Metabolism of prazosin in rat and characterization of metabolites in plasma, urine, faeces, brain and bile using liquid chromatography/mass spectrometry (LC/MS).

1. Prazosin, 2-[4-(2-furanoyl)-piperazin-1-yl]-4-amino-6,7-dimethoxyquinazoline, is an antihypertensive agent that has been used safely since 1976 and is currently being investigated for the treatment of post-traumatic stress disorder. The in vivo metabolism of prazosin in rat was first reported in 1977, although at the time analytical techniques were not as sophisticated, nor were the mass spectrometers as sensitive, as today. Recently, the in vitro metabolism of prazosin in rat liver microsomes and cryopreserved hepatocytes was investigated using liquid chromatography/mass spectrometry (LC/MS), which revealed new metabolic pathways. 2. In the present work, rat in vivo metabolism was reinvestigated using a quadrupole time-of-flight mass spectrometer coupled with ultra-performance liquid chromatography, or chip-based nanoflow electrospray ionization, with the aim of identifying metabolites revealed by the in vitro studies and any new metabolites. 3. It is reported that prazosin was metabolized in rats to produce the metabolites observed in vitro. In addition, new phase I metabolites, M18, M20 and M21, were formed and conjugation with glucose or taurine formed the new phase II metabolites, M16 and M19, respectively. 4. Evidence for bioactivation of prazosin included detection of ring-opened metabolites (M4 and M7) and a cysteinyl-glycine conjugate (M17). Further support to the structure of the ring-opened metabolite M7 was obtained by nuclear magnetic resonance (NMR) experiments on M7 isolated from urine.

Troglitazone quinone formation catalyzed by human and rat CYP3A: an atypical CYP oxidation reaction.

Oxidative ring opening of troglitazone (TGZ)(1) a thiazolidine 2,4-dione derivative used for the treatment of type II diabetes mellitus, leads to the formation of a quinone metabolite. The formation of TGZ quinone was shown to be NADPH dependent and to require active microsomal enzymes. Quinone formation was not affected by co-incubation with catalase or sodium azide and was partially inhibited (25%) by superoxide dismutase (SOD). Kinetic analysis of TGZ quinone formation in human liver microsomes implied single enzyme involvement. CYP3A isoforms were characterized as the primary enzymes involved in quinone formation by several lines of evidence including: (a) troleandomycin and ketoconazole almost completely inhibited microsomal quinone formation when SOD was present, whereas other CYP inhibitors had minimal effects (<20%); (b) TGZ quinone formation was highly correlated with regard to both contents (r(2): 0.9374) and activities (r(2): 0.7951) of CYP3A4 in human liver microsomes (HLM); (c) baculovirus insect cell-expressed human CYP3A4 was able to catalyze TGZ quinone formation at a higher capacity (V(max)/K(m)) than other human CYPs with the relative contribution of CYP3A4 in HLM estimated to be 20-fold higher than that of other CYPs; (d) TGZ quinone formation was increased by 350% in liver microsomes from rats pretreated with dexamethasone (DEX); and (e) plasma concentrations of TGZ quinone were increased by 260-680% in rats pretreated with DEX. The chemical nature of the quinone metabolite suggests an atypical CYP reaction consistent with a one-electron oxidation mechanism where an intermediate phenoxy radical combines with ferryl oxygen to subsequently form the quinone metabolite.

Metabolism and distribution of [2,3-(14)C]acrolein in lactating goats.

The metabolism and distribution of [2,3-(14)C]acrolein were studied in a lactating goat orally administered 0.82 mg/kg of body weight/day for 5 days. Milk, urine, feces, and expired air were collected. The goat was killed 12 h after the last dose, and edible tissues were collected. The nature of the radioactive residues was determined in milk and tissues. All of the identified metabolites were the result of the incorporation of acrolein into the normal, natural products of intermediary metabolism. There was evidence that the three-carbon unit of acrolein was incorporated intact into glucose, and subsequently lactose, and into glycerol. In the case of other natural products, the incorporation of radioactivity appeared to result from the metabolism of acrolein to smaller molecules followed by incorporation of these metabolites into the normal biosynthetic pathways.

Metabolism and distribution of [2,3-14C]acrolein in Sprague-Dawley rats. II. Identification of urinary and fecal metabolites.

