Salvatore J. Salamone

Pharmacokinetically Guided Dose Adjustment of 5-Fluorouracil: A Rational Approach to Improving Therapeutic Outcomes

Chemotherapy dosing of the fluoropyrimidine 5-fluorouracil (5-FU) is currently based on body surface area. However, body surface area-based dosing has been associated with clinically significant pharmacokinetic variability, and as such, dosing based on body surface area may be of limited use. The clinical activity of 5-FU is modest at standard doses, and in general, dosing is limited by the safety profile, with myelosuppression and gastrointestinal toxicity being the most commonly observed side effects. Various strategies have been developed to enhance the clinical activity of 5-FU, such as biochemical modulation, alterations in scheduling of administration, and the use of oral chemotherapy. Studies that have shown an association between plasma concentration with toxicity and clinical efficacy have shown that pharmacokinetically guided dose adjustments can substantially improve the therapeutic index of 5-FU treatment. These studies have shown that only 20%-30% of patients treated with a 5-FU-based regimen have 5-FU levels that are in the appropriate therapeutic range--approximately 40%-60% of patients are underdosed and 10%-20% of patients are overdosed. To date, 5-FU drug testing has not been widely used because of the lack of a simple, fast, and inexpensive method. Recent advances in testing based on liquid chromatography-mass spectroscopy and a nanoparticle antibody-based immunoassay for 5-FU may now allow for routine monitoring of 5-FU in clinical practice. We review the data on pharmacokinetically guided dose adjustment of 5-FU and discuss the potential of this approach to advance therapeutic outcomes.

Effect of Plyometric Training on Swimming Block Start Performance in Adolescents

Bishop, DC, Smith, RJ, Smith, MF, and Rigby, HE. Effect of plyometric training on swimming block start performance in adolescents. J Strength Cond Res 23(7): 2137-2143, 2009-This study aimed to identify the effect of plyometric training (PT), when added to habitual training (HT) regimes, on swim start performance. After the completion of a baseline competitive swim start, 22 adolescent swimmers were randomly assigned to either a PT (n = 11, age: 13.1 ± 1.4 yr, mass: 50.6 ± 12.3 kg, stature: 162.9 ± 11.9 cm) or an HT group (n = 11, age: 12.6 ± 1.9 yr, mass: 43.3 ± 11.6 kg, stature: 157.6 ± 11.9 cm). Over an 8-week preseason period, the HT group continued with their normal training program, whereas the PT group added 2 additional 1-hour plyometric-specific sessions, incorporating prescribed exercises relating to the swimming block start (SBS). After completion of the training intervention, post-training swim start performance was reassessed. For both baseline and post-trials, swim performance was recorded using videography (50Hz Canon MVX460) in the sagital plane of motion. Through the use of Silicon Coach Pro analysis package, data revealed significantly greater change between baseline and post-trials for PT when compared with the HT group for swim performance time to 5.5 m (−0.59 s vs. −0.21 s; p < 0.01) and velocity of take-off to contact (0.19 ms−1 vs. −0.07 ms−1; p < 0.01). Considering the practical importance of a successful swim start to overall performance outcome, the current study has found that inclusion of suitable and safely implemented PT to adolescent performers, in addition to HT routines, can have a positive impact on swim start performance.BACKGROUND 5-Fluorouracil (5-FU) is the most widely used chemotherapy drug, primarily against gastrointestinal, head and neck, and breast cancers. 5-FU has large pharmacokinetic variability resulting in unexpected toxicity or ineffective treatment. Therapeutic drug management of 5-FU minimizes toxicity and improves outcome. A nanoparticle-based immunoassay was developed to provide oncologists with a rapid, cost-effective tool for determining 5-FU plasma concentrations. METHODS Monoclonal antibodies, bound to nanoparticles, were used to develop an immunoassay for the Olympus AU400. Assay precision, linearity, calibration stability, and limit of detection were run at multiple centers; interference, cross-reactivity, lower limit of quantitation and recovery at 1 center. Clinical samples collected from 4 cancer centers were analyzed for 5-FU concentrations by liquid chromatography-tandem mass spectrometry and compared with the immunoassay results. RESULTS With calibrators from 0 to 1800 ng/mL 5-FU and autodilution, concentrations up to 9000 ng/mL could be determined. Time to first result was 10 minutes, and 400 samples per hour could be quantitated from a standard curve stored for >30 days. Imprecision across all laboratories was <5%, and the assay was linear upon dilution over the entire range. Cross-reactivities for dihydro-5-FU, uracil, capecitabine, and tegafur were <1%, 9.9%, 0.05%, and 0.23%, respectively. The limit of detection was 52 ng/mL with a lower limit of quantitation of 86 ng/mL. Assay results of clinical samples (93-1774 ng/mL) correlated with liquid chromatography-tandem mass spectrometry results: (R = 0.9860, slope 1.035, intercept 10.87 ng/mL). CONCLUSIONS This novel immunoassay is suitable for quantitating 5-FU plasma concentrations with advantages of speed, small sample size, minimal sample pretreatment, and application on automated instrumentation. These advantages enable efficient therapeutic drug management of 5-FU in clinical practice.

