Metabolism of the recently encountered designer drug,methylone, in humans and rats

The urinary metabolites of methylone in humans and rats were investigated by analysing urine specimens from its abuser and after administrating to rats with gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-electrospray ionization mass spectrometry (LC-ESI MS), using authentic standards. The time-course excretion profiles of methylone and its three metabolites in rats were further investigated after a single intraperitoneal dosing of 5?mg?kg^?1 methylone hydrochloride. Two major metabolic pathways were revealed for both humans and rats as follows: (1) side-chain degradation by N-demethylation to the corresponding primary amine methylenedioxycathinone (MDC), partly conjugated; and (2) demethylenation followed by O-methylation of either a 3- or 4-OH group on the benzene ring to produce 4-hydroxy-3-methoxymethcathinone (HMMC) or 3-hydroxy-4-methoxymethcathinone (3-OH-4-MeO-MC), respectively, mostly conjugated. Of these metabolites, HMMC was the most abundant in humans and rats. The cumulative amount of urinary HMMC excreted within the first 48?h in rats was approximately 26% of the dose, and the amount of the parent methylone was not more than 3%. These results demonstrate that the analysis of HMMC will be indispensable for proof of the use of methylone in forensic urinalysis.     

Metabolism of the recently encountered designer drug, methylone, in humans and rats

The urinary metabolites of methylone in humans and rats were investigated by analysing urine specimens from its abuser and after administrating to rats with gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-electrospray ionization mass spectrometry (LC-ESI MS), using authentic standards. The time-course excretion profiles of methylone and its three metabolites in rats were further investigated after a single intraperitoneal dosing of 5 mg kg-1 methylone hydrochloride. Two major metabolic pathways were revealed for both humans and rats as follows: (1) side-chain degradation by N-demethylation to the corresponding primary amine methylenedioxycathinone (MDC), partly conjugated; and (2) demethylenation followed by O-methylation of either a 3- or 4-OH group on the benzene ring to produce 4-hydroxy-3-methoxymethcathinone (HMMC) or 3-hydroxy-4-methoxymethcathinone (3-OH-4-MeO-MC), respectively, mostly conjugated. Of these metabolites, HMMC was the most abundant in humans and rats. The cumulative amount of urinary HMMC excreted within the first 48 h in rats was approximately 26% of the dose, and the amount of the parent methylone was not more than 3%. These results demonstrate that the analysis of HMMC will be indispensable for proof of the use of methylone in forensic urinalysis.     

Structure identification and elucidation of mosapride metabolites in human urine,feces and plasma by ultra performance liquid chromatography-tandem mass spectrometry method

Abstract

1.?Mosapride citrate (mosapride) is a potent gastroprokinetic agent. The only previous study on mosapride metabolism in human reported one phase I oxidative metabolite, des-p-fluorobenzyl mosapride, in human plasma and urine using HPLC method. Our aim was to identify mosapride phase I and phase II metabolites in human urine, feces and plasma using UPLC-ESI-MS/MS.

2.?A total of 16 metabolites were detected. To the best of our knowledge, 15 metabolites have not been reported previously in human.

3.?Two new metabolites, morpholine ring-opened mosapride (M15) and mosapride N-oxide (M16), alone with one known major metabolite, des-p-fluorobenzyl mosapride (M3), were identified by comparison with the reference standards prepared by our group. The chemical structures of seven phase I and six phase II metabolites of mosapride were elucidated based on UPLC–MS/MS analyses.

4.?There were two major phase I reactions, dealkylation and morpholine ring cleavage. Phase II reactions included glucuronide, glucose and sulfate conjugation. The comprehensive metabolic pathway of mosapride in human was proposed for the first time.

5.?The metabolites in humans were compared with those in rats reported previously. In addition to M10, the other 15 metabolites in humans were also found in rats. This result suggested that there was little qualitative species difference in the metabolism of mosapride between rats and humans.

6.?In all, 16 mosapride metabolites including 15 new metabolites were reported. These results allow a better understanding of mosapride disposition in human.     

