Effects of branched chain amino acids, l-citrulline, and alpha-glycerylphosphorylcholine supplementation on exercise performance in trained cyclists: a randomized crossover trial.

BACKGROUND: Nutrition plays a key role in training and athletic performance and dietary supplements can make a small, but potentially valuable, contribution to achieving peak athletic performance. This study is the first to investigate the effects of supplementation from the combination of BCAAs, L-citrulline, and A-GPC on exercise performance. METHODS: In this randomized, double-blind, crossover study 30 male trained cyclists (age: 43.7 ± 8.5 years) completed a 20 km cycling time trial (TT) test and a high intensity endurance cycling (HIEC) test following a 7-day supplementation period with either a supplement containing 8 g BCAAs, 6 g L-citrulline, and 300 mg A-GPC or a placebo (15 g maltodextrin). For each trial, mean values for time to completion, peak and average power output, OMNI rating of perceived exertion, and visual analogue scale (VAS) responses on perceived exertion were computed for the 20 km TT test. Mean values for time to fatigue and VAS responses on perceived exertion were computed for the HIEC test. Procedures for dietary intake and exercise patterns were implemented to achieve consistency throughout the study period. RESULTS: There was a significant increase (p = .003) in peak power in the 20 km TT (354.27 ± 87.88 and 321.67 ± 63.65, for supplement and placebo trials, respectively) and a significant increase (p = .001) in time to fatigue in the HIEC test (0:19:49 ± 0:11:13 min and 0:14:33 ± 0:09:59 min, for supplement and placebo trials, respectively) with the test supplement compared to the placebo. With the test supplement, there was an average increase in TT peak power of 11% and an average increase in time to fatigue of 36.2% in the HIEC test compared to the placebo. There was no significant improvement in time to completion, average power, OMNI rating of perceived exertion, or VAS responses on perceived exertion in the TT test and no significant improvement in VAS measures of perceived exertion in the HIEC test. CONCLUSIONS: The combination of BCAAs, L-citrulline, and A-GPC used in this study improves cycling performance and may be useful for individuals seeking to improve athletic performance, particularly in disciplines requiring lower body muscular strength and endurance.

Development and validation of a miniaturized bacteriophage host range screening assay against antibiotic resistant Pseudomonas aeruginosa.

Antimicrobial resistance is a current global health crisis, and the increasing emergence of multidrug resistant infections has led to the resurgent interest in bacteriophages as an alternative treatment. Prior to clinical application, phage suitability is assessed, via susceptibility testing and breadth of host range to bacteriophage, however, these are both large-scale manual processes and labor-intensive. The aim of the study was to establish and validate a scaled down methodology for high-throughput screening to reduce procedural footprint. In this paper, we describe a scaled-down adapted methodology that can successfully screen bacteriophages, isolated and purified from wastewater samples. Furthermore, we describe a miniaturized host range assay against clinical Pseudomonas aeruginosa isolates using a spot test (2 µL/ drop) that was found to be both sensitive (94.6%) and specific (94.7%). It also demonstrated a positive predictive value (PPV) of 86.4% and negative predictive value (NPV) of 98%. The breadth of host range of bacteriophages that exhibited lytic activity on P. aeruginosa isolates was corroborated using the scaled down assay. The high correlation achieved in this study confirms miniaturization as the first step in future automation that could test phage diversity and efficacy as antimicrobials.

K-Ras mutations and treatment outcome in colorectal cancer patients receiving exclusive fluoropyrimidine therapy.

