predictive safety study of ticagrelor's degradation combining lc-ms and in silico approach h....
TRANSCRIPT
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 Sudhttp://www.u-psud.fr/en/index.html
Pharmaceutics & Novel Drug Delivery SystemsMarch 16-18, 2015 Crowne Plaza, Dubai, UAE
2
• Introduction• Ticagrelor Stability Test Design
o Stress testing conditionso Optimisation of LC ans MS conditions o Degradation of the Drug
• DPs structural elucidationo Ticagrelor fragmentation patterno Structural elucidation and degradation pathways
• In Silico toxicological assessment • Summary
Outline of talk
3
• 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?
Introduction
4
Stability studies
API STRESS STUDIES
Major degradation products
Degradation pathwaysAppropriate
analytical strategy for QC and the formal
stab. studiesPotential
degradation products
In solid-stateIn solution
Chemical decomposition Physical stability(polymorphism, hygroscopicity)
Physical stability(pH, ionic strength…)
Relevant specificationsto assess drug stab. via formal
stab. studies
Stability indicating methodsuitable for formal stab.
studies(API & impurity contents)
Formulation strategyManufacturing conditions
Storage conditionsPackaging choice
Tocixity ?
5
Outline of talk• Introduction• Ticagrelor Stability Test Design
o Stress testing conditionso Optimisation of LC ans MS conditions o Degradation of the Drug
• DPs structural elucidationo Ticagrelor fragmentation patterno Structural elucidation and degradation pathways
• In Silico toxicological assessment • Summary
6
• 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%
Stress testing conditions
7
Optimisation of LC conditions Why? o No idea o No database o No standards compounds o Just scan data : std and stressed solutions
How? o Column : C18 Hybrid Xterra MS WATERS (50 mm x 4.6 mm i.d., 2.5
µm), 25 °Co Mobile phase : ACN (solvent A) and ammonium acetate 10 mM
(solvent B), in gradient mode (0-2 min: 1015% A; 2-8 min: 1570% A; 8-10 min: 7015% A; 10-11 min: 1510% A).
o 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)
9
Degradation of the Drug
0.4 0.9 1.4 1.9 2.4 2.9 3.4 3.9 4.40.05
0.1
0.15
0.2
0.25
0.3
f(x) = − 0.000282467300901887 x + 0.284819752601526R² = 0.986074429771919
f(x) = − 0.0405175417201808 x + 0.25002837036941R² = 0.937490808396713
f(x) = − 0.0143000568052568 x + 0.281559060083739R² = 0.996385346166757f(x) = − 0.0143000568052568 x + 0.281559060083739R² = 0.996385346166757
H2O2 0,03%Linear (H2O2 0,03%)Linear (H2O2 0,03%)Linear (H2O2 0,03%)PhotodegradationLinear (Photodegradation)Linear (Photodegradation)Linear (Photodegradation)TemperatureLinear (Temperature)
Time (h)
[Tica
grel
or] (
mol
/L)
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
o Stress testing conditions o Optimisation of LC ans MS conditions o Degradation of the Drug
• DPs structural elucidationo Ticagrelor fragmentation patterno 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
MS2 MS3 MS4 …
• Positive ion mode• ESI – Orbitrap / MSn [C23H29F2N6O4S]+ 523.1926
[C20H23F2N6O4S]+ 481.2612
[C20H23F2N4O4S]+ 453.2088
20140701_ticagrelor_fs_1 #1-20 RT: 0.00-0.26 AV: 20 NL: 6.12E7T: FTMS + p ESI Full ms [150.00-800.00]
200 250 300 350 400 450 500 550 600 650 700 750 800m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
523.1926
545.1741
205.0679
437.2347393.2089
481.2612293.0989 349.1827
194.0708
305.1568569.3130453.2088409.1826
724.2447633.2051
365.1566
261.1302 795.2699585.2869 657.3654185.1146 321.1304
217.1043
701.3915 743.2179
505.2610
14
DPs structural elucidation : Example of DP1 m/z 34520140701_ticagrelor_photo_ms_10 #1-20 RT: 0.01-0.35 AV: 20 NL: 3.52E4F: FTMS + p ESI Full ms2 [email protected] [95.00-800.00]
150 200 250 300 350 400 450 500 550 600 650 700 750 800m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
327.1481
303.1118
285.1013
265.1111
185.0852
247.1007
211.1009
