Aconite alkaloids are the main bioactive ingredients existing in Aconitum, for instance aconitine (AC), which exhibit potent analgesic, antirheumatic and other pharmacological effects. In this study, effects of long-term treatment with liquorice on pharmacokinetics of AC in rats were investigated.
Pharmacokinetics of AC after oral administration of AC at 1.5?mg/kg either with pre-treatment of liquorice water extracts at 0.433 or 1.299?g/kg (crude drug), respectively, for one week or not were studied. Additionally, LS-180 cells and human primary hepatocytes were utilized to explore the potential effects of bioactive ingredients of liquorice on P-glycoprotein (P-gp) and Cytochromes P450 (CYPs), respectively.
The results revealed that exposure of AC after pre-treatment with liquorice was altered remarkably. Area under the concentration-time curve (AUC) decreased from 161?±?37.8 to 58.8?±?8.97 and 44.7?±?8.20?ng/mL*h, respectively. Similarly, Cmax decreased from 26.2?±?5.19 to 11.8?±?1.15 and 6.86?±?0.600?ng/mL, respectively. In addition, expressions of CYPs of human primary hepatocytes were enhanced to various contents after induction. Moreover, accumulation of AC and hypaconitine (HA), not mesaconitine (MA) inside of LS-180 cells were reduced after pre-treatment by comparison with control.
In conclusion, the exposure of AC in vivo declined after pre-treatment with liquorice extract, which may be highly associated with upregulated expression and/or function of CYPs and P-gp.
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.
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.
Ilaprazole is a new proton pump inhibitor, designed for treatment of gastric ulcers, and developed by Il-Yang Pharmaceutical Co (Seoul, Korea). It is extensively metabolised to the major metabolite ilaprazole sulfone.
In the present study, several in vitro approaches were used to identify the cytochrome P450 (CYP) enzymes responsible for ilaprazole sulfone formation. Concentrations of ilaprazole sulfone were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
Incubation of ilaprazole with cDNA-expressed recombinant CYPs indicated that CYP3A was the major enzyme that catalyses ilaprozole to ilaprazole sulfone. This reaction was inhibited significantly by ketoconazole, a CYP3A inhibitor, and azamulin, a mechanism-based inhibitor of CYP3A, while no substantial effect was observed using selective inhibitors for eight other P450s (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP2E1).
In addition, the formation of ilaprazole sulfone correlated well with CYP3A-catalysed testosterone 6β-hydroxylation and midazolam 1′-hydroxylation in 20 different human liver microsome panels. The intrinsic clearance of the formation of ilaprazole sulfone by CYP3A4 was 16-fold higher than that by CYP3A5.
Collectively, these results indicate that the formation of the major metabolite of ilaprazole, ilaprazole sulfone, is predominantly catalysed by CYP3A4/5.
The utility of multivariate analysis in in vitro metabolite identification studies was examined with nefazodone, an antidepressant drug with a well-established metabolic profile.
The chromatographic conditions were purposefully chosen to reflect those utilized in high-throughput screening for microsomal stability of new chemical entities.
Molecular ion, retention time information on groups of human liver microsomal samples with/without nefazodone was evaluated by principal component analysis (PCA). Resultant scores and loadings plots from the PCA revealed the segregation and the ions of interest that designated the drug and its corresponding metabolites. Subsequent acquisition of tandem mass spectrometry (MS/MS) spectra for targeted ions permitted the interrogation and interpretation of spectra to identify nefazodone and its metabolites.
A comparison of nefazodone metabolites identified by PCA versus those found by traditional metabolite identification approaches resulted in very good correlation when utilizing similar analytical methods. Fifteen metabolites of nefazodone were identified in β-nicotinamide adenine dinucleotide phosphate (NADPH)-supplemented human liver microsomal incubations, representing nearly all primary metabolites previously reported.
Of the 15 metabolites, eight were derived from the N-dealkylation and N-dephenylation of the N-substituted 3-chlorophenylpiperazine motif in nefazodone, six were derived from mono- and bis-hydroxylation, and one was derived from the Baeyer Villiger oxidation of the ethyltriazolone moiety in nefazodone.
Toxicity of pyrrolizidine alkaloids (PAs) largely depends on their metabolic activation by hepatic enzymes, including cytochrome P450s, to become chemically reactive pyrrolic derivatives. These then spontaneously release the esterifying acids to generate carbonium ions that form covalent adducts with cellular nucleophiles to exhibit toxicity.
In our investigation, metabolism-mediated toxicity of monocrotaline, retrorsine, lycopsamine, echimidine (retronecine-type PAs), heliotrine (a heliotridine-type PA) and senkirkine (an otonecine-type PA) was studied using an in vitro co-incubation assay.
