Metabolism and disposition of the anticancer quinolone derivative vosaroxin, a novel inhibitor of topoisomerase II.

Background Vosaroxin is a first-in-class anticancer quinolone derivative that is being investigated for patients with relapsed or refractory acute myeloid leukemia (AML). The primary objective of this study was to quantitatively determine the pharmacokinetics of vosaroxin and its metabolites in patients with advanced solid tumors. Methods This mass balance study investigated the pharmacokinetics (distribution, metabolism, and excretion) of vosaroxin in cancer patients after a single dose of 60 mg/m2 14C-vosaroxin, administered as short intravenous injection. Blood, urine and feces were collected over 168 h after injection or until recovered radioactivity over 24 h was less than 1% of the administered dose (whichever was earlier). Total radioactivity (TRA), vosaroxin and metabolites were studied in all matrices. Results Unchanged vosaroxin was the major species identified in plasma, urine, and feces. N-desmethylvosaroxin was the only circulating metabolite detected in plasma, accounting for <3% of the administered dose. However, in plasma, the combined vosaroxin + N-desmethylvosaroxin AUC0-∞ was 21% lower than the TRA AUC0-∞ , suggesting the possible formation of protein bound metabolites after 48 h when the concentration-time profiles diverged. The mean recovery of TRA in excreta was 81.3% of the total administered dose; 53.1% was excreted through feces and 28.2% through urine. Conclusions Unchanged vosaroxin was the major compound found in the excreta, although 10 minor metabolites were detected. The biotransformation reactions were demethylation, hydrogenation, decarboxylation and phase II conjugation including glucuronidation.

Mass balance and metabolism of the antimalarial pyronaridine in healthy volunteers.

This was a single dose mass balance and metabolite characterization study of the antimalarial agent pyronaridine. Six healthy male adults were administered a single oral dose of 720 mg pyronaridine tetraphosphate with 800 nCi of radiolabeled (14)C-pyronaridine. Urine and feces were continuously collected through 168 h post-dose, with intermittent 48 h collection periods thereafter through 2064 h post-dose. Drug recovery was computed for analyzed samples and interpolated for intervening time periods in which collection did not occur. Blood samples were obtained to evaluate the pharmacokinetics of total radioactivity and of the parent compound. Total radioactivity in urine, feces, and blood samples was determined by accelerator mass spectrometry (AMS); parent concentrations in blood were determined with LC/MS. Metabolite identification based on blood, urine, and feces samples was conducted using a combination of LC + AMS for identifying radiopeaks, followed by LC/MS/MS for identity confirmation/elucidation. The mean cumulative drug recovery in the urine and feces was 23.7 and 47.8 %, respectively, with an average total recovery of 71.5 %. Total radioactivity was slowly eliminated from blood, with a mean half-life of 33.5 days, substantially longer than the mean parent compound half-life of 5.03 days. Total radioactivity remained detectable in urine and feces collected in the final sampling period, suggesting ongoing elimination. Nine primary and four secondary metabolites of pyronaridine were identified. This study revealed that pyronaridine and its metabolites are eliminated by both the urinary and fecal routes over an extended period of time, and that multiple, varied pathways characterize pyronaridine metabolism.

Chemiluminescence determination of gemifloxacin based on diperiodatoargentate (III)-sulphuric acid reaction in a micellar medium.

A novel flow-injection chemiluminescence (FI-CL) analysis method for the determination of gemifloxacin in the presence of cetyltrimethylammonium bromide (CTAB) surfactant micelles is described. Strong CL signal was generated during the reaction of gemifloxacin with diperiodatoargentate (III) in a sulfuric acid medium sensitized by CTAB. Under optimum experimental conditions, the CL intensity was linearly related to the concentration of gemifloxacin from 1.0 × 10(-9) to 3.0 × 10(-7) g/mL and the detection limit was 7.3 × 10(-10) g/mL (3σ). The relative standard deviation (RSD) was 1.7 % for a 3.0 × 10(-8) g/mL gemifloxacin solution (11 repeated measurements). The proposed method was successfully applied to the determination of gemifloxacin in pharmaceutical preparations and biological fluids. The possible mechanism of the CL reaction is also discussed briefly.

Absorption, distribution, excretion, and pharmacokinetics of 14C-pyronaridine tetraphosphate in male and female Sprague-Dawley rats.

