrogenase and xanthine oxidase.

Procarbazine, a 1,2-disubstituted hydrazine, is employed therapeutically in the treatment of Hodgkin's disease and a limited number of other neoplasias. The isomeric azoxy metabolites of procarbazine have recently been identified as the precursors of species responsible for both the anti-cancer efficacy and toxic effects mediated by this drug. This study demonstrates that cytosolic enzymes are involved in the metabolism of the azoxy metabolites of procarbazine. Two azoxy procarbazine oxidase activities were resolved by diethylaminoethyl (DEAE)-cellulose chromatography. The activity which did not bind to this column was purified to homogeneity and was identified as a phenobarbital-inducible form of cytosolic aldehyde dehydrogenase. This protein fraction was shown to metabolize only the azoxy 2 procarbazine isomer to yield N-isopropy-p-formylbenzamide (ALD) in a reaction which did not require NAD+ as cofactor. The ALD product formed was also a substrate for a subsequent NAD(+)-dependent reduction reaction catalyzed by that purified protein. The azoxy 2 procarbazine isomer and ALD were shown to be potent inhibitors of both the dehydrogenase and esterase activities of aldehyde dehydrogenase. The second azoxy procarbazine oxidase activity which was retained by the DEAE-cellulose column co-eluted with xanthine oxidase activity. Both the xanthine dehydrogenase/oxidase and azoxy procarbazine oxidase activities of this protein fraction were inhibited by allopurinol, a specific inhibitor of xanthine dehydrogenase. Xanthine dehydrogenase/oxidase was partially purified by an alternative procedure and was shown to metabolize both the azoxy 2 procarbazine isomer and ALD, ultimately producing N-isopropylterephthalamic acid. The ability of xanthine oxidase to metabolize azoxy 2 procarbazine and ALD was confirmed using commercial, purified milk xanthine oxidase.     

In vitro study of 6-mercaptopurine oxidation catalysed by aldehyde oxidase and xanthine oxidase

In spite of over 40 years of clinical use of 6-mercaptopurine, many aspects of complex pharmacology and metabolism of this drug remain unclear. It is thought that 6-mercaptopurine is oxidized to 6-thiouric acid through 6-thioxanthine or 8-oxo-6-mercaptopurine by one of two molybdenum hydroxylases, xanthine oxidase (XO), however, the role of other molybdenum hydroxylase, aldehyde oxidase (AO), in the oxidation of 6-mercaptopurine and possible interactions of AO substrates and inhibitors has not been investigated in more details. In the present study, the role of AO and XO in the oxidation of 6- mercaptopurine has been investigated. 6-mercaptopurine was incubated with bovine milk xanthine oxidase or partially purified guinea pig liver molybdenum hydroxylase fractions in the absence and presence of XO and AO inhibitor/substrates, and the reactions were monitored by spectrophotometric and HPLC methods. According to the results obtained from the inhibition studies, it is more likely that 6- mercaptopurine is oxidized to 6-thiouric acid via 6-thioxanthine rather than 8-oxo-6-mercaptopurine. The first step which is the rate limiting step is catalyzed solely by XO, whereas both XO and AO are involved in the oxidation of 6-thioxanthine to 6-thiouric acid.     

Sensitivity of human leukemic cells to anti-cancer agents in culture

Special-conditioned medium prepared from overgrown cultures of murine leukemic cells was found to support short-term reproduction (24–96 hr) of human leukemic cells in culture. Failure of three preparations to grow in culture may be due to the instability of the special medium when stored for more than 5 days. Leukemic cell preparations from 12 patients had a variable growth response in vitro and also exhibited a different and characteristic sensitivity to anti-cancer agents. The selection of specific anti-cancer agents for treatment of leukemic patients may be possible with the development of this drug-sensitivity test in vitro.     

Role of guinea pig and rabbit hepatic aldehyde oxidase in oxidative in vitro metabolism of cinchona antimalarials.

