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.
5-Dimethylaminopropylamino-8-hydroxytriazoloacridinone, C-1305, being the close structural analogue of the clinically tested imidazoacridinone anti-tumour agent, C-1311, expressed high activity against experimental tumours and is expected to have more advantageous pharmacological properties than C-1311.
The aim of this study was to elucidate the role of selected liver enzymes in the metabolism of C-1305.
We demonstrated that the studied triazoloacridinone was transformed with rat and human liver microsomes, HepG2 hepatoma cells and with human recombinant flavin-containing monooxygenases FMO1, FMO3 but not with CYPs. Furthermore, this compound was an effective inhibitor of CYP1A2 and CYP3A4. The product of FMO catalysed metabolism was shown to be identical to the main metabolite from liver microsomes and HepG2 cells. It was identified as an N-oxide derivative and, under hypoxia, it underwent retroreduction back to C-1305, what was extremely effective with participation of CYP3A4.
In summary, this work revealed that the involvement of the P450 enzymatic system in microsomal and cellular metabolism of C-1305 was negligible, whereas this agent was an inhibitor of CYP1A2 and CYP3A4. In contrast, FMO1 and FMO3 were crucial for metabolism of C-1305 by liver microsomes and in HepG2 cells, which makes C-1305 an attractive potent anti-tumour agent.
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.
Intercellular adhesion molecule (ICAM)-1988 is a small molecule lymphocyte function-associated antigen-1 (LFA-1) antagonist being considered for its anti-inflammatory properties. Following intravenous administration of ICAM1988, clearances in mice, rats, dogs, and monkeys were 17.8, 3.31, 15.4, and 6.85 ml min?1 kg?1, respectively.
In mass balance studies using [14C]-ICAM1988 in rats dosed intravenously, unchanged ICAM1988 contributed to 25.1% of the dose.
In rats, the systemic bioavailability of ICAM1988 was improved to 0.28 when the drug was administered orally as its isobutyl ester, ICAM2660. In rats, this was consistent with the complete in vitro conversion of ICAM2660 to ICAM1988 in plasma, and liver and intestinal S9.
In dogs and monkeys, ICAM2660 did not improve the bioavailability of ICAM1988. This is consistent with limited in vitro conversion of ICAM2660 to ICAM1988 in plasma and liver S9.
In human in vitro studies, ICAM2660 conversion to ICAM1988 in liver was similar to rats while no conversion in plasma and intestinal S9 fractions were observed. Based on the in vitro metabolism similarities of human and rat, it would be anticipated that in human oral administration of ICAM2660 would improve the systemic exposure of ICAM1988.
The stereoselective metabolism of ethofumesate (ETO) and its enantiomers in rabbit and rat liver microsomes have been studied by chiral high-performance liquid chromatography (HPLC) method. Two metabolites were detected in both liver microsomes in the presence of β-nicotinamide adenine dinucleotide phosphate (NADPH).
The T1/2 of (+)-ETO and (?)-ETO in rabbit liver microsomes were 12.2 and 4.7?min of rac-ETO and 25.9 and 6.7 of ETO enantiomers. However, the T1/2 of (+)-ETO and (?)-ETO in rat liver microsomes were 5.3 and 5.9?min of rac-ETO and 7.8 and 10.6 of ETO enantiomers. The stereoselective selectivity is similar to the in vivo study.
After incubation of ETO enantiomers, stereoselectivity was present in the formation of ETO-OH enantiomer in rabbit liver microsomes, but stereoselectivity was not evident in rat liver microsomes.
There was no chiral inversion from the (+)-ETO to (?)-ETO or inversion from (?)-ETO to (+)-ETO in both rabbit and rat liver microsomes.
(R)-3-(4-propylmorpholin-2-yl) phenol (PF-219061) is a potent, selective agonist of the dopamine 3 receptor for the treatment of female sexual dysfunction.
In vivo, PF-219061 exhibits liver blood flow clearance in both rat and dog. Oral bioavailability was 0.7% in dog and less than 5% in rat.
