Mechanism-based inhibition of CYP2C19 in human liver microsomes by the thienopyridine antiplatelet agents clopidogrel, prasugrel and their thiolactone metabolites was investigated by determining the time- and concentration-dependent inhibition of the activity of S-mephenytoin 4′-hydroxylase as typical CYP2C19 activity and compared with ticlopidine and its metabolite.
Clopidogrel was shown to be a mechanism-based inhibitor of CYP2C19 with the inactivation kinetic parameters, kinact and KI, equal to 0.0557?min?1 and 14.3?μM, respectively, as well as ticlopidine (0.0739?min?1 and 3.32?μM, respectively). The thiolactone metabolite of ticlopidine and clopidogrel inhibited CYP2C19 only in a concentration-dependent manner. In contrast, neither prasugrel nor its thiolactone metabolite inhibited CYP2C19 at concentrations up to 100?μM.
The oxidation of the thiophene moiety of clopidogrel to form their respective thiolactones was found to be the critical reaction that produces the chemically reactive metabolites which cause the mechanism-based inhibition of CYP2C19.
Estimation of in vivo drug–drug interaction using in vitro parameters predicted clinically observed data. For clopidogrel, there was no increase in the area under the curve (AUC) at its clinical dose level as predicted by the in vitro parameters, and for ticlopidine the prediction agreed with the clinically observed AUC increase.
In conclusion, clopidogrel is potent mechanism-based inhibitors of CYP2C19 as well as ticlopidine, whereas prasugrel did not inactivate CYP2C19. Administration of prasugrel would not cause a clinically relevant interaction with CYP2C19.
Omacetaxine mepesuccinate (hereafter referred to as omacetaxine) is a protein translation inhibitor approved by the US Food and Drug Administration for adult patients with chronic myeloid leukemia with resistance and/or intolerance to two or more tyrosine kinase inhibitors.
The objective was to investigate the metabolite profile of omacetaxine in plasma, urine and faeces samples collected up to 72?h after a single 1.25-mg/m2 subcutaneous dose of 14C-omacetaxine in cancer patients.
High-performance liquid chromatography mass spectrometry (MS) (high resolution) in combination with off-line radioactivity detection was used for metabolite identification.
In total, six metabolites of omacetaxine were detected. The reactions represented were mepesuccinate ester hydrolysis, methyl ester hydrolysis, pyrocatechol conversion from the 1,3-dioxole ring. Unchanged omacetaxine was the most prominent omacetaxine-related compound in plasma. In urine, unchanged omacetaxine was also dominant, together with 4′-DMHHT. In feces very little unchanged omacetaxine was found and the pyrocatechol metabolite of omacetaxine, M534 and 4′-desmethyl homoharringtonine (4′-DMHHT) was the most abundant metabolites.
Omacetaxine was extensively metabolized, with subsequent renal and hepatic elimination of the metabolites. The low levels of the metabolites found in plasma indicate that the metabolites are unlikely to contribute materially to the efficacy and/or toxicity of omacetaxine.
The article describes and discusses the evolution of strategies to characterize metabolites in support of safety studies over the last 40 years, as well as future trends.
Approaches to derive qualitative and quantitative information on metabolites are described, with a particular focus on the comparison of options to quantify metabolites in the absence of authentic standards.
Current strategies to assess metabolite profiles are summarized into four general approaches and compared against a number of key criteria.
Potential future strategies are discussed, including the use of clinical samples as the starting point for metabolite investigations, minimizing the need for animal radiolabelled studies and establishing metabolite safety without radiolabelled studies in animals or human.
A study of the rates and routes of excretion of 3-fluoro-[U-14C]aniline following intraperitoneal administration to male bile-cannulated rats by liquid scintillation counting (LSC) gave a total recovery of ～ 90% in the 48?h following dosing, with the majority of the dose being excreted in the urine during the first 24?h (～ 49%).
The total recovery as determined by 19F-nuclear magnetic resonance (19F-NMR) was ～ 49%, with the majority of the dose excreted in the first 24?h (～ 41%). The comparatively low recovery in comparison to that obtained from LSC was due to matrix effects in bile and a contribution from metabolic defluorination.
High-performance liquid chromatography with radiometric profiling of urine and bile revealed a complex pattern of metabolites with the bulk of the dose excreted as a single peak.