Phase II in vitro metabolism of 3-methylindole metabolites in porcine liver

1. The Phase II in vitro metabolism of 3-methylindole (3MI) metabolites was investigated in pigs to determine the possible relationship between 3MI Phase II metabolism and 3MI accumulation in fat. Sulphation and glucuronidation of five of the seven major metabolites found to be produced by porcine microsomes was investigated using porcine cytosol and microsomes, respectively. The possible formation of glutathione conjugates was also investigated using microsomally activated 3MI intermediate(s). 2. No sulphation or glucuronidation was observed for metabolites 3-hydroxy-3-methyloxindole, 3-methyloxindole, indole-3-carbinol or 2-aminoacetophenone; however, 5-hydroxy-3-methylindole (5-OH-3MI) was conjugated with both sulphate and glucuronic acid. 3. The enzyme responsible for sulphation of 5-OH-3MI was identified as the thermostable form of phenol-sulphotransferase (TS-PST) based on its susceptibility to TS-PST inhibitors and the correlation between sulphation of 5-OH-3MI and sulphation of the prototype substrate p-nitrophenol (r = 0.94, p < 0.001). 4. A 3MI-glutathione adduct was identified in microsomal incubations containing 3MI and glutathione. 5. Sulphation of 5-OH-3MI was high in pigs with low levels of 3MI in fat. No relationship was observed between 3MI levels in fat and either glutathione transferase or glucuronidation activities in liver.     

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.     

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.     

Study on the metabolism of 5,6-methylenedioxy-2-aminoindane (MDAI) in rats: identification of urinary metabolites

1.?5,6-Methylenedioxy-2-aminoindane (MDAI) is a member of aminoindane drug family with serotoninergic effect, which appeared on illicit drug market as a substitute for banned stimulating and entactogenic drugs.

2.?Metabolism of MDAI, which has been hitherto unexplored, was studied in rats dosed with a subcutaneous dose of 20?mg MDAI.HCl/kg body weight. The urine of rats was collected within 24?h after dosing for analyses by HPLC-ESI-HRMS and GC/MS.

3.?The main metabolic pathways proceeding in parallel were found to be oxidative demethylenation followed by O-methylation and N-acetylation. These pathways gave rise to five metabolites, namely, 5,6-dihydroxy-2-aminoindane, 5-hydroxy-6-methoxy-2-aminoindane, N-acetyl-5,6-methylenedioxy-2-aminoindane, N-acetyl-5,6-dihydroxy-2-aminoindane and N-acetyl-5-hydroxy-6-methoxy-2-aminoindane, which were found predominantly in the form of corresponding glucuronides and sulphates. However, the main portion of administered MDAI was excreted unchanged.

4.?Minor metabolites formed primarily by hydroxylation at various sites include cis- and trans-1-hydroxy-5,6-methylenedioxy-2-aminoindane, 5,6-methylenedioxyindan-2-ol and 4-hydroxy-5,6-methylenedioxy-2-aminoindane.

5.?Identification of all metabolites except for glucuronides, sulphates and tentatively identified 4-hydroxy-5,6-methylenedioxy-2-aminoindane was supported by synthesised reference standards.     

Metabolism of Alkenebenzene Derivatives in the Rat. II. Eugenol and Isoeugenol Methyl Ethers

1. The metabolites of 3,4-dimethoxyallylbenzene (eugenol methyl ether) and 3,4-dimethoxypropenylbenzene (isoeugenol methyl ether) in the rat were identified and quantitatively determined by g.l.c. and g.l.c—mass spectrometry.

2. The major metabolic reactions of 3,4-dimethoxyallylbenzene were oxidation of the allylic side chain to 2-hydroxy-3-(3,4-dimethoxyphenyl)-propionic acid, 3,4-dimethoxybenzoic acid and 3,4-dimethoxycinnamic acid, the two latter being largely excreted as their glycine conjugates. The formation of the hydroxy acid presumably involved epoxidation of the double bond and subsequent hydration to the diol whereas the formation of 3,4-dimethoxycinnamic acid and 3,4-dimethoxybenzoic acid involved migration of the double bond and the formation of cinnamoyl intermediates. Other reactions were O-demethylation to 4-hydroxy-3-methoxyallylbenzene (eugenol) and 3-hydroxy-4-methoxyallylbenzene in equal amounts, oxidation to l-(3,4-dimethoxyphenyl)-2-propen-l-ol, hydroxylation of the benzene ring to a hydroxy-3,4-dimethoxy-allylbenzene and oxidation to 3,4-dimethoxyphenylacetic acid. The formation of l-(3,4-dihydroxyphenyl)propane was found to be carried out by the rat intestinal micro-organisms. A total of at least 63% but as much as 95% dose was accounted for.

