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Keywords:

  • adenosine 5′-diphosphate;
  • clopidogrel;
  • platelet aggregation;
  • P2Y12 receptor;
  • prasugrel;
  • thienopyridine

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

Background and methods: Prasugrel is a novel orally active thienopyridine prodrug with potent and long-lasting antiplatelet effects. Platelet inhibition reflects inhibition of P2Y12 receptors by its active metabolite (AM). Previous studies have shown that the antiplatelet potency of prasugrel is at least 10 times higher than that of clopidogrel in rats and humans, but the mechanism of its higher potency has not yet been fully elucidated.Results: Oral administration of prasugrel to rats resulted in dose-related and time-related inhibition of ex vivo platelet aggregation, and its effect was about 10 times more potent than that of clopidogrel. The plasma concentration of prasugrel AM was higher than that of clopidogrel AM despite tenfold higher doses of clopidogrel, indicating more efficient in vivo production of prasugrel AM than of clopidogrel AM. In rat platelets, prasugrel AM inhibited in vitro platelet aggregation induced by adenosine 5′-diphosphate (ADP) (10 μm) with an IC50 value of 1.8 μm. Clopidogrel AM similarly inhibited platelet aggregation with an IC50 value of 2.4 μm. Similar results were also observed for ADP-induced (10 μm) decreases in prostaglandin E1-stimulated rat platelet cAMP levels. These results indicate that both AMs have similar in vitro antiplatelet activities.Conclusions: The greater in vivo antiplatelet potency of prasugrel as compared to clopidogrel reflects more efficient in vivo generation of its AM, which demonstrates similar in vitro activity to clopidogrel AM.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

Adenosine 5′-diphosphate (ADP), released from activated platelets, is considered one of the key mediators of platelet activation and aggregation, and has been implicated as playing a substantial role in occlusive vascular events [1]. The importance of ADP is supported by the identification of P2Y12, an ADP receptor on platelets, as the target of the clinically effective thienopyridine class of antiplatelet agents, ticlopidine and clopidogrel [2]. Several lines of clinical investigation have clearly demonstrated the efficacy of ticlopidine [3] and clopidogrel [4] in reducing thrombotic events. However, clopidogrel has largely replaced ticlopidine for use in atherothrombotic diseases because of better tolerability [5]. Despite the widespread use of clopidogrel, several important issues, such as a relatively slow onset of action, and significant variability in the response to clopidogrel, remain [6,7]. The concept of clopidogrel ‘resistance’ has led to the concern that some patients may not be adequately protected from platelet activation and aggregation, and are therefore at increased risk for thrombotic events [8,9]. Because of these issues, more effective antiplatelet agents for the treatment of atherothrombotic diseases are under evaluation.

Prasugrel (CS-747, LY640315; Fig. 1) is a novel thienopyridine antiplatelet prodrug that is administered orally and has been shown in preclinical studies to be more potent and have a faster onset of action than clopidogrel [10,11]. Phase 1 studies also showed inhibition of platelet aggregation to be higher with both loading and maintenance doses of prasugrel than with clopidogrel [12,13]. Furthermore, there is evidence in both healthy subjects and stable cardiac patients that a variable response is less frequent following a loading dose of 60 mg of prasugrel than with 300 mg of clopidogrel [14,15]. Following confirmation of the apparent safety of prasugrel in patients in phase 2 studies [16], phase 3 evaluation (TRITON-TIMI 38) is now ongoing in acute coronary syndrome patients undergoing percutaneous coronary intervention [17].

image

Figure 1.  Chemical structures of prasugrel, clopidogrel and their active metabolites (AMs). Prasugrel has one chiral center in its structure and is a racemic agent. However, clopidogrel with the (S)-configuration is an enantiomer. Hydrolysis of the acetate ester moiety of prasugrel, position (a), by esterases leads to a thiolactone metabolite, the precursor to prasugrel AM, which then requires a single-step cytochrome P450 (CYP) oxidation to generate the AM. Clopidogrel is converted to its AM via a two-step CYP oxidation process. Clopidogrel also undergoes rapid hydrolysis at its methyl ester moiety, position (b), to form an inactive carboxylic acid metabolite. Each AM has a reactive thiol group that adds a second chiral center. *, chiral centers.

