DISCLOSURE Elaine Gray, NIBSC

Transcription

1 Monitoring Methods for Direct Thrombin Inhibitors Elaine Gray, PhD Haemostasis Section Biotherapeutics Group NIBSC DISCLOSURE Elaine Gray, NIBSC Relevant Financial Relationship(s) NONE Off Label Usage NONE Inhibition of Thrombin Direct Thrombin Inhibitors Half-life in healthy subjects Argatroban Lepirudin Bivalirudin Synthetic L-arginine derivative min Recombinant hirudin 1.3 hours Semisynthetic hirulog 25 minutes Elimination Hepatic Renal 8% Enzymatic 2% Renal Monitoring needed aptt, ACT aptt aptt, ACT Thrombin binding Reversible Irreversible Partially reversible Antidote None None None Saito H. Disorders of Hemostasis. 3rd ed. 1996;2: Adapted from Presentation on HIT, Jerrold H Levy, MD Methods In theory... Any methods that involve the direct or indirect inhibition of thrombin as endpoint Includes ACT, APTT, PT, Thrombin Time, anti-iia chromogenic assays In practice... APTT used in most of the clinical trials Few reports on Ecarin Clotting Time Ecarin Clotting Time (ECT) Principle of assay DTI inhibits meizothrombin generated by ecarin cleavage of prothrombin Ecarin snake venom from Echis carinatus Generates meizothrombin by cleavage of the arginine 32-isoleucine 321 protein bond of prothrombin. Meizothrombin possesses thrombin-like proteolytic activity and can clot fibrinogen. The active site of meizothrombin is inhibited by direct thrombin inhibitors. 1

2 APTT and ECT Thrombin Inhibitor Monitoring SSC on Control of Anticoagulation set up working group, chaired by Professor Harenberg, to investigate the various methods of monitoring thrombin inhibitor therapies. The working group concluded that a collaborative study is required to study the suitability of the various assay methods. Potzch et al, Thrombos Haemostas 1997 Thrombin Inhibitor Collaborative Study Two licensed thrombin inhibitors: recombinant hirudin and argatroban. In vitro addition of inhibitors to plasma to normal pooled plasma (n=4) to give final concentration:,.31,.63, 1.25, 2.5 μg/ml Methods APTT - local reagents and common reagent Anti-IIa chromogenic assay (S2238) Ecarin clotting time: wet chemistry dry chemistry (TAS analyser, near patient monitors) ECT and TIM II cards ELISA - for Hirudin only Design and Scope of the Study 4 sets of plasma samples for each method. Assay one set of samples per day 4 determinations per plasma sample per day Sensitivity of the methods: ability to detect the lowest and the highest concentrations of inhibitors Robustness of the methods: intralaboratory (day-to-day) and interlaboratory variations Participants Manufacturers of thrombin inhibitors Diagnostic equipment and reagent manufacturers Clinical laboratories Academic Institutes 2

3 APTT Common Reagent - ActinFS 1 laboratories Analysis log log transformation to give linear responses Similar ranges of clotting times for both hirudin and argatroban APTT Local Reagents Various APTT reagents 1 laboratories Analysis log-log transformation to give linear responses One laboratory unable to detect the highest concentrations (2.5 µg/ml) of inhibitors Ecarin Clotting Time Wet Chemistry Ecarin Reagents Professor Nowak 1 laboratories Analysis log-log plots 2 out 1 labs unable to detect clots in the highest concentration (2.5 µg/ml) of hirudin 4 out of 1 labs unable to detect clots in 3 out of the 4 concentrations of argatroban Different ranges of clotting times obtained for the same concentrations of hirudin and argatroban TAS Analyser - ECT 1 laboratories Analysis log-log plots All laboratories detected clots for the hirudin samples All 1 labs unable to detect clots for the highest concentration (2.5 µg/ml) of argatroban Different ranges of clotting times obtained for the same concentrations of hirudin and argatroban TAS Analyser TIM II 1 laboratories Analysis log-log plots All laboratories detected clots for the hirudin and argatroban samples Different ranges of clotting times obtained for the same concentrations of hirudin and argatroban Anti-IIa Chromogenic Anti-IIa chromogenic kit Instrumentation Laboratory/Haemochrom Diagnostica 13 Laboratories Analysis log linear Tendency of non-linearity when 2.5 µg/ml of hirudin included in the analysis Tendency of non-linearity when 2.5 and 1.25 µg/ml of argatroban included in the analysis 3

