Taurine conjugates as metabolites of arylacetic acids in the ferret.

1. The pattern of conjugation in the ferret of 8 arylacetic acids and, for comparison, benzoic acid and 4-nitrobenzoic acid was examined. 2. The arylacetic acids, phenylacetic, 4-chloro- and 4-nitro phenylacetic, alpha-methylphenylacetic (hydratropic), 1- and 2-naphthylacetic and indol-3-ylacetic acids, were excreted in the urine as taurine and glycine conjugates. Diphenylacetic acid did not form an amino acid conjugate and was excreted as a glucuronide. 3. The taurine conjugate was the major metabolite of 4-nitrophenylacetic, alpha-methylphenylacetic, 1- and 2-naphthylacetic and indol-3-ylacetic acids, whereas the glycine conjugate was the major metabolite of phenylacetic and 4-chlorophenylacetic acids. Taurine conjugation did not occur with benzoic and 4-nitrobenzoic acids which were excreted as glycine and glucuronic acid conjugates. 4. Phenacetylglutamine and 4-hydroxyphenylacetic acid were minor urinary metabolites of phenylacetic in the ferret. 5. A number of taurine conjugates of aliphatic and aromatic acids were synthesized and their characterization and properties were studied. The role of taurine as an alternative to glycine in the metabolic conjugation of arylacetic acids is discussed.     

Metabolism of arylacetic acids. 2. The fate of [14C]hydratropic acid and its variation with species.

1. (+/-)-[methyl-14C]-Hydratropic acid was administered to man, rhesus monkey, cat, rabbit and fruit bat. 2. All species excreted 60-100% of administered 14C in the urine in 24 h, and unchanged hydratropic acid accounted for 0-17% of the dose. 3. In man, the urinary 14C consisted of a very small quantity (1%) of unchanged hydratropic acid with the remainder as hydratropylglucuronide. 4. Hydratropylglucuronide was the major urinary excretion product in the 4 animal species, while the glycine conjugate was present in the urine of cat and rat. Additionally, cats excreted the taurine conjugate of hydratropic acid. 5. Bile-duct cannulated rats excreted 20-30% of an injected dose of [14C] hydratropic acid in the bile in 3 h mainly as hydratropylglucuronide.     

Comparative urinary excretion of ethoxyacetic acid in man and rat after single low doses of ethylene glycol monoethyl ether

Male rats were given a single oral dose of ethylene glycol monoethyl ether (EGEE), the dose ranging from plausible human exposures (0.5-1 mg/kg) to doses reported in the literature (100 mg/kg). Urinary excretion of ethoxyacetic acid (EAA) and its glycine conjugate was followed up to 60 h after dosing and compared to data of experimentally exposed human volunteers. In rats, the mean elimination half-life of free as well as conjugated EAA was 7.2 h for all doses. EAA was excreted partly as a glycine conjugate (on average 27%), the extent of conjugation being independent of the dose. The conjugation with glycine showed a clearly diurnal variation, the lowest extent being found during the night. The relative amount of EGEE recovered in urine as EAA was only 13.4% for the lowest dose, but increased as the administered dose of EGEE was higher, indicating that EGEE was metabolised at least in two parallel pathways of which one pathway becomes saturated at relatively low doses. In man, urinary excretion of EAA for equivalent low doses of EGEE differed from that in the rat by a longer elimination half-life (mean 42 h), by the absence of EAA conjugates and by a higher recovery.     

Metabolism of arylacetic acids. 3. The metabolic fate of diphenylacetic acid and its variation with species and dose.

1. [carboxy-14C]Diphenylacetic acid has been administered to seven primate species including man, and four other mammals and the qualitative and quantitative aspects of its elimination determined. 2. In most species, 50-100 percent of the administered 14C was excreted in the urine in 48 h; 2-30 percent of the dose was recovered unchanged in the 24 h urine. 3. In all species the only urinary metabolite detected by radiochromatogram scanning was diphenylacetylglucuronide (10-70 percent of dose). Reverse isotope dilution additionally revealed the formation of trace amounts (less than 1 percent of dose) of the glycine conjugate by four species and of the taurine conjugate by the cat. No evidence was found for the formation of a glutamine conjugate. 4. The influence of dose on the pattern of metabolism and excretion of diphenylacetic acid has been studied in the rat. In this species diphenylacetic acid undergoes extensive elimination and enterohepatic circulation.     