Highlights from: 5-Fluorouracil Drug Management Pharmacokinetics and Pharmacogenomics Workshop; Orlando, Florida; January 2007

In the year of its fiftieth anniversary, 5-fluorouracil (5-FU) continues to be a cornerstone in the treatment of many cancers, including colorectal, head and neck, stomach, and breast carcinomas. The introduction of new targeted therapies represents a promising generation of treatment approaches, but 5-FU remains at the center of all validated protocols. Originally, 5-FU alone was administered as a bolus, but more recently, it is being used with biomodulating agents and/or a change in the schedule of administration of 5-FU to infusion. Because of the preclinical evidence that increased duration of 5-FU exposure improves cytotoxic activity and the fact that 5-FU has a short plasma half-life, continuous infusion schedules were further developed as a strategy to increase the percentage of tumor cells exposed to 5-FU. These regimens have resulted in up to a 2-fold improvement of response rates (RRs) with an improved safety profile when compared with bolus schedules. Capecitabine, an oral fluoropyrimidine, is a 5-FU prodrug drug that closely simulates infusional administration of the drug, delivering 5-FU at the target itself rather than providing systemic 5-FU exposure. Phase III studies incorporating capecitabine (such as the XELOX [capecitabine/oxaliplatin] regimen) demonstrate that XELOX is noninferior to the FOLFOX4 (oxaliplatin/leucovorin [LV]/5-FU) in terms of progression-free survival (PFS), and that adding bevacizumab to FOLFOX4/XELOX improves PFS significantly. It is now well established that infusional 5-FU schedules, as well as delivery of fluoropyrimidines via oral route, serve as the critical backbone for combination regimens and are widely used for the treatment of advanced disease, as well as for the adjuvant therapy of earlystage colorectal cancer (CRC). 5-fluorouracil pharmacokinetics are characterized by a wide interpatient and intrapatient variability and clearance values when equal doses calculated by body surface area are administered to different patients, leading to marked differences in systemic exposures. There is a substantial body of data for colorectal and head and neck cancer (HNC) showing significant relationships between systemic exposure, area under the curve (AUC) concentration at steady state, and pharmacodynamics (toxicity, response, and survival). Several groups reported reduced 5-FU clearance in elderly patients, resulting in an increased rate and degree of toxicity. Some of this risk can be predicted by the MAX2 index, which takes into account the mean percentage of occurrence of the most frequent grade 4 hematologic toxicity and grade 3/4 nonhematologic toxicity. This index might allow for an adjustment factor (on a group basis) for toxicity, when several agents are used in a 5-FU–based regimen. There is a need to individualize 5-FU dosing, and the shift from bolus to continuous infusion administration has created better conditions for dose management. Different approaches are available. A number of studies have demonstrated that the target AUC for 5-FU is the same regardless of tumor type, administration schedule, and drug combination. Also, there have been many successful strategies to monitor 5-FU blood concentrations and adjust individual doses based upon systemic exposure. The clinical utility of this approach has Highlights from: 5-Fluorouracil Drug Management Pharmacokinetics and Pharmacogenomics Workshop Orlando, Florida; January 2007

Determination of busulfan in human plasma using an ELISA format.