The metabolites of [2,3-14C]acrolein in the urine and feces of Sprague-Dawley rats were identified after either intravenous administration in saline at 2.5 mg/kg or oral administration by gavage as an aqueous solution as either single or multiple doses at 2.5 mg/kg or as a single dose of 15 mg/kg. Selected urine and feces samples were pooled by sex and collection interval and profiled by combinations of reverse-phase, anion-exchange, cation-exchange, and ion-exclusion high-performance liquid chromatography (HPLC). Feces were also profiled by size-exclusion chromatography. Metabolites were identified by comparison with well-characterized standards by HPLC and by mass spectrometry. The urinary metabolites were identified as oxalic acid, malonic acid, N-acetyl-S-2-carboxy-2-hydroxyethylcysteine, N-acetyl-S-3-hydroxypropylcysteine, N-acetyl-S-2-carboxyethylcysteine, and 3-hydroxypropionic acid. The fecal radioactivity from the oral dose groups was partitioned into methanol-soluble, water-soluble, and insoluble radioactivity, some of which could be liberated by dilute acid hydrolysis. HPLC analysis of these extracts revealed no discrete metabolites. Size-exclusion chromatography indicated a molecular weight range of 2,000 to 20,000 Da for the radioactivity, which was unaffected by hydrolysis at reflux with 6 M acid or base. This radio-activity was thought to be a homopolymer of acrolein, which was apparently formed in the gastrointestinal tract. The pathways of acrolein metabolism were epoxidation followed by conjugation with glutathione, Michael addition of water followed by oxidative degradation, and glutathione addition to the double bond either following or preceding oxidation or reduction of the aldehyde. The glutathione adducts were further metabolized to the mercapturic acids.

Human spermatozoa produce C16-platelet-activating factor.

Recent data indicate that human spermatozoa produce platelet-activating factor as determined by the rabbit platelet [3H]serotonin release bioassay. In this report, we examined by fast atom bombardment/mass spectrometry the molecular species of platelet-activating factor generated by these germ cells. Extracted spermatozoal samples that contained platelet-activating factor bioactivity underwent straight-phase high-performance liquid chromatography, and fractions which coeluted with authentic C16- and C18-platelet-activating factor standards were subjected to fast atom bombardment/mass spectrometry. Our mass spectral data indicate that human spermatozoa synthesize C16-platelet-activating factor but not C18-platelet-activating factor.

Formation and reversibility of S-linked conjugates of N-(1-methyl-3,3-diphenylpropyl)isocyanate, an in vivo metabolite of N-(1-methyl-3,3-diphenylpropyl)formamaide, in rats.

The metabolic disposition of N-(1-methyl-3,3-diphenylpropyl) formamide was studied in rats. The water-soluble metabolites, N-acetyl-S-[N-(1-methyl-3,3-diphenylpropylcarbamoyl)]cysteine and S-[N-(1-methyl-3,3-diphenylpropylcarbamoyl)]glutathione, were identified in urine and bile, respectively, of rats doses with the secondary formamide. The structures of these metabolites were confirmed by comparison with synthetic standards and by using liquid chromatography mass spectrometry and fast atom bombardment mass spectrometry. Synthetic standards of these metabolites were obtained by reacting the N-(1-methyl-3,3-diphenylpropyl)isocyanate with glutathione or N-acetylcysteine in methanolic solutions. The isocyanate was obtained in high yield by reacting 1-methyl-3,3-diphenylpropylamine with trichloromethyl chloroformate. The S-linked conjugates released the isocyanate in mild alkali, but were stable under acidic conditions. The released isocyanate was characterized by comparison with the synthetic standard using GC/MS and HPLC. A mechanism is proposed for the base-catalyzed elimination of the isocyanate from the thiol conjugates.

Regioisomeric aromatic dihydroxylation of propranolol. Use of monohydroxylated intermediates for structural assignments of the metabolites formed in vitro.

The formation of dihydroxylated propranolol metabolites [(HO)2-Ps], determined as their trimethylsilyl derivatives, was investigated using GC/MS techniques. Tentative structural assignment for the (HO)2-Ps was achieved by matching retention times of the (HO)2-Ps arising from the incubation of synthetic mono-HO-P regioisomers in the presence of the rat liver 9000g supernatant. Seven compounds were identified as (HO)2-Ps: 2,3-, 3,4-, 3,7-, 4,6-, 4,8-, 5,6-, and 7,8-(HO)2-P. Five of these regioisomers were observed as metabolites when propranolol was the substrate: 4,8-, 3,4-, 5,6-, and 4,6-(HO)2-P. The pathway for the formation of (HO)2-Ps was investigated by incubating propranolol in the presence of rat liver microsomes under an 18O2 atmosphere. 18O2 was the source of both hydroxyl group oxygen atoms indicating that sequential hydroxylation occurs. Mono-HO-Ps, not having hydroxyl groups at C-4 or C-5, proved to be the best substrates for the second hydroxylation. Propranolol is a better substrate than is 4-HO-P for formation of (HO)2-Ps. The regioselectivity of the second hydroxylation is predictable on the basis of expected sites of electrophilic substitution on the mono-HO-P intermediates. Substrate stereoselectivity in the formation of (HO)2-Ps was determined.

