Metabolism of 2,6-dichlorobenzamide in rats and mice

1. Oral doses of 2,6-dichlorobenzamide (DCB) were excreted by rats as DCB, two monohydroxy-DCBs, 2-chloro-5-hydroxy-6-(methylthio)benzamide and 2-chloro-5-hydroxy-6-[S-(N-acetyl)cysteinyl]benzamide (mercapturic acid). 2. Biliary excretion (33% of the dose), enterohepatic circulation and intestinal micro-floral metabolism were involved in formation of 2-chloro-5-hydroxy-6-(methylthio)benzamide, and the mercapturic acid served as a precursor. 3. Whole body autoradiography and microautoradiography showed the accumulation of non-extractable residues from DCB in the nasal mucosa and contents of the large intestines of rats and mice dosed with 14C-labelled DCB.     

The fate of 4-cyanoacetanilide in rats and mice; mechanism of formation of a novel electrophilic metabolite

1. The metabolic fate of 4-cyanoacetanilide (CAA), labelled with ¹⁴C and ¹³C in the N-acetyl group, was studied in rats (oral dose, 22.5 mg/kg) and mice (oral dose 21.7 mg/kg).

2. The metabolic profile in the urine of rats was compared with that obtained previously with 4-cyano-N,N-dimethylaniline (CDA) and confirms the intermediacy of CAA in the metabolism of CDA.

3. The precursor of a major metabolite of CDA and CAA (the mercapturic acid N-acetyl-S-[2-keto-2-(4-cyanoanilino)ethyl]cysteine, metabolite C) was identified in the urine of CAA-dosed rats as the O-sulphate conjugate of N-(4-cyanophenyl)glycolamide.

4. Pretreatment of rats with the sulphotransferase inhibitor pentachlorophenol reduced the yield of the mercapturic acid metabolite C, further indicating the intermediacy of a sulphate conjugate.

5. Metabolite C was not formed from CAA by mice; thus, this species difference, also observed with CDA, occurs at the level side-chain (acetyl) hydroxylation as well as at N-acetylation of 4-cyanoaniline as previously proposed.

6. The significance of this pathway as a bioactivation reaction of CDA, CAA and other acetanilides is discussed.     

Biliary excretion and intestinal metabolism in the intermediary metabolism of pentachlorothioanisole

1. Biliary metabolites from rats dosed with pentachlorothioanisole (PCTA) were characterized by fast atom bombardment mass spectrometry and electron impact mass spectrometry.

2. Most of the biliary metabolites from PCTA were mercapturic acid pathway metabolites of methylsulphinyltetrachlorobenzene (51% of the dose); the remaining characterized biliary metabolites (20%) were mainly methylsulphinyltetrachlorothio-phenols excreted as unknown conjugates.

3. Pathways are proposed for the intermediary metabolism of PCTA to bis-(methylthio)tetrachlorobenzene (bis-MTTCB) involving glutathione conjugation, biliary excretion, intestinal metabolism, and enterohepatic circulation.     

Metabolites of 2,4′,5-trichlorobiphenyl in rats

1. Nineteen metabolites of 2,4′,5-trichlorobiphenyl were isolated from rat urine, faeces and bile. These metabolites resulted from one or more of the following transformations: dechlorination, hydroxylation, thiolation, methylthiolation, methylthio oxidation, dihydrodiol formation, mercapturic acid formation and conjugation with glucuronic acid.

2. Mercapturic acid-pathway metabolites were the major metabolites in the bile.

3. Methylthio-, methylsulphinyl- and methylsulphonyl-containing metabolites were the major metabolites in the faeces of control rats.

4. A synthetic pathway for the preparation of sulphur-containing metabolites of trichlorobiphenyl is described.     

Some Applications of Mass Spectrometry in Drug Metabolism Studies

1. Bile secreted from rats given single oral doses of 2-chloro-N-isopropylacetanilide (propachlor) contained 58% dose as metabolites from the mercapturic acid pathway (glutathione, mercapturate, cysteine conjugates, and a sulphoxide of the mercapturate).