Metabolism of Arylacetic Acids: 2. The Fate of [14C]Hydratropic Acid and its Variation with Species

1. (±)-[methyl-¹⁴C]-Hydratropic acid was administered to man, rhesus monkey, cat, rabbit and fruit bat.

2. All species excreted 60-100% oi administered ¹⁴C in the urine in 24?h, and unchanged hydratropic acid accounted for 0-17% of the dose.

3. In man, the urinary ¹⁴C consisted of a very small quantity (1%) of unchanged hydratropic acid with the remainder as hydratropylglucuronide.

4. Hydratropylglucuronide was the major urinary excretion product in the 4 animal species, while the glycine conjugate was present in the urine of cat and rat. Additionally, cats excreted the taurine conjugate of hydratropic acid.

5. Bile-duct cannulated rats excreted 20-30% of an injected dose of [¹⁴C] hydratropic acid in the bile in 3?h mainly as hydratropylglucuronide.     

Drug-protein conjugates--XIII. The disposition of the benzylpenicilloyl hapten conjugated to albumin

The disposition and metabolic fate of benzylpenicillin conjugated to a protein, human serum albumin (HSA), were compared with those of free penicillin in the rat. The conjugate was prepared by in vitro incubation of [3H]-benzylpenicillin and HSA at pH 10.8 for 24 hr at 37 degrees, conditions which favour the formation of penicilloyl-lysine residues. The synthetic conjugate was cleared more slowly from plasma than free penicillin after intravenous administration; thus at 3 hr, concentrations of 5.08 +/- 0.50% dose/ml of the conjugate (0.31 microCi; 2.92 mg protein) was obtained. In an earlier study a concentration of 0.03 +/- 0.01% dose/ml was obtained after administration of free BP (2.7 mmol kg-1). During this time, 1.41 +/- 0.50% of the conjugate dose was excreted in urine while 5.0 +/- 0.2% of the dose was excreted in bile. Tissue analysis indicated that the liver contained 15.3 +/- 0.9% of the dose, while other tissues contained less than 6% of the dose. In long term metabolism studies it was found that 39.5 +/- 1.0% and 46.5 +/- 0.9% of the dose (0.43 microCi; 6.33 mg protein) was excreted in the urine after 3 and 7 days respectively. The principal metabolite (63-68%) excreted in both bile and urine was identified on the basis of cochromatography and fast atom bombardment mass spectrometry as benzylpenicilloic acid, indicating that the conjugate undergoes specific cleavage at the bond between the benzylpenicilloyl moiety and the protein. In vitro degradation studies indicate that the metabolism occurs primarily in the liver. Therefore benzylpenicilloic acid excreted in urine, after administration of free BP, may be formed either by direct hydrolysis of the beta-lactam ring, and/or result from catabolism of protein conjugates formed in vivo.     

Studies on the metabolism of arylacetic acids. 6. Comparative metabolic conjugation of 1- and 2-naphthylacetic acids in the guinea pig, mouse, hamster and gerbil

The patterns of metabolic conjugation of the isomeric 1- and 2-naphthylacetic acids have been compared in guinea pig, mouse, hamster and gerbil. Equimolar doses of [carboxy-14C]1- and 2-naphthylacetic acids were given to these species by i.p. injection, their urine collected and urinary metabolites examined by t.l.c. before and after treatment with beta-glucuronidase or mild alkali. 2. Urinary excretion of 14C following administration of 14C-1-naphthylacetic acid was 76-93% of dose in 72 h, the bulk being eliminated in 24 h. Urinary metabolites comprised 1-naphthylacetic-glycine and -glucuronide together with the unchanged acid. 3. Following administration of 14C-2-naphthylacetic acid, some 68-94% of the 14C dose was recovered in the urine in 72 h, with the majority in the 0-24 h urine. All four species excreted 2-naphthylacetyl-glucuronide and -glycine: additionally, 2-naphthylacetyl-taurine was excreted by mouse, gerbil and hamster and the glutamine conjugate was also present in hamster urine.     

