Predictive safety study of ticagrelor's degradation combining LC-MS and in silico approach H. SADOU YAYE*, B. DO, N. YAGOUBI * Pharmacist at Pitié-Salpêtrière Hospital (Paris) PhD student – University Paris Sud http://www.u-psud.fr/en/index.html syhassane@yahoo.fr Pharmaceutics & Novel Drug Delivery Systems March 16-18, 2015 Crowne Plaza, Dubai, UAE

Outline of talk Introduction Ticagrelor Stability Test Design Stress testing conditions Optimisation of LC ans MS conditions Degradation of the Drug DPs structural elucidation Ticagrelor fragmentation pattern Structural elucidation and degradation pathways In Silico toxicological assessment Summary

Introduction Ticagrelor, a nucleoside analogue, new antiplatelet agent Indicated in the treatment of acute coronary syndromes (ACS) Determination of the intrinsic stability Characterization of degradation products under harsh conditions by LC-MSn / HRMS Rationale of stress testing?

Relevant specifications Stability indicating method Stability studies API STRESS STUDIES Major degradation products Degradation pathways Appropriate analytical strategy for QC and the formal stab. studies Potential degradation products In solid-state In solution Chemical decomposition Physical stability (polymorphism, hygroscopicity) (pH, ionic strength…) Relevant specifications to assess drug stab. via formal Stability indicating method suitable for formal stab. studies (API & impurity contents) Formulation strategy Manufacturing conditions Storage conditions Packaging choice Tocixity ?

Outline of talk Introduction Ticagrelor Stability Test Design Stress testing conditions Optimisation of LC ans MS conditions Degradation of the Drug DPs structural elucidation Ticagrelor fragmentation pattern Structural elucidation and degradation pathways In Silico toxicological assessment Summary

Stress testing conditions ICH, Q1A (R2), Stability Testing of New Drug Substances and Products : stress studies to establish the inherent stability characteristics Thermolysis Photolysis Hydrolysis Oxydation Degradation Threshold < 15%

Optimisation of LC conditions Why? No idea No database No standards compounds Just scan data : std and stressed solutions How? Column : C18 Hybrid Xterra MS WATERS (50 mm x 4.6 mm i.d., 2.5 µm), 25 °C Mobile phase : ACN (solvent A) and ammonium acetate 10 mM (solvent B), in gradient mode (0-2 min: 1015% A; 2-8 min: 1570% A; 8-10 min: 7015% A; 10-11 min: 1510% A). RP-HPLC-UV SIAM developed and validated according to ICH Q2 (R1) for drug quantitation (108 µg mL-1 – 252 µg mL-1) and the impurities (0.108 µg mL-1 – 0.18 µg mL-1)

Degradation of the Drug Ticagrelor standard solution Photolysis Oxidative conditions Thermolysis

Degradation of the Drug Ticagrelor intrinsic stability - 55% 21D storage, t1/2 = 1125 h (DP1, DP3) 12 h-period, t1/2 = 7.45 h (DP2, DP4, DP5) -77 % after 4 h-period exposure, t1/2 = 1.92 h (DP1, DP3, DP6, DP7, DP8, DP9) Zero-order kinetics : k = (A0 - At) / t

Outline of talk Introduction Ticagrelor Stability Test Design Stress testing conditions Optimisation of LC ans MS conditions Degradation of the Drug DPs structural elucidation Ticagrelor fragmentation pattern Structural elucidation and degradation pathways In Silico toxicological assessment Summary

Strategy for the characterization of DPs LC-MS Accurate mass measurements Multistage Mass Spectrometry Studies Fragmentation pattern of the drug

Ticagrelor fragmentation pattern 1/2 Positive ion mode ESI – Orbitrap / MSn [C23H29F2N6O4S]+ 523.1926 [C20H23F2N6O4S]+ 481.2612 [C20H23F2N4O4S]+ 453.2088 MS2  MS3  MS4  …

Ticagrelor fragmentation pattern 2/2 3 1 2 4 5

DPs structural elucidation : Example of DP1 m/z 345 [C14H25N4O4S] + DP1  [C23H29F2N6O4S]+ TICAGRELOR

DPs structural elucidation : Example of DP5 m/z 555 [C23H29F2N6O6S]+ DP5 [C23H29F2N6O4S]+ TICAGRELOR

Proposition of the degradation routes of the drug Photolysis Oxydative Thermolysis

Degradation route of the drug under Light condition

DP3 DP8 DP7

Under Oxidation

Outline of talk Introduction Ticagrelor Stability Test Design Stress testing conditions Optimisation of LC ans MS conditions Degradation of the Drug DPs structural elucidation Ticagrelor fragmentation pattern Structural elucidation and degradation pathways In Silico toxicological assessment Summary

Toxicological assessment : In Silico approach Toxicological endpoints: Mutagenicity, Carcinogenicity, Reproductive toxicity QSAR Toolbox and TEST Software for Ticagrelor and it’s DPs QSAR Toolbox TEST Denomination (m/z) Carcinogenicity / Mutagenicity DNA Binding Protein Binding Ames mutagenicity Developmental Toxicity Ames Mutagenicity TICAGRELOR Negative DP1 DP2 Positive DP3 DP4 DP5 DP6 DP7 DP8 DP9

Toxicological assessment : In Silico approach Toxicological endpoints: Mutagenicity, Carcinogenicity, Reproductive toxicity QSAR Toolbox and TEST Software for Ticagrelor and it’s DPs QSAR Toolbox TEST Denomination (m/z) Carcinogenicity / Mutagenicity DNA Binding Protein Binding Ames mutagenicity Developmental Toxicity Ames Mutagenicity Ticagrelor Negative DP1 DP2 Positive DP3 DP4 DP5 DP6 DP7 DP8 DP9 Benigni, R., et al. (2000). Quantitative structure-activity relationships of mutagenic and carcinogenic aromatic amines. Chem.Revs. 100, 3697-3714.

Toxicological assessment : In Silico approach Toxicological endpoints: Mutagenicity, Carcinogenicity, Reproductive toxicity QSAR Toolbox and TEST Software for Ticagrelor and it’s DPs QSAR Toolbox TEST Denomination (m/z) Carcinogenicity / Mutagenicity DNA Binding Protein Binding Ames mutagenicity Developmental Toxicity Ames Mutagenicity Ticagrelor Negative DP1 DP2 Positive DP3 DP4 DP5 DP6 DP7 DP8 DP9 Benigni, R., et al. (2000). Quantitative structure-activity relationships of mutagenic and carcinogenic aromatic amines. Chem.Revs. 100, 3697-3714.

Summary Stress conditions TICAGRELOR Risk assessment DEGRADATION PRODUCTS Structural elucidation Risk assessment Instrinsic Stability Science Based Data SIAM for drug and impurity determinations In silico toxicological study TICAGRELOR

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