2. Bile secreted from rats given single oral doses of the cysteine conjugate of propachlor contained glucuronide conjugates of hydroxylated 2-methylsulphonyl-N-isopropylacetanilides.

3. In contrast, when the intestinal microflora were bypassed by intravenous administration of the cysteine conjugate of propachlor, the bile contained only the mercapturate and the sulphoxide of the mercapturate.

4. Rats fed an antibiotic-containing diet and given single oral doses of either propachlor or the cysteine conjugate of propachlor excreted only mercapturic acid pathway metabolites in the urine, bile, and faeces, and no methylsulphonyl-containing metabolites. Faecal ¹⁴C from the antibiotic-fed rats given either propachlor or the cysteine conjugate of propachlor was extractable, in contrast to previously reported unextractable faecal ¹⁴C residues from untreated rats given propachlor orally.

5. From these results, we conclude that metabolism by the microflora was necessary for production of the methylsulphonyl-containing metabolites excreted by the rat. Enterohepatic circulation of the xenobiotic moiety of these mercapturic acid pathway metabolites is influenced by the presence of a microbial C-S lyase.     

Disposition of 6-chloro-2-pyridylmethyl nitrate,a new anti-anginal compound,in rats and dogs

1. The absorption, distribution, metabolism and excretion of 6-chloro-2-pyridylmethyl nitrate, a new anti-anginal compound, were investigated in rats and dogs after intravenous and peroral administration of the ¹⁴C-labelled or unlabelled drug.

2. The half-lives of plasma levels for the α and β phase and systemic availability were 6 min, 42 min and 26–50% respectively in rats, and 8 min, 66 min and 5% respectively in dogs.

3. Radioactivity was rapidly distributed in the tissues of rats, and recovered mainly in the 0–24 h urine (95% of dose within 24 h) with no excretion in the expired air.

4. Several metabolites were detected on t.l.c. of rat and dog urine, and four were identified as N-(chloro-2-pyridylcarbonyl)-glycine (M1, 56%), N-acetyl-S-(6-chloro-2-pyridylmethyl)-L-cysteine (M2, 29%), 6-chloro-2-pyridinecarboxylic acid (M3, 5%) and 6-chloro-2-pyridylmethyl. β-D-glucuronate (M4, 7%). No unchanged drug was excreted.     

Excretion of mercapturic acids in human urine after occupational exposure to 2-chloroprene

A pilot study was conducted for human biomonitoring of the suspected carcinogen 2-chloroprene. For this purpose, urine samples of 14 individuals occupationally exposed to 2-chloroprene (exposed group) and of 30 individuals without occupational exposure to alkylating substances (control group) were analysed for six potential mercapturic acids of 2-chloroprene: 4-chloro-3-oxobutyl mercapturic acid (Cl-MA-I), 4-chloro-3-hydroxybutyl mercapturic acid (Cl-MA-II), 3-chloro-2-hydroxy-3-butenyl mercapturic acid (Cl-MA-III), 4-hydroxy-3-oxobutyl mercapturic acid (HOBMA), 3,4-dihydroxybutyl mercapturic acid (DHBMA) and 2-hydroxy-3-butenyl mercapturic acid (MHBMA). In direct comparison with the control group, elevated levels of the mercapturic acids Cl-MA-III, MHBMA, HOBMA and DHBMA were found in the urine samples of the exposed group. Cl-MA-I and Cl-MA-II were not detected in any of the samples, whereas HOBMA and DHBMA were found in all analysed urine samples. Thus, for the first time, it was possible to detect HOBMA and Cl-MA-III in human urine. The mercapturic acid Cl-MA-III could be confirmed as a specific metabolite of 2-chloroprene in humans providing evidence for the intermediate formation of a reactive epoxide during biotransformation. The main metabolite, however, was found to be DHBMA showing a distinct and significant correlation with the urinary Cl-MA-III levels in the exposed group. The obtained results give new scientific insight into the course of biotransformation of 2-chloroprene in humans.     