The main objective of this investigation was to determine the absorption, distribution, excretion, and pharmacokinetics of the antimalarial drug pyronaridine tetraphosphate (PNDP) in Sprague-Dawley rats. Following oral administration of a single dose (10 mg/Kg) of 14C-PNDP, it was observed that the drug was readily absorbed from the small intestine within 1 hour following oral administration and was widely distributed in most of the tissues investigated as determined from the observed radioactivity in the tissues. The peak value of the drug in the blood was reached at around 8 hours postadministration, and radioactivity was detected in most of the tissues from 4 hours onwards. 14C-PNDP showed a poor permeability across the blood-brain barrier, and the absorption, distribution, and excretion of 14C-PNDP were found to be gender-independent as both male and female rats showed a similar pattern of radioactivity. Excretion of the drug was predominantly through the urine with a peak excretion post 24 hours of administration. A small amount of the drug was also excreted in the feces and also in the breath. It was found that the C(max), AUC (0-inf), and T(max) values were similar to those observed in the Phase II clinical trials of pyronaridine/artesunate (Pyramax) conducted in Uganda.

Competitive inhibition of renal tubular secretion of gemifloxacin by probenecid.

Probenecid interacts with transport processes of drugs at several sites in the body. For most quinolones, renal clearance is reduced by concomitant administration of probenecid. The interaction between gemifloxacin and probenecid has not yet been studied. We studied the extent, time course, site(s), and mechanism of this interaction. Seventeen healthy volunteers participated in a randomized, two-way crossover study. Subjects received 320 mg gemifloxacin as an oral tablet without and with 4.5 g probenecid divided in eight oral doses. Drug concentrations in plasma and urine were analyzed by liquid chromatography-tandem mass spectrometry. WinNonlin was used for noncompartmental analysis, compartmental modeling, and statistics, and NONMEM was used for visual predictive checks. Concomitant administration of probenecid increased plasma gemifloxacin concentrations and amounts excreted in urine compared to baseline amounts. Data are average estimates (percent coefficients of variation). Modeling showed a competitive inhibition of the renal tubular secretion of gemifloxacin by probenecid as the most likely mechanism of the interaction. The estimated K(m) and Vmax for the saturable part of renal elimination were 9.16 mg/liter (20%) and 113 mg/h (21%), respectively. Based on the molar ratio, the affinity for the renal transporter was 10-fold higher for gemifloxacin than for probenecid. Since probenecid reached an approximately 200-times-higher area under the molar concentration-time curve from 0 to 24 h than gemifloxacin, probenecid inhibited the active tubular secretion of gemifloxacin. Probenecid also reduced the nonrenal clearance of gemifloxacin from 25.2 (26%) to 21.0 (23%) liters/h. Probenecid inhibited the renal tubular secretion of gemifloxacin, most likely by a competitive mechanism, and slightly decreased nonrenal clearance of gemifloxacin.

A high-performance liquid chromatography-tandem mass spectrometry method for the determination of lifrafenib, a novel RAF kinase and EGFR inhibitor, in human plasma and urine and its application in clinical pharmacokinetic study.

Lifirafenib (BGB-283), a dual inhibitor trageting BRAF kinase and EGFR, showed favorable efficacy and safety in treating patients with different cancer types harboring mutations in BRAF, KRAS and NRAS. In order to support the clinical pharmacokinetic study, a sensitive high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and validated to quantify lifirafenib concentration in human plasma and urine. Plasma samples were purified using protein precipitation. Urine samples were pre-treated by adding tween 80 with the purpose of preventing non-specific adsorption, then extracted by centrifugation. Chromatographic separation was achieved on Phenomenex Luna C18 column with a gradient elution. The mass detection was performed using electrospray ionization (ESI) source under multiple reaction monitoring (MRM) in positive ionization mode. The method was fully validated, and the result of inter-assay and intra-assay precisions were less than 15% and the accuracy within the scope of ±15%. The linear range for plasma and urine covered from 10 to 10,000 ng/mL and 1 to 200 ng/mL, respectively, with correlation coefficients of 0.99. The validation for matrix effect, recovery, stability and carryover were met the acceptance criteria. The method showed robust and sensitive, it successfully fulfilled the requirement of clinical pharmacokinetic study of lifirafenib in Chinese patients with locally advanced or metastatic solid tumors.

New spectrophotometric/chemometric assisted methods for the simultaneous determination of imatinib, gemifloxacin, nalbuphine and naproxen in pharmaceutical formulations and human urine.

