Metabolism of ticagrelor in patients with acute coronary syndromes

Ticagrelor is a state-of-the-art antiplatelet agent used for the treatment of patients with acute coronary syndromes (ACS). Unlike remaining oral P2Y12 receptor inhibitors ticagrelor does not require metabolic activation to exert its antiplatelet action. Still, ticagrelor is extensively metabolized by hepatic CYP3A enzymes, and AR-C124910XX is its only active metabolite. A post hoc analysis of patient-level (n = 117) pharmacokinetic data pooled from two prospective studies was performed to identify clinical characteristics affecting the degree of AR-C124910XX formation during the first six hours after 180 mg ticagrelor loading dose in the setting of ACS. Both linear and multiple regression analyses indicated that ACS patients presenting with ST-elevation myocardial infarction or suffering from diabetes mellitus are more likely to have decreased rate of ticagrelor metabolism during the acute phase of ACS. Administration of morphine during ACS was found to negatively influence transformation of ticagrelor into AR-C124910XX when assessed with linear regression analysis, but not with multiple regression analysis. On the other hand, smoking appears to increase the degree of ticagrelor transformation in ACS patients. Mechanisms underlying our findings and their clinical significance warrant further research.

Two cytochrome P450 enzymes, CYP3A4 and CYP3A5, are responsible for the formation of AR-C124910XX 18 . Strong CYP3A inducers and inhibitors greatly affect pharmacokinetics of ticagrelor and its active metabolite, thus their co-administration with ticagrelor is contraindicated 19,20 . In healthy volunteers the bioavailability of AR-C124910XX ranges from 35% to 40% of ticagrelor plasma concentration, whereas in patients with AMI it may be as low as 21% during the initial hours of treatment 8,9,21 . Although ticagrelor may increase bioavailability of some statins, currently there are no data indicating that any of the medications routinely used for the treatment of ACS may affect metabolism of ticagrelor 22 . This also applies to morphine, the elementary analgesic used to relive chest pain in patients with ACS 21 . Therefore, it remains obscure what causes this discrepancy in the bioavailability of AR-C124910XX between healthy subjects and patients with ACS.
In the current study we sought to identify demographic and clinical factors affecting metabolism of ticagrelor into its active metabolite during the first six hours after ticagrelor loading dose (LD) in patients with ACS.

Methods
Study design. A post hoc analysis of integrated patient-level data pooled from two prospective, phase IV, single center, investigator-initiated trials evaluating pharmacokinetics of ticagrelor in patients with ACS (NCT02612116 -randomized, open-label, active-controlled study, n = 48; NCT02602444 -observational study, n = 73) was performed to determine whether ticagrelor metabolism in patients with ACS is affected by any of the recorded clinical characteristics. Both trials were conducted in accordance with the principles contained in the Declaration of Helsinki and Good Clinical Practice guidelines. The studies were approved by The Ethics Committee of Nicolaus Copernicus University in Toruń, Collegium Medicum in Bydgoszcz, Poland (NCT02612116 -approval number KB 540/2015; NCT02602444 -approval number KB 617/2015). Written informed consent was obtained from each patient prior to any study specific procedures. Study protocols with full lists of inclusion and exclusion criteria, detailed description of methodology, and results of the two included trials were previously published in peer-reviewed journals 14,[23][24][25] .
In line with the available literature 8,9 , the ratio of area under the plasma concentration-time curve for AR-C124910XX (AUC M ) to AUC for ticagrelor (AUC T ) was chosen as a marker of the degree of ticagrelor transformation into its active metabolite ( )   4 . To avoid any potential cofactors affecting hepatic metabolism of ticagrelor, patients on therapy with any strong CYP3A inhibitors or inducers were not included in either of the studies. All subjects were P2Y12 receptor inhibitor-naive at the time of enrolment for each study, and received 180 mg LD of ticagrelor as a part of dual antiplatelet therapy with aspirin. Overall 121 ACS patients were screened, and 117 were eventually included in the analysis (Fig. 1). Patients with no detectable ticagrelor in plasma within the observation period were not included in the analysis.

Concomitant treatment.
During their participation in the trials, all patients were treated according to the European Society of Cardiology (ESC) guidelines 4,27 . Standard therapy included aspirin, beta-blockers, statins, and angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers, if not contraindicated. During the periprocedural period, all study participants received unfractionated heparin in body weight adjusted dose according to the ESC recommendations. To ensure that no potential drug-drug interactions affect the results of this analysis, each patient's individual data were screened to identify any treatment with medications known to interplay with metabolism of ticagrelor. However, after a careful inspection none of the patients from both trials was excluded from the analysis due to this reason.