Use of 19F-nuclear magnetic resonance and gas chromatography-electron capture detection in the quantitative analysis of fluorine-containing metabolites in urine of sevoflurane-anaesthetized patients

1.?The use of fluorine-19 nuclear magnetic resonance (¹⁹F-NMR) and gas chromatography-electron capture detection (GC-ECD) in the analysis of fluorine-containing products in the urine of sevoflurane-exposed patients was explored.

2.?Ten patients were anaesthetized by sevoflurane for 135–660?min at a flow rate of 6 l?min^?1. Urine samples were collected before, directly after and 24?h after discontinuation of anaesthesia.

3.?¹⁹F-NMR analysis of the urines showed the presence of several fluorine-containing metabolites. The main oxidative metabolite, hexafluoroisopropanol (HFIP)-glucuronide, showed two strong quartet signals in the ¹⁹F-NMR spectrum. HFIP concentrations after β-glucuronidase treatment were quantified by ¹⁹F-nuclear magnetic resonance. Concentrations directly after and 24?h after discontinuation of anaesthesia were 131 ± 41 (mean ± SEM) and 61 ± 19?mol?mg^?1 creatinine, respectively. Urinary HFIP excretions correlated with sevoflurane exposure.

4.?Longer scanning times enabled the measurement of signals from two compound A-derived metabolites, i.e. compound A mercapturic acid I (CAMA-I) and compound A mercapturic acid II (CAMA-II), as well as products from β-lyase activation of the respective cysteine conjugates of compound A. The signals of the mercapturic acids, 3,3,3-trifluoro-2-(fluoromethoxy)-propanoic acid and 3,3,3-trifluorolactic acid were visible after combining and concentrating the patient urines. CAMA-I and -II excretions in patients were completed after 24?h.

5.?Since ¹⁹F-nuclear magnetic resonance is not sensitive enough, urinary mercapturic acids concentrations were quantified by gas chromatography-electron capture detection. CAMA-I and -II urinary concentrations were 2.3 ± 0.7 and 1.4 ± 0.4?mol?mg^?1 creatinine, respectively. Urinary excretion of CAMA-I showed a correlation with sevoflurane exposure, whereas CAMA-II did not.

6.?The results show that ¹⁹F-nuclear magnetic resonance is a very selective and convenient technique to detect and quantify HFIP in non-concentrated human urine. ¹⁹F-nuclear magnetic resonance can also be used to monitor the oxidative biotransformation of sevoflurane in anaesthetized patients. Compound A-derived mercapturic acids and 3,3,3-trifluoro-2-(fluoromethoxy)-propanoic acid and 3,3,3-trifluorolactic acid, however, require more sensitive techniques such as gas chromatography-electron capture detection and/or gas chromatography-mass spectrometry for quantification.     

Comparative metabolism of the renal carcinogen 1,4-dichlorobenzene in rat: Identification and quantitation of novel metabolites

1. The metabolism of 1,4-dichlorobenzene has been studied in the male and female Fisher 344 rat over 72 h after oral administration of ¹⁴C-1,4-dichlorobenzene (900 mg = 96.8 μCi/kg). No covalent binding of radioactivity could be detected in samples of liver, kidney, lung and spleen. The major route of excretion was with urine accounting for 41.3% of the dose for male and 37.8% of the dose for female rat within 72 h after dosing.

2. Urinary metabolites of 1,4-dichlorobenzene were identified and quantified. The major metabolites identified in the urine of both the male and female rat, were the sulphate and glucuronide of 2,5-dichlorophenol. Minor amounts of 2,5-dichlorohydroquinone were excreted as an unidentified conjugate.