Ultra-performance liquid chromatography-orthogonal acceleration time of flight mass spectrometry profiling also showed a complex pattern of metabolites, detecting ～ 21 metabolites of 3-fluoroaniline (3-FA) with six of these detected only in urine and four solely in bile.
19F-NMR revealed the presence of the parent compound and 15 metabolites in urine collected during the first 24?h after -dosing. The matrix effects of bile on 19F-NMR spectroscopy made metabolite profiling impractical for this biofluid.
The major metabolite of 3-FA was identified as 2-fluoro-4-acetamidophenol-sulfate.
The pharmacokinetics of ?-acetamidocaproic acid (AACA) were evaluated after the intravenous and oral administration of an antiulcer agent, zinc acexamate (ZAC) at a dose of 20?mg kg?1 (ion pairing between zinc and AACA) in rats with indomethacin-induced acute gastric ulcer (IAGU) or indomethacin-induced small bowel inflammation (ISBI).
In IAGU rats, the area under the curves (AUCs) of AACA were significantly smaller after both the intravenous (551 versus 1270 μg min ml?1) and oral (397 versus 562 μg min ml?1) administration of ZAC than controls, possible due to the significantly faster CLR of AACA. In ISBI rats, however, the AUCs of AACA were comparable with controls after both the intravenous and oral administration of ZAC.
In IAGU rats, the significantly smaller AUCs of AACA were due to the significantly faster CLR (due to the decreased urinary pH by indomethacin treatment) than controls. AACA has a basic secondary amine group. On the other hand, the comparable AUCs of AACA in ISBI rats were due to the comparable CLRs between ISBI and control rats.
AACA was excreted in the urine via active renal tubular secretion in all rats studied.
Pharmacokinetic and metabolism aspects of AMG 222 interaction with target enzyme, dipeptidylpeptidase IV (DPPIV) were investigated.
Inhibition of recombinant human DPPIV by AMG 222 was measured. IC50 decreased as preincubation time increased. koff, kon and Kd were measured. Dilution assay indicated a long dissociation half-life (730?min) relative to DPPIV inhibitor vildagliptin. AMG 222 is a slow-on, tight-binding, slowly reversible inhibitor of DPPIV.
Amide and acid metabolites arising from hydrolysis of AMG 222’s cyano group were formed slowly by rhDPPIV, but not by microsomes or S9. The amide metabolite was converted to the acid metabolite by rhDPPIV, but not by an active site mutant. These metabolites of AMG 222 are formed by target-mediated metabolism of the cyano group, similar to vildagliptin.
Human plasma protein binding of [14C]AMG 222 was saturable and concentration-dependent. After 30?min, [14C]AMG 222 was 80.8% bound at 1?nM and binding decreased to 29.4% above 100?nM. The plasma DPPIV concentration (4.1?nM) and human plasma AMG 222 concentrations that inhibit DPPIV, occurred in the range of concentration-dependent binding. Target-mediated drug disposition influences AMG 222 pharmacokinetics, similar to DPPIV inhibitor, linagliptin.
The metabolism and excretion of a GABAA partial agonist developed for the treatment of anxiety, CP-409,092; 4-oxo-4,5,6,7-tetrahydro-1H-indole-3-carboxylic acid (4-methylaminomethyl-phenyl)-amide, were studied in rats following intravenous and oral administration of a single doses of [14C]CP-409,092.
The pharmacokinetics of CP-409,092 following single intravenous and oral doses of 4 and 15?mg kg?1, respectively, were characterized by high clearance of 169?±?18?ml min?1 kg?1, a volume of distribution of 8.99?±?1.46 l kg?1, and an oral bioavailability of 2.9% ± 3%.
Following oral administration of 100?mg kg?1 [14C]CP-409,092, the total recovery was 89.1% ± 3.2% for male rats and 89.3% ± 0.58% for female rats. Approximately 87% of the radioactivity recovered in urine and faeces were excreted in the first 48?h. A substantial portion of the radioactivity was measured in the faeces as unchanged drug, suggesting poor absorption and/or biliary excretion. There were no significant gender-related quantitative/qualitative differences in the excretion of metabolites in urine or faeces.
