Marianna Lastella

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/m2/day and 5-FU 370 mg/m2/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 × mg/L) with respect to other patients (9.3 ± 4.1 h × mg/L). Furthermore, AUC values lower than 8.4 h × 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 h×mg/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.

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.

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.

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 × 4.6 mm, 5 μm); 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.

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 × 4.6 mm, 10 μm), 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.

Inhibition of protein farnesylation enhances the chemotherapeutic efficacy of the novel geranylgeranyltransferase inhibitor BAL9611 in human colon cancer cells

Proteins belonging to the ras superfamily are involved in cell proliferation of normal and neoplastic tissues. To be biologically active, they require post-translational isoprenylation by farnesyl-transferase and geranylgeranyl-transferase. Enzyme inhibition by drugs may thus represent a promising approach to the treatment of cancer. Therefore, the combined effect of BAL9611, a novel inhibitor of geranylgeranylation, and manumycin, a farnesyl-transferase inhibitor, was evaluated on the SW620 human colon cancer cell line which harbours a mutated K-ras gene. BAL9611 and manumycin dose-dependently inhibited SW620 cell growth with 50% inhibitory concentration (IC50) of 0.47 ± 0.03 and 5.24 ± 1.41 μM (mean ± SE), respectively. The isobologram analysis performed at the IC50 level revealed that the combined treatment was highly synergistic with respect to cell growth inhibition. BAL9611 and manumycin were able to inhibit the geranylgeranylation of p21rhoA and farnesylation of p21ras; both drugs inhibited p42ERK2/MAPK phosphorylation, but their combination was more effective than either drug alone. Moreover, the enhanced inhibition of cell growth in vitro by the BAL9611-manumycin combination was also observed in vivo in CD nu/nu female mice xenografted with SW620 tumours. Finally, both drugs were able to induce cell death by apoptosis in vitro and in vivo, as demonstrated by perinuclear chromatin condensation, cytoplasm budding and nuclear fragmentation, and interoligonucleosomal DNA digestion. In conclusion, the inhibition of protein farnesylation enhances the chemotherapeutic effect of BAL9611 in vitro and in vivo in a synergistic fashion, as a result of the impairment of post-translational isoprenylation of proteins and phosphorylation of p42ERK2/MAPK, whose activation is associated with post-translational geranylgeranylation and farnesylation of p21rhoA and p21ras.

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.

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.