A simplified method to calculate telomere length from Southern blot images of terminal restriction fragment lengths.

Southern blotting of DNA terminal restriction fragment lengths is the gold standard for measuring mean telomere length. Analysis of the final image is a crucial step in this process, however, current techniques are cumbersome and prone to error. Here we present a simple and accurate method for analyzing telomere smears. Basic 2D gel imaging software was used to automatically subtract background, generate standard curves and calculate net intensity and MW at each position (i) along the telomere smear. Our method required no statistical software or major data manipulation and correctly classified >80% of 18 samples as having short, medium or long telomeres compared with 33-72% using other methods.

First-in-human phase I study of infusional and bolus schedules of Deflexifol, a novel 5-fluorouracil and leucovorin formulation, after failure of standard treatment.

BACKGROUND: 5-Fluorouracil (5-FU) is administered with leucovorin (LV) to enhance clinical activity. However, simultaneous administration is not feasible due to their chemical incompatibility, so conditions for the maximum possible beneficial interaction cannot be met. To overcome this, we developed a novel all-in-one, pH neutral stable solution of 5-FU plus LV with ß-cyclodextrin (termed Deflexifol) and assessed its safety and tolerability in a first-in-human phase I trial. METHODS: Patients with advanced solid malignancy received Deflexifol as weekly bolus (375-575 mg/m²) or two-weekly 46 h infusion (1200-3600 mg/m²) for six cycles in a 3+3 dose escalation design. Adverse events, pharmacokinetics and tumor response rates were assessed by standard methods. RESULTS: Forty patients were treated (19 bolus, 21 infusional, median age 67) with no grade 4 adverse events reported. Dose-limiting toxicities of grade 3 diarrhea and myelosuppression were reported for the bolus schedule at 575 mg/m2 (maximum tolerated dose 525 mg/m²), whereas none were reported for the infusional schedule. The recommended phase II infusional dose was declared as 3,000 mg/m², >25% that of 5-FU used in standard-of-care regimens. Pharmacokinetic analyses showed evidence of inter-patient variability, with no evidence of saturation in clearance, and a trend to linear increase in AUC with dose. Disease control rate was 64% despite most patients having failed previous 5-FU regimens. CONCLUSION: Deflexifol is safer and effective in bolus and infusion schedules at higher doses than that permitted by separate infusion of 5-FU and LV. A phase II study evaluating Deflexifol is planned.

A simple HPLC method for plasma level monitoring of mitotane and its two main metabolites in adrenocortical cancer patients.

Mitotane (o,p'-DDD or (1,1-dichloro-2-[o-chlorophenyl]-2-[p-chlorophenyl]ethane, DDD) is the drug of choice for non-resectable and metastatic adrenocortical carcinomas (ACC). Measurement of mitotane and metabolites, o,p'-DDE (1,1-dichloro-2-[p-chlorophenyl]-2-[o-chlorophenyl]ethene, DDE) and o,p'-DDA (1,1-[o,p'-dichlorodiphenyl] acetic acid, DDA) provides a better understanding of mitotane pharmacokinetics and pharmacodynamics. We have developed a simple, robust and efficient high performance liquid chromatography (HPLC) method to measure mitotane and its two main metabolites, DDE and DDA. The method involves a single ethanol extraction of mitotane, DDE, DDA, and an internal standard (int std) p,p'-DDD (1,1-dichloro-2,2-bis(p-chlorophenyl)ethane) with an extraction efficiency of 77-88%. All compounds are measured simultaneously using a reversed-phase phenyl HPLC column with an isocratic elution of mobile phase at a flow rate of 0.6 ml/min followed by UV detection at λ 226 nm. Inter and intra-day validation demonstrates good reproducibility and accuracy. Limits of quantitation are 0.2 µg/ml for mitotane and DDE, and 0.5 µg/ml for DDA. The method has been evaluated in plasma from 23 patients on mitotane therapy, revealing DDA concentrations 1-18 times higher than the parent compound.

Pyridoxine to protect from oxaliplatin-induced neurotoxicity without compromising antitumour effect.

