Development and validation of ultra-performance liquid chromatography-tandem mass spectrometric methods for simultaneous and rapid determination of contezolid and its major metabolite M2 in plasma and urine samples and its application to a study in subjects with moderate liver impairment.

Contezolid is a novel oxazolidinone antibiotic with good antibacterial activity against gram-positive bacteria including methicillin-resistant Staphylococcus aureus. For the purpose to further characterize the pharmacokinetics of contezolid and its major metabolite M2, accurate and rapid ultra-performance liquid chromatography-tandem mass spectrometric assays (UPLC-MS/MS) were developed and validated for simultaneous quantification of contezolid and M2 in human plasma and urine. The plasma samples were pretreated by liquid-liquid extraction. The automated solid phase extraction method was used to preprocess urine samples. ACQUITY UPLC® BEH C8 (2.1 mm × 100 mm, 1.7 µm) column was used to separate the analytes with a gradient mobile phase of acetonitrile and water at a flow rate of 0.4 mL/min. The calibration curves showed good linearity over the concentration ranges of 0.0100-5.00 µg/mL for contezolid in plasma and urine, 0.00200-1.00 µg/mL in plasma and 0.0200-10.0 µg/mL in urine for M2, respectively. For both plasma and urine assays, the intra- and inter-batch accuracy and precision were within 15% for all quality control levels, including the lower limit of quantitation. The methods were fully validated and successfully applied to a pharmacokinetic study of contezolid tablets in subjects with moderate hepatic impairment.

[Pharmacokinetic evaluation of linezolid in patients with major thermal injuries]. / Pharmacocinétique du linézolide chez des patients souffrant de brûlures sévères.

The aims of this multicentre open-label study was to evaluate the pharmacokinetics of linezolid in patients with burn injury above 20 % BSA and to compare them with healthy volunteers, matched in age, sex and weight. After a single 600 mg IV dose of linezolid, multiple blood and urine samples were taken from subjects, in order to determine linezolid concentrations, using a HPLC assay. C(max) and volume of distribution at steady state were not different between the two groups. Values describing clearance were altered in burns, leading to a reduction by half in AUC in these patients (42.5 versus 98.1 mghL(-1)). The enhancement of clearance was due to which of non renal clearance (323+/-191 versus 80.4+/-27.5 mLmin(-1)). We conclude that pharmacokinetics of linezolid are altered in burn patients, in a magnitude sufficient that linezolid concentration may be subtherapeutic in some patients and we suggest that the dosage interval may need to be decreased in this patient population.

Clearance of linezolid via continuous venovenous hemodiafiltration.

BACKGROUND: Linezolid is being used increasingly for life-threatening vancomycin-resistant infections in critically ill patients. Limited data suggest that linezolid is cleared by intermittent hemodialysis. However, information on clearance of linezolid by continuous renal replacement therapy is limited. A patient undergoing continuous venovenous hemodiafiltration (CVVHDF) was evaluated to determine linezolid clearance through CVVHDF. METHODS: A 33-year-old man with necrotizing fasciitis and acute-on-chronic renal failure requiring CVVHDF was treated with linezolid, 600 mg every 12 hours, for a vancomycin-resistant urinary tract infection. The study was performed on day 4 of therapy after a 1-hour infusion of linezolid. A series of blood samples and all urine and diafiltrate were collected during a 12-hour period. Linezolid concentrations were determined by using high-performance liquid chromatography assay. Linezolid clearance through CVVHDF was determined by using 2 methods. Method 1 evaluated the amount of drug recovered in diafiltrate. Method 2 evaluated plasma drug concentrations in prefilter and postfilter (PAN-10 Hemofilter; Asahi Medical Co, Tokyo, Japan) samples. RESULTS: Clearance of linezolid through CVVHDF was 15.6 mL/min by method 1 and 21.6 mL/min by method 2. Total-body clearance was 189 mL/min. The amount of linezolid recovered in diafiltrate was 50 mg (8.3% of the dose). CONCLUSION: Clearance of linezolid through CVVHDF in this patient was marginal. It does not appear that supplemental dosing of linezolid is necessary in patients undergoing CVVHDF.

Excretion and metabolism of DA-7867, a new oxazolidinone, in rats.