PURPOSE: K-Ras mutations predict resistance to anti-epidermal growth factor receptor (EGFR) monoclonal antibodies. Because combinations of anti-EGFR with 5-fluorouracil (5-FU)-based chemotherapy are promising treatments, we analyzed the effect of K-Ras mutations in patients having received exclusive 5-FU therapy. EXPERIMENTAL DESIGN: This study was conducted on 93 stage IV colorectal cancer patients with unresectable measurable liver metastasis receiving 5-FU-leucovorin (56 men and 37 women; 77 cancer deaths). Liver metastases (n = 93) along with primary tumors (n = 48) were analyzed for K-Ras mutations (codons 12 and 13), p53 mutations (exons 4-9), p53 polymorphism (codon 72), thymidylate synthase (TS) polymorphism (28-bp repeats including G>C mutation), methylenetetrahydrofolate reductase polymorphism (677C>T, 1298A>C), thymidylate synthase (TS) activity, dihydropyrimidine dehydrogenase activity, folylpolyglutamate synthase activity, and p53 protein expression. RESULTS: Thirty-six of 93 (38.7%) metastases were K-Ras mutated (30 at codon 12 and 6 at codon 13). Mutated primary tumors (16 of 48) matched perfectly with mutated metastases. The additional analyzed tumor markers were not different between K-Ras mutated and wild-type tumors. The objective response rate was 37%: 44.4% in K-Ras mutated versus 32.1% in wild-type K-Ras metastasis (P = 0.27). Low TS activity in metastasis was the only significant predictor of tumor response (P = 0.047). K-Ras status did not influence specific survival. CONCLUSIONS: The present data indicate a perfect concordance of K-Ras mutations between primary and liver metastasis and suggest that any predictive and/or prognostic value of K-Ras mutations in treatments combining anti-EGFR monoclonal antibodies with 5-FU should be exclusively linked to the anti-EGFR agent.

Optimized blood sampling with cytidine deaminase inhibitor for improved analysis of capecitabine metabolites.

The 5FU prodrug capecitabine undergoes a 3-step enzymatic conversion, including the conversion of 5'DFRC into 5'DFUR by cytidine deaminase (CDA). The presence of CDA activity in blood led us to analyze the possible ex vivo conversion of 5'DFCR into 5'DFUR in blood samples. We thus examined the impact of the addition of a CDA inhibitor (tetrahydrouridine (THU) 1 microM final) in blood. Blood samples from 3 healthy volunteers were taken on tubes containing or not THU. Blood was spiked with 5'DFCR (20 microM final) (T0) and was maintained at room temperature for 2 h. Plasma concentrations of 5'DFRC and 5'DFUR were analyzed with an optimized HPLC assay. In the absence of THU, 5'DFUR was detectable as early as T0. The percent of 5'DFUR produced relative to 5'DFCR increased over time, up to 7.7 % at 2h. In contrast, the presence of THU totally prevents the formation of 5'DFUR. The impact of THU for preventing the conversion of 5'DFCR was confirmed by the analysis of blood samples from 2 capecitabine-treated patients. Addition of THU in the sampling-tube before the introduction of blood is thus strongly recommended in order to guarantee accurate conditions for reliable measurement of capecitabine metabolites in plasma, and thus faithful pharmacokinetic data.

A clinical pharmacokinetic analysis of tegafur-uracil (UFT) plus leucovorin given in a new twice-daily oral administration schedule.

BACKGROUND AND OBJECTIVE: Tegafur is an oral fluorouracil prodrug used in the treatment of colorectal cancer. The aim of this phase II, crossover, bioequivalence study was to compare the pharmacokinetics (primary objective) and tolerability (secondary objective) of tegafur-uracil (UFT) given as three daily doses (tid, reference schedule) with those obtained using a more convenient schedule of two daily doses (bid, new schedule). PATIENT AND METHODS: Twenty-one patients with metastatic colorectal cancer (median age 63 years) received the same oral daily dose of UFT (300 mg/m(2)/day) plus leucovorin (90 mg/day) divided into two or three daily doses. Patients were randomised to receive the first cycle either tid (12 patients) or bid (9 patients). The eligibility criteria included an Eastern Co-operative Oncology Group performance status of < or =1 and adequate bone-marrow, hepatic and renal function. The pharmacokinetics of uracil, fluorouracil and tegafur (high-performance liquid chromatography assays) were evaluated at steady state over 24 hours (area under the plasma concentration-time curve from 0 to 24 hours [AUC(24)], minimum plasma concentration [C(min)] and maximum plasma concentration [C(max)]). The pharmacokinetic parameters were analysed after logarithmic transformation according to a general linear model. RESULTS: The AUC(24)values of fluorouracil (p < 0.0001), uracil (p < 0.0001) and tegafur (p = 0.058) were greater with the bid schedule than the tid schedule. The bid : tid AUC(24) ratio (90% CI) was 1.8 (1.55, 2.10) with fluorouracil, 2.0 (1.59, 2.57) with uracil and 1.2 (1.02, 1.36) with tegafur, indicating that the bid and tid schedules were not bioequivalent. No major toxicity (grade 4) was reported, and grade 3 adverse events accounted for 9% of the total adverse events. Intra-patient comparison of the maximum toxicity grade did not demonstrate a significant difference between the bid and tid schedules (p = 0.18). CONCLUSION: A 2-fold increase in the fluorouracil and uracil AUC values was observed with UFT administered bid compared with tid, without a significant impact on tolerability, suggesting that the more convenient bid schedule may improve the UFT therapeutic index.

