1. 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
Pharmaceutics & Novel Drug Delivery Systems
March 16-18, 2015 Crowne Plaza, Dubai, UAE

2. 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

3. 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?

4. 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 ?

5. 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

6. 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%

7. 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; 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)

8. Degradation of the Drug
Ticagrelor standard solution
Photolysis
Oxidative conditions
Thermolysis

9. 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

10. 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

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

12. Ticagrelor fragmentation pattern 1/2
Positive ion mode
ESI – Orbitrap / MSn
[C23H29F2N6O4S]
[C20H23F2N6O4S]
[C20H23F2N4O4S]
MS2  MS3  MS4  …

13. Ticagrelor fragmentation pattern 2/23 1 2 4 5

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

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

16. Proposition of the degradation routes of the drug
Photolysis
Oxydative
Thermolysis

17. Degradation route of the drug under Light condition

18. DP3
DP8
DP7

19. Under Oxidation

20. 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

21. 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

22. 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,

23. 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,

24. 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

25. Many Thanks for Your Attention