Human hepatocarcinoma (HepG2/C3A) cells were incubated with PAs in the presence and absence of rat liver S9 fraction and the toxicity was assessed as lowered mitochondrial activity.
Bioactivation potential was measured by incubating PAs with rat liver S9 fraction, NADPH and GSH in a cell free system. Pyrrolic metabolites generated were entrapped as glutathione conjugates (7-GSH-DHP and 7,9-di-GSH-DHP) which were quantified using LC-MS-MS analysis.
Our results indicated that PAs were metabolized by rat liver S9 fraction into reactive pyrrolic derivatives which were toxic to HepG2/C3A cells. This approach can be used to determine and compare bioactivation potential and metabolism-mediated toxicity of various PAs.
Human cytochrome P4502B6 (CYP2B6) is predominantly expressed in the liver and it plays a major role in the metabolism of several therapeutically important drugs and environmental toxicants.
The objective was twofold: (1) to determine the role of genetic, physiological, and environmental factors in predicting hepatic CYP2B6 protein expression; and (2) to investigate the role of CYP2B6 in nicotine C-oxidation.
Human livers (n?=?40) were assessed for CYP2B6 protein and genotype.
Linear regression analyses indicated that CYP2B6 genotype (10%), gender (14%), and exposure to inducers (21%), but not age, were predictors of CYP2B6 protein amounts. Livers with at least one CYP2B6*5 or *6 allele were associated with lower CYP2B6. Female livers and livers exposed to inducers (phenobarbital and/or dexamethasone) were associated with higher CYP2B6.
A weak correlation between CYP2B6 and nicotine C-oxidation activity was observed, which was abrogated when controlling for CYP2A6 protein levels. CYP2B6*6 was not associated with different nicotine kinetics.
In summary, CYP2B6 protein expression was associated with genotype, gender, and exposure to inducers, but not with nicotine C-oxidation activity.
The pharmacokinetics (PK) (absorption, distribution, metabolism, excretion) of peginesatide, a synthetic, PEGylated, investigational, peptide-based erythropoiesis-stimulating agent (ESA), was evaluated in rats. The PK profile was evaluated at 0.1–5 mg·kg?1 IV using unlabeled or [14C]-labeled peginesatide. Mass balance, tissue distribution and metabolism were evaluated following IV administration of 5 mg·kg?1 [14C]-peginesatide, with tissue distribution also evaluated by quantitative whole-body autoradiography (QWBA) following an IV dose of 17 mg·kg?1 [14C]-peginesatide.
Plasma clearance was slow and elimination was biphasic with unchanged peginesatide representing >90% of the total radioactivity of the total radioactive exposure. Slow uptake of the radiolabeled compound from the vascular compartment into the tissues was observed.
Biodistribution to bone marrow and extramedullary hematopoietic sites, and to highly vascularized lymphatic and excretory tissues occurred.
A predominant degradation event to occur in vivo was the loss of one PEG chain from the branched PEG moiety to generate mono-PEG.
Renal excretion was the primary mechanism (41%) of elimination, with parent molecule (67%) the major moiety excreted.
In conclusion, elimination of [14C]-peginesatide-derived radioactivity was extended, retention preferentially occurred at sites of erythropoiesis (bone marrow), and urinary excretion was the primary elimination route.
Baicalin was extensively researched for utility in a number of therapeutic areas owing to its anti-inflammatory, anti-oxidant, anti-bacterial, and anti-cancer properties.
A number of preclinical studies, in vitro work, and mechanistic studies were performed to understand the absorption, distribution, metabolism, and excretion profiles of baicalin.
The absorption of baicalin involved several complexities: the restriction to two distant sites; the conversion of baicalin to baicalein; the possible role of transporter(s); and enhanced absorption due to breakdown of conjugates by beta-glucuronidase.
Limited distribution data suggest that baicalin reached several sites such as the brain, eye lens, thymus, etc. Hepatobiliary recycling also served as a distribution phase for sustained delivery of baicalin.
Metabolism data suggest the rapid conversion of baicalin to baicalein, which was extensively subjected to Phase 2 metabolism, conjugates baicalein glucuronide/sulfate have been identified.
Limited excretion data suggest involvement of renal and faecal routes—glucuronide and sulfate conjugates were excreted in urine and faeces (via biliary excretion).
The published data on baicalin suggest imminent challenges for developing baicalin and/or during co-administration with other agents. These challenges are absorption related (transporter or changes in the microenvironment), metabolism related (CYP2B6 induction and/or CYP2E1 inhibition), and excretion/efflux related (competitive biliary pathway and/or OATP1B1 transport).
Reactivity of benzene oxide (BO), a reactive metabolite of benzene, was studied in model reactions with biologically relevant S- and N-nucleophiles by LC-ESI-MS.
