Studies on the metabolism of aristolochic acids I and II

1. After oral administration of aristolochic acid I (AAI) and aristolochic acid II (AAII) to rats, the following metabolites were isolated from the urine and their structures elucidated: aristolactam I, aristolactam Ia, aristolochic acid Ia, aristolic acid I and 3, 4-methylenedioxy-8-hydroxy-1-phenanthrenecarboxylic acid (metabolites of AAI); or aristolactam Ia, aristolactam II and 3,4-methylenedioxy-1-phenanthrenecarboxylic acid (metabolites of AAII). A further metabolite of AAII having a lactam structure has not yet been isolated in pure form.

2. The metabolic pathways have been elucidated by administration of various metabolites.

3. The principal metabolite of AAI in rats was aristolactam Ia; 46% of the dose was excreted in the urine in form of this metabolite and 37% in the faeces. The other substances were minor metabolites. Those metabolites of AAII whose structures have been elucidated were minor metabolites; the largest proportion consisted of aristolactam II, which accounted for 4.6% in the urine and 8.9% in the faeces.

4. The mouse was the only animal which had the same metabolite patterns of AAI and AAII as those found in the rat. Not all the metabolites listed above were found in urine from guinea pigs, rabbits, dogs and man.     

Aristolochic acid I metabolism in the isolated perfused rat kidney

Aristolochic acids are natural nitro-compounds found globally in the plant genus Aristolochia that have been implicated in the severe illness in humans termed aristolochic acid nephropathy (AAN). Aristolochic acids undergo nitroreduction, among other metabolic reactions, and active intermediates arise that are carcinogenic. Previous experiments with rats showed that aristolochic acid I (AA-I), after oral administration or injection, is subjected to detoxication reactions to give aristolochic acid Ia, aristolactam Ia, aristolactam I, and their glucuronide and sulfate conjugates that can be found in urine and feces. Results obtained with whole rats do not clearly define the role of liver and kidney in such metabolic transformation. In this study, in order to determine the specific role of the kidney on the renal disposition of AA-I and to study the biotransformations suffered by AA-I in this organ, isolated kidneys of rats were perfused with AA-I. AA-I and metabolite concentrations were determined in perfusates and urine using HPLC procedures. The isolated perfused rat kidney model showed that AA-I distributes rapidly and extensively in kidney tissues by uptake from the peritubular capillaries and the tubules. It was also established that the kidney is able to metabolize AA-I into aristolochic acid Ia, aristolochic acid Ia O-sulfate, aristolactam Ia, aristolactam I, and aristolactam Ia O-glucuronide. Rapid demethylation and sulfation of AA-I in the kidney generate aristolochic acid Ia and its sulfate conjugate that are voided to the urine. Reduction reactions to give the aristolactam metabolites occur to a slower rate. Renal clearances showed that filtered AA-I is reabsorbed at the tubules, whereas the metabolites are secreted. The unconjugated metabolites produced in the renal tissues are transported to both urine and perfusate, whereas the conjugated metabolites are almost exclusively secreted to the urine.     

Investigation of the metabolism and reductive activation of carcinogenic aristolochic acids in rats.

The metabolic activation of aristolochic acids (AAs) that have been demonstrated to be mutagenic and carcinogenic was investigated. In vitro metabolism study indicated that AAs were metabolized to N-hydroxyaristolactam, which could be either reduced to aristolactams or rearranged to 7-hydroxyaristolactams via the Bamberger rearrangement. In vivo metabolism study is important because the intermediates (aristolactam-nitriumion) of the nitroreduction process are thought to be responsible for the carcinogenicity of AAs. Liquid chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry (MS/MS) were applied to the analyses of a series of positional isomers of hydroxyaristolactams in rat urine samples after the in vivo study of AAs. Three hydroxylated metabolites of aristolactam II and two hydroxylated metabolites of aristolactam I were identified. The structures of the positional isomers were elucidated from the interpretation of MS/MS spectra and theoretical calculations. In addition, several new metabolites were detected in the rat urine by high-resolution mass spectrometry and MS/MS, including those from the decarboxylation of AAs and the conjugations of acetylation, glucuronidation, and sulfation of aristolochic acid Ia.     