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Although the above features stimulated interest in the evaluation of prasugrel for the prevention of thrombotic events, the mechanism explaining its more potent antiplatelet effects requires clarification. Previous reports have clearly shown that prasugrel is inactive in vitro and that the potent and irreversible inhibitory activity after prasugrel dosing reflects P2Y12 inhibition by its active metabolite (AM) [18,19], as also reported previously for clopidogrel [20]. In the present study, we compared the potency of prasugrel vs. clopidogrel, and examined the relationship between ex vivo antiplatelet effects and plasma AM concentrations following dosing. We extended these studies by comparing the effects of both AMs on in vitro platelet aggregation and P2Y12-mediated cAMP decreases in platelets.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

Materials

Prasugrel (free form, CS-747), clopidogrel hydrogensulfate, prasugrel AM [trifluoroacetic acid salt, a mixture of the (R,S)-isomer and the (S,R)-isomer] and clopidogrel AM [free form, (R,S)-isomer] were prepared by Ube Industries Co. (Yamaguchi, Japan). These chemical structures are shown in Fig. 1. Both AMs were dissolved in dimethylsulfoxide (DMSO), resulting in a final DMSO concentration of less than 0.4%, a concentration that, in preliminary experiments, was found not to affect the assays employed in these studies. ADP (sodium salt), apyrase, fibrinogen, gum arabic and 3-isobutyl-1-methylxanthine were purchased from Sigma Chemical Co. (St Louis, MO, USA). Prostaglandin E1 (PGE1) was purchased from Funakoshi Co. (Tokyo, Japan) or Cayman Chemical Co. (St Louis, MI, USA).

As previously reported [19], prasugrel AM is composed of four stereoisomers resulting from the presence of two chiral carbons in its structure (Fig. 1). Our previous report demonstrated that all four stereoisomers have antiplatelet activity, but the (R,S)-isomer is the most potent, being at least 16 times more potent than the other three isomers. On the other hand, clopidogrel AM is composed of two stereoisomers, as the clopidogrel parent drug is a single isomer (Fig. 1). In the present study, the in vitro effects of a mixture of the (R,S)-isomer and (S,R)-isomer of prasugrel AM were compared to those of the (R,S)-isomer of clopidogrel AM. Rapid epimerization makes it very difficult to isolate the pure (R,S)-isomer of prasugrel AM.

Human volunteers and experimental animals

Healthy male volunteers who had not taken any medication for 10 days were used in the present study. Male Sprague–Dawley rats (Japan SLC, Inc., Hamamatsu, Japan) were also used in the present study. Rats were housed in animal quarters maintained at a constant temperature (23 ± 2 °C), humidity (55 ± 5%) and light/dark cycle (12 h). Rats were provided with food (F-2, Funabashi Farm Co., Ltd, Funabashi, Japan) and water ad libitum.

This study was approved by the institutional review board. Informed consent was obtained from all participants.

Ex vivo rat studies

Prasugrel and clopidogrel were suspended in a 5% (w/v) solution of gum arabic. The vehicle (5% gum arabic solution), prasugrel (0.3, 1 and 3 mg mL−1) and clopidogrel (3, 10 and 30 mg mL−1) were orally administered to non-fasted rats (= 5/each time point) in a volume of 1 mL kg−1. At 0.25, 0.5, 1, 2, 4 and 6 h after dosing, 3.6 mL of blood was collected from the abdominal aorta of rats anesthetized with pentobarbital sodium salt (40 mg kg−1, i.p.) for measurement of platelet aggregation using 0.4 mL of 3.8% (w/v) sodium citrate as the anticoagulant. A further 0.5 mL of blood was subsequently collected using a heparinized syringe for the measurement of plasma AMs.