In this paper, novel univariate and multivariate regression methods along with model-updating technique were developed and validated for the simultaneous determination of quaternary mixture of imatinib (IMB), gemifloxacin (GMI), nalbuphine (NLP) and naproxen (NAP). The univariate method is extended derivative ratio (EDR) which depends on measuring every drug in the quaternary mixture by using a ternary mixture of the other three drugs as divisor. Peak amplitudes were measured at 294nm, 250nm, 283nm and 239nm within linear concentration ranges of 4.0-17.0, 3.0-15.0, 4.0-80.0 and 1.0-6.0µgmL-1 for IMB, GMI, NLP and NAB, respectively. Multivariate methods adopted are partial least squares (PLS) in original and derivative mode. These models were constructed for simultaneous determination of the studied drugs in the ranges of 4.0-8.0, 3.0-11.0, 10.0-18.0 and 1.0-3.0µgmL-1 for IMB, GMI, NLP and NAB, respectively, by using eighteen mixtures as a calibration set and seven mixtures as a validation set. The root mean square error of predication (RMSEP) were 0.09 and 0.06 for IMB, 0.14 and 0.13 for GMI, 0.07 and 0.02 for NLP and 0.64 and 0.27 for NAP by PLS in original and derivative mode, respectively. Both models were successfully applied for analysis of IMB, GMI, NLP and NAP in their dosage forms. Updated PLS in derivative mode and EDR were applied for determination of the studied drugs in spiked human urine. The obtained results were statistically compared with those obtained by the reported methods giving a conclusion that there is no significant difference regarding accuracy and precision.

Development and validation of a liquid chromatography-mass spectrometry assay for the determination of pyronaridine in human urine.

A reliable method has been developed for the determination of pyronaridine in human urine using amodiaquine as an internal standard. Liquid-liquid extraction was used for sample preparation. Analysis was performed on a Shimadzu LCMS-2010 in single ion monitoring positive mode using atmospheric pressure chemical ionization (APCI) as an interface. The extracted ion for pyronaridine was m/z 518.20 and for amodiaquine was m/z 356.10. Chromatography was carried out using a Gemini 5 microm C18 3.0 mmx150 mm column using 2 mM perflurooctanoic acid and acetonitrile mixture as a mobile phase delivered at a flow rate of 0.5 mL/min. The mobile phase was delivered in gradient mode. The retention times of pyronaridine and amodiaquine were 9.1 and 8.1 min respectively, with a total run time of 14 min. The assay was linear over a range of 14.3-1425 ng/mL for pyronaridine (R2>or=0.992, weighted 1/Concentration). The analysis of quality control samples for pyronaridine at 28.5, 285, 684 and 1140 ng/mL demonstrated excellent precision with relative standard deviation of 5.1, 2.3, 3.9 and 9.2%, respectively (n=5). Recoveries at concentrations of 28.5, 285, 684 and 1140 ng/mL were all greater than 85%. This LC-MS method for the determination of pyronaridine in human urine has excellent specifications for sensitivity, reproducibility and accuracy and can reliably quantitate concentrations of pyronaridine in urine as low as 14.3 ng/mL. The method will be used to quantify pyronaridine in human urine for pharmacokinetic and drug safety studies.

Quantification of vosaroxin and its metabolites N-desmethylvosaroxin and O-desmethylvosaroxin in human plasma and urine using high-performance liquid chromatography-tandem mass spectrometry.

Vosaroxin is a first-in-class anticancer quinolone derivative topoisomerase II inhibitor that is currently in development in combination with cytarabine for the treatment of acute myeloid leukemia (AML). To investigate vosaroxin pharmacokinetics (PK) in patients, liquid chromatography tandem mass spectrometry (LC-MS/MS) assays to quantify vosaroxin and the two metabolites N-desmethylvosaroxin and O-desmethylvosaroxin in human plasma and urine were developed and validated. Immediately after collection the samples were stored at -80°C. Prior to analysis, the plasma samples were subjected to protein precipitation and the urine samples were diluted. For both assays the reconstituted extracts were injected on a Symmetry Shield RP8 column and gradient elution was applied using 0.1% formic acid in water and acetonitrile-methanol (50:50, v/v). Analyses were performed with a triple quadruple mass spectrometer in positive-ion mode. A deuterated isotope of vosaroxin was used as internal standard for the quantification. The validated assays quantify vosaroxin and N-desmethylvosaroxin in the concentration range of 2-500ng/mL in plasma and urine. For O-desmethylvosaroxin the concentration range of 4-500ng/mL in plasma and urine was validated. Dilution integrity experiments show that samples can be diluted 25 fold in control matrix prior to analysis. The expanded concentration range for plasma and urine for vosaroxin and N-desmethylvosaroxin is therefore from 2 to 15,000ng/mL and in plasma for O-desmethylvosaroxin from 4 to 15,000ng/mL.

Elimination of enoxacin in renal disease.

