A new validated HPLC-UV method for therapeutic monitoring of daptomycin in comparison with reference mass spectrometry.

Daptomycin, a cyclic lipopeptide antibiotic with a broad spectrum of activity against Gram-positive bacteria, is also active against multi-resistant bacterial strains, as well as methicillin-resistant S. aureus, vancomycin-resistant enterococci or penicillin-resistant S. pneumoniae. For these reasons it is a viable alternative for the treatment of persisting infections. However, the therapeutic drug monitoring of daptomycin is recommended because the known variability in drug disposition and the severe clinical conditions of patients. Therefore, we developed a simple and fast UV-HPLC method according to FDA guidelines to monitor plasma concentrations of the drug. Briefly, after a liquid-liquid extraction, plasma calibration samples, quality controls and patients' samples were injected in a HPLC instrument and peaks of daptomycin and gentamicin (internal standard) were resolved by a C18 250 × 4.6 mm, 5 µm stationary phase and peaks were monitored at UV = 262 nm. Mobile phase (isocratic flow of 1 mL/min) consisted of acetonitrile-buffer (KH2PO4 20 mM pH = 3.2) 46:54, vol/vol. Under these conditions, IS and daptomycin peaked at 4.1 and 5.8 min after injection. Values of limits of detection and quantitation accounted for 1.65 and 5.00 (µg/ml), respectively. Values of method linearity (r2) in range 5-100 mg/L were 0.9975 and 0.9956 plasma samples and solvent standard, respectively. Inter- and intra-day variability coefficients were lower than 15 %. The comparison with a reference, commercially-available LC-MS/MS method on 122 patient plasma samples returned excellent correlation (r2 = 0.9474). In conclusion, the present method demonstrated to be reliable and suitable for daptomycin TDM in clinical routine.

A case report of a TDM-guided optimization of mitotane for a safe and effective long-term treatment.

A 43-years old woman was diagnosed an adrenocortical carcinoma (AC) that was excised, whereas two lung metastases were un-operable. Mitotane 6 g/day was started as standard therapy but it was responsible for severe central nervous system (CNS) and gastrointestinal toxicities associated with a 10 kg body weight loss. A therapeutic drug monitoring (TDM) protocol demonstrated that mitotane plasma concentrations (>30 mg/L) exceeded the therapeutic range (14-20 mg/L) and increased even when drug daily dose was reduced by 50%. The increase in drug plasma concentrations was probably due to body slimming. Under continuous TDM control, a reduced mitotane dose (1.5 g/day) was definitively administered and it proved to be tolerable and effective. Indeed, lung metastases were excised and two years later there was no evidence of other neoplastic lesions. In conclusion, the adoption of therapeutic mitotane monitoring allowed the treatment of an AC patient with a reduced, tolerable and effective dose.

Linezolid in the central nervous system: comparison between cerebrospinal fluid and plasma pharmacokinetics.

BACKGROUND: Linezolid is effective against methicillin-resistant Staphylococcus aureus, which may cause central nervous system (CNS) infections, but drug concentrations in plasma are characterized by a large inter-patient variability. Therefore, the present study was aimed at evaluating linezolid pharmacokinetics in plasma and cerebrospinal fluid (CSF) in 7 patients with external ventricular drainage, who received linezolid 600 mg twice daily as 1-h intravenous infusion to prevent CNS infections. METHODS: Plasma and CSF linezolid concentrations were measured by high-performance liquid chromatography (HPLC) after the 1(st) and 5(th) dose, and pharmacokinetics were evaluated by non-compartmental analysis. RESULTS: Values of the CSF area under the time/concentration curve (AUC) (range 18.2-85.5 and 19.6-160.5 h × mg/l at the 1st and 5th dose, respectively) were lower than those calculated in plasma (range 27.6-224.0 and 27.5-166.1 h × mg/l, respectively). For minimum inhibitory concentration (MIC) = 1 mg/l, CSF AUC/MIC values were nearly equal to or greater than 100 only in 2 subjects after the 1st and 5th dose, whereas mean time above the MIC (T > MIC) values were higher than 75% in only 3 patients. Similar results were obtained when pharmacokinetic/pharmacodynamic parameters were evaluated in plasma. CONCLUSION: The present results suggest that changes in linezolid doses and measurement of drug concentrations should be considered as useful strategies to optimize treatment in some patients.