Pharmacokinetic evaluation.
Blood samples for the pharmacokinetic assessment were obtained at prespecified time points before and after 180 mg ticagrelor LD, using an 18-gauge venous catheter inserted into a forearm vein (at baseline, 30 minutes, 1, 2, 3, 4 and 6 hours after ticagrelor LD in both studies, and additionally at 15 and 45 minutes after ticagrelor LD in patients included in NCT02612116 study) ( Fig. 2) 23,24 . The samples were collected into lithium-heparin vacuum test tubes and immediately after collection each sample was placed on dry ice and transferred to the central laboratory. Subsequently, within 20 min from collection, blood specimens were centrifuged at 1500 g for 12 min at 4 °C. Within 10 min postcentrifugation, obtained plasma samples were stored at temperature below −60 °C until analyzed. Ticagrelor and AR-C124910XX plasma concentrations were analyzed with liquid chromatography coupled with tandem mass spectrometry, using a validated method 13,28 . Lower limits of quantification were 4.69 ng/mL for both ticagrelor and AR-C124910XX.
Statistical analysis. Statistical  was evaluated for the period from 0 to 6 hours. Continuous variables were compared between both study arms with the Student's t-test and Mann-Whitney  Data availability. The dataset from NCT02602444 trial analyzed during the current study is available in the figshare.com repository (doi:10.6084/m9.figshare.5396989). The dataset from NCT02612116 trial analyzed during the current study are available from the corresponding author on reasonable request.  Table 1  in patients co-administered with morphine: −3.0%, 95% CI −5.0% to −1.0%, p = 0.004) ( Table 2).