The major metabolic pathways of CP-409,092 were hydroxylation(s) at the oxo-tetrahydro-indole moiety and oxidative deamination to form an aldehyde intermediate and subsequent oxidation to form the benzoic acid. The minor metabolic pathways included N-demethylation and subsequent N-acetylation and oxidation.
The present work demonstrates that oxidative deamination at the benzylic amine of CP-409,092 and subsequent oxidation to form the acid metabolite seem to play an important role in the metabolism of the drug, and they contribute to its oral clearance and low exposure.
The pharmacokinetics and disposition of GDC-0879, a small molecule B-RAF kinase inhibitor, was characterized in mouse, rat, dog, and monkey.
In mouse and monkey, clearance (CL) of GDC-0879 was moderate (18.7–24.3 and 14.5?±?2.1?ml min?1 kg?1, respectively), low in dog (5.84?±?1.06?ml min?1 kg?1) and high in rat (86.9?±?14.2?ml min?1 kg?1). The volume of distribution across species ranged from 0.49 to 1.9?l kg?1. Mean terminal half-life values ranged from 0.28?h in rats to 2.97?h in dogs. Absolute oral bioavailability ranged from 18% in dog to 65% in mouse.
Plasma protein binding of GDC-0879 in mouse, rat, dog, monkey, and humans ranged from 68.8% to 81.9%.
In dog, the major ketone metabolite (G-030748) of GDC-0879 appeared to be formation rate-limited.
Based on assessment in dogs, the absorption of GDC-0879 appeared to be sensitive to changes in gut pH, food and salt form (solubililty), with approximately three- to four-fold change in areas under the curve (AUCs) observed.
Compound A [1-methyl-N-{(1S)-1-[5-(2-naphthyl)-1H-imidazol-2-yl]-7-oxooctyl}piperidine-4-carboxamide is a potent class I histone deacetylase (HDAC) inhibitor that demonstrated good antiproliferative activity against human tumour cell lines of different origin.
This compound showed high in vivo clearance in rats (160?ml min?1 kg?1) due to metabolism. The main metabolite detected in urine after intravenous dosing was characterized as a dihydrohydroxy S-mercapturic acid conjugate. Following oral dosing, however, the mercapturic acid derivative was no longer the main metabolite but the major metabolites were mono- and di-glucuronide conjugates of oxidized species having a mass shift of +34 m/z with respect to the parent.
Comparison of plasma concentration after intra-arterial infusion and intravenous infusion and incubation with microsomes from different tissues (liver, kidney, small intestine and lung) in the presence of β-nicotinamide adenine dinucleotide phosphate (NADPH) indicated that the compound was highly cleared by the lung.
Oxidation of the naphthalene moiety was demonstrated to be the cause of the high in vivo clearance of compound A and the potential for bioactivation of this group was flagged.
Combretastatin A-4 (CA-4), is a natural compound with a potent tubulin polymerization and cell growth inhibitor properties. For these reasons CA-4 is one of the most potent anti-vascular agents that shows strong cytotoxicity against a variety of human cancer cells, including multi-drug-resistant cancer cell lines. In order to complete the knowledge of metabolic fate of CA-4, the in vitro and in vivo phase II metabolism was investigated.
Both in incubation with rat and human liver S9 preparation in the presence of 39-phosphoadenosine-5´-phosphosulfate (PAPS) as a cofactor the formation of a previously no reported sulphate metabolite was demonstrated through liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) data and comparison with a synthetic reference sample.
In incubation of CA-4 using rat and human liver microsomes, the formation of CA-4 glucuronide was observed and chromatographic and mass spectral properties of the metabolite were achieved and compared with those of a synthetic reference sample.
Incubation of CA-4 with rat and human liver S9 preparation in the presence of uridine-5´-diphosphoglucuronic acid trisodium salt (UDPGA) and an β-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-regenerating system as cofactors resulted in the formation of glucuronides arising from phase I CA-4 metabolites.
When CA-4 was administered intraperitoneally to rat at a dose of 30 mg kg?1, both glucuronide and sulphate metabolites were observed in LC-ESI-MS-MS chromatograms and their mass spectral data were identical to those obtained from synthetic standards.