3. 2-(N-acetyl-cysteine-S-yl)- 1,4-dichlorobenzene and 2-(N-acetyl-cysteine-S-yl)-2,3-dihydro-3-hydroxy-1,3-hydroxy-1,4-dichlorobenzene were minor metabolites excreted in the urine of both sexes.

4. A novel biotransformation pathway for 1,4-dichlorobenzene may be postulated, leading to the urinary excretion of a mercapturic acid of chlorophenol.

5. No marked differences in the distribution and excretion of metabolites of 1,4-dichlorobenzene were observed between the male and female Fisher 344 rat.     

Species Differences in Metabolism of Tiaramide Hydrochloride,a New Anti-inflammatory Agent

1. Urinary excretion of the radioactivity in 24?h after oral administration of [¹⁴C]tiaramide hydrochloride was 67% of the dose in mice, 59% in rats, 41% in dogs and 74% in monkeys.

2. The serum half-lives of tiaramide after intravenous administration were approximately 0·2?h in mice, 0·8?h in rats and 0·5?h in dogs.

3. Marked species variations were noted in the composition of metabolites in the serum and urinary radioactivity. The major metabolites found were 1-[(5-chloro-2-oxo-3(2H)-benzothiazolyl)acetyl]-piperazine (DETR) and 4-[(5-chloro-2-oxo-3(2H)-benzothiazolyl)acetyl]-1-piperazineacetic acid (TRAA) in mice, TRAA and 4-[(5-chloro-2-oxo-3(2H)-benzothiazolyl)acetyl]-1-pipera-zineethanol 1-oxide (TRNO) in rats, TRNO and tiaramide-O-glucuronide (TR-O-Glu) in dogs, and TRAA and TR-O-Glu in monkeys.

4. The binding of tiaramide to plasma protein of the various species of animals and human was about 24–34% and the extent of the binding of tiaramide to human plasma protein was independent of drug concentration within the range of 1–100 μM.     

Biotransformation of diethenylbenzenes. III: Identification of metabolites of 1,3-diethenylbenzene in rat

1. Biotransformation of 1,3-diethenylbenzene (1) in rat gave four major metabolites, namely, 3-ethenylphenylglyoxylic acid (2), 3-ethenylmandelic acid (3), N-acetyl-S-[2-(3-ethenylphenyl)-2-hydroxyethyl]-L-cysteine (4) and N-acetyl-S-[1-(3-ethenylphenyl)-2-hydroxyethyl]-L-cysteine (5) were isolated from urine and identified by n.m.r. and mass spectrometry.

2. Four minor metabolites, 3-ethenylbenzoic acid (6), 3-ethenylphenylacetic acid (7), 3-ethenylbenzoylglycine (8) and 2-(3-ethenylphenyl)ethanol (9) were identified by g.l.c.-mass spectrometric analysis of urine extract derivatized in two different ways.

3. All identified metabolites are derived from 3-ethenylphenyloxirane (10), a reactive metabolic intermediate. No product of any metabolic transformation of second ethenyl group has been identified. However, several minor unidentified metabolites were detected by g.l.c.-mass spectrometry.

4. Total thioether excretion in 24h urine after a single i.p. dose of 1 amounted to 28·3±3·5 dose (mean±SD). No significant differences in the thioether fraction were observed in the dose range 100-300mg/kg.

5. Thioether metabolites consisted mainly of mercapturic acids 4 and 5. The ratio of metabolites 5 to 4 was 62:38. Each mercapturic acid consisted of two diastereomers. Their ratio, as determined by quantitative ¹³C-n.m.r. measurement was 95:5 and 79:21 for mercapturic acids 4 and 5, respectively.     

Disposition of 3H-enisoprost,a gastric antisecretory prostaglandin,in healthy humans

1. After oral administration of ³H-enisoprost (450 μg) to five healthy men, as a solution in capsules, peak ³H levels of 5624 ± 566 pg equiv./ml (mean ± S.E.M.) were reached within one hour. No unchanged drug was detected in plasma.