Discussion
Our analysis of patient-level pharmacokinetic data from two prospective trials revealed that ACS patients presenting with STEMI or suffering from DM are more likely to have decreased ticagrelor metabolism rate during the acute phase of ACS. Analgesic treatment with morphine in this setting was found to negatively influence transformation of ticagrelor into AR-C124910XX when assessed with linear regression analysis, but this effect was not significant anymore when evaluated with multiple regression analyses. On the other hand, smoking   in ACS patients. Our study shows that increased bioavailability of crushed ticagrelor tablets does not influence the rate of AR-C124910XX formation following ticagrelor LD in ACS patients 25,32,33 . Admittedly, all STEMI patients included in our study received integral ticagrelor tablets, therefore results of our linear regression analysis in regard to ticagrelor pill formulation may not be applicable to this ACS subtype and require further investigation.
Excessive activation of thrombocytes is one of the main underlying pathomechanisms of ACS. High platelet reactivity (HPR) is a risk factor for adverse thrombotic events in ACS patients, including stent thrombosis, a potentially fatal complication of PCI 2 . Moreover, HPR can also be associated with increased mortality in ACS patients 34 . Thus, obtaining a quick and powerful platelet blockade is necessary from the very early stage of ACS treatment, even in conservatively managed patients. Ticagrelor, the first line P2Y12 receptor inhibitor in ACS, thanks to its favorable pharmacokinetics provides a strong antiplatelet action swiftly after oral ingestion. It has to be noted though, that ticagrelor absorption and onset of action are delayed in patients with ACS compared with healthy volunteers or patients with stable coronary artery disease.
Although ticagrelor is responsible for the majority of the observed antiplatelet effect, its active metabolite is equipotent in P2Y12 receptor inhibition and also plays an important role in limiting excessive platelet activation 9 . Ticagrelor is metabolized and transformed into AR-C124910XX predominantly by CYP3A4 and CYP3A5 enzymes 18 . Therefore, metabolism of ticagrelor is affected by concomitant administration of strong CYP3A inducers or inhibitors. CYP3A inducers may essentially decrease total exposure to ticagrelor and its active metabolite even by 86% and 46%, respectively, as seen in subjects receiving rifampicin 19 . On the other hand, strong CYP3A4 inhibitors, for example ketoconazole, may increase ticagrelor exposure by 632%, simultaneously decreasing bioavailability of AR-C124910XX by 56% 20 . Due to the described drug-drug interactions, use of ticagrelor together with powerful CYP3A4 inducers and inhibitors is contraindicated. On the other hand, although certain genetic variants have been shown to modestly affect ticagrelor and AR-C124910XX pharmacokinetics in the setting of ACS, they were not associated with any detectable effect on efficacy or safety during ticagrelor treatment 35 .
The impact of different clinical characteristics on ticagrelor pharmacokinetics has been evaluated in several previous trials. A study by Teng et al. showed that in healthy volunteers the overall exposure to AR-C124910XX after a single 200 mg loading dose of ticagrelor was 37% higher in women than in men, and 48% higher in elderly than in young individuals, however these differences were proportional to ticagrelor bioavailability between the groups 29 . In a different trial, patients with severe renal impairment (GFR < 30 mL/min) compared with volunteers with normal renal function (GFR > 80 mL/min) had 20% lower ticagrelor and 17% higher AR-C124910XX bioavailability following a single 180 mg LD of ticagrelor. Subsequently, patients with impaired renal function had 48% higher metabolite to parent ratio for AUC 30 . Butler and Teng also demonstrated that patients with severe hepatic impairment (Child-Pugh class A) had 23% and 66% higher total bioavailability of ticagrelor and its metabolite, respectively, than volunteers with normal liver function after single 90 mg ticagrelor dose 36 . Moreover, population pharmacokinetic model developed for ticagrelor indicated that smoking decreases apparent ticagrelor clearance, simultaneously increasing apparent clearance of AR-C124910XX 31 . Despite the pharmacokinetic differences observed in the aforementioned studies a substantial platelet inhibition was obtained regardless of gender, age, presence of severe renal or mild hepatic impairment, or smoking status, and no adjustment in ticagrelor dose was considered necessary in the described populations 11,29,30,36 . Nevertheless, it has to be underlined that these observations were made based on the results obtained from individuals without ACS. Recent pharmacokinetic and pharmacodynamic studies clearly show that presence of ACS, and especially STEMI, delays and decreases bioavailability, onset of action and antiplatelet effect of ticagrelor [13][14][15][16]37 . It is established that presence of HPR, which may be caused by impaired ticagrelor pharmacokinetics, is related to adverse thrombotic events in patients with ACS 2,34 .
In subanalysis of the IMPRESSION study we have reported that although morphine administration did not affect AUC AUC M T in patients with AMI, the observed AUC AUC M T values were lower than these reported for healthy volunteers (21-25% vs. 35-40%) 8,9,21 . Similarly, in the current analysis the mean AUC AUC M T was approximately 20% for the whole study population. It could be speculated that it is due to delayed absorption of ticagrelor in ACS combined with relatively short window of observation (the first six hours after ticagrelor LD) compared with trials in healthy volunteers.
In line with previous studies, the results of the current analysis show that presence of STEMI essentially disturbs pharmacokinetic profile of ticagrelor 13,14,16 . From all examined clinical features STEMI had the greatest negative impact on AUC AUC M T (beta-coefficient for supplementary multiple regression analysis = −0.38). Our results show that the mechanism of impaired ticagrelor pharmacokinetics seen in patients with STEMI during the initial hours after the LD may be multifactorial and related not only to slower intestinal absorption, but also to diminished hepatic metabolism. Interestingly, presence of DM was also found to have significant negative influence on Morphine follows metabolic pathway which does not include CYP3A enzymes, thus clinically relevant interaction with ticagrelor metabolism is rather unlikely 38 . Our supplementary multiple regression analysis model included smoking as a potential factor influencing ticagrelor metabolism, based on a pharmacokinetic model for ticagrelor and its metabolite suggesting that smoking alters apparent clearance of ticagrelor and AR-C124910XX 31 . In our analysis habitual smoking was the only clinical characteristic that positively affects AUC AUC M T (beta-coefficient for supplementary multiple regression analysis = 0.20). The mechanism behind this finding also remains undetermined as smoking does not affect CYP3A-mediated metabolism 31 .
In subgroup analyses of the PLATO study the cardiovascular benefit was consistent in patients treated with ticagrelor compared with those treated with clopidogrel irrespective of type of treatment for STEMI or NSTE-ACS, diabetic status and glycemic control, or smoking status [39][40][41][42] . Thus, the clinical significance of our findings is obscure, uncertain and warrants further research. Study limitations. One could perceive lack of pharmacodynamic data as a limitation of this analysis, however it was the authors' intention to focus on ticagrelor metabolism only. It also has to be noted that the ethnicity of the examined group was homogenous (100% Caucasian), which closely reflects the ethnical structure seen in Poland, where both analyzed studies were conducted. Additionally, smoking status was collected only based on information obtained from each patient, and was not validated objectively.
Moreover, several limitations resulted from the design of the included trials. Neither of two studies evaluated clinical endpoints, thus these data were not available. The information on the time span between the onset of chest pain and ticagrelor loading dose was incomplete, and therefore was not included in the analysis. The liver function was not evaluated at the time of enrolment for the analyzed studies, and the exclusion due to liver function impairment was made based on medical history of severe hepatic disease only.

Conclusions
The degree of ticagrelor hepatic transformation into its active metabolite following a standard 180 mg LD in the setting of ACS is decreased in patients with STEMI and DM. Morphine may be related to smaller, and smoking with greater rate of ticagrelor metabolism in patients with ACS, but these data are not completely conclusive.