Almost negligible hepatic metabolism (minor role of liver for the metabolism) and extensive urinary and fecal excretion of DA-7867 were investigated after intravenous administration at a dose of 10 mg/kg to rats. Pharmacokinetic parameters, especially nonrenal clearances of DA-7867, were very similar between control rats and rats pretreated with SKF 525-A, a nonspecific inhibitor of CYP isozymes, in rats. Similar results were also obtained between control rats and rats with liver cirrhosis induced by dimethylnitrosamine. Hepatic first-pass effect of DA-7867 was almost negligible in rats; the areas under the plasma concentration-time curve from time zero to time infinity of DA-7867 were not significantly different between intravenous and intraportal administration. The above data indicated that liver had almost negligible metabolic activity for DA-7867 in rats. Since metabolism of DA-7867 was not considerable in rats, urinary and fecal excretion of the drug was measured for up to 14 days in ten rats. Fecal excretion was the major route for elimination of DA-7867 in rats; approximately 85.0% of intravenous dose of DA-7867 at 10 mg/kg was recovered from urine (17.0% of intravenous dose), feces (64.0% of intravenous dose), washings of the metabolic cage (3.16% of intravenous dose), and entire gastrointestinal tract (0.421% of intravenous dose).

Effects of protein-calorie malnutrition on the pharmacokinetics of DA-7867, a new oxazolidinone, in rats.

The pharmacokinetic parameters of DA-7867, a new oxazolidinone, were compared after intravenous and oral administration at a dose of 10 mg x kg(-1) to control rats and rats with protein-calorie malnutrition (rats with PCM). After intravenous administration of 10 mg x kg(-1) DA-7867 to rats, metabolism of the drug was not considerable and after 14 days approximately 85.0% of the dose was recovered as unchanged drug from urine and faeces. After intravenous administration to rats with PCM, the area under the plasma concentration-time curve from time zero to time infinity (AUC) was significantly smaller (10800 vs 6990 microg min x mL(-1)) compared with control rats. This may have been due to significantly faster total body clearance (CL, 0.930 vs 1.44 mL x min(-1) x kg(-1)). The faster CL in PCM rats could have been due to significantly faster non-renal clearance (0.842 vs 1.39 mL x min(-1) x kg(-1) due to significantly greater gastrointestinal (including biliary) excretion; the amount of unchanged DA-7867 recovered from the entire gastrointestinal tract at 24 h was significantly greater (1.19 vs 4.28% of intravenous dose)) because the renal clearance was significantly slower in PCM rats (0.0874 vs 0.0553 mL x min(-1) x kg(-1)). After oral administration to PCM rats, the AUC was significantly smaller compared with control rats (7900 vs 4310 microg x min x mL(-1)). This could have been due to a decrease in absorption from the gastrointestinal tract.

Pharmacokinetics, blood partition and protein binding of DA-7867, a new oxazolidinone.

The pharmacokinetics after single intravenous and single and consecutive 2 week oral administration, tissue distribution, in vitro tissue metabolism, stability, blood partition and protein binding of DA-7867, a new oxazolidinone, were evaluated. After intravenous administration at a dose of 10mg/kg to rats, DA-7867 was eliminated slowly with time-averaged total body clearance of 0.915 ml/min/kg. After consecutive 2 week oral administration at a dose of 2 mg/kg/day to rats, DA-7867 was accumulated in rats; the AUC was significantly greater (1430 versus 1880 micro g min/ml) than that after single oral administration at a dose of 2 mg/kg. The rat tissues studied had low affinity to DA-7867; the tissue-to-plasma ratios were smaller than unity after both intravenous and oral administration at a dose of 20 mg/kg. The rat tissues studied had almost negligible metabolic activity for DA-7867 based on 30 min incubation of DA-7867 with 9000 g supernatant fraction of rat tissues. DA-7867 was stable for up to 24 h incubation in various buffer solutions having pHs from 1 to 11, Sørensen phosphate buffer of pH 7.4, and rat plasma, urine and liver homogenate and 3h incubation in five human gastric juices. The binding of DA-7867 to 4% human serum albumin was 50.6% at DA-7867 concentrations ranging from 0.5 to 20 micro g/ml. The equilibrium of DA-7867 between plasma and blood cells of rabbit blood reached fast (within 30 s manual mixing), and the plasma-to-blood cell concentration ratios were independent of initial blood concentrations of DA-7867, 1-20 micro g/ml; the values ranged from 1.39 to 1.63. Protein binding of DA-7867 in five fresh rats plasma was 72.3%.