Cinchona alkaloids (quinine, quinidine, cinchonine, and cinchonidine) were incubated with partially purified aldehyde oxidase from rabbit or guinea pig liver. Reversed-phase HPLC methods were developed to separate the oxidation products from the parent drugs, and the metabolites were identified on the basis of their infrared and mass spectral characteristics. All four alkaloids were oxidized at carbon 2 of the quinoline ring to give the corresponding lactams. In addition, the dihydro contaminants of the cinchona alkaloids were also metabolized by aldehyde oxidase to the 2-quinolone derivatives. Kinetic constants for the oxidation reactions were determined spectrophotometrically and showed that these substrates have a low affinity (KM values of around 10(-5) M) for hepatic aldehyde oxidase, coupled with a relatively low oxidation rate. However, the overall efficiency of the enzyme (Vmax/KM) toward this group of compounds indicates that in vivo biotransformation by aldehyde oxidase will be a significant pathway. Microsomal metabolites were also isolated from quinine and quinidine incubations with rabbit or guinea pig liver fractions. 3-Hydroxyquinine (quinidine) and O-desmethylquinine (quinidine) were identified in microsomal and 10,000g supernatant extracts from quinine and quinidine, respectively. Oxidation of quinine via aldehyde oxidase appeared to be the predominant pathway in rabbit 10,000g fractions, because 2'-quininone was the major metabolite under these conditions with lower concentrations of the microsomal metabolites produced along with a dioxygenated derivative thought to be 3-hydroxy-2'-quininone.     

Potent inhibition of human liver aldehyde oxidase by raloxifene.

The selective estrogen receptor modulator, raloxifene, has been demonstrated as a potent uncompetitive inhibitor of human liver aldehyde oxidase-catalyzed oxidation of phthalazine, vanillin, and nicotine-Delta1'(5')-iminium ion, with K(i) values of 0.87 to 1.4 nM. Inhibition was not time-dependent. Raloxifene has also been shown to be a noncompetitive inhibitor of an aldehyde oxidase-catalyzed reduction reaction of a hydroxamic acid-containing compound, with a K(i) of 51 nM. However, raloxifene had only small effects on xanthine oxidase, an enzyme related to aldehyde oxidase. In addition, several other compounds of the same therapeutic class as raloxifene were examined for their potential to inhibit aldehyde oxidase. However, none were as potent as raloxifene, since IC(50) values were orders of magnitude higher and ranged from 0.29 to 57 micro M. In an examination of analogs of raloxifene, it was shown that the bisphenol structure with a hydrophobic group on the 3-position of the benzthiophene ring system was the most important element that imparts inhibitory potency. The relevance of these data to the mechanistic understanding of aldehyde oxidase catalysis, as well as to the potential for raloxifene to cause drug interactions with agents for which aldehyde oxidase-mediated metabolism is important, such as zaleplon or famciclovir, is discussed.     

Influence of lipophilicity and carboxyl group content on the rate of hydroxylation of methotrexate derivatives by aldehyde oxidase.