N6-adenyl arylation of DNA by aristolochic acid II and a synthetic model for the putative proximate carcinogen.

Aristolochic acid II (AAII), one of the major components of the carcinogenic plant extract aristolochic acid, is known to be mutagenic and to form DNA adducts in vitro and in vivo. The major fluorescent DNA adduct formed upon xanthine oxidase mediated reduction in the presence of calf thymus (CT-) DNA or deoxyadenosine was isolated by means of preparative HPLC and identified by fluorescence, UV/vis absorbance, and 1H NMR spectroscopy as 7-(deoxy-adenosin-N6-yl)aristolactam II. As a model proximate carcinogen, N-chloroaristolactam II was prepared chemically from aristolactam II, the reduction product of AAII. This model compound was spectroscopically characterized and found to react directly with CT-DNA without any activation, forming the same deoxyadenosine adduct. HPLC analysis with fluorescence monitoring detected this adduct in vivo in the liver DNA of Wistar rats treated orally with AAII. These results confirm the anticipated metabolic activation mechanism of AAII as occurring via a cyclic nitrenium ion.     

Differential cytotoxic effects of denitroaristolochic acid II and aristolochic acids on renal epithelial cells

Aristolochic acids (AAs), naturally occurring nephrotoxins and rodent carcinogens, are commonly found in medicinal plants such as Radix aristolochiae. The toxicity of AAs is believed to be associated with the formation of promutagenic AA-DNA adducts, and it has also been suggested that the nitro group in AAs might be important in the process. In order to investigate the role of the nitro group in AA-mediated cytotoxicity, the effects of denitroaristolochic acid II (dN-AAII), aristolochic acid II (AAII) and aristolochic acid I (AAI) on renal tubular epithelial Madin–Darby canine kidney (MDCK) cells were examined and compared. The cytotoxicity of AAI, AAII and dN-AAII was found to be time- and concentration-dependent. As determined by MTT assay, AAI was found to be most toxic in MDCK cells upon exposure for 24, 48 and 72 h, followed by AAII, and dN-AAII showed the least cytotoxicity. The effect of AAI and AAII on the integrity of cell membrane was found to be similar and appeared to be more prominent than that of dN-AAII. Based on the results obtained from the flow cytometric analysis, significant apoptosis in MDCK cells was observed with AAI and AAII at as low as 25 μmol/L following exposure for 24 h, whereas significant apoptosis was induced by dN-AAII at a much higher concentration, 300 μmol/L, suggesting that both AAI and AAII were significantly more cytotoxic than dN-AAII. In addition, the levels of reactive oxygen species (ROS) were increased following treatment with AAI, AAII and dN-AAII at concentrations of 5, 25 and 25 μmol/L, respectively, for 4 h. The results suggest that the nitro group plays an important role in AA-mediated cytotoxicity in MDCK cells and increased intracellular ROS levels may be associated, at least in part, with the cell injury observed in MDCK cells.     

Metabolism of the calcium antagonist gallopamil in man

The metabolism of gallopamil (5-[(3,4-dimethoxyphenyl)methylamino]-2-(3,4,5-trimethoxyphenyl) -2- isopropylvaleronitrile hydrochloride, Procorum, G) was studied after single administration (2 mg i.v., 50 mg p.o.) of unlabelled and labelled G (14G, 2H). TLC, HPLC, GLC, MS and RIA were used for assessment of G and its metabolites in plasma, urine and faeces. G clearance is almost completely metabolic, with only minimal excretion of unchanged drug. Metabolites represent most of the plasma radioactivity after p.o. administration. They are formed by N-dealkylation and O-demethylation with subsequent N-formylation, or glucuronidation, respectively. Compound A, derived by loss of the 3,4-dimethoxyphenethyl moiety of G is the main metabolite in plasma and urine (about 20% of the dose). This metabolite is accompanied by its N-formyl derivative (C), by the N-demethylated compound (H) and the acid (F), formed by oxidative deamination of A. Only 3 unconjugated monphenoles from several O-demthylated products showed distinct plasma levels which were nevertheless lower than metabolite A. These metabolites had no relevance to the pharmacodynamic action. Conjugated monophenolic and diphenolic products represented the major part in plasma and were excreted predominantly via the bile: they represented almost the whole faecal metabolite fraction. Less than 1% of the dose was recovered unchanged in the urine. About 50% of the dose is excreted by urine and 40% by faeces.     