High-performance liquid chromatographic analysis of DA-7867, a new oxazolidinone, in human plasma and urine and in rat tissue homogenates.

An HPLC method was developed for the determination of a new oxazolidinone, DA-7867 (I), in human plasma and urine and in rat tissue homogenates. To 100 microl of biological sample, 300 microl acetonitrile and 50 microl methanol containing 10 microg/ml DA-7858 (the internal standard) were added. After vortex-mixing and centrifugation, the supernatant was evaporated under a gentle stream of nitrogen. The residue was reconstituted in 100 microl of the mobile phase and a 50-microl aliquot was injected directly onto the reversed-phase (C(18)) column. The mobile phase, 20 mM KH2PO4:acetonitrile (75:25, v/v) was run at a flow rate of 1.5 ml/min and the column effluent was monitored by a UV detector set at 300 nm. The retention times of I and DA-7858 were approximately 6.5 and 8.7 min, respectively. The detection limits of I in human plasma and urine and in rat tissue homogenates were 20, 20, and 50 ng/ml, respectively.

The pharmacokinetics of the antimigraine compound zolmitriptan in Japanese and Caucasian subjects.

BACKGROUND: Zolmitriptan is a 5HT(1B/1D) receptor agonist effective in the acute treatment of migraine. Clinical trials in the USA and Europe have demonstrated the optimal oral therapeutic dose to be 2.5 mg. The 2.5-mg oral tablet has recently been licensed in Japan. OBJECTIVE: To compare the pharmacokinetics of zolmitriptan and its metabolites in Japanese and Caucasian subjects and evaluate the effect of gender on these pharmacokinetics in Japanese volunteers. METHODS: In this open, parallel-group study, 30 Japanese and 30 Caucasian volunteers (20-45 years) received a single 2.5-mg zolmitriptan tablet in the fasting state. Blood samples were taken up to 15 h post-dose to determine plasma concentrations of zolmitriptan and its active metabolite, 183C91. Urinary excretion of zolmitriptan, 183C91 and the inactive N-oxide and indole acetic acid metabolites were measured over 24 h. RESULTS: Japanese volunteers were, on average, smaller and lighter than Caucasian volunteers. Plasma-concentration profiles of zolmitriptan and 183C91 were similar in the two groups. Although geometric mean zolmitriptan and 183C91 area under the plasma concentration-time curve (AUC) and maximum plasma concentration (C(max)) were slightly higher in Japanese subjects (up to 20%), these differences were not considered to be of clinical relevance as the 90% confidence interval for the ratio of AUCs fell within pre-specified limits (0.67 to 1.5). Mean zolmitriptan and 183C91 half-lives were around 2.5 h for both populations. Urinary excretion of the four analytes was similar in Japanese and Caucasians. Plasma concentrations of zolmitriptan were higher in Japanese females than males (AUC 40% and C(max) 29% higher), consistent with the results previously obtained in Caucasians. CONCLUSION: Pharmacokinetic parameters of zolmitriptan were similar between Caucasian and Japanese volunteers.

Age and sex effects on the pharmacokinetics of linezolid.

OBJECTIVE: To evaluate the possible effects of age and sex on the pharmacokinetics of linezolid in healthy volunteers. METHODS: A single 600-mg dose of linezolid was administered orally to young (18-40 years) and elderly (> or =65 years) healthy males and females. Blood and urine samples were collected until 48 h after dosing and assayed for linezolid concentrations using a validated high-performance liquid chromatography method. Pharmacokinetic parameters were assessed using noncompartmental methods. Comparisons of the pharmacokinetic parameters for each age and sex group were performed using a two-way analysis of variance model. Pairwise comparisons were done using least-square means analysis. RESULTS: Peak plasma drug concentrations occurred within 1.5 h after linezolid administration for males and females in both age groups. However, the maximum concentration achieved differed significantly between males and females. There was no significant difference between males and females or young and elderly for mean apparent elimination rate constant or half-life. There was no difference in mean apparent oral clearance (CLPO) between the young and elderly; however, there was a significant difference between males and females. Mean CLPO for females was approximately 37% less than mean CLPO for males when not corrected for body weight. Correcting for differences in weight reduced this difference to approximately 20%. Overall, females had a slightly lower volume of distribution than males, but this was not affected by the age of subjects. CONCLUSIONS: A dose adjustment based on age and sex is not warranted due to the wide range of linezolid concentrations that are well tolerated and the relative small difference in linezolid disposition between males and females.