GDC-0449 (2-chloro-N-(4-chloro-3-(pyridin-2-yl)phenyl)-4-(methylsulfonyl)benzamide) is a potent, selective Hedgehog (Hh) signalling pathway inhibitor being developed for the treatment of various cancers.
The in vivo clearance of GDC-0449 was estimated to be 23.0, 4.65, 0.338, and 19.3?ml min?1 kg?1 in mouse, rat, dog and monkeys, respectively. The volume of distribution ranged from 0.490 in rats to 1.68 l kg?1 in mice. Oral bioavailability ranged from 13% in monkeys to 53% in dogs. Predicted human clearance using allometry was 0.096–0.649?ml min?1 kg?1 and the predicted volume of distribution was 0.766 l kg?1.
Protein binding was extensive with an unbound fraction less than or equal to 6%, and the blood-to-plasma partition ratio ranged from 0.6 to 0.8 in all species tested. GDC-0449 was metabolically stable in mouse, rat, dog and human hepatocytes and had a more rapid turnover in monkey hepatocytes.
Proposed metabolites from exploratory metabolite identification in vitro (rat, dog and human liver microsomes) and in vivo (dog and rat urine) include three primary oxidative metabolites (M1–M3) and three sequential glucuronides (M4–M6). Oxidative metabolites identified in microsomes M1 and M3 were formed primarily by P4503A4/5 (M1) and P4502C9 (M3).
GDC-0449 was not a potent inhibitor of P4501A2, P4502B6, P4502D6, and P4503A4/5 with IC50 estimates greater than 20?μM. Ki’s estimated for P4502C8, P4502C9 and P4502C19 and were 6.0, 5.4 and 24?μM, respectively. An evaluation with Simcyp® suggests that GDC-0449 has a low potential of inhibiting P4502C8 and P4502C9. Furthermore, GDC-0449 (15?μM) was not a potent P-glycoprotein/ABCB1 inhibitor in MDR1-MDCK cells.
Overall, GDC-0449 has an attractive preclinical profile and is currently in Phase II clinical trials.
The in vitro metabolism of [14C]methoxychlor (MXC) has been studied using precision-cut liver slices from the Sprague–Dawley male rat, CD-1 male mouse, WE strain male Japanese quail and juvenile rainbow trout (Oncorhynchus mykiss). The results demonstrated integrated phase I and II metabolism of MXC and species differences in the metabolic profiles were observed.
In rat liver slice preparations, MXC was rapidly metabolized to bis-OH-MXC by sequential O-demethylation followed by subsequent O-glucuronidation forming bis-OH-MXC glucuronide. No mono-OH-MXC glucuronide was detected. The doubly conjugated metabolite, bis-OH-MXC 4-O-sulphate 4′-O-glucuronide, was also detected as a rat-specific metabolite.
Formation of mono-OH-MXC and its glucuronide was the main metabolic pathway in the mouse and Japanese quail. In contrast to the rat, only minor amounts of bis-OH-MXC glucuronide were detected. A reductively dehalogenated metabolite, dechlorinated mono-OH-MXC glucuronide, was observed only in mouse preparations.
In rainbow trout, comparative amounts of both mono- and bis-OH-MXC glucuronide were formed as the major metabolites. Unconjugated forms of these metabolites were detected only as minor products.
The different metabolic profiles of MXC observed in the four animal species are possibly due to substrate specificity of contributing CYP450 monooxgenase enzyme(s) in different animal species.
Purpose
Prasugrel is a novel thienopyridine prodrug metabolised to an active metabolite that binds irreversibly to the platelet P2Y12 receptor and inhibits adenosine diphosphate (ADP)-induced platelet aggregation. We compared prasugrel pharmacokinetics, pharmacodynamics, and tolerability in healthy Chinese, Japanese, Korean and Caucasian subjects.Methods
In an open-label, single-centre, parallel-design study, 89 healthy subjects (25 Chinese, 20 Japanese, 22 Korean and 22 Caucasian) aged 20–65 years were given a prasugrel 60-mg loading dose (LD) followed by daily 10-mg maintenance doses (MD) for 7 days and then 5-mg MD for 10 days. Plasma concentrations of prasugrel’s active metabolite and inhibition of ADP-induced platelet aggregation (IPA) were determined.Results
Mean exposure to prasugrel’s active metabolite in all treatment regimens was higher in each of the Asian groups than in the Caucasian group, although there was considerable overlap between individual exposure estimates in Asians and Caucasians. The mean IPA was also higher in Asians than in Caucasians following a prasugrel 60-mg LD, although the difference did not consistently achieve statistical significance. Prasugrel 10-mg or 5-mg MD produced statistically significantly higher IPA in each Asian group compared with that in the Caucasians. Prasugrel was well tolerated during the LD and MD regimens by all groups.Conclusions
Mean exposure to the prasugrel active metabolite following prasugrel 60-mg LD and during daily 10-mg or 5-mg MD was higher in each of the Asian groups than in the Caucasian group, which resulted in greater platelet inhibition. 相似文献The purpose was to investigate whether the pharmacokinetics and pharmacodynamics of prednisolone in the non-human primate was an appropriate surrogate for man.