Ergosta-4,6,8(14),22-tetraen-3-one isolated from Polyporus umbellatus prevents early renal injury in aristolochic acid-induced nephropathy rats

Objectives Aristolochic acid (AA) nephropathy, first reported as Chinese herbs nephropathy, is a rapidly progressive tubulointerstitial nephropathy that results in severe anemia, interstitial fibrosis and end‐stage renal disease. Tubulointerstitial injury was studied in a rat model of AA nephropathy to determine whether ergosta‐4,6,8(14),22‐tetraen‐3‐one (ergone) treatment prevents early renal injury in rats with aristolochic acid I‐induced nephropathy. Methods Early renal injury via renal interstitial fibrosis was induced in rats by administration of aristolochic acid I (AAI) solution intragastrically for 8 weeks. Ninety‐six rats were randomly divided into four groups (n = 24/group): (1) control (2) AAI (3) AAI + ergone (10 mg/kg) and (4) AAI + ergone (20 mg/kg). Blood and urine samples were collected and rat were sacrificed for histological assessment of the kidneys on at the end of weeks 2, 4, 6 and 8. Key findings AAI caused progressive elevation of blood urea nitrogen, creatinine, potassium, sodium, chlorine, proteinuria and urinary N‐acetyl‐β‐D‐glucosaminidase (NAG). Ergone suppressed elevation of blood urea, nitrogen, creatinine, proteinuria and urinary NAG to some degree, but the AAI–ergone‐treated group did not differ from AAI‐treated group for body weight, serum potassium, sodium and chlorine. The progress of the lesions in the kidney after AAI administration was also observed by histopathological examinations, but kidneys from rats of AAI–ergone‐treated group displayed fewer lesions. Conclusions Ergone treatment conferred protection against early renal injury in a rat model of AA nephropathy. Early administration of ergone may prevent the progression of renal injury and the subsequent renal fibrosis in AA nephropathy.     

Acute nephrotoxicity of aristolochic acids in mice

Aristolochic acids (AA), present in Aristolochia plants, are the toxin responsible for Chinese herbs nephropathy (CHN), a rapidly progressive tubulointerstitial nephritis (TIN). To clarify the mechanisms of the development of CHN, we tried to induce TIN in mice using AA. Three strains of inbred mice, BALB/c, C3H/He and C57BL/6, received 2.5 mg kg(-1) of AA or AA sodium salt (AANa) daily by intraperitoneal or oral administration, 5 days a week for 2 weeks. Serum and renal tissue were obtained at sacrifice. Twelve-hour urine samples were individually collected in a metabolic cage at one-week intervals. In the AA-injected groups, severe tubular injury, with the appearance of acute tubular necrosis, and rare cell infiltration into the interstitium, were seen in BALB/c mice. C3H/He mice also developed TIN with prominent cell infiltration into the interstitium and interstitial fibrosis. In C57BL/6 mice, only mild and focal tubulointerstitial changes were seen. Serum creatinine and blood urea nitrogen increased in BALB/c and C3H/He mice. Immunofluorescent study revealed no deposition of immune components in kidneys. In the AANa-treated groups, TIN was also seen in all groups, but even more severe tubulointerstitial changes were induced by intraperitoneal injection. Further examination using purified AAI, AAII, AAIVa and aristolactam I (ALI) revealed that AAI induced strong nephrotoxicity in mice, and that AAII resulted in mild nephrotoxicity. However, AAIVa and ALI caused no nephrotoxicity in this experimental system. There are strain differences in mice in their susceptibility to AA nephropathy. AAI exerted the strongest nephrotoxic effect in mice.     

