The Structure of Physalin T from Physalis alkekengi var. francheti

Abstract

A new steroidal constituent named physalin T (3) was isolated from the aqueous extract of Physalis alkekengi var. francheti. Based on ¹H and ¹³C NMR spectral studies the structure was assigned as 2,3-dihydrophysalin D, i.e., 5α,6β-dihydroxy-2,3,5,6-tetrahydrophysalin B, which is the first example of a natural physalin possessing a saturated ring A moiety. The structure was confirmed by the chemical transformation from the known physalin D (2) to physalin T.     

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.     

Metabolite profiling of anhuienoside C by rat intestinal bacteria using the LC–MS metabolomic approach

Abstract

1.?Anhuienoside C (A-C) is the main active component of the saponin exact of “Di Wu”, an oral drug for rheumatism treatment in China. In this study, we aimed to elucidate the metabolic pathways of A-C by intestinal bacteria using the metabolomic approach.

2.?Four deglycosylated metabolites (M1, M2, M3 and M4) were identified after A-C (50?µM) was incubated with rat fecal lysate. Chemical structures of these metabolites were determined by high-resolution masses and nuclear magnetic resonance (NMR).

3.?A one-compartment pharmacokinetic model was used to describe the formation of bacterial metabolites at a dose of 10?µM A-C. The results revealed that formation of M1 and M2 was rapid, whereas formation of M3 was rather slow. Further, it was found that the metabolites were generated by successive cleavage of the glycosyl residues.

4.?This is the first report that A-C is subjected to efficient bacterial metabolism in the gut with M1 and M2 as main metabolites. Our study should be helpful for a better understanding of in vivo disposition of oral A-C.     

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.     

A microbial model of mammalian metabolism: biotransformation of 4,5-dimethoxyl-canthin-6-one using Cunninghamella blakesleeana CGMCC 3.970

1.?A filamentous fungus, Cunninghamella blakesleeana CGMCC 3.970, was applied as a microbial system to mimic mammalian metabolism of 4,5-dimethoxyl-canthin-6-one (1). Compound 1 belongs to canthin-6-one type alkaloids, which is a major bioactive constituent of a traditional Chinese medicine (the stems of Picrasma quassioides).

2.?After 72?h of incubation in potato dextrose broth, 1 was metabolized to seven metabolites as follows: 4-methoxyl-5-hydroxyl-canthin-6-one (M1), 4-hydroxyl-5-methoxyl-canthin-6-one (M2), canthin-6-one (M3), canthin-6-one N-oxide (M4), 10-hydroxyl-4,5-dimethoxyl-canthin-6-one (M5), 1-methoxycarbonl-β-carboline (M6), and 4-methoxyl-5-O-β-D-glucopyranosyl-canthin-6-one (M7).

3.?The structures of metabolites were determined using spectroscopic analyses, chemical methods, and comparison of NMR data with those of known compounds. Among them, M7 was a new compound.

4.?The metabolic pathways of 1 were proposed, and the metabolic processes involved phase I (O-demethylation, dehydroxylation, demethoxylation, N-oxidation, hydroxylation, and oxidative ring cleavage) and phase II (glycosylation) reactions.

5.?This was the first research on microbial transformation of canthin-6-one alkaloid, which could be a useful microbial model for producing the mammalian phase I and phase II metabolites of canthin-6-one alkaloids.

6.?1, M1?M5, and M7 are canthin-6-one alkaloids, whereas M6 belongs to β-carboline type alkaloids. The strain of Cunninghamella blakesleeana can supply an approach to transform canthin-6-one type alkaloids into β-carboline type alkaloids.     

Isolation and identification of phase I metabolites of butyrolactone I in rats

1.?Butyrolactone I (BL-I), one of the major secondary metabolites of fungus Aspergillus terreus, is a selective cdc2 kinase inhibitor. In the present study, the metabolism of BL-I in male Wistar rats was investigated by characterizing metabolites excreted into feces.

2.?Following an oral dose of 40?mg/kg BL-I, 10 phase I metabolites were isolated from the feces of rats, and their structures were identified on the basis of a range of spectroscopic data and ICD analysis. These metabolites were fully characterized as butyrolactone VI (M1), aspernolide E (M2), 7′′S-hydroxy-9′′-ene-butyrolactone I (M3), 7′′R-hydroxy-9′′-ene-butyrolactone I (M4), 7″S, 8″R-dihydroxy-aspernolide E (M5), 7″R, 8″S-dihydroxy-aspernolide E (M6), 7″R-acetyl-8″S-hydroxy-aspernolide E (M7), 7″S-acetyl-8″R-hydroxy-aspernolide E (M8), 7″R-methoxy-8″S-hydroxy-aspernolide E (M9), butyrolactone V (M10), respectively. It is the first time to describe the metabolites of BL-I in vivo, and metabolites M3 to M9 are new compounds.