Pharmacogenetics of capecitabine in advanced breast cancer patients.

PURPOSE: Germinal gene polymorphisms can explain a part of the interpatient pharmacodynamic variability of anticancer drugs, particularly fluoropyrimidines. Genes for which polymorphisms may potentially influence pharmacodynamics of fluoropyrimidines, including capecitabine, are thymidylate synthase (TS), methylenetetrahydrofolate reductase (MTHFR), and dihydropyrimidine dehydrogenase (DPD). EXPERIMENTAL DESIGN: The aim of this prospective pilot study was to analyze the effect of TS, MTHFR, and DPD gene polymorphisms on toxicity and efficacy in advanced breast cancer patients receiving capecitabine as monotherapy. Germinal polymorphisms of TS (6 bp deletion in the 3' region and 28 bp repeats, including G>C mutation in the 5' region), MTHFR (677C>T and 1298A>C), and DPD (IVS14+1G>A) were determined in 105 consecutive patients. RESULTS: A trend toward a higher global toxicity grade 3 and 4 was observed in patients homozygous for the TS 3RG allele compared with patients heterozygous for the 3RG allele or patients not carrying the 3RG allele (50% versus 19% versus 13% respectively, P=0.064). The sole patient bearing the DPD IVS14+1G>A mutation (heterozygous) deceased from hematologic toxicity. The median response duration was 5.8 months (95% confidence interval, 4.3-7.2). Duration of response was significantly shortened in patients homozygous for the 3RG allele compared with others (P=0.037). CONCLUSIONS: The present data suggest that 3RG3RG breast cancer patients are not good candidates for capecitabine therapy. In addition, attention should be paid to DPD deficiency in breast cancer patients receiving capecitabine. These preliminary data require further confirmation on a larger number of patients.

Prognostic value of tumoral thymidylate synthase and p53 in metastatic colorectal cancer patients receiving fluorouracil-based chemotherapy: phenotypic and genotypic analyses.

PURPOSE: The aim of this multicenter prospective study was to evaluate the role of intratumoral parameters related to fluorouracil (FU) sensitivity in 103 metastatic colorectal cancer patients receiving FU-folinic acid. PATIENTS AND METHODS: Liver metastatic biopsy specimens were obtained for all patients and primary tumor biopsy specimens for 54 patients. Thymidylate synthase (TS), folylpolyglutamate synthetase, and dihydropyrimidine dehydrogenase were measured by radioenzymatic assays; TS promoter polymorphism (2R/2R v 2R/3R v 3R/3R) was determined by polymerase chain reaction; and p53 protein and mutations were analyzed by immunoluminometric assay and denaturing gradient gel electrophoresis, respectively. RESULTS: p53 mutations were observed in 56.7% of metastases. TS activity was significantly higher in 2R/3R tumors as compared with 2R/2R or 3R/3R. TS activity in metastasis was the only parameter linked to clinical responsiveness (responders exhibited the lower TS, P =.047). Univariate Cox analyses demonstrated that TS activity in primary tumor (the greater the TS, the poorer the survival; P =.040), TS promoter polymorphism in primary tumor (risk of death of 2R/3R v 2R/2R, 2.68; P =.035), and p53 stop mutation in metastasis (risk of death of stop mutations v wild type, 3.14; P =.018) were the only significant biologic predictors of specific survival. Stepwise analysis did not discriminate between TS activity and TS polymorphism. CONCLUSION: Present results confirm the value of tumoral TS activity for predicting FU responsiveness, point out the importance of detailed p53 mutation analysis for predicting survival, and suggest that TS genotype in primary tumor carries a prognostic value similar to that of TS activity.