Reaction with N-acetylcysteine (NAC) in aqueous buffer solutions gave N-acetyl-S-(6-hydroxycyclohexa-2,4-dien-1-yl)cysteine (pre-phenylmercapturic acid, PPhMA), which was easily dehydrated in acidic solutions to phenylmercapturic acid (PhMA). The yield of PPhMA + PhMA increased exponentially with pH up to 11% in the pH range from 5.5 to 11.4.
Primary 6-hydroxycyclohexa-2,4-dien-1-yl (HC) adducts were detected also in reactions of purine nucleosides and nucleotides under physiological conditions. After a vigorous acidic hydrolysis, all HC adducts were converted to corresponding phenyl purines, which were identified as 7-phenylguanine (7-PhG), 3-phenyladenine (3-PhA) and N6-phenyladenine (6-PhA). The yield of 7-PhG amounted to 14?±?5 and 16?±?7?ppm for 2′-deoxyguanosine and 2′-deoxyguanosine-5′-monophosphate, respectively, that of 6-PhA was 500?±?70 and 455?±?75?ppm with 2′-deoxyadenosine and 2′-deoxyadenosine-5′-phosphate, respectively, with only traces of 3-PhA.
Reactions with the DNA followed by acidic hydrolysis yielded 26?±?11?ppm (mean ± SD; n?=?9) of 7-PhG as the sole adduct detected.
In contrast to the reactions with S-nucleophiles, the reactivity of BO with nucleophilic sites in the DNA is very low and can therefore hardly account for a significant DNA damage caused by benzene.
The present study was designed to investigate the multiple-dosing pharmacokinetics of antimalarial drugs artemether (ARM), artesunate (ARS), and their metabolite dihydroartemisinin (DHA) in rats.
Rats were randomized into four groups. Two groups of rats received oral doses of ARM or ARS once daily for five consecutive days. And another two groups of rats were given a single oral dose of ARM or ARS. Plasma samples were analysed for artemisinin drugs and their active metabolite DHA, using a validated liquid chromatography/tandem mass spectrometric (LC/MS/MS) method.
ARM and ARS could be biotransformed to metabolite DHA almost immediately after oral administration to rats. The area under the plasma concentration–time curve (AUC0–t) of ARM after 5-day oral doses significantly decreased from 50.3 to 23.4 ng×h/mL (P?<?0.05), and oral clearance (CL/F) of ARM increased from 10.5 to 27.2?L/min/kg (P?<?0.05). The AUC0–t of its metabolite DHA of ARM significantly decreased from 42.1 to 16.4 ng×h/mL (P?<?0.05), and its CL/F increased from 11.7 to 33.4?L/min/kg (P?<?0.05). The 5-day oral doses of ARS did not result in significant changes (P?>?0.05) in pharmacokinetic parameters of ARS, whereas its metabolite DHA exhibited lower AUC (P?=?0.05), compared with that obtained after a single oral administration.
The results showed ARM and its metabolite DHA exhibited time-dependent pharmacokinetic characteristics with decreased plasma drug level after five consecutive days of oral administration to rats, whereas ARS and its metabolite DHA did not show similar characteristics.
As a novel hydrogen sulfide-modulated agent, S-propargyl-L-cysteine (SPRC) is proven to be a potent cardioprotective candidate. Bioavailability and pharmacokinetics of SPRC (20?mg/kg) in beagle dogs after oral and intravenous administrations were investigated in this study. Plasma concentrations of SPRC were measured by a LC-MS/MS method.
Intravenous administration of SPRC (single dose) to beagle dogs gave a mean plasma half-life of 14.7?h, mean clearance of 0.4?ml min?1 kg?1 and mean apparent volume of distribution of 0.56?L/kg. Single oral administration was completely, fast absorbed (Tmax= 0.33?±?0.20?h) with a mean absolute availability of 112% and mean plasma half-life of 16.5?h.
Multiple oral administration (once daily for 10 consecutive days) of SPRC to dogs resulted in steady state plasma drug concentration being reached after seven doses and didn’t cause obvious accumulation. No significant difference was found between the single and multiple pharmacokinetic parameters.
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.
The purpose of this study was to construct a method to predict CYP3A4 induction in the clinical setting from in vitro data using cryopreserved human hepatocytes.
We recently developed an approach with in vitro assays of HepaRG cell lines for predicting CYP3A4 induction by using a novel value, termed the relative factor (RF), determined from the ratio of the concentration of an inducer to the reference standard. In this study, the applicability of the RF approach was expanded to cryopreserved human hepatocytes.
Induction assays were performed in vitro using hepatocytes from four individual donors and eight typical inducers. The obtained RF values were related to the free plasma concentration of each inducer (expressed as Css,u/RF).