2. Enisoprost was rapidly de-esterified to SC-36067 [(±)11α, 16ζ-dihydroxy-16-methyl-9-oxoprost-4Z, 13E-dien-1-oic acid], a pharmacologically active analogue, which reached peak concentrations of 651±200 pg/ml within 20 min of dosing. SC-36067 was eliminated metabolically, with a half-life of 1.61 h, by a combination of β-oxidation, β-oxidation and 9-keto-reduction.

3. After nine days 59.0±2.98% and 17.4±1.57% of the dose was excreted in urine and faeces respectively. The majority of this excretion was complete in two days.

4. Five urinary metabolites were identified by GC-MS. These were (±)3-[2β-(4-hydroxy-4-methyl-1E-octenyl)-3α-hydroxy-5-oxo-1α-cyclopentanyl]propanoic acid (SC-41411; 3.6% dose), (±)3-[3α,5-dihydroxy-2β-(4-hydroxy-4-methyl-1E-octenyl)-1α-cyclopentanyl]propanoic acid (SC-41411 PGF analogue; 4.8% dose), (±)3-[2β-(8-carboxy-4-hydroxy-4-methyl-1E-octenyl)-3α-hydroxy-5-oxo-1α-cyclopentanyl] propanoic acid (SC-41411-16-carboxylic acid; 22% dose), (±)3-[2β-(8-carboxy-4-hydroxy-4-methyl-1E-octenyl)-3α,5-dihydroxy-1α-cylopentanyl]propanoic acid (SC-41411 PGF analogue-16-carboxylic acid; 8.5% dose) and its γ lactone (2.6% dose).

5. These metabolites were also identified chromatographically in plasma, as were SC-36067, (±)3-[2β-(4-hydroxy-4-methyl-1E-octenyl)-5-oxo-1α-cyclopent-3-enyl]propaloic acid and (±)3-[2β-(4-hydroxy-4-methyl-1E-octenyl)-5-oxo-cyclopent-1-enyl]propanoic acid.

6. Some 5-10% of the dose was excreted in urine as tritiated water, indicating that oxidation of the 11α-hydroxy group in SC-36067 or its metabolites also occurred.     

Synthese und Eigenschaften des 3-Hydroxy-1,10-dioxo-5,10-dihydro-1H-pyrido[2,1-b]chinazolin-2-carbonitrils

Synthesis and Properties of 3-Hydroxy-1,10-dioxo-5,10-dihydro-1H-pyrido[2,1-b]quinazoline-2-carbonitrile Anthranilic acid reacts with 2-chloro-5-cyano-4-hydroxypyrid-6-one (3) in glacial acetic acid to yield 3-hydroxy-1,10-dioxo-5,10-dihydro-1H-pyrido[2,1-b]quinazoline-2-carbonitrile (4) . When the reaction is carried out in DMF under Ullmann conditions, 2-(dimethylamino)-5-cyano-4-hydroxypyrid-6-one (5) forms as a by-product. The methylation of 3 with diazomethane affords 2-chloro-5-cyano-2-methoxy-N-methylpyrid-6-one (9) and 2-chloro-5-cynao-4,6-dimethoxypyridine (10) . Under similar conditions compound 4 undergoes an esterifying ring cleavage to furnish methyl 2-(5-cyano-4,6-dimethoxypyrid-2-ylamino)benzoate (7) .     

Identification of goat and mouse urinary metabolites of the pneumotoxin, 3-methylindole

1. Urine from goats dosed i.v. With 3-methylindole (3MI; 15?mg/kg) or [methyl-¹⁴C] 3MI (15?mg/kg, 0.5 μCi/kg) contained at least 11 metabolites of 3MI.