4 ELISA - Hirudin ELISA kit Amercian Diagnostica 1 laboratories Analysis linear-linear plot Good linear responses Analysis of Intra-laboratory Variability Obtained relative potency from each laboratory by random parallel line bioassay, using day 1 results as standard, and day 2, 3, 4 data as tests Geometric mean of the relative potencies were then used to calculate geometric coefficient of variation (%GCV) Intra-Laboratory Variability, % GCV- Hirudin Lab CAPTT LAPTT Anti-IIa Wet ECT Dry ECT TIM ELISA Intra-Laboratory Variability, % GCV- Argatroban Lab CAPTT LAPTT Anti-IIa Wet ECT Dry ECT TIM Analysis of Inter-laboratory Variability With the exception of the ELISA method, ratios of inhibitor plasma responses relative to the negative plasma responses were calculated for each day. This take into account the laboratory s day to day variation The geometric means of the ratios from each lab for each concentration of inhibitors were then used for calculation of % geometric coefficient of variation (%GCV) For the ELISA method, the negative plasma gave essentially no response, therefore ratios were calculated for lowest concentration (.31 μg/ml) relative to the other concentrations of inhibitors. Inter-laboratory Variability, %GCV Hirudin ug/ml L APTT C APTT Wet ECT Dry ECT TIM Anti IIa ELISA

5 Inter-laboratory Variability, %GCV Argatroban ug/ml L APTT C APTT Wet ECT Dry ECT TIM Anti IIa Inter-Laboratory Variability - %GCV Hirudin Argatroban LAPTT CAPTT Wet ECT Dry ECT TIM Anti-IIa ELISA Ratios of Inhibitor Plasma to Negative Plasma Response Ratios of Inhibitor Plasma to Negative Plasma Response Gmean Ratios - Hirudin Ratio Hirudin ug/ml LAPTT CAPTT wet ECT Dry ECT TIM Anti-IIa ELISA Ratio Ratios - Argatroban Argatroban ug/ml LAPTT CAPTT wet ECT Dry ECT TIM Anti-IIa Summary I-I Intra-laboratory Variability Overall, the APTTs and the ECT methods gave low within-laboratory variability %GCV under 5% for most labs. Higher variability for anti-iia assays and hirudin ELISA Summary II Inter-laboratory Variability CAPTT, TIM and dry ECT: lowest variability, %GCV all under 1% LAPTT: Low variability with argatroban samples, but higher variability with hirudin samples. Wet ECT and anti-iia: variability dependent on concentration of inhibitors, the higher the concentration, the higher the variability. Reasonable %GCV (Under 1% for.31 and.63 µg/ml). Hirudin ELISA: High variability 5

6 Summary III - Sensitivity Hirudin ELISA > ECT > APTT > Anti-IIa Argatroban ECT > APTT > Anti-IIa More details cations/anticoagulation/thrombin_inhibitors /CollaborativeStudy_full-length.pdf For ECT, adjustment of concentrations of Ecarin is important to detect both high and low amounts of DTIs Thrombin Generation Test - Argatroban Thrombin Generation Test - Hirudin Thrombin (nm (µg/ml) Lagtime Peak (nm) 25 Argatroban - 5pM rtf & 4µM PL (Pool) Time 2.5µg/ml 1.25µg/ml.625µg/ml.313µg/ml.156µg/ml Pool ETP (nm.min) Thrombin (nm) Hirudin - 5pM rtf & 4µM PL (µg/ml) Lagtime Peak (nm) (Pool) Time 2.5µg/ml 1.2 5µg/ m l.625µg/ml.313µg/ml.156µg/ml Pool ETP (nm.min) Thrombin (nm) (µg/ml) Lagtime Peak (nm) 25 Argatroban - 1pM rtf & 4µM PL (Pool) Time 2.5µg/ml 1.25µg/ml.625µg/ml.313µg/ml.156µg/ml Pool ETP (nm.min) Thrombin (nm) Hirudin - 1pM rtf & 4µM PL (µg/ml) Lagtime (Pool) Time 2.5µg/ml 1.25µg/ m l.625µg/ml.313µg/ml.156µg/ml Pool Peak (nm) ETP (nm.min) Acknowledgement Thanks for Listening! Professor Fareed, Loyola University : Provision of r-hirudin and argatroban Professor Nowak, Friedrich-Schiller- Universität Jena: Ecarin Reagent Dade Behring: APTT Reagent Pharmanetics: TAS analysers and ECT test cards Instrumentation Laboratory/Haemochrom Diagnostica GmbH: S2336 chromogenic kit American Diagnostics: r-hirudin ELISA kit John Hogwood, NIBSC 6