Intranasal dosing was investigated to improve bioavailability. Pre-clinical assessments in rat and dog demonstrated intranasal bioavailabilities of 16–38% in rat and 54–61% in dog with very rapid absorption. It was predicted that an intranasal dose in man would give approximately 25–50% bioavailability.
The clinical data verified the preclinical predictions demonstrating rapid absorption and approximately dose-proportional increases in exposure. The intranasal bioavailability in man was estimated to be 26–38%.
These findings indicate the potential utility of intranasal dosing as a route that circumvents the first-pass effects for PF-219061 resulting in high exposures.
1.?This study investigated the alteration of carboxylesterases in type 2 diabetes. We found that the carboxylesterase 1d (Ces1d) and carboxylesterase 1e (Ces1e) expression and the capacity of hydrolytic activity of liver and intestine decreased, whereas the Akt/mTOR/HIF-1α/ Stra13 (DEC1) signaling was activated in T2D mice. Consistently, high insulin could give rise to the same results in the high-glucose DMEM condition, which mimicked T2D, in primary mouse hepatocytes.
2.?Perifosine or rapamycin almost abolished the decrease of the Ces1d and Ces1e expression and the hydrolytic activity induced by the insulin in the primary mouse hepatocytes.
3.?The responsiveness of human hepatoma (HepG2) cells to high insulin in high-glucose condition was similar to that of primary mouse hepatocytes in terms of the altered expression of carboxylesterases.
4.?The knockdown of HIF-1α or DEC1 with shRNA construct abrogated the decrease of the CES1 and CES2 expression induced by the insulin in high glucose condition in HepG2 cells.
5.?Taken together, the decreased carboxylesterases expression and hydrolytic activity in T2D mice are through the Akt/mTOR/HIF-1α/Stra13 (DEC1) pathway.
The metabolism of six anti-Trypanosoma cruzi 5-phenylethenylbenzofuroxans (PhEBfx) was studied in vitro using rat hepatic microsomal and cytosolic fractions as a mammalian model and whole cells of T. cruzi as a parasitic model.
Some of the expected metabolites were synthesized to provide authentic chromatographic standards.
The metabolites were identified using high-performance liquid chromatography (HPLC) in comparison with the authentic standards and their proportions were determined. Their structures were confirmed using mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy.
The behaviour of the six PhEBfx in the three different systems was similar. The main metabolites, formed by reductive processes, were the corresponding o-nitroanilines.
Two of the test compounds were studied for extended time periods in the rat liver preparations and their terminal metabolites were identified as o-phenylendiamine derivatives.
Scutellarin (SG) is a bioactive flavonoid used to treat cardiovascular disease. Scutellarein (S) is the aglycone form of SG. This study aimed to characterize their intestinal transport and first-pass metabolism by UDP-glucuronosyltransferase-mediated glucuronidation and β-glucuronidase-mediated hydrolysis.
Results showed that S is more readily passed through Caco-2 cell monolayers by passive diffusion than SG. SG was the predominant metabolite of S, which was formed during the transportation of S across Caco-2 cell monolayers or following incubation of S with human microsomes. SG was extensively generated in human liver microsomes (HLMs), which was demonstrated by its higher catalyzing efficiency (Clint) in liver microsomes than in human intestinal microsomes (HIMs).
Enzymatic kinetic analysis indicated that the catalyzing efficiency of UGT1A9 was the highest among the tested UGTs under the present experimental conditions, followed by UGT1A1 and UGT1A3. No significant P450-mediated hydroxylation of S was found. SG may be hydrolyzed into S in both HLMs and HIMs.
The phosphatidylinositol 3-kinase (PI3K) pathway is a major determinant of cell cycling and proliferation. Its deregulation is associated with the development of many cancers.
GDC-0941, a potent and selective inhibitor of PI3K, was characterised preclinically in in vitro and in vivo studies.
Plasma protein binding was extensive, with free fraction less than 7%, and blood-to-plasma ratio ranged from 0.6 to 1.2 among the species tested. GDC-0941 human hepatic clearance was predicted to be moderate by liver microsomal incubations. GDC-0941 had high permeability in Madin-Darby canine kidney cells.