After single intravenous doses of 0.03, 0.3, and 3?mg kg?1, prednisolone demonstrated a dose-dependent clearance and volume of distribution. When corrected for concentration-dependent protein binding, the free clearance was linear at the tested dose levels. The protein binding-corrected volume of distribution was similar across doses. The serum half-life was estimated as being between 2 and 4?h. Prednisolone exhibits near complete inhibition of the cytokines TNF-α, IL-1β, IL-6 and IL-8 with very similar IC50 estimates from 0.09 to 0.16 μg ml?1 (from 0.24 to 0.44 μM).
The monkey demonstrated a similar pharmacokinetics–pharmacodynamics profile of prednisolone when compared with man (from the literature).
We investigated the in vitro metabolism and transport of KR66222 and KR66223, new inhibitors of dipeptidyl peptidase (DPP) 4, using human liver microsomes (HLMs) and a Caco-2 cell monolayer.
Human liver microsomal incubation of KR66222 in the presence of the NADPH-generating system resulted in the formation of two metabolites, identified as S-oxidation (KR68334) and hydrolysis (KR66223) products using liquid chromatography/tandem mass spectrometry. The formation of KR66223 via an esterase and the formation of KR68334 via CYP3A5 and CYP3A4 seem to be major factors in the in vitro metabolism of KR66222 using HLMs. Additionally, KR66222 had a significantly greater basal to apical transport rate (2.5-fold) than apical to basal transport in the Caco-2 cell monolayer, suggesting the involvement of an efflux transport system. Further studies using inhibitors of efflux transporters and P-glycoprotein (P-gp) overexpressed cells revealed that P-gp was involved in the basal to apical transport of KR66222. These findings suggest that KR66222 undergoes a significant first pass effect, which may serve to decrease the bioavailability of KR66222.
The active metabolite, KR66223, was stable for 1?h at 37°C in pooled HLMs (98.9?±?2.6% of control) and did not undergo P-gp-mediated efflux in Caco-2 cells. Apparent permeability of KR66223 (4.96?×?10?6 cm/s) was comparable to that of KR66222 (4.08?×?10?6 cm/s).
In conclusion, considering pharmacokinetic variability and the intestinal first-pass effect caused by the involvement of CYP3A and P-gp, KR66223 seems to have better in vitro metabolism and permeability characteristics than KR66222.
A metabolism study of orally administered 2,2′,4,4′,5,6′-hexabromodiphenyl ether (BDE-154; 11.3 μmoles kg?1) was conducted in conventional and bile duct-cannulated male Sprague–Dawley rats.
In conventional rats, approximately 31% of the radiolabelled dose was retained at 72 h, and lipophilic tissues were the preferred sites for disposition.
Urinary excretion of BDE-154 was very low (1.0%), and parent compound was detected.
Cumulative biliary excretion was 1.3%, and glutathione conjugates were suggested.
Over 62% of the dose in conventional male rats was excreted in faeces, and was composed of parent compound (7.3%), free metabolites (13.1%), and covalently bound residues (41.4%). Faecal metabolites characterized by gas chromatography/mass spectrometry included multiple isomers of monohydroxylated hexa-/penta-/tetrabromodiphenyl ethers, and di-hydroxylated hexa/pentabromodiphenyl ethers.
The adipose tissue 14C was extractable BDE-154, but 40% of liver 14C was bound to macromolecules.