Studies on the Metabolism of Arylacetic Acids: 5. The Metabolic Fate of 2-Naphthylacetic Acid in the Rat,Rabbit and Ferret

1. The fate of [¹⁴C]-2-naphthylacetic acid has been studied in rat, ferret and rabbit and the findings compared with those for the 1-isomer.

2. 2-Naphthylacetic acid was conjugated in all three species with glycine, glutamine, taurine and glucuronic acid.

3. In rat, but not rabbit, 2-naphthylacetic acid is eliminated in the bile (50% dose) and undergoes entergohepatic circulation.

4. The pattern of metabolism and elimination of 2-naphthylacetic acid in rat is dose-dependent. Small doses (5 mg/kg) are excreted in urine and bile mainly as the glucuronic acid conjugate, while larger doses (250 mg/kg) are eliminated in the bile mainly as amino acid conjugates.

5. Metabolic conjugation of 1- and 2-naphthylacetic acids differs in the three species studied. 1-Naphthylacetic acid is conjugated largely with glucuronic acid and amino acid conjugation is minimal, whereas the converse is true of the 2-isomer. These differences in conjugation have been correlated with structural differences between the two acids.

6. 2-Naphthylacetic acid is the first carboxylic acid found to undergo simultaneous amino acid conjugation with glycine, glutamine and taurine in non-primate species. 2-Naphthylacetic acid may be a useful probe for exploring the species occurrence of the various conjugations.     

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.     

Identification of urinary metabolites of 8-methyl-8-azabicyclo- 3,2,1] octan-3-yl 3,5-dichlorobenzoate (MDL 72,222) in the dog and monkey.

The metabolism of 8-methyl-8-azabicyclo- 3,2,1]octan-3-yl 3,5-dichlorobenzoate (MDL 72,222) was studied in the dog and monkey. Four urinary metabolites were detected by HPLC, HPLC/MS, and GC/MS, and were identified by comparison to authentic standards. The major metabolite in the dog, approximately 41% of the administered dose excreted between 0 and 120 hr, was the MDL 72,222-N-oxide. On the other hand, the major metabolite in the monkey was the glycine conjugate of 3,5-dichlorobenzoic acid (greater than 56% of the dose). Seven percent of the dose in the monkey urine was free 3,5-dichlorobenzoic acid. N-Desmethyl MDL 72,222 was present at 2.5 and 1% in the dog and monkey, respectively. Very little (less than 1%) of the parent compound was found in urine. The major pathways of metabolism of MDL 72,222 are N-oxidation, N-demethylation, ester hydrolysis, and amino acid conjugation.     

Application of (1)H- and (19)F-NMR spectroscopy in the investigation of the urinary and biliary excretion of 3,5-, 2,4-ditrifluoromethylbenzoic and pentafluorobenzoic acids in rat

1. The metabolism and excretion of 2,4-, 3,5-ditrifluoromethyl- and pentafluorobenzoic acids were studied in the bile-cannulated rat using (1)H- and (19)F-NMR spectroscopy following intraperitoneal administration at 50 mg kg(-1). 2. Pentafluorobenzoic acid was excreted in the urine entirely unchanged. No detectable compound or metabolites were eliminated in the bile. A total of 63.5 +/- 6.7% of the dose was recovered in the 24-h collection period. 3. In the case of 2,4-ditrifluromethyl benzoic acid, 83.9 +/- 5.2% of the dose was recovered in the 24h after administration, with about 52% being excreted in the urine and 32% in the bile. The majority of the material present in the urine was unchanged parent compound. In bile, some 60% of the compound-related material excreted was present as transacylated ester glucuronide conjugates. 4. For 3,5-ditrifluoromethylbenzoic acid, 49.6 +/- 5.3% of the dose was recovered in the 24-h collection period, with about 22% being excreted in the urine and 28% in the bile. The material excreted in both the urine and bile was a mixture of the parent acid and transacylated ester glucuronides. 5. Urinary excretion in bile-cannulated animals was similar to that found in studies using non-cannulated animals dosed at 100mg kg(-1).     

