3-Hydroxymethyl-s-triazolo[3,4-a]phthalazine, a novel urinary hydralazine metabolite in man.

The elucidation of the structure of a new major metabolic product of hydralazine, 3-hydroxymethyl-s-triazolo[3,4-a]-phthalazine, is described. The structures of several other previously described metabolites of the drug, phthalazone, s-triazolo[3,4-a]phthalazine, and 3-methyl-s-triazolo[3,4-a]phthalazine, are confirmed. A metabolic pathway of hydralazine is also proposed.     

The in vitro metabolism of hydralazine

Hydralazine was metabolized in vitro to phthalazine and phthalazine-1-one by microsomal enzymes requiring NADPH. Hydrazine was not detected as a metabolite under these in vitro conditions. Addition of 1-hydrazinophthalazin-4-one, a possible intermediate metabolite, decreased covalent binding to protein but also decreased metabolism. Phthalazine and phthalazin-1-one also decreased covalent binding to protein to a lesser extent. N-Acetylcysteine significantly decreased the level of phthalazine and phthalazin-1-one detected in microsomal incubations. The results are consistent with a reactive intermediate, but not 1-hydrazinophthalazin-4-one, being responsible for both covalent binding and production of phthalazine.     

High-performance liquid chromatographic studies of reaction of hydralazine with biogenic aldehydes and ketones

To understand hydrazone formation in hydralazine metabolism, the reaction of hydralazine with various biogenic aldehydes and ketones (acetone, pyruvic acid, acetoacetic acid, formaldehyde, and acetaldehyde) in pH 7.4 buffer was studied for potential alterations in hydralazine pharmacokinetics secondary to alcoholism and diabetes. The corresponding hydrazones were isolated, and their structures were characterized. High-performance liquid chromatography was used to monitor the reactions. An aqueous solvent reversed-phase liquid chromatographic system was used to separate hydralazine and its derivatives. Reaction of hydralazine with formaldehyde or acetaldehyde produced the corresponding hydrazones. Formation of an s-triazolo ring system yielded the known s-triazolo[3,4-alpha]phthalazine and 3-methyl-s-triazolo[3,4-alpha]phthalazine metabolites, which also were isolated and characterized and suggested nonenzymatic metabolism.     

Metabolic activation of hydralazine by rat liver microsomes

There is evidence to suggest that the oxidative metabolism of hydralazine (HP), an antihypertensive drug, may represent a toxic pathway which could account for some of the adverse effects of the drug. Experiments were done to determine whether the hepatic oxidative metabolism of HP is associated with the formation of reactive metabolites. In the presence of NADPH, HP was metabolized by rat liver microsomes to three major oxidation products, phthalazine, phthalazinone (PZ), and a dimer compound. Under similar incubation conditions, radioactivity derived from [14C]HP was covalently bound to microsomal protein. Metabolite formation and covalent binding increased following pretreatment of rats with phenobarbital. In contrast, pretreatment with 3-methylcholanthrene or with the monooxygenase inhibitor, piperonyl butoxide, slightly decreased both metabolite formation and covalent binding. Electron spin resonance (ESR) analyses indicated that nitrogen-centered radicals were formed when rat liver microsomes were incubated with HP under conditions similar to those required for covalent binding and for the production of the oxidative metabolites. In addition, reduced glutathione (GSH) caused concentration-dependent decreases in the production of phthalazine, PZ, and the dimer, in the covalent binding of HP to microsomal protein, and in the formation of nitrogen-centered radicals. The results of these investigations indicate that the oxidative metabolism of HP by rat liver microsomes is highly correlated with the formation of nitrogen-centered radicals and the production of metabolites that become covalently bound to microsomal protein. These observations support the hypothesis that the oxidation of HP generates reactive metabolites which may contribute to the toxicity of the drug.     