A good relationship between the Css,u/RF values and the in vivo induction response was found for all donors. Inducers were classified by the Css,u/RF values into three categories for CYP3A4 induction risk (high, medium and low potency), and thereby the degree of CYP3A4 induction in vivo in humans could be predicted from the Css,u/RF values.
The RF approach is applicable to human cryopreserved hepatocytes. Thus, a method to predict the potency of CYP3A4 inducers was constructed using cryopreserved human hepatocytes.
(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.
The aim was to characterize mouse gender and strain differences in the metabolism of commonly used human cytochrome (CYP) P450 probe substrates.
Thirteen human CYP probe substrates (phenacetin, coumarin, 7-ethoxy-4-trifluoromethyl coumarin, amiodarone, paclitaxel, diclofenac, S-mephenytoin, bufuralol, dextromethorphan, chlorzoxazone, p-nitrophenol, testosterone and lauric acid) were used in activity measurements. The metabolism of the probe substrates was compared in liver microsomes from male and female NMRI, CBA, C57bl/6, 129/SvJ and CD1 strains. The expression of proteins identified on Western blots with commonly available antibodies selective for specific human and rat CYP enzymes were compared in the different mouse strains.
Males had higher metabolism than corresponding females for phenacetin O-deethylation (human marker for CYP1A2 activity), and a high correlation was found between phenacetin activity and immunoreactivity in Western blots produced with rat CYP1A2 antibodies.
Protein detected by antibodies cross-reacting with human CYP2B6 and rat CYP2B1/2 antibodies was female specific except for the 129/SvJ strain, where it was absent in both genders.
Females generally had a higher metabolism of bufuralol 1′-hydroxylation and dextromethorphan O-demethylation (human markers for CYP2D activity).
Bufuralol 1′-hydroxylation correlated with a female-dominant mouse CYP, which was detected with antibodies against rat CYP2D4.
p-Nitrophenol 2-hydroxylation correlated better than chlorzoxazone 6-hydroxylation with the protein detected with antibodies against rat CYP2E1, indicating that p-nitrophenol is a more specific substrate for mouse CYP2E1.
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.
Cultured cryopreserved human hepatocytes are extensively used as a model system for studying drug metabolism, although they remain poorly characterized in respect of the major conjugation reactions glucuronidation and sulfation.
Using paracetamol (acetaminophen), we assessed eleven samples of cryopreserved human hepatocytes for their suitability to investigate the simultaneous glucuronidation and sulfation of xenobiotics and evaluated inhibitors of conjugation.
Kinetic characterization showed broadly similar values for paracetamol conjugation by hepatocytes (as reported in the literature for in vitro systems), with Km values of approximately 6 mM and 0.3 mM for glucuronidation and sulfation, respectively. Substantial interindividual differences were observed.
The hepatocytes demonstrated a strong dose-dependent switch from a preponderance of sulfation at low concentrations of paracetamol to glucuronidation at higher doses, consistent with routes of clearance in vivo.
A number of drugs, some of which such as probenecid and sulfinpyrazone are known to interact with paracetamol in vivo, were demonstrated to inhibit the sulfation and/or glucuronidation of paracetamol in hepatocytes, demonstrating the potential application of this model system for studying drug–drug interactions involving conjugation.
This work aimed to investigate plasma pharmacokinetics and tissue distribution of a new acridine derivative 5-acridin-9-ylmethylene-3-(4-methyl-benzyl)-thiazolidine-2,4-dione (AC04) and its 1-oxo-AC04 metabolite disposition in Wistar rats.
After a single AC04 1.5?mg/kg intravenous (i.v.) bolus dose, blood samples were taken up to 120?h. Plasma samples were deproteinization, and AC04 and metabolite were quantified by validated liquid chromatography in tandem with mass spectrometry method. Protein binding was determined by ultrafiltration. AC04 tissue disposition was evaluated after i.v. bolus dose.
Individual AC04 concentration–time profiles were best fitted by a two-compartment model showing CLtot of 3.4?±?3.4?L/h/kg, VdSS of 137.9?±?91.4?L/kg, AUC0–∞ of 788?±?483 ng·h/mL and a t1/2 of 45.5?±?31.5?h. Protein binding was 98.1?±?1.6%. AC04 showed higher penetration into the lung, spleen and liver, with AUC0–96 of 798,443, 263,211 and 303,722 ng·h/mL, respectively. The 1-oxo-AC04 metabolite represented 10% of AC04 plasma concentration, showing a t1/2 of 23.2?±?10.4?h.
These results suggest that, despite the small free plasma fraction, AC04 penetrates extensively reaching high concentrations in most tissues residing for a long time, which is important for its activity on solid tumours. All results combined indicate that AC04 is potentially a good antitumour candidate.