Metabolism of isbufylline in humans. Isolation, identification, and synthesis of plasma and urine metabolites

Isbufylline metabolism after oral administration to humans was studied. The main metabolites detected by the HPLC method, in plasma, were 1-methyl-7-(2-hydroxy-2-methyl-propyl) xanthine (I), 1,3-dimethyl-7-(2-hydroxy-2-methyl-propyl) xanthine (II), and 1-methyl-7-(2-methyl-propyl) xanthine (III). The main metabolites detected in urine were 1-methyl-7-(2-hydroxy-2-methyl-propyl) xanthine (I), 1,3-dimethyl-7-(2-carboxy-propyl) xanthine (IV), and 1,3-dimethyl-7-(2-hydroxymethyl-propyl) xanthine glucuronic acid (V)-Gluc. They were isolated by HPLC, identified by GC/MS, HPLC/MS, or HPLC/MS/MS, and finally synthesized. Recovery of these metabolites, along with the absence of unmetabolized isbufylline in the urine, indicated biotransformation and renal excretion as the main routes of isbufylline elimination in humans. HPLC quantitation of the characterized urine metabolites revealed that 49% of the drug was eliminated as (I), 9% as (V)-Gluc, and 5% as (IV).     

Determination of tripamide and its metabolites in plasma, red blood cells and urine by high-performance liquid chromatography

A procedure for the determination of tripamide and its hydroxylated metabolites in plasma, red blood cells and urine by reversed-phase high-performance liquid chromatography is described. The concentrations in red blood cells showed a monophasic decline and the half-life was 9.5 h. The concentration in red blood cells was markedly higher than that in plasma, showing that 95-98% of the drug is present in whole blood, after a dose of tripamide (90 mg) in man. The specificity and sensitivity of this procedure appear to be satisfactory for pharmacokinetic studies.     

Methylprednisolone-hemisuccinate and its metabolites in serum, urine and bile from two patients with acute graft rejection

A cross-sectional assessment of differences in social class and other sociodemographic variables at hospital admission for patients with psychotic disorders was carried out through a systematic survey of psychotic patients admitted to greater Baltimore psychiatric facilities between 1983 and 1989. Female patients, first-admission patients, and patients with bipolar disorder or other, nonschizophrenic psychosis were more likely to have been admitted to community, university, and private hospitals than to state hospitals. Patients in medium and higher social class categories were 1.29 to 2.57 times more likely to be admitted to community, university, and private hospitals than to state hospitals.     

Determination of nalidixic acid and its two major metabolites in human plasma and urine by reversed-phase high-performance liquid chromatography

This paper describes a precise and sensitive method for analysis of nalidixic acid and its two major metabolites in plasma and urine following the oral administration of a therapeutic dose in humans. After addition of an internal standard (oxolinic acid), 1-ml samples of plasma or urine are extracted at acidic pH with chloroform. The extracts are purified by re-extraction with sodium hydroxide solution and then chloroform. The final extracts are evaporated to dryness, reconstituted in mobile phase and injected into a high-performance liquid chromatograph equipped with RP-8 column and UV detector operating at 254 nm. The limit of sensitivity of the method is lower than 0.5 micrograms/ml of plasma or urine for each compound. The applicability of the method to pharmacokinetic studies of nalidixic acid in humans is demonstrated.     

Amrinone, a phosphodiesterase III inhibitor, and arachidonic acid metabolism in humans

A 59-year-old patient who had photorefractive keratectomy (PRK) to correct high unilateral myopia developed a progressive nuclear cataract. Phacoemulsification and intraocular lens (IOL) implantation were performed. However, determination of IOL power using automated keratometry and computerized videokeratography was not successful in this case of high axial myopia because of a decentered ablation zone, resulting in too-steep keratometric readings. Postoperative hyperopia could only be corrected by an IOL exchange. Because it may not be possible to determine the exact keratometric values for IOL calculation after PRK, subtracting the change in refraction induced by PRK from the preoperative keratometric readings might have been more accurate in this patient.     