Synthesis, formulation, and clinical pharmacological evaluation of hydralazine pyruvic acid hydrazone in two healthy volunteers

Hydralazine pyruvic acid hydrazone [2-(phthalazin-1-yl hydrazono)propionic acid; 1] is a major plasma metabolite of hydralazine in humans. A number of in vitro and animal studies have suggested that this hydrazone may have cardiovascular activity and could account for the prolonged antihypertensive effect of hydralazine in humans in the absence of detectable plasma levels of the parent drug. To study this possibility, the soluble sodium salt of hydralazine pyruvic acid hydrazone (2) was synthesized, its chemical purity and stability was checked, and an intravenous formulation was prepared. Isomeric forms were identified. Doses of 0.3, 0.6, and 1.1 mumol/kg of 2 were administered intravenously to one slow and one heterozygous fast acetylator of sulfamethazine. The slow acetylator received two additional doses of 0.06 and 0.14 mumol/kg. Peak plasma levels of 1 of 18 mumol/L were attained without tachycardia or hypotension in either subject. There was no evidence of nonlinearity in kinetics over the dose range studied and clearance remained constant in both subjects (0.517 +/- 0.033 mL/min/kg in the slow acetylator and 0.744 +/- 0.058 mL/min/kg in the fast acetylator). The distribution of 1 varied unpredictably with dose, and changes were reflected in the terminal half-life (3.47-5.97 h in the slow acetylator and 2.06-5.33 h in the fast acetylator). Only traces of the acetylated metabolite of hydralazine, 3-methyl-s-triazolo[3,4-a]phthalazine (3), were detected in the plasma of the subjects, suggesting that significant metabolism via this route was unlikely. An established and specific assay for hydralazine was further modified to allow measurement of levels as low as 1 nmol/L (0.2 ng/mL).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of inducers and inhibitors of mixed-function oxidases on the biliary excretion of pentacaine metabolites

The biliary excretion of 3H-pentacaine and its metabolites was studied in rats pretreated with an inducer or inhibitor of mixed-function oxidases. Over one-fourth (25.8 per cent) of a 2 mg kg-1 intraportal dose of 3H-pentacaine was excreted in bile in urethaneanaesthetized control rats within 3 h. The radioactivity appeared in the form of the parent drug, basic metabolites, and metabolite conjugates, 3.1, 86.5, and 10.4 per cent of the total radioactivity excreted, respectively. Pretreatment of rats with phenobarbital enhanced only slightly the biliary excretion of basic metabolites, and pretreatment with 3-methylcholanthrene had no effect. Phenobarbital also increased the initial rate of excretion of conjugates, but this effect was not sustained. 3-Methylcholanthrene had a tendency to impair excretion of conjugates by bile. Pretreatment of rats with SKF 525-A decreased the biliary excretion of both basic metabolites and conjugates while cimetidine did not alter significantly the biliary excretion of pentacaine metabolites. These results suggest that the canalicular transport of metabolites may be the most important factor in controlling pentacaine metabolite excretion in bile.     

Interference in assays for hydralazine in humans by a major plasma metabolite, hydralazine pyruvic acid hydrazone.

The present study showed that published spectrophotometric and GLC methods for hydralazine in plasma do not distinguish between the drug and a major plasma metabolite, hydralazine pyruvic acid hydrazone. These methods involve the acid treatment of the sample, which hydrolyzes that hydrazone back to hydralazine. A specific GLC assay for the hydrazone was developed and involves its selective extraction from plasma and transformation to 3-trifluoromethyl-s-triazolo[3,4-a]phthalazine. This derivative could be sensitively measured by GLC using an electron-capture detector. With this procedure, it was shown that most "apparent hydralazine" in plasma is the hydrazone, which forms rapidly from hydralazine and endogenous pyruvic acid. Previous work indicated that the hydrazone was inactive when administered intravenously to rabbits.     

Further evidence for an acetylator phenotype difference in the metabolism of hydralazine in man.

1 The 0-24 h urine from hypertensive patients treated with hydralazine (100 mg twice daily) has been analysed by gas chromatography and high pressure liquid chromatography. 2 4-N-Acetylhydrazinophthalazine-1-one (NAcHPZ), s-triazolo [3, 4-a] phthalazine (TP), phthalazinone (PZ) and hydralazine (free, H; acid-labile hydrazones, HH) were detected and assayed. 3 The results indicate that slow acetylators excrete less NAcHPZ and TP than rapid acetylators but more PZ and HH. 4 Free hydralazine was present in low levels and was only detected in some urine samples. 5 The ratios of the metabolites NAcHPZ/HH; TP/HH; NAcHPZ/PZ and PZ/TP are different in the two acetylator phenotypes. 6 It is possible the ratio PZ/TP may be used for determination of acetylator phenotype. 7 It is concluded that hydralazine metabolism is dependent on the acetylator phenotype.     