High-dose busulfan is an important component of many bone marrow transplantation-preparative regimens. High busulfan plasma levels have been shown to increase the chance of venoocclusive disease and low levels are associated with recurrence of disease or graft rejection. Currently, busulfan levels are monitored by physical methods that are expensive and time consuming, resulting in relatively low overall use of busulfan testing for dose adjustment. Novel highly selective antibodies for busulfan have been generated and a microtiter plate immunoassay capable of quantifying busulfan levels in plasma has been developed. The assay was configured using a busulfan-horseradish peroxidase (HRP) conjugate as the reporter group and busulfan monoclonal antibodies. The assay requires 30 μL of plasma with no sample preparation. The immunoassay has a standard curve based on busulfan with a range of 75-2000 ng/mL. The time to first result is 30 minutes with up to 40 patient samples in duplicate; multiple plates can be run at once. The coefficient of variation (CV) on signal is <5% for an entire plate, and the 95% confidence interval for negative samples (n = 78) is below the lowest calibrator of 75 ng/mL. Cross-reactivity with the major inactive metabolites (tetrahydrothiophene, tetramethyl sulfone, and tetrahydrothiophene-3-ol-1,1-dioxide) was <0.1%. Results generated with clinical samples (n = 35 and n = 70) correlate well to gas chromatography-mass spectrometry (R = 0.976 and 0.985, respectively) with a slope of 1.05 ± 0.05. This immunoassay method is suitable for determining levels of busulfan in human plasma. It offers the advantages of using a smaller sample size, does not require sample preparation, and is less labor intensive than other methods. The ability to make 240 determinations per hour enables effective and timely routine monitoring of busulfan levels in clinical practice.

An automated nanoparticle-based homogeneous immunoassay for determining docetaxel concentrations in plasma.

Background: Docetaxel (Taxotere) (DTX) is a widely used chemotherapy agent used in many regimens for the treatment of solid tumors, for example breast cancer, non–small cell lung cancer, gastric, prostate, and head and neck cancers. This drug meets the criteria for therapeutic dose management, in that it is associated with high pharmacokinetic variability and dose-limiting toxicity; it has a narrow therapeutic window, and there is a significant pharmacokinetic–pharmacodynamic relationship. Measures of exposure and area under the time–concentration curve have been associated with both toxicity and outcomes, making therapeutic dose management for this drug an unmet clinical need. The current methodologies for measuring DTX are based on physical methods, making the analysis less available and costly. An automated immunoassay has been developed to provide greater access to DTX dose management. Methods: A DTX immunoassay (MyDocetaxel) has been developed using a generic nanoparticle turbidimetric method that can be used on a wide variety of automated clinical chemistry analyzers including the Beckman Coulter AU400 and AU640 instruments, which were used in this study. The assay is based on a competitive assay format using a selective DTX monoclonal antibody. Clinical Laboratory Standards Institute protocols for establishing manufacturers claims were used to verify performance. Testing at 3 clinical laboratories was undertaken using the same protocols for laboratory validation of precision, accuracy, and linearity. Method comparison (n = 89) was done using samples collected from patients on DTX therapy. The comparative method was LC–MS/MS validated according to Food and Drug Administration guidance on bioanalytical methods. Institutional review board approval was obtained for prospective collection of samples from patients on DTX therapy. Results: The assay on the AU400 uses 2 &mgr;L of sample, provides the first result in 9.0 minutes and can generate 400 determinations per hour. Internal studies established a lower limit of detection ⩽25 ng/mL and a lower limit of quantitation ⩽30 ng/mL. Additional studies demonstrated no interference from coadministered drugs, major metabolites, or related compounds. Linearity from 50 to 1000 ng/mL was validated. Method comparisons between laboratories and to the physical method gave slopes: 1 ± 0.5, intercepts: < 2.0 ng/mL, R > 0.99, with the range of DTX concentrations measured by the assay 31–9754 ng/mL, with a mean of 689 ng/mL. In all 3 laboratories, the coefficient of variation percentage for repeatability ranged from 0.8% to 6.2% and the within-laboratory precision ranged from 1.4% to 10.1%. Conclusions: This immunoassay is suitable for quantifying DTX in plasma with advantages of small sample size, no sample pretreatment, and the ability to be applied to a wide range of clinical analyzers. With the validation of this method, the application of DTX testing in clinical practice may gain wider acceptance for individualizing patient DTX dosing.