3. The major metabolic pathway of 3,4-dimethoxypropenylbenzene was via the cinnamoyl derivatives, leading to the formation of 4-hydroxy-3-methoxy-cinnamic acid (ferulic acid), 3,4-dimethoxycinnamic acid and 3,4-dimethoxybenzoic acid, the two latter being excreted largely as their glycine conjugates. Other reactions were O-demethylation to 4-hydroxy-3-methoxypropenylbenzene (isoeugenol) and 3-hydroxy-4-methoxypropenylbenzene in equal amounts, and oxidation to 3,4-dimethoxyphenylacetic acid and 4-hydroxy-3-methoxyphenyl-acetone. Epoxidation of the side chain appeared to be a minor metabolic reaction with the propenyl derivative.

4. The biliary metabolites of 3,4-dimethoxyallylbenzene and 3,4-dimethoxypropenylbenzene were identified and most of the urinary metabolites were also found in the bile.     

Characterization of midazolam metabolism in locusts: the role of a CYP3A4-like enzyme in the formation of 1′-OH and 4-OH midazolam

1.?The metabolism of midazolam was investigated in vivo in locusts in order to evaluate the presence of an enzyme with functionality similar to human CYP3A4/5.

2.?Hydroxylated metabolites of midazolam identical to human metabolites were detected in locusts and the apparent affinities (K_m values) were in the same range as reported in humans (in locusts: 7–23 and 33–85?µM for the formation of the 1′-OH and 4-OH metabolites, respectively).

3.?The formation of hydroxylated metabolites could successfully be inhibited by co-administration of ketoconazole, a known CYP3A4/5 inhibitor.

4.?Besides phase I metabolites, a number of conjugated metabolites were detected using high-resolution mass spectrometry. The most abundant metabolites detected were structurally identified by ¹H NMR as two N-glucosides. NMR analysis strongly suggested that the glycosylation occurred at the two nitrogens (either one in each case) of the imidazole ring.

5.?Distribution of midazolam and the glucose conjugates were successfully measured using desorption electrospray mass spectrometry imaging revealing time-dependent changes in distribution over time.

6.?In conclusion, it appears that an enzyme with functionality similar to human CYP3A4/5 is present in locusts. However, it appears that conjugation with glucose is the main detoxification pathway of midazolam in locusts.     

Identification of ginkgolic acid (15:1) metabolites in rats following oral administration by high-performance liquid chromatography coupled to tandem mass spectrometry

1.?In this article, metabolites of ginkgolic acid (GA) (15:1) in rats plasma, bile, urine and faeces after oral administration have been investigated for the first time by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) with the aid of on-line hydrogen/deuterium (H/D) exchange technique and β-glucuronidase hydrolysis experiments.

2.?After oral administration of GA (15:1, M0) to rats at a dose of 10?mg/kg, it was found that metabolites M1-M5 together with parent compound (M0) existed in rat plasma; parent compound (M0) and metabolites M2–M5 were observed in rat bile, and parent compound (M0) with metabolites M1 and M2 were discovered in rat faeces, and there was no parent compound and metabolite detectable in rat urine.

3.?Two oxidative metabolites of GA (15:1, M0) were identified as 2-hydroxy-6-(pentadec-8-enyl-10-hydroxy) benzoic acid (M1) and 2-hydroxy-6-(pentadec-8-enyl-11-hydroxy-13-carbonyl) benzoic acid (M2), respectively. Metabolites M3, M4 and M5 were identified as the mono-glucuronic acid conjugates of parent compound (M0), M1 and M2, respectively.

4.?The results indicated that M1 and M2 with parent compound (M0) were mainly eliminated in faeces and three glucuronide metabolites (M3, M4 and M5) excreted in bile as the predominant forms after oral administration of GA (15:1) to rats.     

Pyrido[3,4-d]pyrimidin-4(3H)-one metabolism mediated by aldehyde oxidase is blocked by C2-substitution

1.?We have previously described C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-one derivatives as cell permeable inhibitors of the KDM4 and KDM5 subfamilies of JmjC histone lysine demethylases.

2.?Although exemplar compound 1 exhibited moderate clearance in mouse liver microsomes, it was highly cleared in vivo due to metabolism by aldehyde oxidase (AO). Similar human and mouse AO-mediated metabolism was observed with the pyrido[3,4-d]pyrimidin-4(3H)-one scaffold and other C8-substituted derivatives.