The influence of lipophilicity and carboxyl group content on the ability of methotrexate (MTX) derivatives to undergo 7-hydroxylation in vitro by partly purified rabbit hepatic aldehyde oxidase was examined. Addition of two to four gamma-glutamyl residues to the MTX molecule caused a progressive decrease in the rate of hydroxylation associated mainly with a decrease in Vmax rather than an increase in Km. These results suggest that the number of carboxyl groups in the side chain has a relatively small effect on affinity for the enzyme active site, but hinders the formation of product. The catalytic efficiency of hydroxylation of MTX tetraglutamate, estimated from Vmax/Km ratios, was 36-fold lower than that of the monoglutamate. In contrast, when the number of carboxyl groups was decreased to one, as in 4-amino-4-deoxy-N10-methylpteroic acid, N alpha-(4-amino-4-deoxy-N10-methylpteroyl)-L-lysine, and gamma-t-butyl-3'-chloromethotexate, enhanced catalytic efficiency was observed, involving both a decrease in Km and an increase in Vmax. The catalytic efficiency of hydroxylation of these three substrates was 88-, 360- and 2100-fold higher than that of MTX. gamma-t-Butyl-3'-chloromethotrexate was a better substrate than gamma-t-butyl-MTX, demonstrating the strong contribution of a lipophilic Cl atom on the phenyl ring. N alpha-(4-Amino-4-deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine, with two carboxyl groups, showed substrate activity similar to that of MTX. The gamma-t-butyl esters of MTX, 3'-chloromethotrexate, and 3',5'-dichloromethotrexate were compared with the parent acids as inhibitors of the growth of cultured human leukemic lymphoblasts (CEM cells) and an MTX-resistant subline (CEM/MTX) defective in MTX transport and polyglutamylation. Although the esters were less effective than the acids against CEM cells except at high concentrations, they were more effective against CEM/MTX cells. This "collateral sensitivity" of CEM/MTX cells to lipophilic MTX esters is consistent with a decreased ability to take up and utilize reduced folates from the culture medium.     

Characterization of aldehyde oxidase enzyme activity in cryopreserved human hepatocytes

Substrates of aldehyde oxidase (AO), for which human clinical pharmacokinetics are reported, were selected and evaluated in pooled mixed-gender cryopreserved human hepatocytes in an effort to quantitatively characterize AO activity. Estimated hepatic clearance (Cl(h)) for BIBX1382, carbazeran, O?-benzylguanine, zaleplon, and XK-469 using cryopreserved hepatocytes was 18, 17, 12, <4.3, and <4.3 ml · min?1 · kg?1, respectively. The observed metabolic clearance in cryopreserved hepatocytes was confirmed to be a result of AO-mediated metabolism via two approaches. Metabolite identification after incubations in the presence of H?1?O confirmed that the predominant oxidative metabolite was generated by AO, as expected isotope patterns in mass spectra were observed after analysis by high-resolution mass spectrometry. Second, clearance values were efficiently attenuated upon coincubation with hydralazine, an inhibitor of AO. The low exposure after oral doses of BIBX1382 and carbazeran (～5% F) would have been fairly well predicted using simple hepatic extraction (f(h)) values derived from cryopreserved hepatocytes. In addition, the estimated hepatic clearance value for O?-benzylguanine was within ～80% of the observed total clearance in humans after intravenous administration (15 ml · min?1 · kg?1), indicating a reasonable level of quantitative activity from this in vitro system. However, a 3.5-fold underprediction of total clearance was observed for zaleplon, despite the 5-oxo metabolite being clearly observed. These data taken together suggest that the use of cryopreserved hepatocytes may be a practical approach for assessing AO-mediated metabolism in discovery and potentially useful for predicting hepatic clearance of AO substrates.     

Stability of isolated hepatic aldehyde dehydrogenases from rats in vitro

Aldehyde dehydrogenases (ALDH) isolated from livers of adult female SPF Sprague-Dawley rats were rapidly (within hours) inactivated by oxygen (7.8 ppm, from air) and simultaneous exposure to light energy (subdued daylight, 5000 lx; direct sunlight, 66,000 lx) at 22 degrees C. Oxygen withdrawal (e.g. by treatment with nitrogen) and darkness prevented the inactivation. An addition of glutathione, dithiothreitol, nicotinic acid-amide-adenine-dinucleotide (NAD) or its reduced form (NADH) to the ALDH preparations preserved the enzyme activity; the above SH-reagents regenerated an already occurred loss of activity rapidly (within minutes) and almost completely. It is concluded that the hepatic ALDH from rats posses in the active centre two SH-groups in close vicinity which can be oxidized slightly to the intramolecular disulfide and reduced again. The protection against inactivation by NAD (oxidized or reduced) may be afforded by occupation of the cosubstrate binding site of the enzyme.     