Single ascending dose tolerability, pharmacokinetic-pharmacodynamic study of dihydropyrimidine dehydrogenase inhibitor Ro 09-4889.

PURPOSE: Ro 09-4889 was designed to enhance the anticancer efficacy of capecitabine (Xeloda) by generating a dihydropyrimidine dehydrogenase inhibitor (DPDi) 5-vinyluracil (5-VU) preferentially in tumor tissues. This study assessed the tolerance to Ro 09-4889 treatment, and related pharmacokinetic and pharmacodynamic data such as inhibition of DPD activity in peripheral blood mononuclear cells (PBMCs) and plasma uracil levels. EXPERIMENTAL DESIGN: This was a single-center, double-blind, placebo-controlled, single-dose escalation study in 64 healthy male volunteers at 1-, 5-, 20-, 50-, 75-, 100-, and 200-mg oral dose of Ro 09-4889. Also, food effect was assessed separately in a group dosed with 20 mg of the compound. RESULTS: No serious adverse effects or significant laboratory and electrocardiogram abnormalities were observed during the study. Ro 09-4889 has a short elimination half-life (t(1/2)) of 0.5 h, followed by metabolites 5'-deoxy-5-vinyluridine (5'-DVUR), 5'-deoxy-5-vinylcytidine (5'-DVCR), and 5-VU with t(1/2) of 1.3, 1.2, and 2 h, respectively. The major metabolite excreted in urine was 5-DVCR (45% of dose). The inhibition of PBMC DPD activity and the increase in plasma uracil were related to Ro 09-4889 dose. DPD inhibition versus dose and uracil AUC (area under the curve) versus dose were modeled using the E(max) model with a baseline effect. The model-predicted ED(50) value was 100 mg. CONCLUSION: Single oral doses of Ro 09-4889 ranging from 1 to 200 mg were well tolerated. On the basis of these findings, a 10-to-30-mg dose range of Ro 09-4889 combined with capecitabine could be appropriate for further evaluation in cancer patients.

ZD1839 (Iressa) modifies the activity of key enzymes linked to fluoropyrimidine activity: rational basis for a new combination therapy with capecitabine.

PURPOSE: The efficacy of new oral fluoropyrimidines, including capecitabine, is improved in cells expressing high levels of thymidine phosphorylase (TP) and low levels of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase. We used a human head and neck cancer cell line (CAL33) to examine the influence of cell cycle modifications on TS, TP, and dihydropyrimidine dehydrogenase activity. EXPERIMENTAL DESIGN: Cells were exposed to the epidermal growth factor receptor tyrosine kinase inhibitor ZD1839 (Iressa(2)) and 5'-deoxy-5-fluorouridine (5'-DFUR), alone and in combination, for up to 96 h, and modifications in cell cycle, enzyme activity, and gene expression were examined. RESULTS: ZD1839 (24- to 72-h exposure) markedly reduced proliferation and caused a rapid increase in G(0)-G(1) and a decrease in S phase; a 40-fold decrease in TS activity at 24 h and a 2.5-fold increase in TP activity at 48 h were observed. A significant link between TP activity and expression was observed (r(2) = 0.98; P = 0.0068). Additional investigations pointed out an increased cellular production of 5-fluorouracil anabolites from 5'-DFUR when cells were preincubated with ZD1839. Dose-effect curves of ZD1839 and 5'-DFUR, alone and in combination, were examined. Combination indices for ZD1839 + 5'-DFUR were 0.58 +/- 0.1 and 0.63 +/- 0.1 for 50% survival and 25% survival, respectively. Additional investigations pointed out an increased cellular production of 5-fluorouracil anabolites from 5'-DFUR when cells were preincubated with ZD1839. CONCLUSIONS: These data demonstrate a strong synergistic interaction between ZD1839 and 5'-DFUR when ZD1839 is applied before or concurrently with 5'-DFUR. Such a drug combination would have two advantages: (a) the theoretical advantage of tumor selectivity of epidermal growth factor receptor-targeted therapy; and (b) the practical advantage of a combination therapy that could be administered p.o.