The effects of an experimental hepatitis on the metabolic disposition of 3-o-(+)-[14C]methylcatechin in the rat

The induction of an experimental hepatitis did not affect the overall ability of the rat to metabolize the flavanol 3-O-(+)-[14C]methylcatechin by methylation or glucuronidation. The induction of hepatitis did cause a significant increase in metabolite excretion in urine (from 52% of the dose in control rats to 88% in hepatitis). Fecal excretion was correspondingly depressed (44 to 4% of the dose). In bile duct-cannulated rats, the induction of hepatitis prior to 3-O-(+)-[14C]methylcatechin administration resulted in low 14C excretion (38%) in bile (cf. 58% in bile of controls). The data obtained indicate that following induction of hepatitis biliary metabolites reabsorbed from the intestine are not reexcreted in bile in an enterohepatic cycle as in the normal rat but are excreted via the kidney. Induction of hepatitis did not affect the fast clearance of unchanged 3-O-methyl-(+)-catechin from plasma but plasma clearance of the metabolites was reduced from 112 to 89 ml/hr.     

Effect of inducers of cytochrome P-450 on the metabolism of [3-14C]coumarin by rat hepatic microsomes

1. The metabolism of [3-14C]coumarin has been studied in rat hepatic microsomes and with two purified cytochrome P-450 isoenzymes. 2. [3-14C]Coumarin was converted by liver microsomes to several polar products including 3- and/or 5-hydroxycoumarin, omicron-hydroxyphenylacetic acid and a major unidentified novel coumarin metabolite. 3. [3-14C]Coumarin was also converted to reactive metabolite(s) as indicated by covalent binding to proteins, and by the depletion of reduced glutathione added to the microsomal incubations. 4. [3-14C]Coumarin metabolism to polar and covalently bound metabolites by rat liver microsomes was induced by pretreatment with phenobarbitone, 3-methylcholanthrene, beta-naphthoflavone, Aroclor 1254 and isosafrole; but not by dexamethasone or nafenopin. 5. The profile of [3-14C]coumarin metabolism to polar products was similar in control and pretreated liver microsomes and in incubations with purified cytochrome P450 IA1 and P450 IIB1 isoenzymes. 6. The results indicate that coumarin is a substrate for isoenzymes of the cytochrome P450 IA and P450 IIB subfamilies. The bioactivation of coumarin by rat hepatic microsomes is postulated to result in the formation of a coumarin 3,4-epoxide intermediate which may rearrange to 3-hydroxycoumarin, be further metabolized to a coumarin 3,4-dihydrodiol, or form a glutathione conjugate.     

Kinetic studies of hydralazine reaction with acetaldehyde

In vitro kinetic studies of the reaction of hydralazine with acetaldehyde at physiological concentrations and pH were conducted. This reaction, which leads to the formation of 3-methyl-S-triazolo[3,4-a]phthalazine, may occur in the plasma and may represent an alternative pathway for hydralazine metabolism. The reaction of hydralazine with acetaldehyde followed second-order kinetics with an activation energy of 16.9 kcal/mole. At 37 degrees, the half-life of the reaction for a colution containing 2.3 microgram of acetaldehyde/ml and 1 microgram of hydralazine/ml was 4.5 hr. The rate increased with increasing acetaldehyde concentrations.     

Characterization of metabolites of benz(j)aceanthrylene in faeces, urine and bile from rat

