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

syhassane@yahoo.fr

Pharmaceutics & Novel Drug Delivery SystemsMarch 16-18, 2015 Crowne Plaza, Dubai, UAE

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• 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

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• 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

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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 ?

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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

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• 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

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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)

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Degradation of the Drug

Ticagrelor standard solution Photolysis

Oxidative conditions Thermolysis

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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

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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

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Strategy for the characterization of DPs

• LC-MS• Accurate mass measurements• Multistage Mass Spectrometry Studies• Fragmentation pattern of the drug

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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

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Ticagrelor fragmentation pattern 2/2

3

1

2

4

5

3

3

3

3

3

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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 345.20@cid35.00 [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

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DPs structural elucidation : Example of DP5 m/z 555

[C23H29F2N6O4S]+ TICAGRELOR[C23H29F2N6O6S]+ DP5

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• Photolysis

• Oxydative

• Thermolysis

Proposition of the degradation routes of the drug

Degradation route of the drug under Light condition

DP3

DP8DP7

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Under Oxidation

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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

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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

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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.

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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

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Many Thanks for Your Attention