Point of care assay for blood aripiprazole concentrations: development, validation and utility.

BACKGROUND: The antipsychotic aripiprazole is often used in the treatment of first-episode psychosis. Measuring aripiprazole blood levels provides an objective measure of treatment adherence, but this currently involves taking a venous blood sample and sending to a laboratory for analysis. AIMS: To detail the development, validation and utility of a new point of care (POC) test for finger-stick capillary blood concentrations of aripiprazole. METHOD: Analytical performance (sensitivity, precision, recovery and linearity) of the assay were established using spiked whole blood and control samples of varying aripiprazole concentration. Assay validation was performed over a 14-month period starting in July 2021. Eligible patients were asked to provide a finger-stick capillary sample in addition to their usual venous blood sample. Capillary blood samples were tested by the MyCare™ Insite POC analyser, which provided measurement of aripiprazole concentration in 6 min, and the venous blood sample was tested by the standard laboratory method. RESULTS: A total of 101 patients agreed to measurements by the two methods. Venous blood aripiprazole concentrations as assessed by the laboratory method ranged from 17 to 909 ng/mL, and from 1 to 791 ng/mL using POC testing. The correlation coefficient between the two methods (r) was 0.96 and there was minimal bias (slope 0.91, intercept 4 ng/ml). CONCLUSIONS: The MyCare Insite POC analyser is sufficiently accurate and reliable for clinical use. The availability of this technology will improve the assessment of adherence to aripiprazole and the optimising of aripiprazole dosing.

Evaluation of a Nanoparticle-Based Busulfan Immunoassay for Rapid Analysis on Routine Clinical Analyzers.

BACKGROUND: Busulfan is an alkylating agent used in allogeneic hematopoietic stem cell transplantation for various malignant and nonmalignant disorders. Therapeutic drug monitoring of busulfan is common because busulfan exposure has been linked to veno-occlusive disease, disease relapse, and failed engraftment. The authors developed an automated immunoassay, along with stable calibrators and controls, and quantified busulfan in sodium heparin plasma. METHODS: The authors evaluated a homogenous nanoparticle immunoassay, the MyCare Oncology Busulfan Assay Kit (Saladax Biomedical, Inc), for precision, sensitivity, accuracy, and linearity on an open channel clinical chemistry analyzer; they compared the method with 2 mass spectrometry methods (liquid chromatography-tandem mass spectrometry and gas chromatography/mass spectrometry), using anonymized, remnant patient samples. RESULTS: The coefficients of variation for repeatability and within-laboratory precision were ≤9.0%. The linear range was 150-2000 ng/mL; samples up to 6000 ng/mL can be measured with sample dilution. Measured values deviated by ≤14% from assigned values. Comparison between validated mass spectrometry methods resulted in a correlation coefficient R ≥ 0.995. CONCLUSIONS: The MyCare Busulfan Assay Kit shows the precision, accuracy, linearity, and test range for performing busulfan concentration measurements in sodium heparin plasma on routine clinical chemistry analyzers.

Randomized study of individualized pharmacokinetically-guided dosing of paclitaxel compared with body-surface area dosing in Chinese patients with advanced non-small cell lung cancer.

AIMS: This prospective, randomized study was initiated to assess the impact of pharmacokinetically (PK)-guided paclitaxel (PTX) dosing on toxicity and efficacy compared with body-surface area (BSA)-based dosing in Chinese non-small cell lung cancer patients. METHODS: A total of 319 stage IIIB/IV non-small cell lung cancer patients receiving first-line chemotherapy were enrolled. Patients were randomized to receive 3-weekly carboplatin plus PTX at a starting dose of 175 mg/m2 with subsequent PTX dosing based on either BSA or PK-guided dosing targeting time above a PTX plasma concentration of 0.05 µmol/L (PTXTc > 0.05 ) between 26 and 31 hours. The primary safety endpoint was grade 4 haematological toxicity. The secondary endpoints were neuropathy, objective response rate, progression-free survival and overall survival. RESULTS: In total, 275 (86%) patients completed ≥2 cycles of chemotherapy (140 in BSA arm and 135 in PK arm). In cycle 1, with the same PTX dose, average PTXTc > 0.05 was 37 hours (range = 18-57 hours). Over cycles 2-4, patients in the PK arm had significantly lower average PTX doses and exposure compared with the BSA arm (128 vs 161 mg/m2 , P < .0001 and 29 vs 35 hours, P < .0001). PK-guided dosing significantly reduced the cumulative incidence of grade 4 haematological toxicity (15% vs 24%, P = .004), grade 4 neutropenia (15% vs 23%, P = .009) and grade ≥ 2 neuropathy (8% vs 21%, P = .005). Objective response rate (32% vs 26%, P = .28) and overall survival (21.0 vs 24.0 months, P = .815) were similar in PK and BSA arms. Progression-free survival was slightly improved in PK arm (4.67 vs 4.17 months, P = .026). CONCLUSION: PK-guided PTX dosing significantly reduced grade 4 haematological toxicities and grade ≥ 2 neuropathy without an adverse impact on clinical outcomes.