3.?We identified the C2-position as the oxidation site by LC-MS and ¹H-NMR and showed that C2-substituted derivatives are no longer AO substrates.

4.?In addition to the experimental data, these observations are supported by molecular modelling studies in the human AO protein crystal structure.     

The metabolism and drug–drug interaction potential of the selective prostacyclin receptor agonist selexipag

1.?The metabolism of selexipag has been studied in vivo in man and the main excreted metabolites were identified. Also, metabolites circulating in human plasma have been structurally identified and quantified.

2.?The main metabolic pathway of selexipag in man is the formation of the active metabolite ACT-333679. Other metabolic pathways include oxidation and dealkylation reactions. All primary metabolites undergo subsequent hydrolysis of the sulphonamide moiety to their corresponding acids. ACT-333679 undergoes conjugation with glucuronic acid and aromatic hydroxylation to P10, the main metabolite detected in human faeces.

3.?The formation of the active metabolite ACT-333679 is catalysed by carboxylesterases, while the oxidation and dealkylation reactions are metabolized by CYP2C8 and CYP3A4. CYP2C8 is the only P450 isoform catalysing the aromatic hydroxylation to P10. CYP2C8 together with CYP3A4 are also involved in the formation of several minor ACT-333679 metabolites. UGT1A3 and UGT2B7 catalyse the glucuronidation of ACT-333679.

4.?The potential of selexipag to inhibit or induce cytochrome P450 enzymes or drug transport proteins was studied in vitro. Selexipag is an inhibitor of CYP2C8 and CYP2C9 and induces CYP3A4 and CYP2C9 in vitro. Also, selexipag inhibits the transporters OATP1B1, OATP1B3, OAT1, OAT3, and BCRP. However, due to its low dose and relatively low unbound exposure, selexipag has a low potential for causing drug–drug interactions.     

Stereoselective hydroxylation by CYP2C19 and oxidation by ADH4 in the in vitro metabolism of tivantinib

1.?In prior studies, it has been shown that tivantinib is extensively metabolized in humans to many oxidative metabolites and glucuronides. In order to identify the responsible enzymes, we investigated the in vitro metabolism of tivantinib and its four major circulating metabolites.

2.?The primary isoforms involved in the elimination of tivantinib were CYP2C19 and CYP3A4/5. CYP2C19 showed catalytic activity for the formation of M5 (hydroxylated metabolite), but not for M4 (a stereoisomer of M5), whereas CYP3A4/5 catalyzed the formation of both metabolites. For the elimination of M4, M5 and M8 (keto-metabolite), CYP3A4/5 was the major cytochrome P450 isoform and UGT1A9 was mainly involved in the glucuronidation of M4 and M5.

3.?ADH4 was identified as one of the major alcohol dehydrogenase isoforms contributing to the formation of M6 (sequential keto-metabolite of M4 and M5) and M8. The substrate preference of ADH for M4, and not M5, was observed in the formation of M6.

4.?In conclusion, CYP2C19, CYP3A4/5, UGT1A9 and ADH4 were the primary drug metabolizing enzymes involved in the in vitro metabolism of tivantinib and its metabolites. The stereoselective hydroxylation by CYP2C19 and substrate stereoselectivity of ADH4-catalyzed oxidation in the in vitro metabolism of tivantinib was discovered.     

Metabolism and excretion of benzo[a]pyrene in the rabbit

1. Following i.v. administration of [¹⁴C]benzo[a]pyrene (3 μmol/kg) to rabbits, 30% of the ¹⁴C dose appeared in bile and 12% in urine, within six hours.

2. Biliary and urinary metabolites were mainly conjugated; <12% of the ¹⁴C was extractable with ethyl acetate, but after treatment with β-glucuronidase or aryl sulphatase 30–40% became extractable.

3. H.p.l.c. analysis of the extracts indicated that the major non-polar metabolite was benzo[a]pyrene, 9,10-diol (18% of ¹⁴C in bile and 24% of ¹⁴C in urine, mainly conjugated with glucuronic acid). Smaller amounts of the 4,5-diol, the 3,6-quinone, and the 9-hydroxy- and 3-hydroxybenzo[a]pyrene were also found in bile (total <10%), together with 9-hydroxybenzo[a]pyrene and two unknown metabolites (X and Y) in urine (total <4%).

