Identification of the major degradation pathways of ticagrelor

doi:10.1016/j.jpba.2014.11.046

Journal of Pharmaceutical and Biomedical Analysis

Volume 105, 25 February 2015, Pages 74-83

https://doi.org/10.1016/j.jpba.2014.11.046 Get rights and content

Highlights

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Stability-indicating assay method (SIAM) was developed for API and impurity determination.
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Degradation kinetics was studied.
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Degradations products structures were elucidated using LC/ESI-MSⁿ and HR-MS.
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Degradation pathways were proposed.

Abstract

Ticagrelor is a direct-acting and reversible P2Y₁₂-adenosine diphosphate (ADP) receptor blocker used as antiplatelet drug. Forced degradation under various stress conditions was carried out. The degradation products have been detected and identified by high-pressure liquid chromatography multistage mass spectrometry (LC-MSⁿ) along with high-resolution mass spectrometry. C₁₈ XTerra MS column combined with a linear gradient mobile phase composed of a mixture of 10 mM acetate ammonium/acetonitrile was shown suitable for drug and impurity determinations and validated as a stability indicating method. Structural elucidation of the degradation products relied on MSⁿ studies and accurate mass measurements giving access to elemental compositions. Up to nine degradation products resulting from oxidation/auto-oxidation, S-dealkylation and N-dealkylation have been identified, covering a range of possible degradation pathways for derivatives with such functional groups. Kinetics was also studied in order to assess the molecule's shelf-life and to identify the most important degradation factors.

Graphical abstract

Introduction

Ticagrelor, (1S,2S)-3-[7-[[(1R,2S)-2-(3,4-difluorophényl)cyclopropyl]amino]-5-propylsulfanyltriazolo[4,5-d]pyrimidin-3-yl]-5-(2-hydroxyéthoxy)cyclopentane-1,2-diol is a new antiplatelet agent, the first reversible P2Y12-adenosine diphosphate (ADP) receptor blocker indicated in the treatment of acute coronary syndromes (ACS) [1], [2]. Until recently, clopidogrel was the key P2Y12 antagonist advocated, but recent large-scale randomized trials have demonstrated net clinical benefit of the new antiplatelet agents (ticagrelor and prasugrel) with respect to clopidogrel [2], [3]. Therefore, the European guidelines recommend ticagrelor and prasugrel as first-line treatment in patients presenting with non-ST segment elevation ACS and ST-segment elevation ACS [4], [5]. Ticagrelor provides fast, great and more consistent ADP-receptor inhibition.

Even properly designed, marketed drugs undergo environmental stresses, which may result in a loss of activity and cause adverse effects owing to the advent of degradation products. One of the best ways to apprehend the phenomenon and to anticipate risks is to get access to degradation pathways in order to determine intrinsic stability of drugs, according to ICH recommendations [6]. LC-MSⁿ in conjunction with accurate mass measurements has been increasingly used for structural characterization of degradation products [7]. The literature shows scant information on stability and degradation behaviour of ticagrelor. Its metabolites have been described in plasma using hyphenated LC-MS [8], [9]. An LC stability indicating method was developed to determine ticagrelor in presence of its degradation products, but structural elucidation was not described [10].

The pharmaceutical importance of ticagrelor as an antiplatelet drug has prompted us to carry out a characterization study of its degradation products generated under harsh conditions, by using LC–MSⁿ along with high-resolution MS. A stability indicating method intended to drug quantification and impurity determination was also developed and validated. Kinetics was performed in order to assess the molecule's shelf-life and to identify the most important degrading factors.

Section snippets

Instrumentation

A reversed-phase liquid chromatography (LC) method with mass spectrometry (MS) detection was implemented to study the impurity profile of ticagrelor. LC-UV allowed for routinely drug quantification in presence of its potential degradation products and for impurity determination.