1. The excretion of benz[j]aceanthrylene (B[j]A) and the biotransformation products found in faeces, urine and bile of rat exposed to [3H]-labelled B[j]A have been studied. 2. About 95% of the administered radioactivity was excreted within 7 days, 79% via faeces and 16% via urine, and most of the radioactivity in urine and faeces was excreted within 2 days. 3. The B[j]A metabolites excreted between days 1 and 2, including those excreted in bile during the first 5.5 h in a separate experiment, were further characterized by HPLC, UV and electrospray/atmospheric pressure chemical ionization mass spectrometry. 4. In faeces, bile and urine, hydroxylated B[j]A metabolites predominated. The major metabolites in faeces were B[j]A-1,2-dihydrodiol-8-hydroxy and B[j]A-1,2-dihydrodiol-10-hydroxy. These metabolites were found as conjugated metabolites in the bile. The glucuronide conjugate of B[j]A-1,2-dihydrodiol-10-hydroxy was also a major metabolite in urine. Two sulphate conjugates of oxidized B[j]A were detected in bile, a sulphate conjugate of a B[j]A-dihydrodiol-phenol and B[j]A-1,2-dihydrodiol-10-sulphate. Trans-B[j]A-1,2-dihydrodiol was detected in urine, faeces and bile. 5. These findings support the hypothesis that epoxidation at the cyclopenta ring is an important biotransformation pathway for B[j]A in vivo. In addition to the characterized metabolites, a large fraction of polar compounds, possibly glutathione conjugates, was also excreted in urine and bile.     

The metabolism of tolamolol in the mouse, rat, guinea-pig, rabbit and dog.

1. [3H, 14C]Tolamolol was well absorbed after oral administration to mice, rats, guinea-pigs, rabbits and dogs. 2. The major route for excretion of radioactivity by mice, rats and guinea-pigs was the faeces; in rabbits the major route was the urine. Dogs excreted similar amounts of radioactivity by both routes. Biliary excretion of radioactivity by the rat and guinea-pig was demonstrated. 3. Tolamolol was extensively metabolized by all five species. The major metabolite in mice, rats, guinea-pigs and rabbits was the product of hydroxylation of the tolyl ring, which was excreted as such as the glucuronide and sulphate conjugates. 4. In the dog the major metabolite was the acid resulting from hydrolysis of the carbamoyl group. This acid was also excreted by the rabbit, but was only a minor metabolite in the other species studied.     

Effect of microsomal enzyme inducers on biliary and urinary excretion of acetaminophen metabolites in rats. Decreased hepatobiliary and increased hepatovascular transport of acetaminophen-glucuronide after microsomal enzyme induction

Treatment of rats with phenobarbital (PB), 3-methylcholanthrene, and pregnenolone-16 alpha-carbonitrile increased the total (biliary plus urinary) excretion of thioether and glucuronic acid conjugates of acetaminophen (AA) without influencing AA-sulfate excretion, suggesting that these microsomal enzyme inducers enhance both cytochrome P-450-mediated toxication and UDP-glucuronosyltransferase-mediated detoxication of AA. However, induction with transstilbene oxide (TSO) did not increase the total excretion of AA-thioethers or AA-glucuronide and decreased AA-sulfate excretion. In addition, all inducers increased the ratio of AA metabolites excreted into urine over that excreted into bile. The extent of this shift from biliary to urinary excretion was dependent on both the AA metabolite and the inducer. The largest shift in the excretory route was seen with AA-glucuronide and induction with PB and TSO as inducers. Specifically, PB and TSO treatments decreased biliary excretion of AA-glucuronide by 70 and 89%, respectively, and increased its blood concentration up to 6- and 11-fold and urinary excretion 3- and 3.6-fold, respectively. Galactosamine depletes UDP-glucuronic acid from the liver only, thereby inhibiting hepatic but not extrahepatic glucuronidation. Galactosamine treatment prevented the PB-induced increase in AA-glucuronide in blood and urine. This suggests that the PB-induced increases in AA-glucuronide in blood and urine originated from the liver. Thus, microsomal enzyme inducers not only influence xenobiotic biotransformation, but may also after the contribution of the excretory routes (i.e. bile and urine) in the elimination of xenobiotic metabolites by changing the direction of hepatic transport.     