Positive interaction between lapatinib and capecitabine in human breast cancer models: study of molecular determinants.

The combination of lapatinib and capecitabine is approved in Her2+ metastatic breast cancer. However, the pharmacological mechanisms for this association have not been fully elucidated. In this non-clinical study, we evaluated the efficacy of this association on a panel of six human breast cancer cell lines as a means to identify the molecular determinants of response to this combination. Cell viability was evaluated after concomitant/sequential exposure, and response/resistance determinants for each drug such as dihydropyrimidine dehydrogenase (DPD), thymidylate synthase (TS), thymidine phosphorylase, Bax, Bcl2, P21 levels, and phospho p42/44 and HER1/2 signaling pathway were studied. Lapatinib proved to markedly downregulate TS activity, thus suggesting a subsequent better efficacy of capecitabine. Capecitabine optimized the downregulation of p-AKT and p-P42/44 expression by lapatinib. Consequently, we observed an increase in the Bax/Bcl2 ratio and p21 protein expression in cells exposed to the combination. Overall, our data showed that whatever the schedule and the cell line were, additive to synergistic interaction was achieved in our models. The optimal in vitro combination was finally tested in tumor-bearing mice. Our results fully confirmed that associating both drugs led to a 77% reduction in tumor growth as compared with control animals in BT474-xenografted models. Taken together, this non-clinical study shows that lapatinib and capecitabine modulate each other's molecular determinants of response and that concomitant dosing seems to be the optimal way to combine these drugs. Besides, modulation of TS expression by lapatinib makes its association with capecitabine a promising way to overcome breast cancers resistant in relation with TS overexpression.

Pharmacokinetics of trabectedin on hemodialysis: an application for the management of cancer patients with end-stage renal disease.

PURPOSE: The pharmacokinetics of trabectedin has never been reported in patients with impaired renal function or in patients on hemodialysis. METHODS: We examined trabectedin PK in a patient on hemodialysis, starting trabectedin therapy at a standard dose for recurrence of a retroperitoneal myxoid liposarcoma that had occurred under immunosuppressive drugs for kidney transplant. RESULTS: As compared with a population with normal renal function, the study patient presented a higher C (max) and AUC, with lower clearance, terminal half-life, and volume of distribution. The low dialysis clearance, accounting for a minor part of the total body clearance and the absence of detectable trabectedin in the dialysate samples, suggests that hemodialysis does not efficiently clear trabectedin. Trabectedin tolerance was good. CONCLUSIONS: This case reports for the first time the feasibility of trabectedin therapy in a hemodialyzed patient. Given the rising incidence of cancer in patients with end-stage renal disease, it is crucial to provide data that improve the management of anticancer drugs in dialyzed patients.

Tipifarnib plus tamoxifen in tamoxifen-resistant metastatic breast cancer: a negative phase II and screening of potential therapeutic markers by proteomic analysis.

PURPOSE: Tipifarnib, a farnesyltransferase inhibitor, has antitumor activity in heavily pretreated metastatic breast cancer patients. Preclinical data suggest that FTIs could restore tamoxifen responsiveness in tamoxifen-resistant disease. Thus, combining FTIs and tamoxifen may be a promising clinical approach after relapse or progression on tamoxifen. EXPERIMENTAL DESIGN: Postmenopausal patients with measurable estrogen receptor- and/or progesterone receptor-expressing metastatic breast cancers were enrolled. Only patients with disease progression on tamoxifen were eligible, but there was no limitation regarding prior chemotherapy or hormone therapy regimens. Patients were immediately treated with 300 mg (n = 12) or 200 mg (n = 10) tipifarnib twice daily for 21 of 28-day cycles plus tamoxifen once daily. Serum was collected at baseline and after 8 weeks of treatment to enable proteomic comparison and identify possible predictive response markers. RESULTS: Twenty patients were enrolled and evaluated for efficacy: one patient had an objective response (liver metastasis) and nine had stable disease after 6 months for a clinical benefit rate of 50%; median duration of benefit was 10.3 (range, 7.4-20.2) months. The proteomic analysis by SELDI-TOF and LTQ-FT-Orbitrap identified a known peptide of fibrinogen alpha, the intensity of which was significantly increased in patients with progression compared with patients who benefited from the combined treatment after 8 weeks. CONCLUSIONS: Because the primary end point of efficacy (three objective responses) was not achieved, the study is negative. Nevertheless, the identified peptide could be of interest in discriminating, at 8 weeks of treatment, responders from nonresponders.