Excel-Based Tool for Pharmacokinetically Guided Dose Adjustment of Paclitaxel.

Background: Neutropenia is a frequent and severe adverse event in patients receiving paclitaxel chemotherapy. The time above a paclitaxel threshold concentration of 0.05 &mgr;mol/L (Tc > 0.05 &mgr;mol/L) is a strong predictor for paclitaxel-associated neutropenia and has been proposed as a target pharmacokinetic (PK) parameter for paclitaxel therapeutic drug monitoring and dose adaptation. Up to now, individual Tc > 0.05 &mgr;mol/L values are estimated based on a published PK model of paclitaxel by using the software NONMEM. Because many clinicians are not familiar with the use of NONMEM, an Excel-based dosing tool was developed to allow calculation of paclitaxel Tc > 0.05 &mgr;mol/L and give clinicians an easy-to-use tool. Methods: Population PK parameters of paclitaxel were taken from a published PK model. An Alglib VBA code was implemented in Excel 2007 to compute differential equations for the paclitaxel PK model. Maximum a posteriori Bayesian estimates of the PK parameters were determined with the Excel Solver using individual drug concentrations. Concentrations from 250 patients were simulated receiving 1 cycle of paclitaxel chemotherapy. Predictions of paclitaxel Tc > 0.05 &mgr;mol/L as calculated by the Excel tool were compared with NONMEM, whereby maximum a posteriori Bayesian estimates were obtained using the POSTHOC function. Results: There was a good concordance and comparable predictive performance between Excel and NONMEM regarding predicted paclitaxel plasma concentrations and Tc > 0.05 &mgr;mol/L values. Tc > 0.05 &mgr;mol/L had a maximum bias of 3% and an error on precision of <12%. The median relative deviation of the estimated Tc > 0.05 &mgr;mol/L values between both programs was 1%. Conclusions: The Excel-based tool can estimate the time above a paclitaxel threshold concentration of 0.05 &mgr;mol/L with acceptable accuracy and precision. The presented Excel tool allows reliable calculation of paclitaxel Tc > 0.05 &mgr;mol/L and thus allows target concentration intervention to improve the benefit–risk ratio of the drug. The easy use facilitates therapeutic drug monitoring in clinical routine.

Development and Evaluation of a Nanoparticle-Based Immunoassay for Determining Paclitaxel Concentrations on Routine Clinical Analyzers

Background: Paclitaxel (PTX; Taxol, Abraxane) is used in many regimens for breast cancer, non–small cell lung cancer (NSCLC), and ovarian cancer. Multiple studies have demonstrated that PTX exhibits a greater than 10-fold interpatient variability of clearance rates when patients are dosed according to body surface area (BSA). Pharmacokinetic and pharmacodynamic relationships have been elucidated from BSA-based dosing. PTX is a candidate for dose management, and studies have shown that therapeutic dose management (TDM) is feasible and may provide improved outcomes for patients undergoing treatment. Methods: A PTX immunoassay (MyPaclitaxel) has been developed, which employs a novel PTX monoclonal antibody in a nanoparticle-based turbidimetric assay in a competitive format. Precision, accuracy, and linearity were evaluated by Clinical Laboratory Standards Institute protocols at 3 laboratories on the Olympus AU400 analyzer. Method comparison was done versus a validated high-performance liquid chromatography–tandem mass spectroscopy method using samples (n = 119) collected from patients on PTX therapy. Results: The assay requires 8 &mgr;L of plasma sample and can produce 400 determinations per hour. The response curve is based on a 6-point nonlinear curve fit and has a range of 0-320 ng/mL, extended to 3200 ng/mL with 10-fold autodilution. Three controls and 4 patient pools were used in precision studies. For all samples across 3 sites, repeatability coefficient of variation percentages ranged 0.9%-4.9%, and within-laboratory coefficient of variation percentages were 1.0%-4.2% with standard curve stability up to 24 days. Linearity was demonstrated over the linear range. Lower limits of detection and quantitation were 11 and 19 ng/mL, respectively. Method comparison results were analyzed by Deming regression, demonstrating a slope = 1.002 and intercept = −3.029 and an R = 0.996. The PTX samples ranged from 24 to 3164 ng/mL with a mean of 745 ng/mL. Conclusions: The analytical performance of an automated immunoassay for PTX has been validated and may serve as a useful tool for TDM of this drug.