3.?BL-I and metabolites M2 to M10 were evaluated for their antimicrobial activity and in vitro antiproliferative activities. Only M-3 and M-4 showed inhibitory effect against staphylococcus aureus both with MIC of 125?μg/ml. BL-I and metabolites M-4 and M-5 exhibited potent cancer cell growth inhibitory activities against HL-60 (human leukemia) cell lines with the IC₅₀ values of 13.2, 28.8 and 35.7?μM, respectively.

4.?On the basis of metabolites profile, a possible metabolism pathway for BL-I in rats has been proposed. This is the first systematic study on the phase I metabolites of BL-I.     

Identification of the cytochrome P450 enzymes involved in the metabolism of domperidone

1.?The objective was to identify the major cytochrome P450 enzyme(s) involved in the metabolism of domperidone.

2.?Experiments were performed using human liver microsomes (HLMs), recombinant human cytochrome P450 enzymes, cytochrome P450 chemical inhibitors and monoclonal antibodies directed against cytochrome P450 enzymes.

3.?Four metabolites were identified from incubations performed with HLMs and excellent correlations were observed between the formation of domperidone hydroxylated metabolites (M1, M3 and M4), N-desalkylated domperidone metabolite (M2) and enzymatic markers of CYP3A4/5 (r²?=?0.9427, 0.951, 0.9497 and 0.8304, respectively).

4.?Ketoconazole (1?μM) decreased the formation rate of M1, M2, M3 and M4 by 83, 78, 75 and 88%, respectively, whereas the effect of other inhibitors (quinidine, furafylline and sulfaphenazole) was minimal. Important decreases in the formation rate of M1 (68%), M2 (64%) and M3 (54%) were observed with anti-CYP3A4 antibodies.

5.?Formation of M1, M2 and M3 in HLMs exhibited Michaelis–Menten kinetics (K_m: 166, 248 and 36?μM, respectively). Similar K_m values were observed for M1, M2 and M3 when incubations were performed with recombinant human CYP3A4 (K_m: 107, 273 and 34?μM, respectively).

6.?The data suggest that CYP3As are the major enzymes involved in the metabolism of domperidone.     

Species differences in metabolism of EPZ015666, an oxetane-containing protein arginine methyltransferase-5 (PRMT5) inhibitor

Abstract

1.?Metabolite profiling and identification studies were conducted to understand the cross-species differences in the metabolic clearance of EPZ015666, a first-in-class protein arginine methyltransferase-5 (PRMT5) inhibitor, with anti-proliferative effects in preclinical models of Mantle Cell Lymphoma. EPZ015666 exhibited low clearance in human, mouse and rat liver microsomes, in part by introduction of a 3-substituted oxetane ring on the molecule. In contrast, a higher clearance was observed in dog liver microsomes (DLM) that translated to a higher in vivo clearance in dog compared with rodent.

2.?Structure elucidation via high resolution, accurate mass LC-MSⁿ revealed that the prominent metabolites of EPZ015666 were present in hepatocytes from all species, with the highest turnover rate in dogs. M1 and M2 resulted from oxidative oxetane ring scission, whereas M3 resulted from loss of the oxetane ring via an N-dealkylation reaction.

3.?The formation of M1 and M2 in DLM was significantly abrogated in the presence of the specific CYP2D inhibitor, quinidine, and to a lesser extent by the CYP3A inhibitor, ketoconazole, corroborating data from human recombinant isozymes.

4.?Our data indicate a marked species difference in the metabolism of the PRMT5 inhibitor EPZ015666, with oxetane ring scission the predominant metabolic pathway in dog mediated largely by CYP2D.     

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.     

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.     

The metabolism of 4-bromoaniline in the bile-cannulated rat: application of ICPMS (79/81Br), HPLC-ICPMS & HPLC-oaTOFMS

Abstract

1.?An excretion balance study was performed following i.p. administration of 4-bromoaniline (50?mg kg^?1) to bile-cannulated rats, using bromine-detected (^79/81Br) ICPMS for quantification. Approximately 90% of the dose was recovered in urine (68.9?±?3.6%) and bile (21.4?±?1.4%) by 48?h post-administration.

