Presentation on theme: "Development of Assays for Use in Patient Response Measurement in Phase 0 Trials Robert Kinders Ralph Parchment Pharmacodynamic Assay Development and Implementation."— Presentation transcript:
Development of Assays for Use in Patient Response Measurement in Phase 0 Trials Robert Kinders Ralph Parchment Pharmacodynamic Assay Development and Implementation Section, and Laboratory of Human Toxicology & Pharmacology SAIC-Frederick, Inc., NCI-Frederick, Frederick, Maryland 21702 Funded by Contract N01-CO-12400
Assay Design Parameters for Phase 0 Quantitative assay readouts are essential to meet protocol endpoints –Accuracy by recovery, interfering substances, cross-reactivity and dilution linearity –LLQ, slope and dynamic range must be consistent with expected signal level in an 18-gauge needle biopsy or 2.5-10 E5 PBMC –Specimen turnaround in 24 hours Assay must yield consistent results over at least a year of clinical testing Controls and calibrators are critical –Calibrators should be available, stable, quantified, characterized –Controls should mimic specimen and be run in every assay PAR assay uses cell extracts Specific antibody-stained biopsies used in γ-H2AX assay Reagent sources and an alternate vendor should be identified
Tumor Extract Dilution Analysis and Recovery Validated PAR Immunoassay
Assay Validation Most Critical Aspect: Demonstrate that the assay is fit for purpose –An assay with a 20% interday/instrument/operator CV cannot detect a 20% modulation of signal –Must know accuracy and precision to assess performance –This does not capture biological variation, which is large Analytical sensitivity yields real-world benefits: –Allows repeat determinations on the same specimen –Adapted for the size of the specimen to be used in the clinic: e.g., an 18-gauge needle biopsy Robustness: Must be transferable to other labs
PAR Immunoassay Development Key changes made included: –Specimen treatment step added –Specimen diluent and dilution level –Specimen incubation time and temperature –Substrate incubation time and temperature Other Changes –Use of a shaking step before the read –Blockers –Carrier protein –Read time –Instrument settings –Conjugate, conjugate concentration, and diluent [PAR] Total %CV Previous New 10 46.4 ? 30 30.2 8.1 100 24.6 7.4 300 19.5 6.9 1000 13.9 6.4
Completion of a Validated Assay Format Selection of assay format Production of assay controls (QC samples) Adequate instrumentation (calibrated and validated) Selection of standards and the standards matrix –Will the standards matrix be a cell extract? –How will that affect stability of the standards? Adequate assay sensitivity for multiple determinations on the same clinical specimen Specific optimization of handling by specimen type Validation issues: –Use common reagents (master lot) –Precision validation (operator, day, instrument) –Accuracy validation (dilution linearity, spike recovery) –Assay transfer between sites NOW WE CAN START!
Now Start Application in Preclinical Modeling Establish the dynamic range of response to treatment Time window of sampling consistent with clinical realities Tie the dose/response curve in tumor growth inhibition to marker modulation –What is the minimum detectable signal? –Is that in the microdose range? –Can the assay report whether an effective dose is delivered to the tumor?
Intratumoral γ- H2AX Response to 4.7 mg/kg Topotecan +2 Hours, Mouse #22, All 3 Fields The biopsy assay counts pixels to determine the fraction of nuclear area that is γ- H2AX-positive over three 200x fields. It uses the DAPI signal to identify fields containing mostly live cells. It matters where the needle goes!
Comparison of H2AX Response to Topotecan or Indenoisoquinoline Drug Dose, +2-Hour Timepoint Plot is vehicle- corrected Ln/Ln with plateau response points omitted for topotecan (TPT) and NSC 725776 Range of vehicle reads was the same across the 3 experiments; means & SDs overlapped
Helpful Links and References http://www.westgard.com/lesson.htm http://www.nihs.go.jp/drug/validation/q2bwww.html http://www.fda.gov/cder/guidance/4252fnl.htm http://www.clinchem.org/info_ar/anal_meth.shtml http://www.cbrlabs-inc.com/assay-validation.html Validation of Immunoassays for Bioanalysis: a Pharmaceutical Industry Perspective. Findlay J., Smith W., Lee D., Nordblom G., Das I., DeSilva B., Khan M., and Bowsher R. Journal of Pharmaceutical and Biomedical Analysis. 2000; 21: 1249-1273. Immunoassay, A Practical Guide. Ed. Dan Chan and Marie Pearlstein. Academic Press, 1987, 1992.
Operations and Technical Support Contractor for The National Cancer Institute at Frederick