Preparation of platelet-rich plasma (PRP) and washed platelets, and measurements of aggregation

PRP and washed platelet preparations were obtained, and subsequent aggregation studies were performed, as described previously [10,18]. Platelet aggregation was measured in a light transmission aggregometer (PAM-8C, Mebanix Co., Tokyo, Japan; HEMA TRACER 313 M, MC Medical, Inc., Tokyo, Japan), and the maximum aggregation response was recorded.

Measurement of cAMP levels

PGE1-stimulated cAMP levels in rat platelets were determined as previously described, with slight modifications [18]. PGE1 (10 μm) was added to washed platelet suspensions, and the mixture was incubated at 37 °C for 3 min, when ADP (10 μm) was added. Aliquots were taken from the mixture before and at 3 and 6 min after the PGE1 addition. These samples were quenched with HCl in EDTA solution and boiled. After neutralization with CaCO3, the final supernatants were assayed for cAMP levels using a commercially available enzyme immunoassay kit (Amersham Pharmacia Biotech, Inc., Piscataway, NJ, USA).

Measurement of prasugrel and clopidogrel AMs in plasma

The assays were performed as previously described [21,22]. Heparinized blood was centrifuged at 10 000 × g for 3 min at 4 °C to obtain plasma. Fifty microliters of sample plasma was derivatized with 4 μmol of 3′-methoxyphenacyl bromide for 10 min at room temperature, and was extracted with an SPE column. The eluate was subjected to high-pressure liquid chromatography, which was conducted using an Alliance 2690 Separations Module (Waters Corporation, Milford, MA, USA) with a reverse-phase column. Analytes were ionized in the positive ionization mode at the electrospray ionization interface, and detected in the selected reaction monitoring mode on a Finnigan TSQ API2 MS/MS system (ThermoQuest Co., San Jose, CA, USA). Data acquisition and analysis were performed using xcaliber 1.2 software (Finnigan, San Jose, CA, USA). Postacquisition quantitative analyses were performed using lcquan software (Finnigan). The lower limits of quantification were 20 ng mL–1 both for prasugrel AM and for clopidogrel AM.

Statistical analysis

Results are expressed as mean ± SEM. Statistical analysis was performed by Dunnett’s tests using a statistical software package, sas® (SAS Institute Inc., Cary, NC, USA). IC50 and ED50 values were calculated by the least squares method. The area under the plasma concentration–time curve (AUC0–t, h*ng mL−1) was calculated with winnonlin (Pharsight Corporation, Mountain View, CA, USA), using the mean AM concentration at each point. The relationship between the mean concentration of the plasma AM or AUC0–t and the mean inhibition of platelet aggregation was determined by linear regression analysis and the Pearson correlation coefficient using sas, where log AUC0–t and logit % inhibition of platelet aggregation values were used. A P-value less than 0.05 was regarded as statistically significant.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

Effects of prasugrel and clopidogrel on ex vivo platelet aggregation in rats

Prasugrel (0.3, 1 and 3 mg kg−1) and clopidogrel (3, 10 and 30 mg kg−1) were orally administered once to rats, and ex vivo platelet aggregation induced by ADP (3 μm) was measured 0.25, 0.5, 1, 2, 4 and 6 h after each dose. Prasugrel inhibited platelet aggregation induced by ADP in a dose-related manner, and the maximum inhibition of platelet aggregation was observed between 2 and 4 h after dosing, with an ED50 value 4 h postdose of 1.2 mg kg−1 (p.o.) (Fig. 2A). Following oral administration of clopidogrel, a slower onset of action was noted; in addition, the antiplatelet potency of clopidogrel was 13 times less than that of prasugrel at 4 h after dosing (ED50 = 16 mg kg−1, p.o.) (Fig. 2B). These results confirmed previous rat and human data on the relative onset and potency of prasugrel’s antiplatelet action [10,13,14].