2.?HPLC-ICPMS (^79/81Br) was used to selectively detect and profile the major urinary and biliary-excreted metabolites and determined that the 0–12?h urine contained at least 21 brominated metabolites with 19 bromine-containing peaks observed in the 6–12?h bile samples.

3.?The urinary and biliary metabolites were subsequently profiled using HPLC-oaTOFMS. By exploiting the distinctive bromine isotope pattern ca. 60 brominated metabolites were detected in the urine in negative electrospray ionisation (ESI) mode while bile contained ca. 21.

4.?While a large number of bromine-containing metabolites were detected, the profiles were dominated by a few major components with the bulk of the 4-bromoaniline-related material in urine accounted for by 4-bromoanaline O-sulfate (～75% of the total by ICPMS, 84% by TOFMS). In bile a hydroxylated N-acetyl compound was the major metabolite detected, forming some ～65% of the 4-bromoaniline-related material by ICPMS (37% by TOFMS).     

Pharmacokinetics,metabolism and excretion of [14C]-lanicemine (AZD6765), a novel low-trapping N-methyl-d-aspartic acid receptor channel blocker,in healthy subjects

Abstract

1.?(1S)-1-phenyl-2-(pyridin-2-yl)ethanamine (lanicemine; AZD6765) is a low-trapping N-methyl-d-aspartate (NMDA) channel blocker that has been studied as an adjunctive treatment in major depressive disorder. The metabolism and disposition of lanicemine was determined in six healthy male subjects after a single intravenous infusion dose of 150?mg [¹⁴C]-lanicemine.

2.?Blood, urine and feces were collected from all subjects. The ratios of C_max and AUC_(0–∞) of lanicemine to plasma total radioactivity were 84 and 66%, respectively, indicating that lanicemine was the major circulating component with T_1/2 at 16?h. The plasma clearance of lanicemine was 8.3?L/h, revealing that lanicemine is a low-clearance compound. The mean recovery of radioactivity from urine was 93.8% of radioactive dose.

3.?In urine samples, 10 metabolites of lanicemine were identified. Among which, an O-glucuronide conjugate (M1) was the most abundant metabolite (～11% of the dose in excreta). In plasma, the circulatory metabolites were identified as a para-hydroxylated metabolite (M1), an O-glucuronide (M2), an N-carbamoyl glucuronide (M3) and an N-acetylated metabolite (M6). The average amount of each of metabolite was less than 4% of total radioactivity detected in plasma or urine.

4.?In conclusion, lanicemine is a low-clearance compound. The unchanged drug and metabolites are predominantly eliminated via urinary excretion.     

In vitro metabolism of gefitinib in human liver microsomes

1.?The in vitro metabolism of gefitinib was investigated by incubating [¹⁴C]-gefitinib, as well as M537194, M387783 and M523595 (the main metabolites of gefitinib observed in man), at a concentration of 100?μM with human liver microsomes (4?mg?ml^?1) for 120?min. These relatively high substrate and microsomal protein concentrations were used in an effort to generate sufficient quantities of metabolites for identification.

2.?HPLC with ultraviolet light, radiochemical and mass spectral analysis, together with the availability of authentic standards, enabled quantification and structural identification of a large number of metabolites. Although 16 metabolites were identified, metabolism was restricted to three regions of the molecule.

3.?The major pathway involved morpholine ring-opening and step-wise removal of the morpholine ring and propoxy side chain. O-demethylation of the quinazoline methoxy group was a quantitatively less important pathway, in contrast to the clinical situation, where O-desmethyl gefitinib (M523595) is the predominant plasma metabolite. The third metabolic route, oxidative defluorination, was only a minor route of metabolism. Some metabolites were formed by a combination of these processes, but no metabolism was observed in other parts of the molecule.

4.?Incubation of gefitinib produced ten identified metabolites, but the use of the three main in vivo metabolites as additional substrates enabled a more comprehensive metabolic pathway to be constructed and this has been valuable in supporting the more limited data available from the human in vivo study.     

Preclinical metabolism and pharmacokinetics of NVS-CRF38, a potent and orally bioavailable corticotropin-releasing factor receptor 1 antagonist

Abstract

1.?The pharmacokinetic properties and metabolism of NVS-CRF38 [7-(3,5-dimethyl-1H-1,2,4-triazol-1-yl)-3-(4-methoxy-2-methylphenyl)-2,6-dimethylpyrazolo[5,1-b]oxazole], a novel corticotropin-releasing factor receptor 1 (CRF₁) antagonist, were determined in vitro and in animals.