Augmentation of clozapine with aripiprazole in severe psychotic bipolar and schizoaffective disorders: a pilot study.

AIM: To evaluate the efficacy and safety of the augmentation of clozapine with aripiprazole in patients with treatment-resistant schizoaffective and psychotic bipolar disorders in a retrospective manner. Pharmacodynamic and pharmacokinetic interactions between the two drugs were also investigated. PATIENTS: Three men and 4 women (median age 36 and 40 years, respectively) who had mean scores at BPRS and CGI-Severity of 59.1+/-12.0 and 5.4+/-0.5, respectively, were treated with clozapine (mean dose 292.9+/-220.7 mg/day). Patients received an adjunctive treatment with aripiprazole (mean dose 6.8 +/- 3.7 mg/day). Clozapine, norclozapine and aripiprazole plasma levels were measured by means of a high performance liquid chromatograpy with UV detection. RESULTS: Total scores at BPRS decreased significantly (from 59.1+/-12.0 to 51.1+/-15.6, p=0.007) after aripirazole augmentation. In particular, the factors "thought disorder" (from 10.4+/-4.4 to 9.0+/-4.5, p=.047) and "anergia" (from 10.0+/-2.7 to 8.0+/-2.4, p=.018) significantly improved. Concomitant administration of aripiprazole and clozapine did not result in an increase in side effects over the period of treatment. Dose-normalized plasma levels of both clozapine and norclozapine and the clozapine/norclozapine metabolic ratio in all patients did not vary as well. CONCLUSION: The augmentation of clozapine with aripirazole was safe and effective in severe psychotic schizoaffective and bipolar disorders which failed to respond to atypical antipsychotics. A possible pharmacokinetic interaction between clozapine and aripiprazole does not account for the improved clinical benefit obtained after aripiprazole augmentation.

A rapid high-performance liquid chromatography method to measure linezolid and daptomycin concentrations in human plasma.

Daptomycin and linezolid, recently introduced to treat severe Gram-positive infections, are effective against multidrug-resistant Gram-positive microorganisms such as methicillin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis, and vancomycin-resistant Enterococci bacteria that are less sensitive or frankly resistant, including methicillin-resistant S. aureus. However, alteration of their plasma profile has been described in some patients and this may be associated with toxicities or selection of resistant strains. The measurement of plasma concentrations of both drugs may allow the identification of those subjects at major risk of adverse events. Therefore, a rapid and sensitive high-performance liquid chromatography method for the analysis of daptomycin and linezolid was developed and applied in clinical settings. Drugs were extracted from plasma by adding methanol and, after centrifugation, clear supernatants were injected into the high-performance liquid chromatography system. Isocratic elution (1.5 mL/min) was performed using a mobile phase consisting of ammonium phosphate buffer 40 mM, pH 4.0, acetonitrile (70:30, vol/vol) through a BDS C8 Hypersil stationary phase (250 x 4.6 mm, 5 mum); ultraviolet detection was used at 214 nm. Linezolid and daptomycin eluted within 20 minutes from the injection, and mean recoveries ranged between 95.4% and 112.1%, respectively. The method was linear (coefficient of linearity, 0.998-0.999) over the full range of concentrations assayed, from 0.78125 mg/L (limit of quantitation) to 100 mg/L for both drugs. The Sy.x values were equal to 0.25 +/- 0.10 and 0.29 +/- 0.18 mg/L for daptomycin and linezolid, respectively. Precision values were lower than 20% over the entire range of calibration standard, and accuracy was within the range of 80% to 120% for all concentrations. The present method proved to be sensitive and specific to measure daptomycin and linezolid plasma concentrations in patients affected by severe Gram-positive infections, allowing therapeutic drug monitoring in those patients at major risk of severe adverse events.