The clearance of GDC-0941 was high in mouse (63.7?mL/min/kg), rat (49.3?mL/min/kg) and cynomolgus monkey (58.6?mL/min/kg), and moderate in dog (11.9?mL/min/kg). The volume of distribution ranged from 2.52?L/kg in rat to 2.94?L/kg in monkey. Oral bioavailability ranged from 18.6% in monkey to 77.9% in mouse.
Predicted human clearance and volume of distribution using allometry were 6?mL/min/kg and 2.9?L/kg, respectively. The human efficacious doses were predicted based on results from preclinical pharmacokinetic studies and xenograft models.
GDC-0941 preclinical characterisation and predictions of its properties in human supported its progression towards clinical development. GDC-0941 is currently in phase II clinical trials.
PAP-1 (5-(4-phenoxybutoxy)psoralen), a potent small-molecule blocker of the voltage-gated potassium Kv1.3 channel, is currently in preclinical development for psoriasis. This study was undertaken to identify the major phase I metabolites of PAP-1 in Sprague-Dawley (SD) rats.
Five phase I metabolites, that is 5-(oxybutyric-acid)psoralen (M1), 5-[4-(4-hydroxybutoxy)]psoralen (M2), 5-[4-(4-hydroxyphenoxy)butoxy]psoralen (M3), 5-[4-(3-hydroxyphenoxy)butoxy]psoralen (M4), and 8-hydroxyl-5-(4-phenoxybutoxy)psoralen (M5), were isolated from the bile of rats and identified by mass spectrometry and NMR spectroscopy. The last four metabolites are new compounds.
Incubation of PAP-1 with SD rat liver microsomes rendered the same five major metabolites in a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent manner suggesting that cytochrome P450 (CYP) enzymes are involved in PAP-1 metabolism. Inhibitors of rat CYP1A1/2 (alpha-naphthoflavone) and CYP3A (ketoconazole) but not CYP2D6 (quinidine), CYP2E (diethyldithiocarbamate), or CYP2C9 (sulphaphenazole) blocked the metabolism of PAP-1 in rat microsomes.
Of the five metabolites M3, M4, and M5 were found to inhibit Kv1.3 currents with nanomolar IC50s, while M1 and M2 were inactive. Our results identified the Kv1.3-inactive M1 as the major phase I metabolite, and suggest that hydroxylation and O-dealkylation are the major pathways of PAP-1 metabolism.
We further conducted a 6-month repeat-dose toxicity study with PAP-1 at 50?mg/kg in both male and female Lewis rats and did not observe any toxic effects.
The stereoselective degradations of racemate metalaxyl (rac-MX) and its single enantiomers in rat and rabbit hepatic microsomes were assayed by a chiral high-performance liquid chromatography method.
The t1/2 of (+)-S-MX in rat liver microsomes was between 7–8?min tested by rac-MX and the individual (+)-S-enantiomer, respectively, and that for (?)-R-MX was 15–16?min. In contrast, t1/2 in rabbit liver microsomes was much longer and showed great difference when using racemate and single enantiomer, which was similar to the results of in vivo study.
The enantioselectivity in rat hepatic microsomes was more evident and the degradations of MX enantiomers in rat and rabbit hepatic microsomes were Nicotinamide adenine dinucleotide phosphate-dependent.
Michaelis constant (Km) and intrinsic metabolic clearance (CLint) of (+)-S-MX were larger than that of (?)-R-MX and there was no chiral inversion from (+)-S-MX to (?)-R-MX or vice versa in both rat and rabbit hepatic microsomes.
Multidrug resistance is a major problem in hepatocellular carcinoma. Hedyotiscone A, a compound isolated from Chinese herbal medicine Hedyotis corymbosa (HC, family Rubiaceae), was used as the chemical marker to distinguish between HC and an anticancer herb Hedyotis diffusa (HD) in our previous study.
The present study aimed to investigate whether HA exhibited antiproliferative activities in multidrug-resistant hepatocellular carcinoma cells R-HepG2 and the parental cells HepG2 using MTT assay and [3H]-thymidine incorporation assay.