The elimination of enoxacin was investigated in 15 subjects, 10 of whom were hospital outpatients with renal disease and varying degrees of renal impairment. Each was given enoxacin orally (200 mg b.i.d.) for 7 days. Blood specimens collected over 24 hours after the final dose of enoxacin and urine collected during the 12-hour dose interval after the final dose were assayed for enoxacin by HPLC. The elimination half-life of enoxacin increased with worsening renal function. In general, patients with diminished renal function had lower plasma enoxacin clearance values than had normal subjects, and a statistically significant correlation between apparent oral clearance and creatinine clearance was observed. Excretion of enoxacin by the kidney accounted for 26% to 72% of the apparent plasma clearance in normal subjects. This was markedly reduced in patients with severe renal failure.

In vitro and in vivo metabolism of pyronaridine characterized by low-energy collision-induced dissociation mass spectrometry with electrospray ionization.

The in vitro and in vivo metabolism of pyronaridine, an antimalarial agent, was investigated in rats and humans. In vitro incubation of pyronaridine with rat and human liver microsomes resulted in the formation of 11 metabolites, with pyronaridine quinoneimine (M3) as the major metabolite. The structures of pyronaridine metabolites were characterized on the basis of the product ion mass spectra obtained under low-energy collision-induced dissociation (CID) ion trap mass spectrometry. Both pyronaridine (m/z 518) and M3 (m/z 516) produced the same product ion (m/z 447). These results could be explained by the characteristic neutral loss of a 69 Da fragment from M3 via gamma-H rearrangement and 1,7 sigmatropic shift, whereas the neutral loss of a 71 Da fragment from the pyronaridine occurred by charge site-initiated heterolytic cleavage. These fragmentations were further supported by the tandem mass spectrum of D(3)-pyronaridine. Other metabolites generated in the microsomal incubations were carbonylated, hydroxylated and O-demethylated derivatives. Pyronaridine and its metabolites were detected in both feces and urine after intraperitoneal administration to rats. The in vivo metabolic profile in rats was different from the in vitro profile. M3, a chemically reactive quinonimine, was not detected whereas O-demethylated derivatives (M14, M15, M16, and M19) were identified in fecal and urinary extracts. The role of quinoneimine metabolites in pyronaridine-caused toxicity should be further evaluated, although these metabolites or their conjugates were not detected in urine and feces.

Urinary concentrations and bactericidal activities of newer fluoroquinolones in healthy volunteers.

Eleven healthy male subjects participated in a crossover study to compare the urine concentrations and bactericidal activities of newer fluoroquinolones against common uropathogens. Each volunteer received a single oral dose of gatifloxacin (400 mg), levofloxacin (250 mg), moxifloxacin (400 mg) and trovafloxacin (200 mg), and a urine sample was obtained at 2, 6, 12 and 24 h after the dose. Urine concentrations were highest with gatifloxacin and levofloxacin and lowest with trovafloxacin. Each drug concentration was studied against a levofloxacin susceptible and moderately-susceptible strain of Escherichia coli (minimal inhibitory concentration, MICs: 0.125 and 4 mg/l), K. pneumoniae (MICs: 0.125 and 4 mg/l), Pseudomonas aeruginosa (MICs: 0.5 and 4 mg/l) and Enterococcus faecalis (MICs: 0.25 and 4 mg/l). The duration of urine bactericidal activity (UBA) was based upon the median bactericidal titre at each time period. Both gatifloxacin and levofloxacin exhibited prolonged (> or = 6 h) UBA against all of the study isolates. Moxifloxacin exhibited prolonged UBA against both isolates of E. coli, K. pneumoniae and E. faecalis but not against either strain of P. aeruginosa. Prolonged UBA was not observed for trovafloxacin against the moderately-susceptible strains with the exception of E. faecalis. Furthermore, UBA was not observed for trovafloxacin against the susceptible strain of P. aeruginosa. Although these newer fluoroquinolones exhibited similar in vitro activity against these uropathogens, only those compounds with the highest urinary concentrations (gatifloxacin and levofloxacin) produced prolonged UBA against both strains of P. aeruginosa. The findings from this study suggest that both microbiological activity and urinary concentrations are important parameters to consider when choosing a fluoroquinolone for empirical treatment of urinary tract infections (UTIs).

On-line sample cleanup and chiral separation of gemifloxacin in a urinary solution using chiral crown ether as a chiral selector in microchip electrophoresis.