Glutathione conjugation of the alpha-bromoisovaleric acid enantiomers in the rat in vivo and its stereoselectivity. Pharmacokinetics of biliary and urinary excretion of the glutathione conjugate and the mercapturate

The glutathione (GSH) conjugation of (R)-and (S)-alpha-bromoisovaleric acid (BI) in the rat in vivo, and its stereoselectivity, have been characterized. After administration of racemic [1-14C]BI two radioactive metabolites were found in bile: only one of the possible diastereomeric BI-GSH conjugates, (R)-I-S-G (35 +/- 2% of the dose), and an unidentified metabolite "X" (6 +/- 1%). In urine, only one of the possible BI-mercapturates, (R)-I-S-MA (14 +/- 1%), minor unidentified polar metabolites (5 +/- 1%) and unchanged BI (13 +/- 2%) were excreted. When (R) or (S)-BI were administered separately, the same metabolites were found. However, a ten-fold difference in excretion half lives of the biliary metabolites was observed following (S)-and (R)-BI administration, (S)-BI being more rapidly excreted. The excretion of the mercapturate in urine shows the same difference in excretion rate: its half life after administration of (R)-BI was more than 10 times longer than after a dose of (S)-BI. More of the dose of (S)-BI was excreted after 5 hr in bile and urine: 58% and 23% respectively for (S)- and (R)-BI. Therefore, a pronounced stereoselectivity in GSH conjugation exists for the (R) and (S) enantiomers of BI in the rat in vivo, which is a major determinant of their pharmacokinetics. The results suggest that (slow) inversion of the chiral centre of BI occurred in the rat in vivo.     

Drug-protein conjugates—II: An investigation of the irreversible binding and metabolism of 17α-ethinyl estradiol in vivo

Irreversible binding of radiolabelled material derived from [6,7-³H]17α-ethinyl estradiol ([³H]EE₂) to rat liver microsomal and soluble proteins occurred in vivo. It was measured by exhaustive solvent extraction and equilibrium dialysis. Three hours after administration of 5.0 μg kg^?1, 0.27 ± 0.14% (mean ± S.D., N = 4) of the dose was irreversibly bound to hepatic microsomes and 0.24 ± 0.16% to soluble protein. Induction of hepatic microsomal cytochrome P-488 and cytochrome P-450 by β-naphthoflavone (BNF) and phenobarbitone, respectively, did not significantly alter the irreversible binding of [³H]EE₂. Although enzyme induction did not affect the extent of binding, treatment with BNF, but not phenobarbitone, altered the quantitative pattern of the sulphated biliary metabolites of [³H]EE₂. The sulphated metabolites excreted by BNF-dosed rats comprised a significantly (P < 0.005) greater proportion of 2-hydroxyEE₂ than those excreted by vehicle-dosed controls. The increase was principally due to a decline in the proportion of 2-methoxyEE₂. It was suspected that the biliary metabolites of [³H]EE₂ insusceptible to hydrolysis might include conjugates formed by reactions between thiols and the reactive metabolite(s) of [³H]EE₂. Therefore l-[³⁵S]cysteine was administered prior to EE₂ in an attempt to label on or more of them. However, Except for an ³⁵S-labelled component also excreted by rats given only l-[³⁵S]cysteine, none of the sulphur-labelled biliary components co-eluted with a major [³H]EE₂ metabolite.     

Biotransformation of zeranol: disposition and metabolism in the female rat, rabbit, dog, monkey and man

The disposition of [3H]zeranol has been studied in the female Wistar rat, New Zealand rabbit, beagle dog, rhesus monkey and man. The blood elimination half-life of total radioactivity in rabbit was 26 h, monkey 18 h and man 22 h. In all species studied the drug was absorbed, oxidized and/or conjugated, and was extensively excreted via the bile in all species except rabbit and man, in which urinary excretion predominated. Blood total radioactivity in man probably consisted entirely of conjugates of zeranol and/or its metabolites. Urinary metabolites in all species included conjugates (beta-glucuronides and/or sulphates) of zeranol and the major metabolite zearalanone. A more polar minor metabolite was isolated from human urine and was shown to be hydroxy-zeranol. Taleranol (7 beta-zearalanol, the lower-melting diastereoisomer), a probable metabolite of zeranol (7 alpha-zearalanol, the higher-melting diastereoisomer) in animals and in man, was shown to be a urinary metabolite in a female New Zealand white rabbit which had received [3H]zeranol (8 mg/kg per day) for seven days. A reverse isotope dilution method was developed for the quantification of both diastereoisomers of zearalanol, and also zearalanone, in urine.     