The LC system (Dionex, Les Ulis, France) consists of a quaternary pump, a vacuum degasser, a photo-diode array (PDA) detector and an autosampler. Thereof is piloted by Chromeleon® software version 6.80 SR11 (Dionex, Les

LC-MS conditions and performance

The main objective was to separate ticagrelor from its degradation products and the major degradation products from each other. After several trials with different columns, C18 XTerra MS column (50 mm × 4.6 mm i.d., 2.5 μm) was found suitable for the separation of the aforementioned compounds. During the optimization process using this column, several conditions with various mobile phases like methanol/water and acetonitrile/water in different proportions were tested in isocratic mode. Optimal

Conclusion

The degradation behaviour of ticagrelor under hydrolytic (acid, base), oxidative, photolytic and thermal stress conditions was studied as per ICH guidelines. A stability-indicating LC method was developed and has shown suitable for drug quantification as well as for impurity determination. Kinetics results showed that ticagrelor was extremely fragile with respect to light and oxidative conditions. A total of nine degradation products were highlighted. Based on the fragmentation schemes and some

Acknowledgments

The authors thank Audrey Solgadi (Mass Spectrometry Platform–Institut Paris-Sud d’Innovation Thérapeutique) and Edouard Burtet (Department of Pharmacy, Pitié-Salpêtrière Hospital–APHP) for their highly appreciated contributions to this work and their advices.

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Starting from preliminary results, it was possible to gather information related to the chromatographic behaviour of compounds to be analysed. Although TIC susceptibility to stressed media has already been reported (Bueno et al., 2017; Kumar et al., 2016; Yaye et al., 2015), a new study was conducted in order to standardize the experimental conditions and to evaluate the main degradation products formed in each condition. One important feature verified during initial LC analyses was the necessity of working with mobile phase at a pH higher than 8.0.
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In the case of TIC, a single patent exists describing four polymorphs called I, II, III and IV melting at 128, 135, 139 and 150 °C respectively (Bohlin et al., 2003). Previously, the chemical behaviour of TIC under several stress conditions has been investigated, which resulted in the elucidation of nine main degradation products formed through oxidation, S-dealkylation and N-dealkylation mechanisms (Sadou Yaye et al., 2015). Taking into account the physical and chemical properties of the pure compounds, mixtures of ASA and TIC will be investigated using differential scanning calorimetry (DSC) and liquid chromatography-high resolution-multistage mass spectrometry (LC-HR-MSn) and as complementary techniques Fourier Transform Infrared measurements (FTIR) and thermogravimetric analysis (TGA).
The mainstay treatment for patients with acute coronary syndrome is an oral route dual antiplatelet therapy with a P2Y12-receptor antagonist and Aspirin (ASA). To improve patient adherence to such treatments, combination therapies (polypill) are envisioned. Physicochemical solid-state studies have been carried out to develop a preformulation strategy of ASA with the P2Y12-receptor antagonist Ticagrelor (TIC). The investigations were carried out using differential scanning calorimetry, liquid chromatography-high resolution-multistage mass spectrometry (LC-HR-MSⁿ) and as complementary techniques Fourier transform infrared measurements and thermogravimetric analysis. A simple eutectic transition at 98 °C with a mole fraction for the eutectic liquid of 0.457 has been observed and the mixing of ASA and TIC molecules in each other's crystal structures appears to be limited. No cocrystals of TIC and ASA have been found. The appearance of the eutectic liquid was linked with a clear onset of chemical instability of the two pharmaceuticals. The decomposition mechanism in the liquid phase involves prior decomposition of ASA, whose residues react with well-identified TIC interaction sites. Seven interaction products were observed by LC-HR-MSⁿ linked to corresponding degradation products. The most important degradation pathway is N-dealkylation. In conclusion, polypills of ASA and TIC are a viable approach, but the decomposition of ASA should be avoided by eliminating high temperatures and high humidity.
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Literature data describes few studies about analytical methods for ticagrelor impurities control. Sadou Yaye and collaborators (Sadou et al., 2015) developed a stability-indicating analytical method by HPLC for the quantification of ticagrelor and determination of its impurity profile with ultraviolet detection. In the study, the authors elucidated nine related substances, which were formed after exposure ticagrelor tablets to different stress conditions (Sadou et al., 2015).
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¹: Both authors contributed equally to this study and are therefore considered as first authors.

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