Metabolism of phenylbutazone in rats

The metabolism of phenylbutazone has been investigated in female rats dosed with the drug by gavage. The major route of excretion is via the urine; 50% of the dose being excreted in the first 24 h. A small percentage of the dose is excreted in the faeces. Following administration of 14C-phenylbutazone, five labelled, unconjugated hydroxy compounds were identified in the urine by t.l.c. and autoradiography; both hydrolysable and non-hydrolysable conjugates were found. Aqueous extracts of faeces contained O conjugates of oxyphenbutazone and 4-hydroxy-oxyphenbutazone (which may be a decomposition product). Urine metabolites soluble in organic solvents were quantified by inverse isotope dilution assay and spectrophotometric analysis. The major metabolite is the gamma-hydroxy derivative of phenylbutazone present both as the lactone and as the straight-chain compound, while oxyphenbutazone and p, gamma-dihydroxyphenylbutazone are minor metabolites.     

Predicting points of departure for risk assessment based on in vitro cytotoxicity data and physiologically based kinetic (PBK) modeling: The case of kidney toxicity induced by aristolochic acid I

Aristolochic acids are naturally occurring nephrotoxins. This study aims to investigate whether physiologically based kinetic (PBK) model-based reverse dosimetry could convert in vitro concentration-response curves of aristolochic acid I (AAI) to in vivo dose response-curves for nephrotoxicity in rat, mouse and human. To achieve this extrapolation, PBK models were developed for AAI in these different species. Subsequently, concentration-response curves obtained from in vitro cytotoxicity models were translated to in vivo dose–response curves using PBK model-based reverse dosimetry. From the predicted in vivo dose–response curves, points of departure (PODs) for risk assessment could be derived. The PBK models elucidated species differences in the kinetics of AAI with the overall catalytic efficiency for metabolic conversion of AAI to aristolochic acid Ia (AAIa) being 2-fold higher for rat and 64-fold higher for mouse than human. Results show that the predicted PODs generally fall within the range of PODs derived from the available in vivo studies. This study provides proof of principle for a new method to predict a POD for in vivo nephrotoxicity by integrating in vitro toxicity testing with in silico PBK model-based reverse dosimetry.     

Metabolism of thymoxamine. II. Studies with 14C-thymoxamine in man

Thymoxamine is rapidly and completely absorbed in man. Rapid biotransformation is observed after intravenous and oral administration of 40 mg 14C-thymoxamine HCl. No unchanged compound is found in the body. More than 90% of plasma and urine radioactivity could be ascribed to six metabolites: the desacetyl compound (metabolite I), the monodemethylated metabolite I (metabolite II), the sulfate conjugates of I and II (metabolites III and IV) and the glucuronides of I and II (metabolites V and VI). The unconjugated metabolites are observed in plasma only after intravenous administration. Similar patterns for polar metabolites are found in plasma and urine for both routes of administration. The sulfate fraction amounts to about 50-60% and the glucuronide fraction to about 30-40% of the radioactivity, the conjugates of metabolite I being more abundant than those of metabolite II. The elimination of the metabolites is rapid, the half-life of radioactivity elimination being 1.5 h during the first 12 hours and 12 h thereafter. 80% of the radioactivity dose is recovered in the urine within 4 hours. Recovery after four days amounts to 99.8% (i.v.) and 97.7% (oral). The results are discussed with regard to the application of the drug in man, taking into account that not only the unconjugated metabolites but also the sulfate conjugates are pharmacologically active.     

Metabolism of thymoxamine. I. Studies with 14C-thymoxamine in rats

Thymoxamine is rapidly and completely absorbed in rats. It is a prodrug which does not enter the systemic circulation in its unchanged form. After either oral or intravenous administration it undergoes rapid and intense metabolism involving four biotransformation reactions: Enzymatic hydrolysis to the corresponding phenol (metabolite I), Monodemethylation to metabolite II, Sulfate conjugation of I and II (metabolites III and IV) and Conjugation of I and II with glucuronic acid (metabolites V and VI). With these 6 metabolites identified approximately 95% of the radioactivity can be accounted for in plasma, urine and bile. Whereas the systemic availability of I and II is low, III and IV show high bioavailability. Metabolites I to IV are pharmacologically active, while III and IV are less potent than I and II. The radioactivity distribution in tissues is different after oral and intravenous administration consistent with the higher portion of unconjugated metabolites in the body after administration by parenteral route. Although 60% of the labelled compounds is eliminated via bile, the radioactive compounds are almost completely excreted in the urine after both routes of administration. This demonstrates complete reabsorption of the biliary metabolites. Secondary peaks of radioactivity in plasma and organs at 4 hours are explained by the participation of the metabolites in the enterohepatic circulation.     