Our results showed that HA could significantly inhibit cell proliferation in R-HepG2 and HepG2 (IC50?=?43.7 and 56.3 µg/mL, respectively), but not in normal human liver cells WRL-68 (IC50 > 100 µg/mL) cells, suggesting its selective cytotoxic effects. Besides, HA induced apoptosis in R-HepG2 cells, as confirmed by annexin-V & propidium iodide staining, and DNA fragmentation assay. The caspase cascade was activated as shown by a significant increase of cleaved caspases-3, -7 and -9 in HA-treated R-HepG2 cells. The activities and protein expression of P-glycoprotein as well as mRNA expression of MDR1 were also decreased in HA-treated R-HepG2 cells.
Our study demonstrated for the first time the antiproliferative activities of hedyotiscone A in multidrug-resistant R-HepG2 cells. The findings revealed the potential of this compound in treating multidrug-resistant tumor.
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.
assess multimodal therapies
account historical data
safeguard ethics and efficacy during the course of long-term trials
study drugs, before well-established toxicity information is available
account the possibility of therapeutic equivalence between test and reference treatment
study multiple treatments in one trial
adjust change scores for baseline levels.
factorial designs
historical controls designs
group-sequential interim analysis designs
sequential designs for continuous monitoring
therapeutic equivalence designs
multiple crossover-periods / multiple parallel-groups design
increased precision designs through multivariate adjustment.
Phenobarbitone and related compounds induce hepatic microsomal cytochrome P450 (CYP) 2B forms (mediated by the constitutive androstane receptor), whereas peroxisome proliferators induce CYP4A forms (mediated by the peroxisome proliferator-activated receptor alpha) in rats and mice.
A number of non-genotoxic CYP2B and CYP4A inducers have been shown to produce liver tumours in rats and mice.
The hepatic effects of CYP2B and CYP4A inducers are reviewed and evaluated with respect to their established modes of action for rodent liver tumour formation and species differences in response. While CYP2B and CYP4A inducers stimulate replicative DNA synthesis in rodent liver, they do not appear to be mitogenic agents in human hepatocytes.
Epidemiological studies have demonstrated that phenobarbitone and rodent peroxisome proliferators do not increase the incidence of liver tumours in humans.
It is concluded that rodent CYP2B and CYP4A inducers do not pose a hepatocarcinogenic hazard for humans.
The objective of this study was to characterize cytochrome P450 (CYP) activities in both intestinal and hepatic microsomes from Wistar and Sprague–Dawley rats.
Specific probes for measuring CYP activities were selected using rat recombinant CYP.
The intestinal microsome preparation was optimized getting a more relevant and reproducible abundance of CYPs to measure CYP activities.
Testosterone, propranolol, diclofenac, and midazolam were determined as specific substrates of rat CYP2C11, CYP2D2, CYP2C6, and CYP3A, respectively. Ethoxyresorufin and pentoxyresorufin were not specific substrates of CYP1A2 and CYP2B1, respectively. Hepatic and intestinal microsomes expressed active CYP1A1, CYP1A2, CYP2B1, and CYP3A2. Only liver expressed active CYP2C6, CYP2C11, and CYP2D2. Wistar liver expressed more active CYP1A and CYP3A2, but less active CYP2B1 than Wistar intestine. Sprague–Dawley liver expressed more active CYP2B1 and CYP3A2, but less active CYP1A than Sprague–Dawley intestine.
In conclusion, CYP activities were qualitatively equivalent but not quantitatively in both strains.
Carvacrol (2-methyl-5-(1-methylethyl)-phenol), one of the main components occurring in many essential oils of the family Labiatae, has been widely used in food, spice and pharmaceutical industries.
The carvacrol glucuronidation was characterized by human liver microsomes (HLMs), human intestinal microsomes (HIMs) and 12 recombinant UGT (rUGT) isoforms.
One metabolite was identified as a mono-glucuronide by liquid chromatography/mass spectrometry with HLMs, HIMs, rUGT1A3, rUGT1A6, rUGT1A7, rUGT1A9 and rUGT2B7.
The study with a chemical inhibition, rUGT, and kinetics study demonstrated that rUGT1A9 was the major isozyme responsible for glucuronidation in HLMs, and rUGT1A7 played a major role for glucuronidation in HIMs.
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.