Determination of bupivacaine and three of its metabolites in rat urine by capillary electrophoresis

A capillary electrophoretic (CE) method for the analysis of urinary extracts of the local anesthetic, bupivacaine, and its three main metabolites, desbutylbupivacaine, 3'-hydroxybupivacaine, and 4'-hydroxybupivacaine, in rat urine has been developed. The limits of detection were 0.22 microM for desbutylbupivacaine and bupivacaine, 0.15 microM for 3'-hydroxybupivacaine, and 0.16 microM for 4'-hydroxybupivacaine. The linear range was from 0.7 microM to 16.8 microM for all four compounds. Migration time and peak height reproducibilities, and extraction efficiencies were determined for all four compounds. Peak height reproducibilities (n = 5) for the overall method were improved through the use of prilocaine as an internal standard. Peak height reproducibilities were 5.6% RSD for desbutylbupivacaine and bupivacaine, and 9.9% RSD for 3'-hydroxybupivacaine and 4'-hydroxybupivacaine. Migration time reproducibilities (n = 5) were 2.4% for all compounds. Urine samples were collected from rats administered therapeutic doses of bupivacaine and extracted using a solid-phase extraction method (SPE). Separation of bupivacaine and its metabolites was achieved in 15 min.     

Pharmacokinetics of dilevalol and its conjugates in man. Assay method for plasma, blood, urine and bile samples and preliminary pharmacokinetic studies

The renal and biliary excretion of the beta-adrenoceptor blocking agent dilevalol (CAS 75659-07-3) and its conjugates was examined in a preliminary pharmacokinetic study. Plasma, urine and bile dilevalol concentrations were determined with a simplified procedure that is based on alkaline liquid-liquid extraction using diethyl ether and subsequent reversed-phase HPLC separation of the reconstituted samples (on a PRP-1 stationary phase using a mixture of methanol and pH 9.8 carbonate buffer as mobile phase). Triamterene was used as internal standard. The quantification of the conjugates was accomplished indirectly via enzymatic hydrolysis (glusulase) with and without addition of the beta-glucuronidase inhibitor 1,4-saccharolactone (at a final concentration of 5.5 mmol/l). In the pharmacokinetic study healthy volunteers and cholecystectomised patients with a T-drain received a single oral dose of 200 mg dilevalol. Furthermore, to healthy volunteers an i.v. dose of 60 mg dilevalol was given in order to estimate the absolute bioavailability. From the obtained data the systemic plasma clearance was calculated to be 1708 ml/min. The oral bioavailability was calculated to be 16%. The log concentration-time curves of the metabolites paralleled those of dilevalol in the terminal section with average terminal half-lives of approx. 5 h. In volunteers the fractions of the dose excreted renally were 0.5% for parent drug, 23% for the glucuronide(s) and 8% for the sulfate. The corresponding values found for the patients were not significantly different. In the patients' bile only 1.2% of the total dose were found (0.03% dilevalol, 1.1% dilevalol glucuronide(s), 0.1% dilevalol sulfate).(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of Ag ions in water, urine and blood

Isolated human polymorphonuclear leukocytes were submitted to long wave ultraviolet light (UVA) with and without preincubation of the cells with 8-methoxypsoralen (8-MOP). Leukocyte chemotaxis was determined in modified Boyden chambers using caseine as the attractant. Combined treatment (8-MOP + UVA) significantly inhibited the chemotactic activity with 8-MOP concentrations above 0.1 mug/ml and 2 J/cm(2). However, with high dosage of UVA (5 J/cm(2)) with and without 8-MOP still 25-30% cells migrated through the filters. Also, cell viability as determined by trypan blue exclusion was only moderately affected by combined treatment. The results indicate that these nonreplicating cells are comperatively insensitive to UVA and 8-MOP.     