2. Goat metabolized 3MI to sulfate conjugates of 4- or 7-hydroxy-3-methyloxindole, 5- or 6-hydroxy-3-methyloxindole, and 3,5- or 6-dihydroxy-3-methyloxindole; glucuronic acid conjugates of indole-3-carboxylic acid and 4- or 7-hydrexy-3-methyl-oxindole; and unconjugated 3-hydroxy-3-methyloxindole. Diastereoisomeric glucuronic acid conjugates of 3-hydroxy-3-methyloxindole were also identified in goat urine.

3. Urine from mice dosed i.p. With 3MI (400mg/kg) or [ring-UL-¹⁴C] 3MI (400?mg/kg, 125 μCi/kg) contained at least six metabolites of 3MI.

4. Mice metabolized 3MI to glucuronic acid conjugates of 3,5- or 6-dihydroxy-3-methyloxindole, 5- or 6-hydroxy-3-methyloxindole, and indole-3-carboxylic acid; and unconjugated indole-3-carboxylic acid. Unconjugated 3-hydroxy-3-methyloxindole was identified in mouse urine in a previous report.

5. Both goats and mice metabolized 3MI to a mercapturate, 3-[(N-acetyl-L-cystein-S-yl)methyl]indole, which has been previously identified and was confirmed in this study.

6. 3-Methyloxindole was not identified in the urine of either goats or mice.

7. The major pathways of 3MI biotransformation in goats and mice is the formation of mono- and dihydroxy-3-methyloxindoles and their subsequent conjugation with glucuronic acid or sulfate.

8. There are no apparent qualitative differences in the biotransformation of 3MI between goats and mice that can account for their different sensitivities to 3MI induced lung injury.     

Glutathione conjugation of chlorobenzylidene malononitriles in vitro and the biotransformation to mercapturic acids in rats

The glutathione conjugation of 2-chloro-, 3-chloro-, 4-chloro- and 2,6-dichlorobenzylidene malononitrile (chloroBMNs) was investigated in vitro. In incubation mixtures containing rat liver cytosol (9000 g), the decrease in the initial amount of glutathione due to the various chloroBMNs ranged from 40 to 60% and occurred both enzymatically and spontaneously at physiological conditions (37°C, pH7.4). 2,6-DichloroBMN, however, depleted glutathione largely spontaneously (38±3%). The steric hindrance of the two chlorosubstituents probably plays an important role during the glutathione-S-transferase catalyzed reaction.The hydrolysis of the chloroBMNs to the corresponding chlorobenzaldehydes and malononitrile was studied in a mixture of buffer pH 7.4 and ethanol. The rate of hydrolysis of 2,6-dichloroBMN was slower than those of the related chloroBMNs. This means that 2,6-dichloroBMN will be the most stable compound in the presence of water.Only IP administration of 2-chloroBMN (CS) to adult male Wistar rats gave enhancement of urinary thioether excretion. A thioether could be isolated and was identified as the N-acetyl-S-[2-chlorobenzyl]-L-cysteine. The quantity of this benzylmercapturic acid in the urine of rats amounted to 4.4% dose (0.07 mmol/kg, n=12).After IP administration of 2-chloro- and 3-chlorobenzaldehyde to rats benzylmercapturic acid excretion in the urine was found to be 7.6 and 1.1% of the dose, respectively. Administration of the related 4-chloro- and 2,6-dichlorobenzaldehyde, however, resulted in _no urinary mercapturic acid excretion.It is very likely that in rats the initial biotransformation of chloroBMNs is mainly hydrolysis to corresponding chlorobenzaldehydes, leading in the case of 3-chloro-, 4-chloro- and 2,6-dichloroBMN to no mercapturic acid excretion in the urine.Nevertheless, 2,6-dichloroBMN will be the most reactive compound with proteins and therefore the best haptene in comparison with the related chloroBMNs.This work was financially supported by a grant from the Dutch Foundation for Medical Research FUNGO, grant no. 13-28-57     

The Biotransformation of 8-Chloro-6-phenyl-4H-s-triazolo[4, 3-a][1,4] benzodiazepine (D-40TA), a New Central Depressant,in Man,Dog and Rat