The study demonstrated the importance of performing individual polybrominated diphenyl ether (PBDE) metabolism studies to understand fully PBDE pharmacokinetics.
Self-administration of complementary products concurrently with conventional medication is increasingly common. The potential for cytochrome P450 (CYP) inhibition requires investigation. The N-in-one assay with ten probe substrates for nine CYPs was used with human liver microsomes to investigate ten products. CYP inhibition was measured in a single liquid chromatography-tandem mass spectrometry (LC/MS-MS) analysis. Estimated IC50-values were determined for the extracts that produced significant inhibition (less than 100 μg ml?1).
Inhibition of CYP2C19 by dong quai (IC50?=?13.7–14.3 μg ml?1 for the methanolic extract) and CYP2D6 by goldenseal (IC50?=?6.7 and 6.3 μg ml?1 for the aqueous and methanolic extracts, respectively), are of particular concern as the potential for adverse interactions is high. The inhibition of CYP2C8 by horsetail (IC50?=?93 μg ml?1 for the aqueous extract) requires further investigation, as the potential for concurrent use with products that require CYP2C8 for metabolism is significant. CYP3A4 inhibition varied depending on the probe reaction being monitored.
The earlier reported findings of inhibition by black cohosh, goldenseal and gotu kola were confirmed. The present work has shown that the N-in-one cocktail is a rapid and reliable method that can be used as an initial screen to help prioritize products that require more detailed investigations and it can also be applied to monitor product variability.
Dexamethasone cipecilate (DX-CP, 9-fluoro-11β,17,21-trihydroxy-16α-methylpregna-1,4-diene-3,20-dione 21-cyclohexanecarboxylate 17-cyclopropanecarboxylate) is a novel synthetic corticosteroid used to treat allergic rhinitis. The pharmacological effect of DX-CP is considered to be mainly due to its active de-esterified metabolite (DX-17-CPC). To investigate the in vitro metabolism of DX-CP in human liver, DX-CP was incubated with human liver microsomes and S9. In addition, a metabolism study of DX-CP with human nasal mucosa was carried out in order to elucidate whether DX-17-CPC is formed in nasal mucosa, the site of action of DX-CP.
DX-17-CPC was the major metabolite in both liver microsomes and S9. Two new epoxide metabolites, UK1 and UK2, were detected in liver S9, while only UK1 was detected in liver microsomes. This suggests that cytosol enzymes are responsible for the formation of UK2.
In human nasal mucosa, DX-CP was mainly transformed into DX-17-CPC. By using recombinant human carboxylesterases (CESs), the reaction was shown to be catalyzed by CES2.
These results provide the evidence that the active metabolite DX-17-CPC is the main contributor to the pharmacological action after the intranasal administration of DX-CP to humans.
The aim was to identify the individual human cytochrome P450 (CYP) enzymes responsible for the in vitro N-demethylation of hydromorphone and to determine the potential effect of the inhibition of this metabolic pathway on the formation of other hydromorphone metabolites.
Hydromorphone was metabolized to norhydromorphone (apparent Km = 206?? 822?μM, Vmax = 104 ? 834?pmol?min?1?mg?1 protein) and dihydroisomorphine (apparent Km = 62 ? 557?μM, Vmax = 17 ? 122?pmol?min?1?mg?1 protein) by human liver microsomes.
In pooled human liver microsomes, troleandomycin, ketoconazole and sulfaphenazole reduced norhydromorphone formation by an average of 45, 50 and 25%, respectively, whereas furafylline, quinidine and omeprazole had no effect. In an individual liver microsome sample with a high CYP3A protein content, troleandomycin and ketoconazole inhibited norhydromorphone formation by 80%.
The reduction in norhydromorphone formation by troleandomycin and ketoconazole was accompanied by a stimulation in dihydroisomorphine production.
Recombinant CYP3A4, CYP3A5, CYP2C9 and CYP2D6, but not CYP1A2, catalysed norhydromorphone formation, whereas none of these enzymes was active in dihydroisomorphine formation.
In summary, CYP3A and, to a lesser extent, CYP2C9 catalysed hydromorphone N-demethylation in human liver microsomes. The inhibition of norhydromorphone formation by troleandomycin and ketoconazole resulted in a stimulation of microsomal dihydroisomorphine formation.
