Daptomycin Population Pharmacokinetics in Patients Affected by Severe Gram-Positive Infections: An Update.

Daptomycin pharmacokinetics may not depend on renal function only and it significantly differs between healthy volunteers and severely ill patients. Herein, we propose a population pharmacokinetics model based on 424 plasma daptomycin concentrations collected from 156 patients affected by severe Gram-positive infections during a routine therapeutic drug monitoring protocol. Model building and validation were performed using NONMEM 7.2 (ICON plc), Xpose4 and Perl-speaks-to-NONMEM. The final pop-PK model was a one-compartment first-order elimination model, with a 2.7% IIV for drug clearance (Cl), influence of creatinine clearance on drug clearance and of sex on distribution volume. After model validation, we simulated 10,000 patients with the Monte-Carlo method to predict the efficacy and tolerability of different daptomycin daily dosages. For the most common 6 mg/kg daily dose, the simulated probability of overcoming the toxic minimum concentration (24.3 mg/L) was 14.8% and the efficacy (expressed as a cumulative fraction of response) against methicillin-resistant S. aureus, S. pneumoniae and E. faecium was 95.77%, 99.99% and 68%, respectively. According to the model-informed precision dosing paradigm, pharmacokinetic models such as ours could help clinicians to perform patient-tailored antimicrobial dosing and maximize the odds of therapy success without neglecting toxicity risks.

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.

Methods: for studying pharmacogenetic profiles of combination chemotherapeutic drugs.

INTRODUCTION: Molecular and genetic analysis of tumors and individuals has led to patient-centered therapies, through the discovery and identification of genetic markers predictive of drug efficacy and tolerability. Present therapies often include a combination of synergic drugs, each of them directed against different targets. Therefore, the pharmacogenetic profiling of tumor masses and patients is becoming a challenge, and several questions may arise when planning a translational study. AREAS COVERED: The review presents the different techniques used to stratify oncology patients and to tailor antineoplastic treatments according to individual pharmacogenetic profiling. The advantages of these methodologies are discussed as well as current limits. EXPERT OPINION: Facing the rapid technological evolution for genetic analyses, the most pressing issues are the choice of appropriate strategies (i.e., from gene candidate up to next-generation sequencing) and the possibility to replicate study results for their final validation. It is likely that the latter will be the major obstacle in the future. However, the present landscape is opening up new possibilities, overcoming those hurdles that have limited result translation into clinical settings for years.

Comparative pharmacokinetic and pharmacodynamic evaluation of branded and generic formulations of meloxicam in healthy male volunteers.

PURPOSE: The primary aim of the present study was to assess the pharmacokinetic bioequivalence between a generic formulation of meloxicam 15 mg tablets (Meloxicam Hexal) and its respective brand product (Mobic), in order to verify whether the generic product conforms to the regulatory standards of bioequivalence in the postmarketing setting. As a secondary exploratory aim, the pharmacodynamic effects of the two formulations were also evaluated by means of rating scales following hyperalgesia induced by cutaneous freeze injury. SUBJECTS AND METHODS: A single 15 mg dose of generic or branded meloxicam tablets was administered to 24 healthy male volunteers in a crossover fashion. Plasma samples, collected for 24 hours after dosing, were assayed for meloxicam concentration by a validated highperformance liquid chromatography method. RESULTS: THE ANALYSIS OF PHARMACOKINETIC PARAMETERS DID NOT SHOW ANY SIGNIFICANT DIFFERENCE BETWEEN THE TWO MELOXICAM FORMULATIONS: the 90% confidence intervals fell within the acceptance range of 80%-125% (0.84-1.16 for area under the curve [0-24], and 0.89-1.23 for peak concentration). No difference in the pharmacodynamic end point was observed between the two groups. CONCLUSION: The pharmacokinetic profiles of the two meloxicam formulations confirm the regulatory criteria for bioequivalence; pharmacodynamic data indicate a similar antihyperalgesic effect. The two formulations can be used interchangeably in the clinical setting.

Pharmacokinetic and pharmacogenetic predictive markers of irinotecan activity and toxicity.

After the rapid development of new classes of antineoplastic drugs, research activities have focused their efforts to the identification of predictive markers of drug activity and tolerability. Irinotecan (CPT-11) may induce severe toxicities (diarrhea, neutropenia) that limit its clinical use, but the increasing knowledge of its pharmacokinetics offered a potential approach to treatment optimization. Pharmacokinetics, the first area of investigation, has identified markers such as biliary index, the relative extent of conversion and the glucuronidation ratio, which are capable to define the risk for severe adverse effects. Because of the existence of some issues concerning the adoption of pharmacokinetic strategies to optimize CPT-11 dose and schedule, analyses of genetic polymorphisms seemed to offer a more reliable and safer approach for the identification of patients at risk than pharmacokinetics. In this view, the uridine diphosphate glucuronosil transferase isoform 1A1 (UGT1A1) was associated with significant changes in disposition of CPT-11 and its metabolites, and consequently with treatment-induced toxicities. However, the complex pharmacokinetics of irinotecan and the involvement of several enzymes other than UGT (i.e., carboxyl estherases, CYP450 isoforms), and transmembrane transporters (ABCB1, ABCC1, ABCG2, SLCO1B1) make difficult the identification of patients with an optimal sensitivity and specificity, and a large part of variability among patients still remains unexplained. Furthermore, prospective clinical studies that should demonstrate the reliability of those pharmacokinetic and pharmacogenetic markers are still lacking. In the present review, pharmacokinetic and pharmacogenetic markers will be discussed.

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.

5-fluorouracil pharmacokinetics predicts disease-free survival in patients administered adjuvant chemotherapy for colorectal cancer.