1. Metabolic studies of 8-chloro-6-phenyl-4H-^s-triazolo [4,3-a] [1,4] benzodiazepine (D—40TA) in man, dog and rat led to identification of the following metabolites: six hydroxylation products; 8-chloro-2,4-dihydro-6-phenyl-1H-s-triazolo[4,3-a] [1,4]benzodiazepin-1-one (I), 8-chloro-6-(4-hydroxyphenyl)-4H-s -triazolo[4,3-a][1,4]benzodiazepine (II), 8-chloro-6- (3-hydroxyphenyl)-4H-s-triazolo[4,3-a] [1,4] benzodiazepine (III), 8-chloro-4-hydroxy-6-phenyl-4H-s- triazolo[4,3-a][1,4]benzodiazepine (IV), 8-chloro-2,4-dihydro-6- (4-hydroxy-phenyl)-1H-s-triazolo[4,3-a] [1,4] benzodiazepin-1-one (V) and 8-chloro-2,4-dihydro-6-(3-hydroxyphenyl)- 1H-s- triazolo[4,3-a][1,4]benzodiazepin-1-one (VI); five ring-opened metabolites; 5-chloro-2-(4H-1,2,4-triazol-4-yl) -benzophenone (VII), 5-chloro-2- (2,3-dihydro-3-oxo- 4H -1,2,4- triazol-4-yl)benzophenone (VIII), 5-chloro-2- (2,3-dihydro-3-oxo-4H - 1,2,4-triazol-4-yl)-2-hydroxybenzophenone (IX), 5-chloro-2- (2,3-dihydro-3-oxo-4H-1,2,4-triazol-4-yl)-4'-hydroxybenzophenone (X) and 5-chloro-2-(3,5-dioxo-2,3,4,5-tetrahydro-1H-1,2,4-triazol-4-yl) benzophenone(XI).

2. In man, metabolites I, IV, VII and VIII are present as the free form in the urine, and I, II, IV and VIII are present as conjugates. In the dog, all the metabolites are present. The rat transforms the compound mainly to metabolites I, II, IV and V. None of the ring-opened metabolites are observed in the rat.     

Pharmacokinetics and tissue distribution of 3-((5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide; a novel ALK5 inhibitor and a potential anti-fibrosis drug

The authors investigated the pharmacokinetics and metabolism of 3-((5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide (IN-1130), a novel ALK5 inhibitor, which suppresses renal and hepatic fibrosis, and also exerts anti-metastatic effects on breast cancer-bearing MMTV-cNeu mice model. Plasma half-lives of orally administered IN-1130 were 62.6 min in mice, 76.6?±?10.6 min in dogs, 156.1?±?19.3 min in rats, and 159.9?±?59.9 min in monkeys. IN-1130 showed a high apparent permeability coefficient (P_app) of (45.0?±?2.3)?×?10^?6 cm s^?1 in in vitro permeability tests in a Caco-2 cell monolayer model. The bioavailability of orally administered IN-1130 was 84.9% in dogs and 34.4% in monkeys (oral dose, 5.5 mg kg^?1), 11.4% in rats and 8.95% in mice (oral dose, 50.3 mg kg^?1), respectively. Orally given IN-1130 was readily distributed into liver, kidneys and lungs. The major metabolite of IN-1130 (M1) was detected in the systemic circulation of rat and mouse and was purified and tentatively identified as 3-((4-(3-hydroxyquinoxaline-6-yl)-5-(6-methylpyridine-2-yl)-1H-imidazol-2-yl)methyl)benzamide or 3-((4-(2-hydroxyquinoxalin-6-yl)-5-(6-methylpyridine-2-yl)-1H-imidazol-2-yl)methyl)benzamide. The highest levels of M1 were found in liver. The results of this study suggest that IN-1130 has the potential to serve as an effective oral anti-fibrotic drug.     