345.1585 667.3963544.1845 715.3857 791.6190512.7149 588.5270390.5319 448.1610
[C14H25N4O4S] + DP1
[C23H29F2N6O4S]+ TICAGRELOR
N
N
NH
N
N
N
OOH
SCH3
OH OH
F
F
15
DPs structural elucidation : Example of DP5 m/z 555
[C23H29F2N6O4S]+ TICAGRELOR[C23H29F2N6O6S]+ DP5
20
Outline of talk• Introduction• Ticagrelor Stability Test Design
o Stress testing conditionso Optimisation of LC ans MS conditions o Degradation of the Drug
• DPs structural elucidationo Ticagrelor fragmentation patterno Structural elucidation and degradation pathways
• In Silico toxicological assessment • Summary
• Toxicological endpoints: Mutagenicity, Carcinogenicity, Reproductive toxicity
• QSAR Toolbox and TEST Software for Ticagrelor and it’s DPs
21
Toxicological assessment : In Silico approach
QSAR Toolbox TEST Denomination
(m/z)Carcinogenicity /
MutagenicityDNA
BindingProtein Binding
Amesmutagenicity
Developmental Toxicity
Ames Mutagenicity
TICAGRELOR Negative Negative Negative Negative Negative Negative
DP1 Negative Negative Negative Negative Negative Negative
DP2 Negative Negative Positive Negative Positive Negative
DP3 Positive Negative Negative Positive Negative Positive
DP4 Positive Positive Negative Positive Negative Negative
DP5 Negative Negative Negative Negative Positive Negative
DP6 Negative Negative Negative Negative Positive Positive
DP7 Positive Negative Negative Positive Negative Positive
DP8 Positive Negative Negative Positive Negative Positive
DP9 Positive Negative Negative Positive Negative Positive
22
Toxicological assessment : In Silico approach
QSAR Toolbox TEST Denomination
(m/z)Carcinogenicity /
MutagenicityDNA
BindingProtein Binding
Amesmutagenicity
Developmental Toxicity
Ames Mutagenicity
Ticagrelor Negative Negative Negative Negative Negative Negative
DP1 Negative Negative Negative Negative Negative Negative
DP2 Negative Negative Positive Negative Positive Negative
DP3 Positive Negative Negative Positive Negative Positive
DP4 Positive Positive Negative Positive Negative Negative
DP5 Negative Negative Negative Negative Positive Negative
DP6 Negative Negative Negative Negative Positive Positive
DP7 Positive Negative Negative Positive Negative Positive
DP8 Positive Negative Negative Positive Negative Positive
DP9 Positive Negative Negative Positive Negative Positive
• Toxicological endpoints: Mutagenicity, Carcinogenicity, Reproductive toxicity
• QSAR Toolbox and TEST Software for Ticagrelor and it’s DPs
Benigni, R., et al. (2000). Quantitative structure-activity relationships of mutagenic and carcinogenic aromatic amines. Chem.Revs. 100, 3697-3714.
23
Toxicological assessment : In Silico approach
QSAR Toolbox TEST Denomination
(m/z)Carcinogenicity /
MutagenicityDNA
BindingProtein Binding
Amesmutagenicity
Developmental Toxicity
Ames Mutagenicity
Ticagrelor Negative Negative Negative Negative Negative Negative
DP1 Negative Negative Negative Negative Negative Negative
DP2 Negative Negative Positive Negative Positive Negative
DP3 Positive Negative Negative Positive Negative Positive
DP4 Positive Positive Negative Positive Negative Negative
DP5 Negative Negative Negative Negative Positive Negative
DP6 Negative Negative Negative Negative Positive Positive
DP7 Positive Negative Negative Positive Negative Positive
DP8 Positive Negative Negative Positive Negative Positive
DP9 Positive Negative Negative Positive Negative Positive
• Toxicological endpoints: Mutagenicity, Carcinogenicity, Reproductive toxicity
• QSAR Toolbox and TEST Software for Ticagrelor and it’s DPs
Benigni, R., et al. (2000). Quantitative structure-activity relationships of mutagenic and carcinogenic aromatic amines. Chem.Revs. 100, 3697-3714.
Stress conditions
DEGRADATION PRODUCTS
Structural elucidation
Risk assessment
N
N
NH
N
N
N
OOH
SCH3
OH OH
F
F
Instrinsic StabilityScience Based Data
SIAM for drug and impurity
determinations
In silico toxicological
study
TICAGRELOR
Summary