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.

Rapid Homogeneous Immunoassay to Quantify Gemcitabine in Plasma for Therapeutic Drug Monitoring.

BACKGROUND: Gemcitabine (2',2'-difluoro-2'-deoxycytidine) is a nucleoside analog used as a single agent and in combination regimens for the treatment of a variety of solid tumors. Several studies have shown a relationship between gemcitabine peak plasma concentration (Cmax) and hematological toxicity. An immunoassay for gemcitabine in plasma was developed and validated to facilitate therapeutic drug monitoring (TDM) by providing an economical, robust method for automated chemistry analyzers. METHODS: A monoclonal antibody was coated on nanoparticles to develop a homogenous agglutination inhibition assay. To prevent ex vivo degradation of gemcitabine in blood, tetrahydrouridine was used as a sample stabilizer. Validation was conducted for precision, recovery, cross-reactivity, and linearity on a Beckman Coulter AU480. Verification was performed on an AU5800 in a hospital laboratory. A method comparison was performed with (LC-MS/MS) liquid chromatography tandem mass spectrometry using clinical samples. Selectivity was demonstrated by testing cross-reactivity of the major metabolite, 2',2'-difluorodeoxyuridine. RESULTS: Coefficients of variation for repeatability and within-laboratory precision were <8%. The deviation between measured and assigned values was <3%. Linear range was from 0.40 to 33.02 µ/mL (1.5-125.5 µM). Correlation with validated LC-MS/MS methods was R = 0.977. The assay was specific for gemcitabine: there was no cross-reactivity to 2',2'-difluorodeoxyuridine, chemotherapeutics, concomitant, or common medications tested. Tetrahydrouridine was packaged in single-use syringes. Gemcitabine stability in whole blood was extended to 8 hours (at room temperature) and in plasma to 8 days (2-8°C). CONCLUSIONS: The assay demonstrated the selectivity, test range, precision, and linearity to perform reliable measurements of gemcitabine in plasma. The addition of stabilizer improved the sample handling. Using general clinical chemistry analyzers, gemcitabine could be measured for TDM.

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.

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.

Point-of-care measurement of clozapine concentration using a finger-stick blood sample.

BACKGROUND: The use of clozapine demands regular monitoring of clozapine plasma concentrations and of white blood cell parameters. The delay between sending blood samples for analysis and receiving the results hinders clinical care. Point-of-care testing (POCT) can provide drug assay results within a few minutes. AIM: This study aimed to investigate the utility of a novel point-of-care device that can measure clozapine concentrations using capillary blood samples collected via a finger stick. METHOD: During a five-week period starting in June 2019 eligible patients were asked to provide a finger-stick capillary sample in addition to their usual venous blood sample. Samples were analysed by the novel point-of-care device and by the standard laboratory method. Capillary blood samples were tested by the MyCare™ Insite POCT analyser, and a quantitative measurement of clozapine concentration was provided within six minutes. RESULTS: A total of 309 patients agreed to measurements by the two methods. Analysis revealed clozapine concentrations in venous blood as determined by the laboratory method ranged from 20 to 1310 ng/mL and by POCT from 7 to 1425 ng/mL. There was a strong positive correlation (R = 0.89) between the results from the venous and the capillary sample methods. The slope of the association between standard assay and MyCare™ Insite was 1.0 with an intercept of -21 ng/mL, indicating minimal bias. CONCLUSION: Clozapine concentrations can be accurately measured at the point of care using capillary blood samples collected via a finger stick. This approach may be more acceptable than venous sampling to patients and, with almost instant results available, more useful to clinicians.

Engineering of recombinant antibody fragments to methamphetamine by anchored periplasmic expression.

The detection of methamphetamine and other chemically related illicit drugs relies extensively on immunoassays. Here we report the cloning and affinity maturation of an anti-methamphetamine antibody which is being employed in the current commercial assays. An anti-methamphetamine scFv was cloned from hybridoma cells, expressed in bacteria and its affinity towards methamphetamine and N-ethylamphetamine (ethamphetamine) was determined by Surface Plasmon Resonance (SPR). The anti-methamphetamine scFv gene was subjected to random mutagenesis by error prone PCR and variants with improved affinity were isolated from the resulting library by a novel screening methodology termed Anchored Periplasmic Expression (APEx) [Harvey, B.R., Georgiou, G., Hayhurst, A., Jeong, K.J., Iverson, B.L., Rogers, G.K. (2004). Anchored periplasmic expression, a versatile technology for the isolation of high-affinity antibodies from Escherichia coli-expressed libraries. Proc. Natl. Acad. Sci. U. S. A. 101, 9193.]. The isolated clones exhibited improved affinity to these illicit drugs, yet maintained low cross-reactivity to over-the-counter drugs. In addition, all clones displayed improved expression characteristics in Escherichia coli. The affinity improved scFv antibodies are thus likely to be useful in methamphetamine class immunodiagnostics.