Biotransformation of trithiozine. III - Isolation and identification of further metabolites in rat urine

The metabolism of a new antisecretory and antiulcer drug, trithiozine (I.S.F. 2001, T), was studied in 4 hr rat urine samples after i.p. administration. After extraction at pH 7 with chloroform, the urine was either incubated with beta-glucuronidase or acidified to pH 2 and subsequently extracted with chloroform. The organic layers were evaporated to dryness and the residues used for TLC analysis. The neutral extracts revealed five spots, not present in control rat urine, corresponding to the unchanged drug T and to four metabolites. Two of the metabolites had been previously identified as the 4-(3,4,5-trimethoxybenzoyl)tetrahydro-1,4-oxazine (TBO) and the 4-(3,4,5-trimethoxythiobenzoyl)tetrahydro-1,4-oxazine-S-oxide (TO). Three other metabolites were found in the extracts after beta-glucuronidase incubation. TLC, U.V. and M.S. data were consistent with the structure 4-(3,5-dimethoxy-4-hydroxythiobenzoyl)tetrahydro-1,4-oxazine (HT), the corresponding S-oxide (HO) and the 4-(3,5-dimethoxy-4-hydroxybenzoyl)tetrahydro-1,4-oxazine (HBO). The acidic extracts revealed two spots structurally identified as the 3,4,5-trimethoxybenzoic acid (TBA) and the previous HBO. On the basis of present knowledge, a possible metabolic pathway of T is reported, consisting in a rapid metabolic oxidation on the sulfur atom and a slower demethylation on the para methoxy group. The presence of TBA is indicative of subsequent enzymatic hydrolysis of TBO. The intense and long-lasting inhibitory effect of T on gastric secretion is tentatively correlated with the pharmacological activities of some of its metabolites.     

Studies of metabolism of tripamide, a new antihypertensive agent. II. Metabolism by the hepatic microsomal enzymes

1. The metabolism of tripamide, N-(4-aza-endo-tricyclo[5.2.1.0(2.6)]decan-4-yl)-4-chloro-3-sulphamoylbenzamide, has been studied with rat liver microsomal preparations. 2. Hydrolysis of tripamide was induced by phenobarbitone pretreatment and inhibited by O-ethyl O-p-nitrophenyl phenylphosphonothioate (EPN), a classical inhibitor of hepatic microsomal arylamidase. The hydrolysis was also catalysed by partially purified rabbit liver microsomal arylamidase. 3. The hydroxylation of tripamide was induced by 3-methylcholanthrene and inhibited by CO. 4. Inhibition of the hydroxylation of tripamide by antibodies of cytochrome P-450 and P-448 was studied. The 8-hydroxylation was inhibited by both antibodies, but 3-hydroxylation was inhibited by neither.     

Effects of ursodeoxycholic acid on conjugated bile acids and progesterone metabolites in serum and urine of patients with intrahepatic cholestasis of pregnancy

This study examined the effect of ambient temperature and feeding on brown adipose tissue (BAT) function and thermoregulation in lambs born either vaginally at term or by Caesarean section close to term. Immediately after birth lambs were placed in a warm (30 degrees C) or cool (15 degrees C) ambient temperature and measurements of colonic temperature and heat production recorded for 6 h. Lambs were fed 50 ml of colostrum when 5 h old. The amount of uncoupling protein and level of guanosine 5'diphosphate (GDP) binding in BAT was higher in vaginally delivered lambs than in lambs delivered by Caesarean section. For each delivery group, GDP binding was greater in lambs maintained at 30 degrees C than in lambs maintained at 15 degrees C. O2 consumption, CO2 production and colonic temperature only increased after feeding in lambs born by Caesarean section and maintained at 30 degrees C, a response that was accompanied by a decreased incidence of shivering. Irrespective of delivery temperature, plasma thyroid hormone concentrations and noradrenaline content of BAT were lower in lambs born by Caesarean section than in those born vaginally. Plasma cortisol concentrations were higher in lambs delivered by Caesarean section, as was adrenaline content of BAT in these lambs maintained at 30 degrees C. It is concluded that the thermoregulatory response to feeding in terms of changes in both recruitment of shivering and colonic temperature were observed only in lambs delivered by Caesarean section.     