2.?NVS-CRF38 undergoes near complete absorption in rats and dogs. In both species the compound has low hepatic extraction and is extensively distributed to tissues.

3.?In rat and human hepatic microsomes and cryopreserved hepatocytes from rat, dog, monkey and human, NVS-CRF38 was metabolised to form O-desmethyl NVS-CRF38 (M7) and several oxygen adducts (M1, M3, M4, M5 and M6). In hepatocytes further metabolites were observed, specifically the carboxylic acid (M2) and conjugates (sulphate and glucuronide) of M7.

4.?Formation of primary metabolites in hepatocytes was blocked by the cytochrome P450 enzyme (P450) suicide inhibitor 1-aminobenzotriazole, implicating P450 enzymes in the primary metabolism of this compound.

5.?NVS-CRF38 is weakly bound to plasma proteins from rat (fu_b?=?0.19), dog (fu_b?=?0.25), monkey (fu_b?=?0.20) and humans (fu_b?=?0.23). Blood-to-plasma partition for NVS-CRF38 approaches unity in rat and human blood.

6.?The hepatic clearance of NVS-CRF38 in humans is predicted to be low (extraction ratio?～?0.2) based on scaling from drug depletion profiles in hepatic microsomes.     

Mechanism-based inhibition of CYPs and RMs-induced hepatoxicity by rutaecarpine

Abstract

1.?Rutaecarpine, a quinolone alkaloid isolated from the unripe fruit of Evodia rutaecarpa, is one of the main active components used in a variety of clinical applications, including the treatment of hypertension and arrhythmia. However, its hepatotoxicity has also been reported in recent years.

2.?Reactive metabolites (RMs) play a vital role in drug-induced liver injury. Rutaecarpine has a secondary amine structure that may be activated to RMs. The aim of the study was to investigate the inhibition of rutaecarpine on CYPs and explore the possible relationship between RMs and potential hepatotoxicity.

3.?A cell counting kit-8 cytotoxicity assay indicated that rutaecarpine can decrease the primary rat hepatocyte viability, increase lactate dehydrogenase and reactive oxygen species, reduce JC-1, and cause cell stress and membrane damage. The indexes were significantly restored by adding ABT, an inhibitor of CYPs. A cocktail assay showed that CYP1A2, CYP2C9, CYP2C19, CYP2E1 and CYP3A4 can be inhibited by rutaecarpine in human liver microsomes. The IC₅₀ values of CYP1A2 with and without NADPH were 2.2 and 7.4?μM, respectively, which presented a 3.3 shift. The results from a metabolic assay indicated that three mono-hydroxylated metabolites and two di-hydroxylated metabolites were identified and two GSH conjugates were also trapped.

4.?Rutaecarpine can inhibit the activities of CYPs and exhibit a potential mechanism-based inhibition on CYP1A2. RMs may cause herb–drug interactions, providing important information for predicting drug-induced hepatotoxicity.     

Endogenous and xenobiotic metabolite profiling of liver extracts from SCID and chimeric humanized mice following repeated oral administration of troglitazone

Abstract

1.?Metabonomic analysis, via a combination of untargeted and targeted liquid chromatography-mass spectrometry (LC-MS) and untargeted ¹H NMR spectroscopy-based metabolite profiling, was performed on aqueous (AQ) and organic liver extracts from control (SCID) and chimeric humanized (PXB) mice dosed with troglitazone at 0, 300 and 600?mg/kg/day for seven days.

2.?LC-MS analysis of AQ liver extracts showed a more “human-like” profile for troglitazone metabolites for PXB, compared with SCID, mice.

3.?LC-MS detected differences in endogenous metabolites, particularly lipid species in dosed mice, including elevated triacylglycerols and 1-alkyl,2-acylglycerophosphates as well as lowered diacylglycerophosphocholines and 1-alkyl,2-acylglycerophosphocholines for PXB compared with SCID mouse liver extracts. Following drug administration changes in the relative proportions of the ions for various unsaturated fatty acids were observed for both types of mouse, some of which were specific to PXB or SCID mice.