Metabolism of 1-[14C]nitropyrene in isolated perfused rat livers

1-Nitropyrene (1-NP), a constituent of diesel exhaust, is carcinogenic to rats and is a bacterial and mammalian mutagen. Biliary and fecal excretion of 1-NP metabolites are the major routes of excretion in rats, suggesting that hepatic metabolism plays a dominant role in determining the biological fate of 1-NP. The purpose of this investigation was to quantitate 1-[14C]NP metabolites formed in isolated perfused rat livers and excreted in bile from rats. Perfused rat livers displayed a capacity for oxidation, reduction, acetylation, and conjugation of 1-NP (or its metabolites). Reduction of 1-NP followed by N-acetylation was the major metabolic pathway observed in the perfused livers. Acetylaminopyrene (AAP) was the major metabolite detected, with total quantities (150 nmol) accounting for about 60% of the total 1-[14C]NP dose (258 nmol) added to the perfusate. Considerably smaller quantities of aminopyrene and hydroxynitropyrenes were also detected. Livers perfused with 1-[14C]NP excreted about 36 nmol equivalents of 1-[14C]NP (12% of the total 1-NP dose) in bile after 60 min. Some of the biliary metabolites were tentatively identified as metabolites of the mercapturic acid pathway. The spectrum of biliary metabolites was qualitatively identical to that seen in bile from intact rats. Quantities of 14C covalently bound to hepatic macromolecules from perfused livers were 0.4 nmol 1-NP eq/g liver. The data from this study indicate that the liver may be an important site for metabolism of 1-NP.     

Mercapturic acid formation in the marmoset (Callithrix jacchus)

Benzylmercapturic acid is a major metabolite of [methylene-14C]benzyl chloride in the marmoset, as in the rat. The excretion of the minor metabolites benzylmercapturic acid sulphoxide and benzylcysteine accounted for a greater proportion of the dose than in the rat. Excretion of hippuric acid as a metabolite of benzyl chloride was variable in the marmoset. Acetylation of S-benzyl- and S-pentyl-L-cysteine to the corresponding mercapturic acids was extensive in the marmoset. Trace amounts of the sulphoxides of these acids were also excreted.     

Oxidation and glucuronidation of valproic acid in male rats--influence of phenobarbital, 3-methylcholanthrene, beta-naphthoflavone and clofibrate

The influence of phenobarbital, clofibrate, 3-methylcholanthrene and beta-naphthoflavone on omega- and beta-oxidation as well as on glucuronidation of valproic acid (n-dipropylacetic acid) was evaluated in male Sprague-Dawley rats by determination of urinary excretion of its metabolites by GC-MS after administration of 100 mg/kg. In controls 12% of the dose was excreted within 24 hours, primarily as glucuronides; metabolites formed by oxidation amounted to about 4%. Phenobarbital treatment led to stimulation of 4-hydroxyvalproic acid [(omega-1)-oxidation], 5-hydroxyvalproic acid and n-propylglutaric acid (omega-oxidation) excretion. Clofibrate enhanced the excretion of 4-hydroxyvalproic acid and 3-keto-valproic acid, a product of peroxisomal beta-oxidation. beta-Naphthoflavone slightly increased the excretion of 5-hydroxyvalproic acid. The most specific effect was found for 3-methylcholanthrene, which was effective in stimulating the formation of 3-hydroxyvalproic acid which might be formed by (omega-2)-oxidation. The addition of fatty acids (olive oil in which 3-methylcholanthrene and beta-naphthoflavone were suspended) led to increased excretion of 3-keto-valproic, 4-hydroxyvalproic and n-propylglutaric acid. The excretion of 3-hydroxyvalproic acid was completely suppressed by olive oil. Such specific effects were not observed for glucuronidation of valproic acid and its metabolites, although stimulation was attained after phenobarbital, clofibrate and 3-methylcholanthrene treatment, because of instability of glucuronide conjugates. Stimulation of valproic acid metabolism was also shown by increased plasma clearance after treatment with phenobarbital. In contrast, clofibrate given once 1 hr before valproic acid inhibited excretion of valproic acid, possibly by competition during renal tubular secretion. Determination of valproic acid metabolites in urine provides a useful tool for evaluation of inducer specificity of short chain fatty acid metabolism and differentiation between microsomal and peroxisomal enzyme activity.