Marialuisa Polillo

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.

The c.480C>G polymorphism of hOCT1 influences imatinib clearance in patients affected by chronic myeloid leukemia.

The aim of the study was to investigate any possible influence of polymorphisms of transmembrane transporters human organic cation transporter 1 (hOCT1), ABCB1, ABCG2 on imatinib pharmacokinetics in 33 men and 27 women (median age and range, 56 and 27–79 years, respectively) affected by chronic myeloid leukemia. A population pharmacokinetic analysis was performed to investigate imatinib disposition in every patient and the role of transporter polymorphisms. Results showed that the α1-acid glycoprotein and the c.480C>G genotype of hOCT1 had a significant effect on apparent drug clearance (CL/F) being responsible, respectively, for a 20% and 10% decrease in interindividual variability (IIV) of CL/F (from 50.1 up to 19.6%). Interestingly, 25 patients carrying at least one polymorphic c.480 G allele had a significant lower CL/F value with respect to the 35 c.480CC individuals (mean±s.d., 9.6±1.6 vs 12.1±2.3 l h−1, respectively; P<0.001). In conclusion, the hOCT1 c.480C>G SNP may significantly influence imatinib pharmacokinetics, supporting further analyses in larger groups of patients.

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.

Population pharmacokinetics of daptomycin in patients affected by severe Gram-positive infections

A population pharmacokinetic analysis of daptomycin was performed based on therapeutic drug monitoring (TDM) data from 58 patients receiving doses of 4-12 mg/kg for the treatment of severe Gram-positive infections. At a daily dose of 8 mg/kg, daptomycin plasma concentrations (mean ± S.D.) were 76.9 ± 9.8 mg/L at the end of infusion and 52.7 ± 15.4 mg/L and 11.4 ± 5.4 mg/L at 0.5 h and 23 h after drug administration, respectively. The final model was a one-compartmental model with first-order elimination, with estimated clearance (CL) of 0.80 ± 0.14 L/h and a volume of distribution (V(d)) of 0.19 ± 0.05 L/kg. Creatinine clearance (CL(Cr)) was identified as having a significant influence on daptomycin CL, and a decrease in CL(Cr) of 30 mL/min from the median value (80 mL/min) was associated with a reduction of daptomycin CL from 0.80 L/h to 0.73 L/h. These results confirm that the presence of severe infection may be associated with an altered disposition of daptomycin, with an increased Vd. MICs were available in 41 patients and results showed that 38 and 31 subjects achieved AUC/MIC values associated with bacteriostatic (>400) and bactericidal effects (>800), respectively. Of note, 31 of these 41 subjects experienced a clinical improvement or were cured. Although daptomycin pharmacokinetics may be influenced by infections, effective AUC/MIC values were achieved in the majority of patients. The present model may be applied in clinical settings for a TDM routine on the basis of a sparse blood sampling protocol.

Case Report of a Successful Treatment of Methicillin-Resistant Staphylococcus aureus (MRSA) Bacteremia and MRSA/Vancomycin-Resistant Enterococcus faecium Cholecystitis by Daptomycin

ABSTRACT A 72-year-old man, receiving 8 mg daptomycin/kg body weight/day for methicillin-resistant Staphylococcus aureus (MRSA) bacteremia, was diagnosed with MRSA/vancomycin-resistant Enterococcus faecium (VRE) cholecystitis (daptomycin MIC values, 1 and 2 mg/liter, respectively). After the fifth drug dose, the bile concentration of daptomycin was 66 mg/liter 5 min after drug administration, with the biliary concentration/MIC values higher than 30 for both bacterial strains. Therefore, daptomycin achieved therapeutic levels in bile, hence suggesting a role for the drug in the treatment of MRSA/VRE cholecystitis.

Pharmacogenetics of BCR/ABL Inhibitors in Chronic Myeloid Leukemia

Chronic myeloid leukemia was the first haematological neoplasia that benefited from a targeted therapy with imatinib nearly 15 years ago. Since then, several studies have investigated the role of genes, their variants (i.e., polymorphisms) and their encoded proteins in the pharmacokinetics and pharmacodynamics of BCR-ABL1 tyrosine kinase activity inhibitors (TKIs). Transmembrane transporters seem to influence in a significant manner the disposition of TKIs, especially that of imatinib at both cellular and systemic levels. In particular, members of the ATP-binding cassette (ABC) family (namely ABCB1 and ABCG2) together with solute carrier (SLC) transporters (i.e., SLC22A1) are responsible for the differences in drug pharmacokinetics. In the case of the newer TKIs, such as nilotinib and dasatinib, the substrate affinity of these drugs for transporters is variable but lower than that measured for imatinib. In this scenario, the investigation of genetic variants as possible predictive markers has led to some discordant results. With the partial exception of imatinib, these discrepancies seem to limit the application of discovered biomarkers in the clinical settings. In order to overcome these issues, larger prospective confirmative trials are needed.

Polycomb genes are associated with response to imatinib in chronic myeloid leukemia.