In chiral capillary electrophoresis of primary amine enantiomers using (+)-18-crown-6-tetracarboxylic acid (18C6H4) as a chiral selector, the presence of alkaline metal ions in the sample solution as well as in the run buffer is undesirable due to their strong competitive binding with 18C6H4. A channel-coupled microchip electrophoresis device was designed to clean up alkaline metal ions from a sample matrix for the chiral analysis of amine. In the first channel, the metal ions in the sample were monitored by indirect detection using quinine as a chromophore and drained to the waste. In the second separation channel, gemifloxacin enantiomers, free of the alkaline metal ions, were successfully separated using only a small amount of the chiral selector (50 microM 18C6H4).

Determination of trovafloxacin in human body fluids by high-performance liquid chromatography.

For the quantitative determination of trovafloxacin (a new naphthyridinone antibacterial agent) in serum and urine a simple isocratic HPLC method with fluorimetric detection is described. Serum was deproteinised with a mixture of acetonitrile and perchloric acid. The protein-free extract was separated on a reversed-phase column (Nucleosil 100-5 C18) and quantified by means of fluorescence (excitation 275 nm, emission 405 nm). The mobile phase consisted of a mixture of 250 ml acetonitrile and 750 ml distilled water containing 10 mmol/l tetrabutylammonium phosphate. Urine was diluted with 0.25 mol/l phosphoric acid 1:20 (v/v) which was adjusted to pH 3.6 with sodium hydroxide solution. Diluted urine samples were separated on a cation-exchange column (Nucleosil 100-5 SA) and also detected by means of fluorescence. Trovafloxacin was sufficiently separated from endogenous compounds. Results of validation are given. The method was applied successfully to a study of healthy volunteers.

Determination of the antibacterial trovafloxacin by differential-pulse adsorptive stripping voltammetry.

A differential-pulse adsorptive stripping voltammetric method for the determination of trace amounts of the antibacterial trovafloxacin (TRFLX) is proposed. The optimal experimental parameters for the drug assay were: accumulation potential=-0.30 V (vs. Ag/AgCl), accumulation time=120 s, pulse amplitude=50 mV and scan rate=5 mV s(-1) in Britton-Robinson buffer (pH 4.5). The linear concentration range of application was 2.0-20.0 ng ml(-1) of TRFLX, with a relative standard deviation of 3.6% (for a level of 5.0 ng ml(-1)) and a detection limit of 0.6 ng ml(-1). The method was applied to determination of TRFLX in human urine and serum samples. It was validated using HPLC as a reference method. Recovery levels of the method reached 100% in all cases

Metabolism of a dopamine receptor partial agonist in rats, including an unusual N-dearylation reaction.

The metabolism of 3,4-dihydro-7-[4-(1-naphthalenyl)-1-piperazinyl]butoxy]-1,8-naphthyridin-2(1H)-one (NPBN) was investigated in rats. Animals were administered 30 mg/kg NPBN that was labeled with both tritium and carbon-14. The mass recovery of drug-related material was 96-98%, with almost all material excreted in feces. Metabolism occurred by oxidation reactions followed by conjugation. The main route of metabolism of NPBN occurred via oxidation of the naphthylene ring, which led to naphthol and dihydrodiol metabolites as well as a relatively novel N-dearylated metabolite in which the naphthylene ring was removed. In vitro investigation in rat liver microsomes also showed a glutathione adduct on the naphthalene and a glutathione adduct of naphthoquinone, which, along with the dihydrodiol metabolite, is consistent with the initial generation of an epoxide. A mechanism is proposed whereby the N-dearylation arises via epoxidation, followed by formation of an exocyclic iminium ion intermediate that is hydrolyzed to yield the N-dearylated metabolite. An additional mechanism involves oxidation of the naphthol metabolite via a radical mechanism, since this metabolite was also shown to undergo N-dearylation.

Analysis of blood and urine samples from Macaca mulata for pyronaridine by high-performance liquid chromatography with electrochemical detection.

We describe a high-performance liquid chromatographic method with electrochemical detection for quantifying pyronaridine in rhesus monkey (Macaca mulata) blood and urine samples. The detection limit is 20 ng/ml at a signal-to-noise ratio of 4 in 0.5-ml samples of blood or urine. Blood analysis includes a liquid-liquid extraction and a subsequent solid-phase extraction that removes an interferent present in blood. For urine, a back-extraction is substituted for the solid-phase extraction step. The method uses an analogue of amodiaquine as internal standard, a 10-microns rigid macroporous styrene-divinylbenzene copolymer column and a mobile phase of 1% (v/v) triethylamine in methanol-water (34:66, v/v). The method was applied to samples of blood and urine from a monkey after a single intramuscular dose of pyronaridine tetraphosphate (160 mg as base).