PURPOSE: To evaluate 5-fluorouracil (5-FU) and 5-fluoro-5,6-dihydrouracil (5-FDHU) pharmacokinetics and disease-free survival (DFS) in colorectal cancer patients given 5-FU-based adjuvant chemotherapy within a nonrandomized, retrospective, pharmacokinetic study. EXPERIMENTAL DESIGN: One hundred fifteen patients including 72 men (median age, 63 years; range, 36-79 years) and 43 women (median age, 60 years; range, 36-73 years) received 6 cycles of l-leucovorin 100 mg/m(2)/day and 5-FU 370 mg/m(2)/day i.v. boluses (5 days every 4 weeks). Individual plasma concentrations of 5-FU and 5-FDHU were determined on day 1 of the first cycle with a validated high performance liquid chromatography method, and the main pharmacokinetic variables were determined. Follow-up of all patients was extended up to 5 years after the end of adjuvant chemotherapy, and DFS was recorded. Univariate and multivariate analyses were conducted to evaluate any correlation among 5-FU pharmacokinetics, clinical and pathologic variables, and DFS. RESULTS: The area under the time/concentration curve (AUC) of 5-FU was significantly lower in 58 subjects who recurred (7.5 +/- 2.9 h x mg/L) with respect to other patients (9.3 +/- 4.1 h x mg/L). Furthermore, AUC values lower than 8.4 h x mg/L together with lymph node involvement and the interruption of treatment or reduction of doses were identified as risk factors at univariate analysis. The completion of 6 cycles of adjuvant treatment without dosage modifications was the only independent risk factor at multivariate analysis, despite a trend toward significance for 5-FU AUC values (cutoff value, 8.4 hxmg/L) was observed (P = 0.06). CONCLUSIONS: Pharmacokinetics of 5-FU should be regarded as an important factor for predicting disease recurrence in colorectal cancers.

Thymidylate synthase, dihydropyrimidine dehydrogenase and thymidine phosphorylase expression in colorectal cancer and normal mucosa in patients.

OBJECTIVE: To compare thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) and thymidine phosphorylase (TP) gene polymorphism and expression in colorectal cancer (CRC), and normal mucosa in chemotherapy-naïve patients. METHODS: TS, DPD and TP mRNA expression was analysed by real-time reverse-transcription polymerase chain reaction in primary CRC and adjacent normal tissues from 53 patients with glyceraldehyde-3-phosphate dehydrogenase as housekeeping gene. TS promoter (TSER and C/G SNP) and DPD IVS14+1G>A genotypes were determined by polymerase chain reaction and restriction fragment length polymorphism assays. Moreover, the correlation between TS, DPD and TP expression and cytotoxicity of 5-fluorouracil was evaluated in Colo 320, HT-29, CaCo-2 and SW620 human CRC cell lines. RESULTS: TP and DPD mRNA expression was significantly different in tumour and normal tissue (7.51+/-13.50 vs. 1.10+/-0.57, P<0.05 and 0.60+/-0.63 vs. 1.17+/-0.55, P<0.0001, respectively), whereas no differences were observed in TS mRNA levels. High-grade, undifferentiated tumours (WHO grade 3) had significantly higher mRNA levels of TS with respect to moderately differentiated (WHO grade 2) carcinomas (0.38+/-0.37 vs. 0.00+/-0.44, respectively; P<0.05). Noteworthy, TS mRNA expression was significantly decreased (P<0.05) in homozygous TSER*3G/3G (-0.35+/-0.35) with respect to pooled homozygous TSER*2/2 and heterozygous TSER*2/3 genotypes (0.14+/-0.41). In-vitro results showed a higher sensitivity to 5-FU of cell lines with the lowest TS expression. CONCLUSIONS: The present results demonstrated significant differences in DPD and TP gene expression between normal mucosa and tumour samples, while TSER*3G/3G and high-grade histology were associated with significant variation in TS gene expression in tumour samples.

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.

Drug therapeutic failures in emergency department patients. A university hospital experience.

Drug therapeutic failure (DTF) could be considered as an adverse drug reaction in which the expected drug effects do not occur following a prescribed pharmacological treatment, including any clinical event that could be related to a low prescribed dose or lack of compliance. DTFs are responsible for increasing disease length, hospitalisation time and social costs, with the worsening of patient quality of life. The aims of the present study are: (1) to estimate the frequency of DTFs among cases of adverse drug events referred to the emergency department; (2) to identify drug classes implicated in DTFs; (3) to analyse the putative causes underlying DTFs. Data presented in this paper were obtained from the Pronto Soccorso and Adverse Drug Events (PSADE) study carried out to analyse drug-related emergency department admissions in several Italian hospitals. Patients, admitted to the emergency department throughout two periods of 10 days each, were interviewed to gain information on their medical status and drug intake during the last two weeks. The present study analysed the patient questionnaires collected in the emergency department of Pisa University Hospital. Among 123 recorded cases of adverse drug event, 41 cases (19:22 male:female ratio; age range: 17-98 years, median age: 75 years) were identified as suspect DTF, resulting in a frequency of 33.33%. A statistical analysis was performed to evaluate the influence of two variables, class of patient age and number of drugs assumed, on DTFs. In accordance with the present findings, showing that the number of drugs assumed by a patient may increase the risk of DTF more than advanced age (odds ratio: 1.371, P<0.02; 1.295, P<0.03, respectively), the prescription of pharmacological combinations might be proposed as a main risk factor for DTF occurrence. In conclusion, our results suggest that DTFs represent an important cause of emergency department admission, particularly in elderly subjects treated with pharmacological associations.