Parsalmide: a new anti-inflammatory agent. III. Study of the analgesic and anti-inflammatory activity

When human chorionic gonadotropin (hCG) was treated with a 20-fold molar excess of N-acetylimidazole in aqueous solution, two tyrosyls were acetylated, resulting in a 50% reduction in in vivo biological activity. With increased quantities of reagent, the number of tyrosyls acetylated increased but with no further decrease in the biological activity. In vitro biological activity (binding to hCG receptors) of the hormone was not affected at all. Acetylation in urea increased tyrosyl modification, accompanied by acetylation of some lysyls, yielding a product in which all seven tyrosyls were acetylated and both in vivo and in in vitro biological activities were completely abolished. Deacylation with hydroxylamine partially restored biological activity of some but not all of the modified products. When individual hCG subunits were treated with the same reagent, the number of tyrosyls acetylated in each subunit again increased with increasing amounts of reagent, up to three in the alpha subunit and two in the beta subunit in the absence of urea. The tyrosyls in the beta subunit appeared less reactive to the reagent than those in the alpha subunit. Subunits modified to these extents retained ability to recombine as examined by gel electrophoresis, but the recombined products varied considerably in both in vivo and in vitro biological activities. A completely tyrosyl-acetylated product of hCG-alpha did not combine with intact hCG-beta, while fully modified hCG-beta did with intact hCG-alpha.     

Determination and confirmation of identities of flumequine and nalidixic, oxolinic, and piromidic acids in salmon and shrimp

A previously published liquid chromatographic (LC) method for determining residues of flumequine (FLU) and nalidixic (NAL), oxolinic (OXO), and piromidic (PIR) acids in catfish tissue was applied to salmon and shrimp muscle. Identities of all 4 residues in salmon and shrimp were confirmed by gas chromatography/mass spectrometry (GC/MS). The tissue is homogenized with acetone, the acetone extract is defatted with hexane, and the quinolones are extracted into chloroform. The extract is further purified by first partitioning into base and then back-extracting from a solution acidified to pH 6.0. Analytes are determined by LC with simultaneous UV and fluorescence detection. Muscle tissue was fortified with each quinolone at 5, 10, 20, 40, and 80 ng/g. Average recoveries and relative standard deviations (RSDs) for salmon, which represent an average of the 5 levels for each analyte, ranged from 75.9 to 90.8% and from 2.25 to 6.40%, respectively. Average recoveries and RSDs for shrimp ranged from 81.3 to 91.2% and from 7.34 to 10.7%, respectively. Identities of OXO, FLU, NAL, and PIR were confirmed in extracts of salmon and shrimp tissue fortified at 10 ng/g by determination of decarboxylated quinolones by GC/MS. Four diagnostic ions were monitored for OXO, FLU, and PIR, and 5 ions were monitored for NAL. All ion relative abundances were within 10% of those calculated for standard decarboxylated quinolones. Optimum conditions for decarboxylation and GC/MS confirmation are given.     

Isolation and identification of major urinary metabolites of rifabutin in rats and humans

