Validation of a Commercial Assay and Decision Support Tool for Routine Paclitaxel Therapeutic Drug Monitoring (TDM).

BACKGROUND: The value of therapeutic drug monitoring (TDM) for paclitaxel (PTX) was recently demonstrated in the largest TDM trial ever conducted in oncology. The trial demonstrated significant reduction in neuropathy when using TDM. Dose adjustment for PTX was based on time above a threshold concentration (Tc>0.05). Tc>0.05 must be calculated with a pharmacokinetic model and complex nonlinear mixed-effects software. The use of the software and chromatographic methods to measure PTX requires specialized expertise. User-friendly methods to quantitate PTX and calculate Tc>0.05 could simplify the introduction of TDM into routine clinical practice. METHODS: The immunoassay (MyPaclitaxel) was used to quantitate PTX in samples from the clinical trial; the results were used to calculate Tc>0.05 using a stand-alone computer program with a simple, friendly graphical user interface for nonlinear mixed-effects pharmacokinetic calculations (MyCare Drug Exposure Calculator). The resulting dose recommendations from the calculated Tc>0.05 were compared with those using liquid chromatography-ultraviolet detection and NONMEM to examine the efficacy of the simpler tools for TDM. RESULTS: There was a good agreement between the immunoassay and liquid chromatography-ultraviolet detection: Passing-Bablok regression slope was 1.045 and intercept was -6.00, R was 0.9757, and mean bias was -1.77 ng/mL (-2.07 nmol/L). Dosing recommendations were identical for 70% of the cycles and within 10% for 89% of the samples. All Tc>0.05 values were at the same or adjacent medical decision points. CONCLUSIONS: MyPaclitaxel assay and MyCare Drug Exposure Calculator are convenient, user-friendly tools that may be suitable for routine TDM of PTX in clinical care.

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 manufacturer's 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 µL 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.

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 µL 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.

Inhibition of Escherichia coli RecA by rationally redesigned N-terminal helix.

Bacterial RecA promotes the development and transmission of antibiotic resistance genes by self-assembling into an ATP-hydrolyzing filamentous homopolymer on single-stranded DNA. We report the design of a 29mer peptide based on the RecA N-terminal domain involved in intermonomer contact that inhibits RecA filament assembly with an IC50 of 3 microM.

Effects of As(III) binding on beta-hairpin structure.

While arsenic(III) compounds can exert profound toxicological and pharmacological effects, their modes of action and, in particular, the structural consequences of their binding to cysteinyl side chains in proteins, remain poorly understood. To gain an understanding of how arsenic binding influences beta-structure, pairs of cysteines were introduced into a model monomeric beta-hairpin to yield a family of peptides such that coordination occurs either across the strands or within the same strand of the beta-hairpin. Circular dichroism, NMR, UV-vis spectroscopy, and rapid-reaction studies were used to characterize the binding of monomethylarsonous acid or p-succinylamidephenyl arsenoxide (PSAO) to these peptides. Placement of cysteines at non-hydrogen bond (NHB) positions across the beta-hairpin, such that they occupy the same face of the sheet, was found to enhance the structure as assessed by CD. Cross-strand cysteine residues that project on opposite faces close to the termini of the hairpin can still bind arsenic tightly and show modestly increased beta-sheet content. NMR and modeling studies suggest that arsenic can be accommodated at this locus without disrupting the core interactions stabilizing the turn. However, As(III) binding to nonopposed cysteines, or to cysteines at HB and NHB positions along one strand of the hairpin, caused loss of structure. UV-vis titrations show that all these hairpin peptides bind PSAO stoichiometrically with K(d) values from 13 to 106 nM. Further, binding is moderately rapid, with second-order rate constants for association of 10,000-22,000 M(-1) s(-)1 irrespective of the placement of the cysteines within the hairpin and the consequent extent of structural reorganization required as a result of binding. These studies complement recent work with alpha-helices and further demonstrate that capture of a pair of thiols by As(III) may result in significant changes in local secondary structure in the protein targets of these potent bioactive agents.

Structure-based design of a fluorimetric redox active peptide probe.

Structure-based iterative design was used to prepare a disulfide-containing nonapeptide as a fluorimetric probe for chemical and biochemical disulfide forming and breaking reactions. The peptide is composed entirely of natural amino acids and exhibits a marked (42%) change in fluorescence between its oxidized and its reduced states. The probe is easily synthesized and highly water soluble and exhibits well-behaved kinetics on reduction with the reductant tris-carboxyethylphosphine. The reduced peptide is an excellent substrate of the enzyme quiescin-sulfhydryl oxidase and may find utility in the characterization of other disulfide oxidoreductases.

Effects of As(III) binding on alpha-helical structure.

As(III) displays a wide range of effects in cellular chemistry. Surprisingly, the structural consequences of arsenic binding to peptides and proteins are poorly understood. This study utilizes model alpha-helical peptides containing two cysteine (Cys) residues in various sequential arrangements and spatial locations to study the structural effects of arsenic binding. With i, and i + 1, i + 2, or i + 3 arrangements, CD spectroscopy shows that As(III) coordination causes helical destabilization when Cys residues are located at central or C-terminal regions of the helix. Interestingly, arsenic binding to i, i + 3 positions results in the elimination of helical structure and the formation of a relatively stable alternate fold. In contrast, helical stabilization is observed for peptides containing i, i + 4 Cys residues, with corresponding pseudo pairwise interaction energies (Delta G(pw) degrees) of -1.0 and -0.7 kcal/mol for C-terminal and central placements, respectively. Binding affinities and association rate constants show that As(III) binding is comparatively insensitive to the location of the Cys residues within these moderately stable helices. These data demonstrate that As(III) binding can be a significant modulator of helical secondary structure.

New water-soluble phosphines as reductants of peptide and protein disulfide bonds: reactivity and membrane permeability.

Tris(2-carboxyethyl)phosphine (TCEP) is a widely used substitute for dithiothreitol (DTT) in the reduction of disulfide bonds in biochemical systems. Although TCEP has been recently shown to be a substrate of the flavin-dependent sulfhydryl oxidases, there is little quantitative information concerning the rate by which TCEP reduces other peptidic disulfide bonds. In this study, mono-, di-, and trimethyl ester analogues of TCEP were synthesized to evaluate the role of carboxylate anions in the reduction mechanism, and to expand the range of phosphine reductants. The effectiveness of all four phosphines relative to DTT has been determined using model disulfides, including a fluorescent disulfide-containing peptide (H(3)N(+)-VTWCGACKM-NH(2)), and with protein disulfide bonds in thioredoxin and sulfhydryl oxidase. Mono-, di-, and trimethyl esters exhibit phosphorus pK values of 6.8, 5.8, and 4.7, respectively, extending their reactivity with the model peptide to correspondingly lower pH values relative to that of TCEP (pK = 7.6). At pH 5.0, the order of reactivity is as follows: trimethyl- > dimethyl- > monomethyl- > TCEP >> DTT; tmTCEP is 35-fold more reactive than TCEP, and DTT is essentially unreactive. Esterification also increases lipophilicity, allowing tmTCEP to penetrate phospholipid bilayers rapidly (>30-fold faster than DTT), whereas the parent TCEP is impermeant. Although more reactive than DTT toward small-molecule disulfides at pH 7.5, all phosphines are markedly less reactive toward protein disulfides at this pH. Molecular modeling suggests that the nucleophilic phosphorus of TCEP is more sterically crowded than the thiolate of DTT, contributing to the lower reactivity of the phosphine with protein disulfides. In sum, these data suggest that there is considerable scope for the synthesis of phosphine analogues tailored for specific applications in biological systems.