An Automated Homogeneous Immunoassay for Quantitating Imatinib Concentrations in Plasma.

Background: Imatinib pharmacokinetic variability and the relationship of trough concentrations with clinical outcomes have been extensively reported. Although physical methods to quantitate imatinib exist, they are not widely available for routine use. An automated homogenous immunoassay for imatinib has been developed, facilitating routine imatinib testing. Methods: Imatinib-selective monoclonal antibodies, without substantial cross-reactivity to the N-desmethyl metabolite or N-desmethyl conjugates, were produced. The antibodies were conjugated to 200 nm particles to develop immunoassay reagents on the Beckman Coulter AU480 analyzer. These reagents were analytically validated using Clinical Laboratory Standards Institute protocols. Method comparison to liquid chromatography tandem mass spectrometry (LC-MS/MS) was conducted using 77 plasma samples collected from subjects receiving imatinib. Results: The assay requires 4 µL of sample without pretreatment. The nonlinear calibration curve ranges from 0 to 3000 ng/mL. With automated sample dilution, concentrations of up to 9000 ng/mL can be quantitated. The AU480 produces the first result in 10 minutes and up to 400 tests per hour. Repeatability ranged from 2.0% to 6.0% coefficient of variation, and within-laboratory reproducibility ranged from 2.9% to 7.4% coefficient of variation. Standard curve stability was 2 weeks and on-board reagent stability was 6 weeks. For clinical samples with imatinib concentrations from 438 to 2691 ng/mL, method comparison with LC-MS/MS gave a slope of 0.995 with a y-intercept of 24.3 and a correlation coefficient of 0.978. Conclusions: The immunoassay is suitable for quantitating imatinib in human plasma, demonstrating good correlation with a physical method. Testing for optimal imatinib exposure can now be performed on routine clinical analyzers.

A UNIFIED PRE-ANALYTICAL PROTOCOL FOR HANDLING OF CSF SAMPLES BEFORE ANALYSES OF AD BIOMARKER LEVELS

O2-09-01 CSF, PLASMA AND MRI BIOMARKER TRAJECTORIES DURING THE DEVELOPMENT OFALZHEIMER’S DISEASE Sebastian Palmqvist, Niklas Mattsson, Olof Strandberg, Philip Insel, Shorena Janelidze, Erik Stomrud, Oskar Hansson, Department of Neurology, Sk ane University Hospital, Lund, Sweden; Clinical Memory Research Unit, Lund University, Malm€o, Sweden; Department of Neurology, Lund, Sweden; Clinical Memory Research Unit, Lund University, Lund, Sweden; Center for Imaging of Neurodegenerative Diseases, San Francisco Veterans Administration Medical Center, San Francisco, CA, USA; Lund University, Lund, Sweden. Contact e-mail: sebastian.palmqvist@med.lu.se

BIOMARKER STABILITY IN CSF: PRE-ANALYTICAL FACTORS IN A PROSPECTIVE COLLECTION

of the optical densities in ten repetitions of the standard curves was 5% for all standards. In the clinical part, at the cut off value 691 pg/mL, Ab 1-42 showed the sensitivity and the specificity of 69.3 %, and 88.9%, respectively, whereas at the cut off value 0.06, Ab 42/40 ratio showed even better sensitivity and specificity of 93.3%, and 100%, respectively. The area under the ROC curve for Ab 42/40 (0.974) was highly significantly larger compared to the AUC of the Ab 1-42 concentration ROC curve (0.827, p<0.0001). Conclusions: (a) the novel Ab1-40 and Ab1-42 ELISA assays characterize with very good analytical performance; (b) we reconfirm that the CSF Ab42/40 concentration ratio shows significantly better diagnostic performance compared to the CSF Ab1-42 concentration alone.