4.?¹H NMR spectroscopy revealed that AQ PXB mouse liver extracts had elevated amounts of inosine, fumarate, creatine, aspartate, trimethylamine N-oxide, glycerophosphocholine, phosphocholine, choline, glutamine, glutamate, acetate, alanine and lactate relative to SCID mice and decreased histidine, glycogen, α- and β-glucose, taurine, and glutathione. Increased uracil and tyrosine concentrations were detected for PXB mice on troglitazone administration.

5.?Metabonomic profiling thus showed clear differences between humanized and SCID mice, including after administration of troglitazone.     

In vitro inhibitory effects of sophocarpine on human liver cytochrome P450 enzymes

Abstract

1.?Sophocarpine is a biologically active component isolated from the foxtail-like sophora herb and seed that is often orally administered for the treatment of cancer and chronic bronchial asthma. However, whether sophocarpine affects the activity of human liver cytochrome P450 (CYP) enzymes remains unclear.

2.?In this study, the inhibitory effects of sophocarpine on the eight human liver CYP isoforms (CYP1A2, 3A4, 2A6, 2E1, 2D6, 2C9, 2C19, and 2C8) were investigated in vitro using human liver microsomes (HLMs).

3.?The results indicate that sophocarpine could inhibit the activity of CYP3A4 and 2C9, with the IC₅₀ values of 12.22 and 15.96?μM, respectively, but that other CYP isoforms were not affected. Enzyme kinetic studies showed that sophocarpine is not only a noncompetitive inhibitor of CYP3A4 but also a competitive inhibitor of CYP2C9, with K_i values of 6.74 and 9.19?μM, respectively. Also, sophocarpine is a time-dependent inhibitor of CYP3A4 with K_inact/K_I value of 0.082/21.54?μM^?1?min^?1.

4.?The in vitro studies of sophocarpine with CYP isoforms suggested that sophocarpine has the potential to cause pharmacokinetic drug interactions with other co-administered drugs metabolized by CYP3A4 and 2C9. Further clinical studies are needed to evaluate the significance of this interaction.     

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.     

Species differences in metabolism of ripasudil (K-115) are attributed to aldehyde oxidase

1.?We examined the metabolism of ripasudil (K-115), a selective and potent Rho-associated coiled coil-containing protein kinase (ROCK) inhibitor, by in vitro and in vivo studies.

2.?First, we identified metabolites and metabolic enzymes involved in ripasudil metabolism. Species differences were observed in metabolic clearance and profiles of metabolites in liver S9 fraction and hepatocytes. In addition, ripasudil was metabolised in humans and monkey S9 without nicotinamide adenine dinucleotide phosphate (NADPH). Studies using specific inhibitors and human recombinant enzyme systems showed that M1 (main metabolite in humans) formation is mediated by aldehyde oxidase (AO).

3.?Therefore, we developed ripasudil as an ophthalmic agent. First, we compared the pharmacokinetic profiles of ripasudil in humans and rats. The results indicated rapid disappearance of ripasudil from the circulation after instillation in humans and its level remained relatively high only in M1. In contrast, we found six metabolites from M1 to M6 in plasma after oral administration to rats.

4.?Analysis of enzyme kinetics using S9 showed that the formation of M1 is the major metabolic pathway of ripasudil in humans even though CYP3A4/3A5 and CYP2C8/3A4/3A5 were associated with the formation of M2 and M4, respectively. In conclusion, AO causes differences in ripasudil metabolism between species.     

Biotransformation of fluorophenyl pyridine carboxylic acids by the model fungus Cunninghamella elegans

1.?Fluorine plays a key role in the design of new drugs and recent FDA approvals included two fluorinated drugs, tedizolid phosphate and vorapaxar, both of which contain the fluorophenyl pyridyl moiety.

2.?To investigate the likely phase-I (oxidative) metabolic fate of this group, various fluorinated phenyl pyridine carboxylic acids were incubated with the fungus Cunninghamella elegans, which is an established model of mammalian drug metabolism.

3.?¹⁹F NMR spectroscopy established the degree of biotransformation, which varied depending on the position of fluorine substitution, and gas chromatography–mass spectrometry (GC–MS) identified alcohols and hydroxylated carboxylic acids as metabolites. The hydroxylated metabolites were further structurally characterised by nuclear magnetic resonance spectroscopy (NMR), which demonstrated that hydroxylation occurred on the 4′ position; fluorine in that position blocked the hydroxylation.

4.?The fluorophenyl pyridine carboxylic acids were not biotransformed by rat liver microsomes and this was a consequence of inhibitory action, and thus, the fungal model was crucial in obtaining metabolites to establish the mechanism of catabolism.