4. The proximate carcinogen, the 7,8-diol, was not detected in any extract.

5. After intraduodenal administration of biliary metabolites of [¹⁴C]benzo[a]pyrene (approx. 0·3 μmol), ¹⁴C was excreted in the bile (21% dose) and urine (14%) within 23?h, indicating that metabolites can undergo enterohepatic circulation in the rabbit.     

Studies on the Metabolism of Sympathomimetic Amines. The Metabolism of (±)-[14C] Adrenaline in the Horse

Abstract

1. The metabolism of (±)-[¹⁴C]adrenaline has been studied in two horses after intravenous administration.

2. The plasma half-life of ¹⁴C was 228 min (Horse A) and 100 min (Horse B).

3. Excretion of ¹⁴C in the urine in 20 h was 90% (A) and 70% (B). Over 94% of the excreted radioactivity has been identified.

4. The two horses differed in their metabolism of adrenaline. Unchanged drug accounted for 11% (A) and 20% (B), and for A and B respectively, metabolites excreted were 3-methoxyadrenaline (10%, 21%), 4-hydroxy-3-methoxymandelic acid (16%, 13%), 3,4-dihydroxyphenylglycol (6%, 0%), 4-hydroxy-3-methoxyphenylglycol (10%, 3%), 3,4-dihydroxybenzoic acid (5%, 0%), 3,4-dihydroxymandelic acid (7%, 3%) and 4-hydroxy-3-methoxybenzoic acid (18%, 4%).     

In vitro metabolism of monensin A: microbial and human liver microsomes models

Abstract

1.?Monensin A, an important antibiotic ionophore that is primarily employed to treat coccidiosis, selectively complexes and transports sodium cations across lipid membranes and displays a variety of biological properties.

2.?In this study, we evaluated the fungi Cunninghamella echinulata var. elegans ATCC 8688A, Cunninghamella elegans NRRL 1393 ATCC 10028B and human hepatic microsomes as CYP-P450 models to investigate the in vitro metabolism of monensin A and compare the products with the metabolites produced in vivo.

3.?Mass spectrometry analysis of the products from these model systems revealed the formation of three metabolites: 3-O-demethyl monensin A, 12-hydroxy monensin A and 12-hydroxy-3-O-demethyl monensin A. We identified these products by tandem mass spectrometry and through comparison with the in vivo metabolites.

4.?This analysis demonstrated that the model systems produce the same metabolites found in in vivo studies, thus they could be used to predict the metabolism of monensin A. Furthermore, we verified that liquid chromatography coupled to mass spectrometry is a powerful tool to study the in vitro metabolism of drugs, because it allows the successful identifications of several derivatives from different metabolic models.     

Thyroid hormone-like and estrogenic activity of hydroxylated PCBs in cell culture

The thyroid hormone-disrupting activity of hydroxylated PCBs was examined. 4-Hydroxy-2,2',3,4',5,5'-hexachlorobiphenyl (4-OH-2,2',3,4',5,5'-HxCB), 4-hydroxy-3,3',4',5-tetrachlorobiphenyl (4-OH-3,3',4',5-TCB) and 4,4'-dihydroxy-3,3',5,5'-tetrachlorobiphenyl (4,4'-diOH-3,3',5,5'-TCB), which have been detected as metabolites of PCBs in animals and humans, and six other 4-hydroxylated PCBs markedly inhibited the binding of triiodothyronine (1x10(-10)M) to thyroid hormone receptor (TR) in the concentration range of 1 x 10(-6) to 1 x 10(-4) M. However, 4-hydroxy-2',4',6'-trichlorobiphenyl (4-OH-2',4',6'-TCB), 3-hydroxy-2,2',5,5'-tetrachlorobiphenyl, 4-hydroxy-2,2',5,5'-tetrachlorobiphenyl, 4-hydroxy-2,3,3',4'-tetrachlorobiphenyl, 2,3',5,5'-tetrachlorobiphenyl and 2,3',4',5,5'-pentachlorodiphenyl did not show affinity for TR. The thyroid hormonal activity of PCBs was also examined using rat pituitary cell line GH3 cells, which grow and release growth hormone in a thyroid hormone-dependent manner. 4-OH-2,2',3,4',5,5'-HxCB, 4,4'-diOH-3,3',5,5'-TCB and 4-OH-3,3',4',5-TCB enhanced the proliferation of GH3 cells and stimulated their production of growth hormone in the concentration range of 1 x 10(-7) to 1 x 10(-4) M, while PCBs which had no affinity for thyroid hormone receptor were inactive. In contrast, only 4-OH-2',4',6'-TCB exhibited a significant estrogenic activity using estrogen-responsive reporter assay in MCF-7 cells. However, the 3,5-dichloro substitution of 4-hydroxylated PCBs markedly decreased the estrogenic activity. These results suggest that, at least for the 17 PCB congeners and hydroxylated metabolites tested, a 4-hydroxyl group in PCBs is essential for thyroid hormonal and estrogenic activities, and that 3,5-dichloro substitution favors thyroid hormonal activity, but not estrogenic activity.     