The hepatic reductase null mouse as a model for exploring hepatic conjugation of xenobiotics: application to the metabolism of diclofenac

The distribution, metabolism, excretion and hepatic effects of diclofenac were investigated following a single oral dose of 10?mg/kg to wild type and hepatic reductase null (HRN) mice. For the HRN strain the bulk of the [(14)C]-diclofenac-related material was excreted in the urine/aqueous cagewash within 12?h of administration (~82%) with only small amounts eliminated via the faeces (~2% in 24?h). Wild type mice excreted the radiolabel more slowly with ca. 52 and 15% of the dose recovered excreted in urine and faeces, respectively, by 24?h post dose. The metabolic profiles of the HRN mice were dominated by acyl conjugation to either taurine or glucuronic acid. Wild type mice produced relatively small amounts of the acyl glucuronide. Whole Body Autoradiography (WBA) of mice sacrificed at 24?h post dose indicated increased retention of radioactivity in the livers of HRN mice compared to wild type mice. Covalent binding studies showed no differences between the two strains. Metabolism of diclofenac in HRN mice involved mainly acyl glucuronide formation and taurine amide conjugation. This mouse model may find utility in understanding the impact of reactive metabolite formation via routes that involve the production of acyl-CoA or acyl glucuronides of acidic drugs.     

Bioavailability, metabolism, and renal excretion of benzoic acid in the channel catfish (Ictalurus punctatus)

The pharmacokinetics and metabolism of the model compound benzoic acid were examined after intravascular (iv) and po administration at 10 mg/kg in the channel catfish (Ictalurus punctatus). A two-compartment pharmacokinetic model best described the plasma disposition of parent benzoic acid after iv dosing. The following pharmacokinetic values were estimated: elimination half-life, 5.9 hr; total body clearance, 61 ml/hr/kg; and volume of distribution (steady-state), 369 ml/kg. Plasma protein binding of [14C]benzoic acid was 18%. Benzoic acid was rapidly and extensively absorbed after po administration; absorption half-life was 0.8 hr and bioavailability was 95%. Renal excretion was the primary route of elimination of benzoic acid and metabolites. More than 80% of the iv-administered 14C was recovered in the urine in 24 hr. Unchanged benzoic acid comprised more than 90% of the urinary radiolabel. The major urinary metabolite was benzoyltaurine, which comprised 6-7% of the excreted 14C. Channel catfish were qualitatively similar to other teleost fishes in the formation of the taurine conjugate of benzoic acid. In contrast, the primary mammalian metabolite is the glycine conjugate, hippuric acid.     

Systemic lanthanum is excreted in the bile of rats

Lanthanum carbonate is a non-calcium-based oral phosphate binder for the control of hyperphosphataemia in patients with chronic kidney disease Stage 5. As part of its pre-clinical safety evaluation, studies were conducted in rats to determine the extent of absorption and routes of excretion. Following oral gavage of a single 1500 mg/kg dose, the peak plasma lanthanum concentration was 1.04+/-0.31 ng/mL, 8 h post-dose. Lanthanum was almost completely bound to plasma proteins (>99.7%). Within 24h of administration of a single oral dose, 97.8+/-2.84% of the lanthanum was recovered in the faeces of rats. Comparing plasma exposure after oral and intravenous administration of lanthanum yielded an absolute oral bioavailability of 0.0007%. Following intravenous administration of lanthanum chloride (0.3 mg/kg), 74.1+/-5.82% of the dose (96.9+/-0.50% of recovered lanthanum) was excreted in faeces in 42 days, and in bile-duct cannulated rats, 10.0+/-2.46% of the dose (85.6+/-2.97% of recovered lanthanum) was excreted in bile in 5 days. Renal excretion was negligible, with <2% of the intravenous dose recovered in urine. These studies demonstrate that lanthanum undergoes extremely low intestinal absorption and that absorbed drug is predominantly excreted in the bile.     

