Mario Del Tacca

Correlation of CDA, ERCC1, and XPD polymorphisms with response and survival in gemcitabine/cisplatin-treated advanced non-small cell lung cancer patients.

Purpose: Selecting patients according to key genetic characteristics may help to tailor chemotherapy and optimize the treatment in non–small cell lung cancer (NSCLC). Polymorphisms at the xeroderma pigmentosum group D (XPD), excision repair cross-complementing 1 (ERCC1), and cytidine deaminase (CDA) genes have been associated with alterations in enzymatic activity and may change sensitivity to the widely used cisplatin-gemcitabine regimen. Experimental Design: Analyses of CDA, XPD, and ERCC1 polymorphisms were done on blood samples of 65 chemotherapy-naïve, advanced NSCLC patients treated with cisplatin-gemcitabine. Furthermore, CDA enzymatic activity was evaluated by high-performance liquid chromatography analysis. Association between XPD Asp312Asn and Lys751Gln, ERCC1 C118T, and CDA Lys27Gln polymorphisms and response, clinical benefit, toxicity, time to progression (TTP), and overall survival (OS) was estimated using Pearsons χ2 tests, the Kaplan-Meier method, the log-rank test, and the Cox proportional hazards model. Results: The CDA Lys27Lys polymorphism significantly correlated with better clinical benefit (P = 0.04) and grade ≥3 neutropenia and thrombocytopenia, as well as with longer TTP and OS (P = 0.006 and P = 0.002, respectively), whereas no significant associations were found among ERCC1 and XPD polymorphisms and both response and clinical outcome. Finally, the enzymatic activity assay showed a significant lower mean in subjects harboring the CDA Lys27Lys polymorphism. Conclusions: Our data suggested the role of CDA Lys27Lys polymorphism as a possible predictive marker of activity, toxicity, TTP, and OS in advanced NSCLC patients treated with cisplatin and gemcitabine. These results may be explained by the lower enzymatic activity associated with the Lys27Lys CDA and offer a potential new tool for treatment optimization.

Synergistic cytotoxicity and pharmacogenetics of gemcitabine and pemetrexed combination in pancreatic cancer cell lines

Purpose: Gemcitabine is an inhibitor of ribonucleotide reductase (RR) and DNA synthesis and is an effective agent in the treatment of pancreas cancer. The present study investigates whether the multitargeted antifolate pemetrexed would be synergistic with gemcitabine against MIA PaCa-2, PANC-1, and Capan-1 pancreatic cancer cell lines. Experimental Design: Cells were treated with gemcitabine and pemetrexed, and the type of drug interaction was assessed using the combination index. Cytotoxicity of gemcitabine was examined with inhibitors of (a) deoxycytidine kinase (dCK), which activates gemcitabine by phosphorylation, and (b) 5′-nucleotidase (drug dephosphorylation) and cytidine deaminase (drug deamination), the main inactivating enzymes. The effects of gemcitabine and pemetrexed on cell cycle were analyzed by flow cytometry, and apoptosis was examined by fluorescence microscopy. Finally, quantitative, real-time PCR was used to study the pharmacogenetics of the drug combination. Results: Synergistic cytotoxicity and enhancement of apoptosis was demonstrated, mostly with the sequence pemetrexed→gemcitabine. Pemetrexed increased cells in S phase, the most sensitive to gemcitabine, and a positive correlation was found between the expression ratio of dCK:RR and gemcitabine sensitivity. Indeed, pemetrexed significantly enhanced dCK gene expression (+227.9, +86.0, and +135.5% in MIA PaCa-2, PANC-1, and Capan-1 cells, respectively), and the crucial role of this enzyme was confirmed by impairment of gemcitabine cytotoxicity after dCK saturation with 2′-deoxycytidine. Conclusions: These data demonstrate that the gemcitabine and pemetrexed combination displays schedule-dependent synergistic cytotoxic activity, favorably modulates cell cycle, induces apoptosis, and enhances dCK expression in pancreatic cancer cells.