PURPOSE: Oxaliplatin (OHP) in combination with 5-fluorouracil/leucovorin (FOLFOX) is clinically used as frontline therapy in patients with advanced colorectal carcinoma (CRC), with response rates ranging from 46 to 71%. This combination is now considered a standard treatment for metastatic CRC and also in the post-operative adjuvant setting. Reversible, cumulative, peripheral sensory neuropathy is the principal dose-limiting toxicity of OHP therapy. Pyridoxine (vitamin B6) has been shown to reduce cisplatin and fluoropyrimidine-related neurotoxicity but its administration with OHP has not yet been studied. Low doses of pyridoxine are free of side effects; it can be given orally. If pyridoxine administration with oxaliplatin has no adverse effect on OHP cytotoxicity effects, it will be a simple and cost-effective way to minimise OHP-induced neurotoxicity. METHODS: In vitro simultaneous combination of OHP and pyridoxine was studied in 6 CRC cell lines (HT29, Widr, SW480, HCT116, H630 and SW1116), in an ovarian cancer cell line (A2780) and its cisplatin-resistant subline (ADDP) and in an oestrogen-dependent breast cancer cell line (MCF-7). Three fixed concentrations of pyridoxine: 1, 10 and 25 µM were combined with varying concentrations of OHP, and the growth inhibitory effects were evaluated using the MTT cell growth assay. RESULTS: Oxaliplatin induced consistent cytotoxicity in all cell lines with GI(50) values between 0.23 and 7.6 µM. Addition of pyridoxine at concentrations of 1-25 µM does not affect OHP cytotoxicity. CONCLUSIONS: Administration of pyridoxine, at concentrations extending across possible therapeutic plasma levels in humans, does not antagonise OHP antitumour effects in a range of relevant tumour cell lines. This study provides a foundation for clinical studies to test whether pyridoxine can minimise OHP-related neurotoxicity, and clinicians can be confident that pyridoxine is very unlikely to reverse the antitumour effects of OHP, as seems to be the case with Ca/Mg infusions. This could prove to be a cost-effective way to minimise OHP-related neurotoxicity, allowing more effective less toxic treatment and better outcomes in patients.

Identification of a novel single nucleotide polymorphism in the first tandem repeat sequence of the thymidylate synthase 2R allele.

Thymidylate synthase (TS) activity is an important determinant of response to chemotherapy with fluoropyrimidine prodrugs and its expression is largely determined by the number of functional upstream stimulatory factor (USF) E-box consensus elements present in the 5'regulatory region of the TYMS gene. Two known polymorphisms in this area, a variable number of tandem repeat (VNTR) consisting of 2 or 3 repeats (2R/3R) of a 28-bp sequence and a further G > C single nucleotide substitution within the second repeat of the 3R, result in genotypes with between 2 and 4 functional repeats in most humans. Here, we identify a further G > C SNP in the first repeat of the TYMS 2R allele, which effectively abolishes the only functional USF protein binding site in this promoter. The frequency of the new allele was found to be 4.2% (95% CI = 1.4-9.6%), accounting for 8.8% (95% CI = 2.9-19.3%) of all 2R alleles in our patient cohort. Thus, we observed that the lowest number of inherited functional binding sites is 1 instead of 2 as previously thought, and could potentially be 0 in a homozygous individual. This would severely decrease TS expression and may have implications for predicting efficacy and toxicity of therapy with commonly used fluorouracil-based therapy regimes.

Predictors of vinorelbine pharmacokinetics and pharmacodynamics in patients with cancer.