Fate of silvex following oral administration to humans

The fate of silvex [2-(2,4,5-trichlorophenoxy)propionic acid] was defined in seven men and in one woman following oral administration of 1 mg/kg. No adverse effects were observed. Samples of blood plasma, urine, and feces were collected at designated time intervals through 168 hr. Plasma samples were analyzed for silvex only, while samples of urine and feces were analyzed for silvex, silvex conjugate(s), 2,4,5-trichlorophenol, and 2,4,5-trichlorophenol conjugate(s). Apparent first-order kinetics described the biphasic clearance of silvex from plasma and excretion in urine. The half-life values for clearance of silvex from plasma were 4.0 +/- 1.9 and 16.5 +/- 7.3 hr for the initial and terminal phases, respectively. Peak plasma levels of silvex occurred within 2-4 hr after dosage. Within 24 hr after administration, 65% of the administered dose had been excreted in urine. Silvex was excreted in urine as silvex and silvex conjugate(s). The half-life values for excretion of silvex per se in urine were 5.0 +/- 1.8 and 25.9 +/- 6.3 hr for the two phases, respectively. Small amounts (3.2% or less) of silvex and/or silvex conjugate(s) were eliminated in feces. Recovery of silvex and its conjugate(s) in urine and feces through 168 hr ranged from 66.6 to 95.1% of the administered dose, with a mean value and standard deviation of 80.3 +/- 10.5%. In humans, silvex is readily absorbed after ingestion and subsequently readily excreted, predominantly via the urine.     

Isolation and identification of the polar metabolites of chlorpheniramine in the dog

The metabolites of chlorpheniramine were isolated from dog urine. After daily repeated dosing with chlorpheniramine, [methylene-14C]chlorpheniramine maleate was given as a tracer and urine was collected until less than 1% of the labeled dose was excreted daily. An average of 54% of the oral radioactive dose was recovered in the urine. In addition to the N-demethylated metabolites, one very polar metabolite accounting for about 18% and two less polar metabolites accounting for a total of about 30% of the total urine radioactivity were isolated. Hydrolysis studies of the most polar metabolite indicated that it was a conjugate, though not a glucuronide or sulfate. The metabolite identified after hydrolysis was 3-(p-chlorobenzyl)-3-(2-pyridyl)propionic acid. One of the two less polar metabolites was identified as the corresponding alcohol. The least abundant metabolite could not be identified.     

Meeting of the Drug Metabolism Group: Significance of Drug Metabolism and Disposition in Patient Therapy

1. ¹⁴C-Labelled benzoic acid, salicylic acid and 2-naphthylacetic acid were administered orally to horses, and urinary metabolites investigated by chromatographic and mass spectral techniques.

2. [¹⁴C] Benzoic acid (5?mg/kg) was eliminated rapidly in the urine, and quantitatively recovered in 24?h. The major urinary metabolite was hippuric acid (95% of dose) with much smaller amounts of benzoic acid, benzoyl glucuronide and 3-hydroxy-3-phenylpropionic acid. Administration of [ring-D₅]benzoic acid together with [¹⁴C]benzoic acid to a pony permitted the mass spectral determination of metabolites of the exogenous benzoic acid metabolites in the presence of the same endogenous compounds.

3. [¹⁴C]Salicylic acid (35?mg/kg) was eliminated rapidly in the urine, 98% of the ¹⁴C dose being excreted in 24?h. The major excretion product was unchanged salicylate (94% of dose). Gentisic acid, salicyluric acid and the ester and ether glucuronides of salicylic acid were very minor metabolites.

4. 2-Naphthyl[¹⁴C]acetic acid (2?mg/kg) was excreted very slowly in the urine, with 53 and 77% of the ¹⁴C dose being recovered in six days. 2-Naphthylacetylglycine was the major metabolite (26 and 38% dose) and in addition, the glucuronic acid and taurine conjugates were excreted together with unchanged 2-naphthylacetic acid.