Pharmacogenetics of anticancer drug sensitivity in pancreatic cancer

Chemotherapy has produced unsatisfactory results in pancreas cancer and novel approaches, including treatment tailoring by pharmacogenetic analysis and new molecular-targeted drugs, are required. The scarcity of effective therapies may reflect the lack of knowledge about the influence of tumor-related molecular abnormalities on responsiveness to drugs. Advances in the understanding of pancreas cancer biology have been made over the past decade, including the discovery of critical mutations in oncogenes (i.e., K-Ras) as well as the loss of tumor suppressor genes, such as TP53 and p16INK4. Other studies showed the dysregulation of the expression of proteins involved in the control of cell cycle, proliferation, apoptosis, and invasiveness, such as Bcl-2, Akt, mdm2, and epidermal growth factor receptor. These characteristics might contribute to the aggressive behavior of pancreatic cancer and influence response to treatment. Indeed, the inactivation of p53 may explain the relative resistance to 5-fluorouracil, whereas Bcl-2 overexpression is associated with reduced sensitivity to gemcitabine. However, the future challenge of pancreas cancer chemotherapy relies on the identification of molecular markers that help in the selection of drugs best suited to the individual patient. Recent pharmacogenetic studies focused on genes encoding proteins directly involved in drug activity, showing the role of thymidylate synthase and human equilibrative nucleoside transporter-1 as prognostic factor in 5-fluorouracil- and gemcitabine-treated patients, respectively. Finally, inhibitors of signal transduction and angiogenesis are under extensive investigation, and several prospective trials have been devoted to this area. Pharmacogenetics is likely to play a central role in the personalization of treatment, to stratify patients based on their likelihood of response to both standard agents (i.e., gemcitabine/nucleoside transporters) and targeted treatments (i.e., epidermal growth factor receptor gene mutations and/or amplification and tyrosine kinase inhibitors), Thus, molecular analysis should be implemented in the optimal management of the patient affected by pancreatic adenocarcinoma. [Mol Cancer Ther 2006;5(6):1387–95] [Mol Cancer Ther 2006;5(6):1387-95]

Clinical and Pharmacodynamic Evaluation of Metronomic Cyclophosphamide, Celecoxib, and Dexamethasone in Advanced Hormone-refractory Prostate Cancer

Purpose: The aims of the present study were to evaluate the clinical activity and the pharmacodynamic profile of the novel schedule of a single i.v. standard dose of cyclophosphamide (CTX) immediately followed by an oral metronomic CTX regimen with celecoxib (CXB) and dexamethasone (DEX) in advanced hormone-refractory prostate cancer patients. Experimental Design: Twenty-eight patients (68% docetaxel-resistant) received 500 mg/m2 CTX i.v. bolus on day 1 and, from day 2, 50 mg/day CTX p.o. plus 200 mg/twice a day CXB p.o. and 1 mg/day DEX p.o. until disease progression. Plasma vascular endothelial growth factor (VEGF) and thrombospondin-1 were detected by ELISA, and real-time reverse transcription-PCR of VEGF and thrombospondin-1 gene expression on peripheral blood mononuclear cell and of VE-cadherin (VE-C) in blood samples was done. Results: A confirmed prostate-specific antigen decrease of ≥50% from baseline was observed in 9 of 28 patients (32%). Median progression-free survival and overall survival were 3 months (95% confidence interval, 2.2-4.2 months) and 21 months (95% confidence interval, 12.4-29.4 months), respectively. Toxicity was mild and no grade 3 to 4 toxicities occurred. A significant relationship was found between plasma VEGF and prostate-specific antigen values (r = 0.4223; P < 0.001). VEGF levels significantly increased in nonresponders, whereas the responder patients maintained significantly lower levels of VE-C gene expression after the beginning of the treatment if compared with nonresponder ones. Conclusion: Metronomic CTX plus CXB and DEX showed favorable toxicity and activity profile in patients. VE-C gene expression and VEGF levels represent potentially useful pharmacodynamic markers for the clinical response.

Pharmacological issues of linezolid: an updated critical review.

Linezolid is the first oxazolidinone agent introduced into clinical practice for use against Gram-positive bacteria that are resistant to β-lactams and glycopeptides, including methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). An optimal antibacterial effect is achieved when plasma drug concentrations are above the minimum inhibitory concentration (MIC) [T>MIC] for the entire length of treatment and the ratio between the area under the plasma concentrationtime curve (AUC) and the MIC (AUC/MIC) is greater than 100, as is most commonly obtained with administrationof the standard dosage of linezolid 600 mg twice daily. A wide tissue distribution, including the CNS and respiratory tract, nearly linear pharmacokinetics and good tolerability are additional characteristics of linezolid. However, variability in the drug pharmacokinetics associated with clinical conditions (e.g. sepsis, burn injuries, end-stage renal disease, cystic fibrosis), haemodialysis and/or young age may lower the T>MIC and the AUC/MIC ratio, thus impairing both antibacterial activity and prevention of mutants. In most cases, changes in the dosage or in the schedule of administration (e.g. an additional [third] daily dose) may improve the effectiveness of linezolid. It is worth noting that linezolid could affect its own metabolism as a result of protein synthesis inhibition in mitochondria, and this could lead to high plasma concentrations and an increased risk of non-negligible toxicities. The latter may be reported during long-term administration of linezolid or in the presence of some pathological conditions (e.g. renal disease or kidney transplantation) associated with high plasma drug concentrations. Therefore, treatment optimization should be considered a requirement for more effective and tolerable use of the drug, particularly in special populations.