A routine feasible HPLC analysis for the anti-angiogenic tyrosine kinase inhibitor, sunitinib, and its main metabolite, SU12662, in plasma.

Sunitinib is an oral inhibitor of multiple tyrosine kinase receptors with antitumor activity in metastatic renal cell carcinoma. So far, published methods for analysis of sunitinib and its active metabolite (SU12662) in plasma are exclusively based on mass spectrometry. In the context of a large-scale feasibility pharmacokinetic analysis, we developed an original, simple, high-performance liquid chromatography (HPLC) assay with UV detection. A stability study of sunitinib and SU12662 in different light exposure conditions is presented. Due to photo-instability of the compounds, blood sampling and the whole handling procedure have to be performed quickly and with minimal light exposure (6-7 lx). Following single organic extraction with tert-butyl methyl ether, HPLC analysis was performed on an ODS column and UV detection was monitored at 369 nm (run time 15 min). This assay was selective and sensitive enough (limit of detection approximately 1 ng/ml) to quantify minimal concentrations at steady state (Css min) of sunitinib and SU12662 in treated patients.

Dihydropyrimidine dehydrogenase activity and the IVS14+1G>A mutation in patients developing 5FU-related toxicity.

AIMS: To examine retrospectively the relationship between DPD phenotype/genotype and the intensity of 5FU toxicity. METHODS: One hundred and thirty-one case-reports (81 women, 50 men) with 5FU-related toxicity were analyzed. RESULTS: The lower the DPD activity (10-504 pmol min(-1) mg(-1)), the higher the toxicity grade was scored (P < 0.01). Toxicity-related deaths occurred in nine patients (eight women) who significantly expressed lower DPD activity than other patients. Two of the deceased patients had normal DPD activity. The IVS14+1G>A mutation, analyzed in 93 patients, was detected in two patients (nonlethal toxicity). CONCLUSIONS: The IVS14+1G>A mutation may not help prevent toxicity and patients with normal DPD activity may develop life-threatening 5FU toxicity.

Taxotere-5'-deoxy-5-fluorouridine combination on hormone-refractory human prostate cancer cells.

Single-agent docetaxel (Taxotere) treatment has recently demonstrated promising clinical activity in patients with advanced hormone-refractory prostate cancer. Taxanes were recently found to upregulate the tumoral activity of thymidine phosphorylase (TP), a key cellular enzyme [transformation of 5'-deoxy-5-fluorouridine (5'-DFUR) into 5-fluorouracil] in the activation cascade of capecitabine (Xeloda). We tested (cytotoxic effects and molecular mechanisms) the Taxotere-5'-DFUR combination on hormone-refractory prostate cancer cell lines (DU145 and PC3). Cells were exposed to Taxotere and/or 5'-DFUR in three different sequences: Taxotere was given alone for 48 h, then 5'-DFUR was added for 48 h; Taxotere and 5'-DFUR together during 96 h or 5'-DFUR was given alone for 48 h then Taxotere was added for 48 h. The drug sequence Taxotere applied first followed by 5'-DFUR led to synergistic cytotoxic effects on both cell lines; the other sequences resulted in simple additivity. Taxotere did not modify TP activity while it decreased thymidylate synthase activity. There was an increase in CD95 cellular membrane levels following exposure to Taxotere-5'-DFUR, which is in agreement with the supra-additive cytotoxic combination. This observation may serve as a preclinical rationale for a next step testing the Taxotere-capecitabine combination at the clinical level in prostate cancer patients.