Regioisomeric aromatic dihydroxylation of propranolol. Synthesis and identification of 4,6- and 4,8-dihydroxypropranolol as metabolites in the rat and in man.

The formation of the three prominent dihydroxylated propranolol [(HO)2-P] metabolites, 4,6-, 4,8-, and 3,4-(HO)2-Ps, was investigated in vivo in rat and in man. Authentic O,O-dibenzyl ethers of 4,6- and 4,8-(HO)2-P were synthesized from the corresponding dihydroxy-l-naphthaldehydes. The l-carboxyaldehyde group was used as the latent side chain l-naphthol ether available by Baeyer-Villiger oxidation and subsequent side chain elaboration. The benzyl ethers were cleaved, and the resulting dihydroxynaphthalenes were immediately derivatized with bis-N,O-(trimethylsilyl)trifluoroacetamide. After ip administration of P-3,3-d2 (20 mg/kg) to rats, 4,6-, 5,6-, 3,7-, 3,4-, and 4,8-(HO)2-Ps were identified by GC/MS as urinary metabolites. After administering pseudoracemic propranolol [(2R)-P-d0/(2S)-P-3,3-d2] ip to rats (20 mg/kg), parent ions of the (HO)2-Ps as trimethylsilyl derivatives were monitored by GC/MS. While 4,6- and 3,4-(HO)2-P arose stereoselectively from (2S)-propranolol, 4,8-, 4,7-, and 5,6-(HO)2-P arose stereoselectively from (2R)-propranolol. About 3.3% of the dose was converted to (HO)2-Ps. Three (HO)2-Ps, 4,6-, 4,8-, and 3,4-(HO)2-P, were identified as urinary metabolites of propranolol in man after a single oral dose of 80 mg of P-3,3-d2. Less than 1% of this dose was converted to urinary (HO)2-Ps in 24 hr.

Synthesis and identification of 3-(4-hydroxy-1-naphthoxy)lactic acid as a metabolite of propranolol in the rat, in man, and in the rat liver 9000 g supernatant fraction.

The formation of 3-(4-hydroxy-1-naphthoxy)lactic acid (4-HO-NLA) from propranolol was investigated. Authentic 4-HO-NLA was synthesized from 4-methoxy-1-naphthol using methods previously used for preparation of naphthoxylactic acid (NLA). Cleavage of the 4-methyl ether was accomplished using iodotrimethylsilane in the presence of cyclopentene. After ip administration of propranolol-3,3-d2 (P-3,3-d2) (20 mg/kg) to rats, 4-HO-NLA-d2 was identified by GC-MS as a urinary metabolite. After administering pseudoracemic propranolol (S-P-d0/R-P-3,3-d2) ip to rats (20 mg/kg), parent ions of 4-HO-NLA-d0 and -d2 as methyl ester-trifluoroacetyl derivatives were monitored by GC-MS. 4-HO-NLA arose stereoselectively from S-propranolol (S/R ratio 2.6). After administering approximately equimolar quantities of 4-HO-P and P-3,3-d2 (10 mg/kg each, ip), only 4-HO-NLA-d2 arising from metabolism of P-3,3-d2 was observed by GC-MS indicating that 4-HO-P is probably not an important metabolic intermediate in vivo. In vitro experiments in the presence of the rat liver supernatant fraction performed with P-3,3-d2, NLA, and 4-HO-P showed that only NLA led to 4-HO-NLA. Incubation of NLA also produced two other hydroxylated NLA regioisomers. Incubation of 4-methoxypropanolol, a more lipophilic congener of 4-HO-P, produced no lactic acid metabolite in the presence of the rat liver supernatant fraction, indicating that poor lipophilicity is not the only deterrent to N-dealkylation of the side chain of 4-HO-P.(ABSTRACT TRUNCATED AT 250 WORDS)

Microbiological Systems in Organic Synthesis: Preparative-Scale Resolution of (RS)-Glaucine by Fusarium solani and Stereospecific Oxidation of (R)-(-)-Glaucine by Aspergillus flavipes.

The destructive resolution of (6aR,S)-glaucine (Ic) was accomplished by oxidation of the (6aS)-(+)-enantiomer (Ia), using Fusarium solani ATCC 12823 to yield the unnatural alkaloid (6aR)-(-)-glaucine (Ib). Eighteen cultures were examined for their ability to metabolize the (6aR)-(-)-enantiomer (Ib), and Aspergillus flavipes ATCC 1030 was found to catalyze the stereoselective oxidation of this substrate to didehydroglaucine. Thus, it has been demonstrated that "R" and "S" organisms exist with regard to the oxidation of aporphines to didehydroaporphines.