Lack of pharmacokinetic bioequivalence between generic and branded amoxicillin formulations. A post‐marketing clinical study on healthy volunteers

AIMSnThere are concerns about the quality of generic drugs in the postmarketing setting. The aim was to establish whether two generic formulations of amoxicillin, available on the Italian market, fulfil the criteria for clinical pharmacokinetic bioequivalence vs. the branded drug.nnnMETHODSnTwo generic amoxicillin products (generic A and B) were selected among four fast-release tablet formulations available on the Italian market. Twenty-four healthy adult volunteers of either sex participated to a single-dose, randomized, three-treatment, crossover, single-blind bioequivalence study designed to compare generic A and B with branded amoxicillin. Plasma samples were collected at preset times for 24 h after dosing, and assayed for amoxicillin levels by high-performance liquid chromatography.nnnRESULTSnNinety percent confidence intervals of AUC ratios were 0.8238, 1.0502 (ratio 0.9302) and 0.8116, 1.1007 (ratio 0.9452) for generic A and B vs. branded amoxicillin, respectively. Ninety percent confidence intervals of C(max) ratios were 0.7921, 1.0134 (ratio 0.8960) and 0.8246, 1.1199 (ratio 0.9610) for generic A and B vs. branded amoxicillin, respectively. The mean pharmacokinetic profiles showed that the AUC value of branded amoxicillin was 8.5 and 5.4% greater than that estimated for generic A and B, respectively. Few adverse events were recorded; these were not serious and occurred without apparent relationship to any specific amoxicillin formulation.nnnCONCLUSIONSnThese results indicate that one of the two marketed amoxicillin generics analysed in the present study is not bioequivalent to the brand leader product for C(max) on the basis of single-dose pharmacokinetic assessment.

Vascular endothelial growth factor pharmacogenetics: a new perspective for anti-angiogenic therapy

The pharmacogenetic approach to anti-angiogenic therapy should be considered a possible strategy for many pathological conditions with high incidence in Western countries, including solid tumors, age-related macular degeneration or endometriosis. While pharmacogenetic studies are building stronger foundations for the systematic investigations of phenotype-genotype relationships in many research and clinical fields of medicine, pharmacogenetic data regarding anti-angiogenic drugs are still lacking. Here we review preclinical and clinical genetic studies on angiogenic determinants such as vascular endothelial growth factor and vascular endothelial growth factor receptor-2. We suggest that pharmacogenetic profiling of patients who are candidates for the currently available anti-angiogenic agents targeting vascular endothelial growth factor and vascular endothelial growth factor receptor-2 may aid the selection of patients on the basis of their likelihood of responding to the drugs or suffering from toxicity.

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.

Epigenetic mechanisms of irinotecan sensitivity in colorectal cancer cell lines

Irinotecan is a topoisomerase-I (Top-I) inhibitor used for the treatment of colorectal cancer. DNA demethylating agents, including 5-azacytidine (5-aza), display synergistic antitumor activity with several chemotherapy drugs. 5-Aza may enhance irinotecan cytotoxicity by at least one of the following mechanisms: (a) Top-I promoter demethylation, (b) activation of genes involved in Top-I transcriptional regulation (p16 or Sp1), and (c) modulation of the cell cycle and apoptosis after DNA damage. The growth-inhibitory effects of SN38, the active metabolite of irinotecan, 5-aza, and their combinations, were studied in four colorectal cancer cell lines. The effects of treatments on cell cycle were analyzed by flow cytometry, and apoptosis was measured by fluorescence microscopy. Top-I, Sp1, and p53 expression modulated by 5-aza were measured by real-time PCR. Methylation of Top-I, p16, 14-3-3σ, and hMLH1 promoters before and after 5-aza treatment were measured by MethyLight PCR and DNA bisulfite sequencing. Low-dose 5-aza significantly enhanced the apoptotic effect of irinotecan in all colorectal cancer cells, whereas a synergistic cytotoxic effect was observed only in p53-mutated cells (HT29, SW620, and WiDr). This synergistic effect was significantly correlated with Top-I up-regulation by 5-aza, and coupled to p16 demethylation and Sp1 up-regulation. p16 demethylation was also associated with enhanced cell cycle arrest after irinotecan treatment. In contrast, 5-aza down-regulated Top-I expression in the p53 wild-type LS174T cells in a p53-dependent manner, thereby reducing SN38 cytotoxicity. In conclusion, 5-aza modulates Top-I expression by several mechanisms involving Sp1, p16, and p53. If confirmed in other models, these results suggest that p16 and p53 status affects the 5-aza–irinotecan interaction. [Mol Cancer Ther 2009;8(7):1964–73]

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

AIMSnABCB1 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.nnnMATERIALS & METHODSnc.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.nnnRESULTSnA 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.nnnCONCLUSIONnc.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.