Determination of linezolid in human serum and urine by high-performance liquid chromatography.

An HPLC method is described for the determination of the new oxazolidinone antibiotic linezolid (I) in human biofluids. After precipitation of serum proteins with perchloric acid the protein free supernatant was separated by isocratic reversed-phase chromatography on a Nucleosil-100 5C18 column. The mobile phase consisted of a mixture of acetonitrile: sodium acetate buffer: water (180:100:720, v/v) adjusted to pH 3.7. Urine was diluted with aqueous buffer solution. The column eluate was monitored at 250 nm. Validation of the method yielded satisfactory results for serum (and urine); detection limit 0.07 mg/l (2.4), lower limit of quantitation 0.14 mg/l (4.7), linear range 20 mg/l (500), imprecision within series (c.v.) 1.8-2.5% (0.8-1.0), imprecision between series (c.v.) 1.8-9.3 (0.4-9.3), recovery 99-102% (93-103). Comparison of HPLC results with results obtained using a quantitative microbiological assay yielded acceptable agreement both for serum and urine. The method was successfully used in a pharmacokinetic study with human volunteers.

Pharmacokinetics, metabolism, and excretion of linezolid following an oral dose of [(14)C]linezolid to healthy human subjects.

Linezolid (Zyvox), the first of a new class of antibiotics, the oxazolidinones, is approved for treatment of Gram-positive bacterial infections, including resistant strains. The disposition of linezolid in human volunteers was determined, after a 500-mg (100-microCi) oral dose of [(14)C]linezolid. Radioactive linezolid was administered as a single dose, or at steady-state on day 4 of a 10-day, 500-mg b.i.d. regimen of unlabeled linezolid (n = 4/sex/regimen). Mean recovery of radioactivity in excreta was 93.8 +/- 1.1% (range 91.2-95.2%, n = 15), of which 83.9 +/- 3.3% (range 76.7-88.4%) was in urine and 9.9 +/- 3.4% (range 5.3-16.9%) was in feces. There was no major difference in rate or route of excretion of radioactivity by dose regimen. Linezolid was excreted primarily intact, and as two inactive, morpholine ring-oxidized metabolites, PNU-142586 and PNU-142300. Other minor metabolites were characterized by high-performance liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry and (19)F NMR spectroscopy. After the single radioactive dose, linezolid was the major circulating drug-related material accounting for about 78% (male) and 93% (female) of the radioactivity area under the curve (AUC). PNU-142586 (T(max) of 3-5 h) accounted for about 26% (male) and 9% (female) of the radioactivity AUC. PNU-142300 (T(max) of 2-3 h) accounted for about 7% (male) and 4% (female) of the radioactivity AUC. Overall, mean linezolid and PNU-142586 exposures at steady-state were similar across sex. In conclusion, linezolid circulates in plasma mainly as parent drug. Linezolid and two major, inactive metabolites account for the major portion of linezolid disposition, with urinary excretion representing the major elimination route. Formation of PNU-142586 was the rate-limiting step in the clearance of linezolid.

A new and rapid method for monitoring the new oxazolidinone antibiotic linezolid in serum and urine by high performance liquid chromatography-integrated sample preparation.

A sensitive and rapid HPLC-assay for determining the new oxazolidinone antibiotic linezolid in serum and urine is described. HPLC-integrated sample preparation permits the direct injection of serum and urine samples without any pre-treatment. The in-line extraction technique is realized by switching automatically from the extraction column to the analytical column. After the matrix has passed the extraction column the retained analyte will be quantitatively transferred to the analytical column where separation by isocratic HPLC will be performed. Linezolid is detected according to its absorption maximum at 260 nm. The quantification limits are estimated to be 0.3 and 0.5 microg/ml in serum and urine samples, respectively. The described procedure allows sample clean-up and determination of the antibiotic within 20 min, thereby facilitating drug-monitoring in clinical routine.