Hydralazine: a potent inhibitor of aldehyde oxidase activity in vitro and in vivo

The interaction of the vasodilator, hydralazine, with the molybdenum hydroxylases, aldehyde oxidase and xanthine oxidase has been investigated. A potent progressive inhibition of rabbit liver aldehyde oxidase, in the presence of substrate, by low concentrations of hydralazine (0.1-1 microM) was observed in vitro but no effect was seen with bovine milk xanthine oxidase. This activity was mirrored in vivo when levels of aldehyde oxidase were significantly decreased in rabbits administered hydralazine (10 mg/kg/day for seven days) whereas hepatic xanthine oxidase activity was unaltered by hydralazine treatment. Various metabolites of hydralazine were synthesized but found to be devoid of in vitro inhibitory activity. Aldehyde oxidase prepared from either guinea pig or baboon liver was inhibited in a similar way to that of rabbit liver.     

A quantitative approach to hepatic clearance prediction of metabolism by aldehyde oxidase using custom pooled hepatocytes

We describe the usability of human pooled hepatocytes for non-CYP metabolism evaluation and an in vivo-in vitro correlation analysis for aldehyde oxidase (AO) substrate compounds using pooled hepatocytes. By comparing intrinsic clearance values of 18 compounds primarily metabolized by AO, UDP-glucuronosyltransferase, carbonyl/aldo-keto reductase, flavin-containing monooxygenase, and monoamineoxidase in individual hepatocytes and pooled hepatocytes from the same individual donors, intrinsic clearance in the pooled hepatocytes was ± 30% of the average clearance value in individuals for 15 of 18 compounds, suggesting that pooled hepatocytes maintained the average activity of the individual hepatocytes. Although the results of an in vivo-in vitro correlation analysis for AO substrate compounds showed a trend toward under-prediction, the underestimation ratios for all AO substrates were nevertheless comparable (7.2- to 14.9-fold), suggesting that hepatic clearance prediction for these compounds can be quantified using empirical scaling. These observations enabled us to obtain specific pooled hepatocytes that showed the expected non-CYP enzyme activities by pre-characterization and to quantify hepatic clearance prediction for AO compounds using an empirical scaling factor.     

The participation of human hepatic P450 isoforms, flavin-containing monooxygenases and aldehyde oxidase in the biotransformation of the insecticide fenthion

Although fenthion (FEN) is widely used as a broad spectrum insecticide on various crops in many countries, very scant data are available on its biotransformation in humans. In this study the in vitro human hepatic FEN biotransformation was characterized, identifying the relative contributions of cytochrome P450 (CYPs) and/or flavin-containing monooxygenase (FMOs) by using single c-DNA expressed human enzymes, human liver microsomes and cytosol and CYP/FMO-specific inhibitors.Two major metabolites, FEN-sulfoxide and FEN-oxon (FOX), are formed by some CYPs although at very different levels, depending on the relative CYP hepatic content. Formation of further oxidation products and the reduction of FEN-sulfoxide back to FEN by the cytosolic aldehyde oxidase enzyme were ruled out. Comparing intrinsic clearance values, FOX formation seemed to be favored and at low FEN concentrations CYP2B6 and 1A2 are mainly involved in its formation. At higher levels, a more widespread CYP involvement was evident, as in the case of FEN-sulfoxide, although a higher efficiency of CYP2C family was suggested.Hepatic FMOs were able to catalyze only sulfoxide formation, but at low FEN concentrations hepatic FEN sulfoxidation is predominantly P450-driven. Indeed, the contribution of the hepatic isoforms FMO₃ and FMO₅ was generally negligible, although at high FEN concentrations FMO's showed activities comparable to the active CYPs, accounting for up to 30% of total sulfoxidation. Recombinant FMO₁ showed the highest efficiency with respect to CYPs and the other FMOs, but it is not expressed in the adult human liver. This suggests that FMO₁-catalysed sulfoxidation may represent the major extra-hepatic pathway of FEN biotransformation.