Metabolism and excretion of imrecoxib in rat

The metabolism and excretion of imrecoxib, a novel and moderately selective cyclooxygenase-II inhibitor, were investigated in rat. The structures of metabolites were identified by mass spectrometry (MSn) and nuclear magnetic resonance. Metabolic profiles of imrecoxib in urine, bile and faeces were obtained by HPLC and LC/MSn, and cumulative excretion was determined by LC/MSn. Imrecoxib was extensively metabolized in rat after intravenous administration, with less than 2% of the dose excreted as parent drug in either urine or faeces. The major metabolic pathway was that the 4'-methyl group of imrecoxib was first oxidized to the 4'-hydroxymethyl metabolite (M4), followed by additional oxidation to 4'-carboxylic acid metabolite (M2). The dihydroxylated metabolite, 4'-hydroxymethyl-5-hydroxyl imrecoxib (M3), was further oxidized to 4'-hydroxymethyl-5-carbonyl metabolite (M5), and glucuronide conjugates of M2-4 were formed. After intravenous (5 mg kg-1) administration, the majority of the dose was recovered in the faeces. The dose was primarily excreted as the carboxylic acid metabolite in addition to the 4'-hydroxymethyl metabolite. The carboxylic acid metabolite was mainly excreted in faeces, while the 4'-hydroxymethyl metabolite was mainly excreted in urine.     

Excretion and metabolism of lorcainide in rats, dogs and man

After p.o. or i.v. administration of 3H-lorcainide, excretion of the radioactivity was almost complete within four days. In rats and dogs, about 35% of the dose was excreted in the urine and about 60% in the faeces. However, in humans, 62% was excreted in the urine and 35% in the faeces. In rats, about 70% of the orally administered radioactivity was excreted in the bile within 24 hours. Enterohepatic circulation was proven by "donor-acceptor" coupling in rats. Lorcainide was extensively metabolized. Urinary and faecal metabolites were isolated by extraction and high pressure liquid chromatography (HPLC), and characterized by chromatographic comparison with reference compounds, by mass spectrometry, and NMR. The mass balance for unchanged lorcainide and its major metabolites (determined by radio-HPLC) was very similar in the urine and faeces. Only minor quantitative differences were observed between intravenously and orally dosed animals, and between male and female rats. Major biotransformation pathways in the three species were: hydroxylation, O-methylation and glucuronidation. 4-Hydroxy-3-methoxy-lorcainide was the main metabolite. alpha-Oxidation resulting in alpha, 4-dihydroxy-3-methoxy-lorcainide, was observed in dogs only. Minor pathways were: oxidative N-dealkylation and amide hydrolysis. A remarkable 5-hydroxy-3,4-dimethoxy-metabolite was identified unambiguously in the three species.     

Biotransformation of BOF-4272, a sulfoxide-containing drug, in the cynomolgus monkey.

BOF-4272, (+/-)-8-(3-methoxy-4-phenylsulfinylphenyl) pyrazolo[1,5-a]-1,3,5-triazine-4(1H)-one), is a new drug intended for the treatment of hyperuricemia. This report describes the pharmacokinetics and detailed metabolic pathways of BOF-4272 in the cynomolgus monkey, which were investigated using the metabolites found in plasma, urine, and faeces after intravenous and oral administration. M-4 was the main metabolite in plasma after intravenous administration. M-3 and M-4 were the main metabolites in plasma after oral administration. The Cmax and AUC(0-t) of M-4 were the highest of all the metabolites after intravenous administration. The Cmax and AUC(0-t) of M-3 were the highest of all the metabolites, and those of M-4 were the second highest, after oral administration. M-4 and M-3 were the main metabolites detected in urine and faeces, respectively, after intravenous administration, with M-4 and M-3 at 47.2% in urine and 19.1% in faeces, respectively, within 120 h after administration. M-4 was the only metabolite detected in urine after oral administration, at about 5% within 120 h after administration. M-3 was detected in faeces at 17.0% within 120 h after oral administration. These results suggest that, in the cynomolgus monkey, BOF-4272 is rapidly biotransformed to a main metabolite (M-4, a sulphoxide-containing metabolite of BOF-4272) and that M-4 is mainly excreted in urine and possibly also in bile, with subsequent conversion to M-3 by the intestinal flora. It is expected that the biotransformation of BOF-4272 would be similar in healthy human volunteers.     