The antimycobacterial drug rifabutin is extensively metabolized in humans and laboratory animals. About 40% of the dose is excreted in urine as unchanged drug, and lipophilic (extractable with 1-chlorobutane) and polar metabolites. Polar metabolites accounted for 59.1 +/- 2.5% and 88.8 +/- 4.4% of radioactivity in urine collected over 96 hr after intravenous administration of 25 and 1 mg/kg of [14C]rifabutin to Sprague-Dawley rats, respectively. After 48 hr, all urinary radioactivity consisted of polar metabolites. The most abundant polar metabolite, identified by electrospray ionization-MS, collision-induced dissociation-MS, and comparison of HPLC retention times with the synthetic standard, was N-isobutyl-4-hydroxy-piperidine. Lipophilic metabolites accounted for <20% of urinary radioactivity. Major lipophilic metabolites, 25-O-deacetyl-rifabutin, 27-O-demethyl-rifabutin, 31-hydroxy-rifabutin, 32-hydroxy-rifabutin, and 20-hydroxy-rifabutin were isolated from both human and rat urine by HPLC and identified by electrospray ionization-MS, collision-induced dissociation-MS, and NMR spectrometry. In addition, two metabolites formed by the oxidation of the N-isobutyl-piperidyl group of rifabutin were found in the urine of rats, but not humans.     

Determination of n-hexane metabolites by liquid chromatography/mass spectrometry. 2. Glucuronide-conjugated metabolites in untreated urine samples by electrospray ionization

A liquid chromatography atmospheric pressure electrospray mass spectrometry (ESI-LC/MS) system was evaluated for the identification and characterization of n-hexane conjugated metabolites (glucuronides) in untreated urine samples. Chromatography of glucuronides was obtained under ion-suppressed reversed-phase conditions, by using high-speed (3 cm, 3 microns) columns and formic acid (2 mM) as modifier in the mobile phase. The mass spectrometer was operated in negative ion (NI) mode. For the first time, four glucuronides were identified by ESI-LC/MS in untreated urine samples of rats exposed to n-hexane: 2-hexanol-glucuronide, 5-hydroxy-2-hexanone-glucuronide, 2,5-hexanediol-glucuronide and 4,5-dihydroxy-2-hexanone-glucuronide. Confirmation of the conjugated metabolites was obtained by LC/MS/MS experiments. Gas chromatography/mass spectrometry (GC/MS) and atmospheric pressure chemical ionization (APCI) LC/MS analyses were performed on the same samples. An integrated approach GC/MS-LC/MS for the semi-quantitative analysis of n-hexane glucuronides, whose standards are not commercially available, is discussed and proposed here. In order to understand the fate of the metabolites during sample pre-treatment, a study about the effects of enzymatic and acid hydrolysis on urine samples was conducted on glucuronides isolated by solid-phase extraction. Combined analyses by GC/MS and LC/MS enabled us to distinguish 'true' n-hexane metabolites from compounds resulting from sample treatment and handling (i.e. enzymatic and acid hydrolysis, extraction and GC injection).     

Biotransformation of linogliride, a hypoglycemic agent in laboratory animals and humans

Following oral administration of linogliride, a hypoglycemic agent, to rat (50 mg kg-1), dog (30 mg kg-1), and man (100 mg per subject), plasma, urine, and fecal extract sample pools were obtained. Nine metabolites plus unchanged linogliride were isolated and identified. The number of metabolites identified were: rat (5), dog (9), and man (1). In each species, more than 78% of the administered dose was recovered in the urine pools. Identified metabolites were estimated to account for > 82% of the total amounts of drug-related sample in urine pools and > 50% in plasma and fecal extract pools. Formation of linogliride metabolites in the three species can be described by four proposed pathways: pyrrolidine hydroxylation, aromatic hydroxylation, morpholine hydroxylation, and imino-bond cleavage. Comparison of the proposed metabolic pathways among species reveals a similarity between rat and dog. In these two species, pyrrolidine hydroxylation was quantitatively the most important pathway, with 5-hydroxylinogliride and dominant hypoglycemic active metabolite in all sample pools. Further oxidation of 5-hydroxylinogliride resulted in the formation of five minor metabolites. The other three pathways appeared to be quantitatively unimportant. Metabolism of linogliride in man occurred to a very limited extent. More than 90% of the total linogliride-related material in plasma was the unchanged drug. Greater than 76% of the administered dose was excreted unchanged in the urine. Only 5-hydroxylinogliride was identified in minor amounts in human samples.