Comparative metabolism of the cysteine and homocysteine conjugates of propachlor by in situ perfused kidneys of rats

1. ¹⁴C-Cysteinyl- and homocysteinylpropachlor were metabolized to their respective mercapturic acids by rat kidneys in situ. First-pass elimination of ¹⁴C in urine was 47·5% for the cysteine conjugate and 36% for the homocysteine conjugate.

2. About half of the perfused ¹⁴C-labelled material isolated from urine from kidneys perfused with homocysteinylpropachlor was unchanged homocysteinylpropachlor and about half was the corresponding mercapturic acid. However, only the corresponding mercapturic acid and the S-oxide of this mercapturic acid (31·4% and 1·7% of the dose) were found in urine from kidneys perfused with cysteinylpropachlor, indicating that rat kidneys more efficiently acetylated the natural substrate, the cysteine conjugate.     

Metabolism of 6-Chloro-5-cyclohexylindane-1-carboxylic Acid (TAI-284), a New Non-steroidal Anti-inflammatory Agent. II. Isolation and Identification of Metabolites in the Perfusate of the Isolated Liver Perfusion in Rats

Abstract

1. Biotransformation of 6-chloro-5-cyclohexylindane-1-carboxylic acid labelled with tritium and deuterium was studied in isolated perfused rat liver. Five metabolites were isolated by column and thin-layer chromatography.

2. The metabolites and their methyl esters were characterized by mass and proton magnetic resonance spectrometry with aid of a shift reagent, tris(dipivalomethanato)europium. The following metabolites were identified : 6-chloro-5-(4′-oxocyclohexyl)indane-1-carboxylic acid(metabolite I),6-chloro-5-(cis-4′-hydroxycyclohexyl)indane-1-carboxylic acid (metabolite IIa), 6-chloro-5-(trans-4′-hydroxycyclohexyl)indane-1-carboxylic acid (metabolite III), 6-chloro-5-(cis-3′-hydroxycyclohexyl)indane-1-carboxylic acid (metabolite IV or IIb). Metabolites IV and IIb are diastereoisomers.

3. The compound was metabolized primarily by hydroxylation producing metabolites IIa (cis-4′-ol), IIb (cis-3′-ol), III (trans-4′-ol) and IV (cis-3′-ol). Metabolites IIa and III seemed to be further metabolized to I (4′-oxo).     

Metabolism of N-[4-chloro-2-fluoro-5-[(1-methyl-2-propynyl)oxy]phenyl]-3,4,5,6-tetrahydrophthalimide (S-23121) in the rat. II. Absorption,disposition, excretion and biotransformation

1. To examine the metabolic fate of N-[4-chloro-2-fluoro-5-[(1-methyl-2-propynyl)oxy]phenyl]-3,4,5,6-tetrahydrophthalimide (S-23121), rats were given a single oral dose of [phenyl-¹⁴C]S-23121 at 1 or 250mg/kg.

2. The radiocarbon was almost completely eliminated from the rat within 7 days after administration for both dose groups. Faecal ¹⁴C-excretion was major (71-86% of the dose) and urinary ¹⁴C-excretion was minor (18-30%).

3. ¹⁴C-tissue residues on the seventh day after administration were generally very low. Peak ¹⁴C-concentrations in the kidney and liver occurred 4h after administration and decreased rapidly thereafter. Amounts (percentage of dose) of the parent compound in faeces were 13-26% for low dose, and 22-35% for high dose.

4. The major metabolites in faeces were sulphonic acid conjugates (13-20% of the administered dose), formed by incorporation of a sulphonic acid group into the double bond of the tetrahydrophthalimide. The major metabolites in urine were sulphates and glucuronides of 4-chloro-2-fluoro-5-hydroxyaniline, amounting to 5-7 and 2-3% of the administered dose, respectively. Sulphonic acid conjugates were not detected in urine, blood, kidney or liver.