Studies on drug metabolism by use of isotopes XXVII: urinary metabolites of rutin in rats and the role of intestinal microflora in the metabolism of rutin

Analysis of urinary metabolites of orally administered rutin (I) labeled with deuterium [( 2',5',6'-2H]rutin, rutin-d) was carried out by GLC-MS. In rat urine, 3-hydroxyphenylacetic acid (III), 3-methoxy-4-hydroxyphenylacetic acid (IV), 3,4-dihydroxyphenylacetic acid (V), 3,4-dihydroxytoluene (VI), and 3-(m-hydroxyphenyl)propionic acid (VIII) were identified as rutin metabolites and were differentiated from the corresponding endogeneous compounds. Unchanged I and quercetin (II) were not present in the urine. Rutin-d was injected intraperitoneally in rats, administered orally to neomycin-treated rats, and incubated in vitro with the intestinal contents of rats. The experiments suggested the involvement of intestinal microflora in the metabolism of orally administered I.     

HPLC-ESI/ITMSn法分析大鼠体内4-甲基哌嗪-1-二硫代甲酸-(3-氰基-3，3-二苯基)丙酯盐酸盐及其主要代谢物

目的研究新化合物4-甲基哌嗪-1-二硫代甲酸-(3-氰基-3，3-二苯基)丙酯盐酸盐(TM208)在大鼠体内的主要代谢产物。方法大鼠ig TM208 500 mg·kg^-1后，收集粪样、尿样和血样，用液相色谱-电喷雾离子阱质谱法测定。根据TM208及其代谢产物的色谱保留时间和电喷雾离子阱质谱(ESI-ITMSⁿ )电离规律及生物体内药物代谢转化规律，推导代谢物的结构。结果在粪样中发现8种I相代谢产物，在尿样和血样中发现5种I相代谢产物，未发现II相代谢产物。结论本法操作简便、快速、灵敏度高、专属性强，是一种研究TM208体内代谢产物的有效方法。     

Metabolism of the thiocarbamate herbicide SUTAN® in rats

1. The metabolism of the thiocarbamate herbicide SUTAN° (butylate) was studied after administration of single oral doses of [isobutyl-1-¹⁴C]SUTAN to male and female rats.

2. The radiolabelled dose was rapidly absorbed and excreted, with 79% of the dose excreted in the urine in 72?h. The small percentages of radioactivity excreted in the faeces and as ¹⁴CO₂ were significantly higher (P≤0.05) in males than in females.

3. SUTAN was extensively metabolized, and no unmetabolized SUTAN was found in the urine. A total of 18 of the 29 urinary metabolites were identified, and identified metabolites represented 83–88% of the urinary radioactivity.

4. Diisobutylamine was the major urinary metabolite in both males and females, averaging 51% of the urinary radioactivity.

5. Other significant urinary metabolites included primary hydroxylated and tertiary hydroxylated diisobutylamines and a series of mercapturic acid pathway metabolites, including an S-glucuronide and several hydroxylated and unhydroxylated mercapturates.

6. Oxidations at the three alkyl groups produced a variety of minor urinary metabolites, and hydroxylation of the primary or tertiary carbon on the isobutyl groups, followed by an intramolecular reaction, generated a series of minor cyclized metabolites.     

The metabolism of benzyclane [1-benzyl-1-(3-N,N-dimethylaminopropoxy)cycloheptane] in rat and man.

1. Two metabolites, isolated from the urine of rats dosed with bencyclane fumarate, were characterized as cis-1-benzyl-1-(3-N,N-dimethylaminopropoxy)-4-hydroxycycloheptane (metabolite I) and 1-benzyl-1-(3-N,N-dimethylaminopropoxy)-4-oxocycloheptane (metabolite II). 2. Bencyclane and the two metabolites were determined in the urine of rats and volunteers by g.l.c. Metabolite I was a major metabolite in men, being excreted in urine to the extent of 23.5% dose in the first 24 h.