In vitro models of xenobiotic metabolism in trout for use in environmental bioaccumulation studies

1.?In vitro screens are sought as informative, alternatives to the use of animals in vivo and to improve upon the current use of fish liver 9000?g supernatants (S9) in environmental risk assessment.

2.?The rates of ethoxyresorufin-O-deethylation (relative to S9 protein) measured under different conditions of culture of rainbow trout hepatocytes were significantly higher than those detected in S9, in the order of freshly isolated hepatocytes > 10-day spheroid cultures > primary hepatocytes in culture > S9. The percentage of conjugated metabolites was also similar between freshly isolated and spheroid cultured hepatocytes (9.9 and 13.5%).

3.?The rate of oxidation was enhanced (1.7 fold) when S9 was supplemented with cofactors for phase II conjugation but this was only approximately one tenth of the rate in freshly isolated hepatocytes (7.1 pmol/min/mg S9 protein equivalent).

4.?Hepatocytes also hydroxylated ibuprofen, producing two metabolites, in contrast to only one (identified as the 1-hydroxy derivative) using hepatic S9 fractions.

5.?Since the bioaccumulation potential of chemicals is often based on un-supplemented S9 in incubations ≥1?h when activity declines, it is recommended that predictability would be greatly improved through the use of hepatocyte spheroids, due to their maintenance of activity and longevity.     

The metabolism of the dual endothelin receptor antagonist macitentan in rat and dog

Abstract

1.?The metabolism of the endothelin receptor antagonist macitentan has been characterized in bile duct-cannulated rats and dogs.

2.?In both species, macitentan was metabolized along five primary pathways, i.e. conjugation with glucose (M9), oxidative depropylation (M6), aliphatic hydroxylation (M7), oxidative cleavage of the ethylene glycol linker (M4) and hydrolysis of the sulfamide moiety (M3). Most of the primary metabolites underwent subsequent biotransformation including conjugation with glucuronic acid or glucose, hydrolysis of the sulfamide group or secondary oxidation of the ethylene glycol moiety.

3.?Though there were species differences in their relative importance, all metabolic pathways were present in rat and dog. The depropylated M6 was the only metabolite present in plasma of both species.

4.?Metabolism was a prerequisite for macitentan excretion as relevant amounts of parent drug were neither detected in bile nor urine. Biliary excretion was the major elimination pathway, while renal elimination was of little importance.     

Identification of human cytochrome P450 enzymes involved in the metabolism of IN-1130, a novel activin receptor-like kinase-5 (ALK5) inhibitor

1.?The in vitro metabolism of 3-((5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide (IN-1130), a selective activin receptor-like kinase-5 (ALK5) inhibitor and a candidate drug for fibrotic disease, was studied.

2.?The cytochrome P450s (CYPs) responsible for metabolism of IN-1130 in liver microsomes of rat, mouse, dog, monkey and human, and in human CYP supersomes?, were identified using specific CYP inhibitors. The order of disappearance of IN-1130 in various liver microsomal systems studied was as follows: monkey, mouse, rat, human, and dog.

3.?Five distinct metabolites (M1–M5) were identified in all the above microsomes and their production was substantially inhibited by CYP inhibitors such as SKF-525A and ketoconazole. Among nine human CYP supersomes? examined, CYP3A4, CYP2C8, CYP2D6*1, and CYP2C19 were involved in the metabolism of IN-1130, and the production of metabolites were significantly inhibited by specific CYP inhibitors. IN-1130 disappeared fastest in CYP2C8 supersomes. CYP3A4 produced four metabolites of IN-1130 (M1–M4), whereas supersomes expressing human FMO cDNAs, such as FMO1, FMO3, and FMO5, produced no metabolites.

4.?Hence, it is concluded that metabolism of IN-1130 is mediated by CYP3A4, CYP2C8, CYP2D6*1, and CYP2C19.