PURPOSE: Marked interindividual variation in drug disposition and toxicity pose an ongoing challenge to chemotherapy dosage individualization. The aim of this study was to evaluate pretreatment clinical features, genotype and functional indicators of drug clearance as predictors of vinorelbine clearance, and myelotoxicity that could inform dosage optimization. PATIENTS AND METHODS: Forty-one patients with cancer received a 60 mg intravenous dose of vinorelbine. Pretreatment routine body size measurements and blood tests were performed. Midazolam clearance and hepatic technetium labeled sestamibi (99mTc-MIBI) clearance were used to investigate CYP3A and ABCB1 (MDR1, P-glycoprotein) phenotype respectively and selected single nucleotide polymorphisms in CYP3A and ABCB1 were documented. A limited blood sampling strategy was employed and vinorelbine concentrations were determined by high-performance liquid chromatography. Posterior Bayesian estimates of vinorelbine clearance were obtained for each patient using population pharmacokinetic modeling. Myelotoxicity was estimated from the fractional survival of neutrophils post-treatment. RESULTS: There was 4.3-fold variation in vinorelbine clearance across the cohort. In a multivariable analysis, pretreatment estimated creatinine clearance (P < .01) and hepatic (99m)Tc-MIBI clearance (P = .01) were independent predictors of vinorelbine clearance. Fractional survival of neutrophils ranged from 1.3% to 100% and was significantly correlated with vinorelbine clearance (P < .01). Body-surface area was the only pretreatment predictor of fractional survival of neutrophils independent of vinorelbine clearance (P = .02). CONCLUSION: Specific indicators of drug clearance provide predictive information about vinorelbine pharmacokinetics, and body-surface area, probably reflecting normal bone marrow reserve, provides an additional pharmacodynamic indicator. Use of a fixed dose of vinorelbine with modifications guided by pretreatment measures is worthy of prospective evaluation.

Telomere length in haemopoietic stem cells can be determined from that of mononuclear blood cells or whole blood.

Telomere length can be used to predict the replicative capacity of haematological progenitor cells and may be an important prognostic factor for the onset of cellular immune dysfunction. However, such measurements require invasive bone marrow (BM) biopsies and laborious stem cell isolations that are impractical in a clinical setting. Previous studies have used peripheral blood (PB) cells as an indicator of stem cell telomere length without demonstrating a correlation. In this study, we examined the telomere length in PB, isolated mononuclear cells (MNC) and BM aspirates from each of 19 patients ranging in age from 45 to 81 years. Correlation analysis confirmed that mean telomere length of BM aspirates was equivalent to that of PB (r = 0.85, P < 0.001), or MNC (r = 0.94, P < 0.001). Since BM is a heterogeneous population of cells, we have also shown in 13 separate patients that the mean telomere length in isolated peripheral blood stem cell (PBSC) harvests was equivalent to that of isolated CD34+ stem cells (r = 0.83, P < 0.001). Thus, telomere length in haemopoietic stem cells can be determined from that of whole or fractionated PB in future studies of haematological disorders.

Simple liquid chromatographic method for the determination of uracil and dihydrouracil plasma levels: a potential pretreatment predictor of 5-fluorouracil toxicity.

5-Fluorouracil (5-FU) is a commonly used anti-cancer drug with notable activity in clinical practice, yet it causes significant unpredictable and often serious toxicity. Both 5-FU and uracil (U) are catabolised by dihydropyrimidine dehydrogenase (DPD) to form dihydrofluorouracil (FUH(2)) and dihydrouracil (UH(2)), respectively. A means of predicting toxicity before treatment would be more valuable. Variations in dihydropyrimidine dehydrogenase (DPD) activity between patients are at least partly responsible for variable toxicity. Measurement of the UH(2) to U ratio may be a measure of pyrimidine catabolism and thus be utilised to predict subsequent toxicity. We have developed an efficient extraction and detection method using HPLC for the simultaneous measurement of UH(2) and U in plasma. A single C(18) Spherisorb ODS2 (25 cm) column using isocratic elution was utilised. U, UH(2) and the internal standard 4-chlorouracil were detected at wavelengths of 257, 220, and 268 nm, respectively. The chromatographic run time was 45 min which is half that of other methods. The detection limit was 0.02 microM for U and 0.1 microM for UH(2) using only 0.5 ml of plasma for both compounds. The basal plasma concentrations of U and UH(2) in 23 individuals ranged from 0.025 to 0.27 microM and 0.4-1.7 microM, respectively. This simple method may permit the assessment of pyrimidine catabolism, and therefore allow prediction of the toxicities associated with the use of fluorinated pyrimidines.