AIM Imatinib is a tyrosine kinase inhibitor that has revolutionized the treatment of chronic myeloid leukemia (CML). Despite its efficacy, about a third of patients discontinue the treatment due to therapy failure or intolerance. The rational identification of patients less likely to respond to imatinib would be of paramount clinical relevance. We have shown that transmembrane transporter hOCT1 genotyping predicts imatinib activity. In parallel, Polycomb group genes (PcGs) are epigenetic repressors implicated in CML progression and in therapy resistance. PATIENTS & METHODS We measured the expression of eight PcGs in paired pre- and post-imatinib bone marrow samples from 30 CML patients. RESULTS BMI1, PHC3, CBX6 and CBX7 expression was significantly increased during imatinib treatment. Post-treatment levels of CBX6 and CBX7 predicted 3-month response rate. Measurement of post-treatment BMI1 levels improved the predictive power of hOCT1 genotyping. CONCLUSION These results suggest that the expression levels of PcGs might be useful for a more accurate risk stratification of CML patients.

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

Abstract 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 1st and 5th 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.

Association of the hOCT1/ABCB1 genotype with efficacy and tolerability of imatinib in patients affected by chronic myeloid leukemia

PurposeThe present study was aimed at investigating whether imatinib pharmacogenetics is related to its pharmacodynamics in patients affected by chronic myeloid leukemia.MethodsThrough a procedure based on a sequence of classical statistics methods, we investigated the possible relationships between treatment efficacy/tolerability and combinations of time-independent variables as gender and genetic covariates in the form of single nucleotide polymorphisms (SNPs) or combinations thereof. Moreover, since the drug tolerability has a strong incidence on the discontinuation of the therapy, we investigated whether the time of manifestation of the most frequent toxic effects can be related to time-independent patients’ characteristics or not.ResultsWe found that a combination of two polymorphisms, namely hOCT1 c.480C>G (rs683369) and ABCB1 c.3435C>T (rs1045642), seems to play the role of predictor for imatinib in both efficacy and toxicity. Furthermore, the time of manifestation of edema toxicity is found to be associated to a combination of gender and ABCB1 c.3435C>T, whereas the time of manifestation of cramp toxicity appears related to gender.ConclusionsThe novelty of this study is dual: the achievement of results that potentially have a significant clinical interest and the demonstration that the adoption of composed covariates may represent a unique tool to study different aspects of the treatment with imatinib.

Reduced circulating B-lymphocytes and altered B-cell compartments in patients suffering from chronic myeloid leukaemia undergoing therapy with Imatinib

*p versus normals and versus CML-1= 0.001. Data are expressed as means ± SD. To the Editor Imatinib mesylate, an inhibitor of several tyrosine kinases, such as Abelson gene (ABL), Breakpoint Cluster RegionAbelson gene (BCR-ABL1), c-KIT and platelet-derived growth factor receptors [1] is used to treat chronic myeloid leukaemia (CML) in chronic phase and is able to interfere with some immunologic pathways. Imatinib modifies some biological aspects of B-lymphocyte, being able to induce hypogammaglobulinemia and to alter the phenotype of bone marrow plasma cells [2,3]. A recent report by De Lavallade et al. [4] has shed new light on the mechanisms by which Imatinib and other tyrosine kinase inhibitors can affect B-lymphocyte immune response. We report our experience on 34 patients with CML undergoing Imatinib therapy. We evaluated the circulating B-lymphocyte compartments by means of multiparameter flow cytometry. Samples from 34 CML patients (23 men, 11 women, age 37–83 years), undergoing Imatinib treatment (Glivec, Novartis Pharmaceuticals Corporation, East Hanover, NJ, USA), 400mg/daily) as the first line therapy, were studied at the time of periodical (every 3months) monitoring of response to therapy. At the time of the study, the patients were not undergoing therapy with other drugs. None of the patients had a clinical history of recurrent infections. Twenty-three normal subjects (age 19–65 years) were evaluated as control group. The following blood parameters were registered: white blood cells (WBC), total lymphocyte percentage and absolute number, γ-globulin levels. The disease was monitored by conventional chromosome analysis (quinacrine and Giemsa banding), FISH analysis (LSI BCR-ABLdual-colour dual-fusion translocation probe), polymerase chain reaction (PCR) for the BCR/ABL1 translocation using the standardized PCR protocol of the European BIOMED-1 project and the standard operating procedures of LabNet guidelines [5]. Responders were defined as patients who obtained at least a three-log reduction in the BCR-ABL1messenger RNA level (MMR> 3). Major response was defined as MMR> 4 and complete molecular response as MMR> 5. Immunophenotyping was carried out by a FacsCanto II cytometer equipped with two lasers (488 and 633 nm), using a five-colour method with the following monoclonal antibodies: CD20/PerCP-Cy5.5, CD19/PE-Cy.7, CD27/APC and CD45/APC-Cy7.7 (Becton Dickinson, Franklin Lakes, NJ, USA). The antibody panel was completed by a F(ab′)2