5. This study has shown that the horse can utilize glycine, taurine and glucuronic acid for conjugation of xenobiotic carboxylic acids, and that the relative extents of these pathways are governed by the structure of the carboxylic acid.     

Species differences in the metabolism of suprofen in laboratory animals and man

The metabolism of the oral anti-inflammatory agent suprofen (S), 2-4-(2-thienylcarbonyl)phenyl)propionic acid, has been studied in mice, rats, guinea pigs, dogs, monkeys, and human volunteers. The major metabolites of S in the serum, urine, and feces of these species were determined by GC/MS and HPLC techniques. The metabolic pathways of S in these species involved reduction of the ketone group to an alcohol (S-OH), hydroxylation of the thiophene ring (T-OH), elimination of the thiophene ring to a dicarboxylic acid (S-COOH), and conjugation with glucuronic acid or taurine. In 72-hr urine and feces of these species after po dosing of 1.6 to 2 mg/kg of S, S and these metabolites accounted for 46 to 92% of the dose and were mainly excreted in the urine. S was present as a major product (excreted mainly in conjugated form) in all species. S-OH was a major component in guinea pig and dog but a minor one in other species. T-OH was identified as a major metabolite in monkey, rat, mouse, and man, but a minor one in guinea pig, and it was absent in the dog. S-COOH was present as the minor metabolite in mouse and rat, and present at trace levels in dog, monkey, and man. Conjugation of the propionic acid functionality with taurine was observed only in the dog; in the other species, conjugation with glucuronic acid was extensive. Absorption parameters of S in the rat and monkey were similar to those in man; however, other species were very different from man.     

Disposition and metabolism of letosteine in rats

The metabolism and disposition of letosteine, labeled either with 14C or 35S, has been investigated in Sprague-Dawley rats. In separate experiments, rats received 20 mg/kg, iv or orally, [14C]letosteine or [35S]letosteine. Radioactivity was rapidly excreted, mainly in urine, after iv and oral administration. Recovery of radioactivity from 0-72-hr excreta averaged 95% after both routes of [14C]letosteine administration, whereas only 50% was recovered when [35S]letosteine was administered. 14CO2 accounted for about 7.3% (iv) and 5.1% (po) of the dose of [14C]letosteine. Comparison of the iv and oral areas under the plasma 14C radioactivity concentration-time curves suggested that oral absorption of letosteine was complete. Analysis of the radioactivity content of urine showed that letosteine undergoes rapid and extensive metabolism. Several metabolites were identified by TLC, HPLC, and MS. The findings are consistent with a splitting of the ester group of letosteine and subsequent cleavage of the thiazolidinyl ring, yielding cysteine, hypotaurine, taurine, and inorganic sulfate. The metabolite derived from the side chain was identified in the urine as 3-(hydroxycarbonylmethylthio)propanoic acid. It undergoes further oxidation into sulfoxide and sulfone derivatives, which are also present in the urine.     

In vivo metabolism of the anti-inflammatory agent 2-(5-ethylpyridin-2-yl)benzimidazole

The metabolic fate of anti-inflammatory agent 2-(5-ethylpyridin-2-yl)benzimidazole (KB-1043) was studied in rats after oral administration. An average of 12.2 +/- 1.5% of the dose was excreted in the urine in the course of 0-48 h; 56.7 +/- 2.6% with feces. Two metabolites were also detected in the urine and isolated by reverse phase HPLC. Structures have been given after identification by comparison with authentic samples. The more abundant metabolite proved to be 2-(5-(1-hydroxyethyl)pyridin-2-yl)benzimidazole, a benzylic oxidation product, which was also excreted as glucuronic acid conjugate; the other metabolite was confirmed to be 2-(5-acetylpyridin-2-yl)benzimidazole. Carrageenin edema and gastric ulcerogenic activity were also tested for the two identified metabolites.