ABCB1 polymorphisms are associated with clozapine plasma levels in psychotic patients.

AIMS: ABCB1 is a transmembrane transporter that is expressed in excretory organs (kidneys and liver), in intestine mucosa and on the blood-brain barrier. Because of the particular distribution of the protein, the activity of ABCB1 may significantly affect drug pharmacokinetics during absorption and distribution. Of note, several SNPs of ABCB1 are known and many of them affect transporter activity and/or expression. In this view, changes in the pharmacokinetics of drugs that are ABCB1 substrates could be clinically relevant and the evaluation of ABCB1 SNPs should deserve particular attention. Therefore, the aim of the present study was to investigate the possible association between ABCB1 polymorphisms and clozapine plasma levels in psychotic patients. MATERIALS & METHODS: c.1236C>T (exon 12), c.2677G>T (exon 21) and c.3435C>T (exon 26) SNPs of ABCB1 were evaluated by PCR techniques, while plasma levels of clozapine and norclozapine were measured by HPLC in 40 men (aged, 47.6 +/- 16.6 years, median: 42 years) and 20 women (aged 40.7 +/- 11.4 years, median: 38 years) 1 month after the start of clozapine administration. RESULTS: A total of three SNPs were in Hardy-Weinberg equilibrium, with a calculated frequency of the wild-type alleles of 0.54, 0.55 and 0.45 for SNPs on exons 12, 21 and 26, respectively. Patients with c.3435CC or c.2677GG genotypes had significantly lower dose-normalized clozapine levels than those who were heterozygous or TT carriers. More interestingly, c.3435CC patients (15 subjects) needed significantly higher daily doses of clozapine (246 +/- 142 mg/day) compared with the remaining 24 CT and 21 TT patients (140 +/- 90 mg/day) in order to achieve the same clinical benefit. CONCLUSION: c.3435CC patients require higher clozapine doses to achieve the same plasma concentrations as CT or TT patients, and ABCB1 genotyping should be considered as a novel strategy that should improve drug use.

Improved analysis of 5-Fluorouracil and 5,6-dihydro-5-Fluorouracil by HPLC with diode array detection for determination of cellular dihydropyrimidine dehydrogenase activity and pharmacokinetic profiling.

Administration of 5-fluorouracil (5-FU) may be associated with severe toxicities in patients who are deficient of dihydropyrimidine dehydrogenase (DPD) activity. For this reason, a sensitive HPLC method for the analysis of 5-FU and 5-fluoro-5,6-dihydrouracil (5-FDHU) was developed in the present study for the determination of DPD activity in nucleated cells of peripheral blood and pharmacokinetic analysis of 5-FU and 5-FDHU in humans. 5-FU and 5-FDHU were extracted from biologic matrices by adding sodium acetate, sodium sulfate, and diethyl ether/propanol. Dried samples were reconstituted in a mobile phase (KH2PO4 35 mmol/L, pH 4.0), isocratically eluted with a Hypersil C18 stationary phase (25 cm x 4.6 mm, 10 microm), and detected by a diode array detector (measurement and reference wavelengths, 215 and 360 nm, respectively). 5-Fluorocytosine (internal standard), 5-FDHU, and 5-FU were eluted within 13 minutes of the injection without interferences. Recoveries ranged between 81% to 85% for all compounds, and the method proved to be linear, with a coefficient of linearity of 0.999. The limits of detection and quantification were 3.2 and 16 ng/mL, respectively, and the within-day and between-day CV were less than 10% for both 5-FU and 5-FDHU. The present assay proved to be sufficiently sensitive and specific to evaluate cellular DPD activity and measure 5-FU and 5-FDHU plasma concentrations in cancer patients, thus allowing therapeutic 5-FU monitoring in patients and identification of DPD-deficient subjects at major risk of severe toxicities.