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Figure 2. Ex vivo effects of prasugrel and clopidogrel on platelet aggregation induced by 3 μm ADP in rats. Platelet aggregation in platelet-rich plasma (PRP) was measured at 0.25, 0.5, 1, 2, 4 and 6 h after oral administration of prasugrel and clopidogrel. Results are expressed as mean ± SEM (= 5).

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Plasma concentration of prasugrel AM and clopidogrel AM in rats

To better understand the basis for the differences between the platelet inhibitory profiles of prasugrel and clopidogrel, plasma concentrations of both AMs were measured after oral administration of prasugrel (0.3, 1 and 3 mg kg−1) and clopidogrel (3, 10 and 30 mg kg−1) to rats. The plasma concentrations of prasugrel AM and clopidogrel AM increased in a dose-related manner (Fig. 3). At the lowest dose of prasugrel (0.3 mg kg−1) and clopidogrel (3 mg kg−1), AM levels were generally below the lower detection limits of the plasma AM assay. Calculated AUC0–6 h values of prasugrel were 82.0 and 356 ng*h mL−1 at 1 and 3 mg kg−1, respectively, and those of clopidogrel were 40.3 and 150 ng*h mL−1 at 10 and 30 mg kg−1, respectively. The Cmax and AUC0–6 h of prasugrel AM following 1 and 3 mg kg−1 were higher than those of clopidogrel AM following 10 and 30 mg kg−1, respectively, despite the tenfold higher doses of clopidogrel. These findings are consistent with more efficient AM formation following prasugrel administration as compared to clopidogrel.

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Figure 3.  Plasma concentrations of active metabolites of prasugrel and clopidogrel after oral administration of each agent in rats. Plasma concentrations of the active metabolites were measured at 0.25, 0.5, 1, 2, 4 and 6 h after oral administration of prasugrel (1 and 3 mg kg−1) and clopidogrel (10 and 30 mg kg−1) to rats. Results are expressed as mean ± SEM (= 5).

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We further analyzed the relationship between pharmacodynamics and pharmacokinetics. Upon comparison of the plasma concentrations of the AM at all fixed time points with platelet aggregation at the corresponding time points, there was no statistically significant overall correlation between the plasma concentration of either AM and inhibition of platelet aggregation: = 0.21 (= 0.5958) for prasugrel, and = −0.42 (= 0.2215) for clopidogrel. This no doubt reflects persistent, irreversible inhibition of platelet P2Y12 and aggregation after clearance of free plasma AM at the later time points. In contrast, there was a statistically significant positive correlation between the total exposure calculated as the AUC0–t (at 0.25, 0.5, 1, 2, 4 and 6 h) of the plasma AM concentration and the percentage inhibition of platelet aggregation at corresponding time points (Fig. 4): = 0.96 (< 0.0001) for prasugrel, and = 0.92 (< 0.0001) for clopidogrel. These results indicate that the antiplatelet effects of prasugrel and clopidogrel are dependent on the amount of AMs generated in vivo.

image

Figure 4.  Correlation between the AUC0–t of plasma active metabolite (AM) concentration and percentage inhibition of platelet aggregation by prasugrel and clopidogrel in rats. Platelet aggregation and plasma concentrations of AMs were measured at 0.25, 0.5, 1, 2, 4 and 6 h after oral administration of prasugrel (0.3, 1 and 3 mg kg−1) and clopidogrel (3, 10 and 30 mg kg−1). There was a statistically significant positive correlation between the AUC0–t and inhibition of platelet aggregation for (A) prasugrel (< 0.0001) and (B) clopidogrel (< 0.0001).

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In vitro effects of prasugrel AM and clopidogrel AM on rat and human platelet aggregation

Aggregation of washed rat platelets was induced by ADP (10 μm). Both prasugrel AM and clopidogrel AM inhibited the ADP-induced platelet aggregation in a concentration-related manner, with maximum inhibition being achieved between 3 and 10 μm (Fig. 5). The IC50 values were 1.8 μm for prasugrel AM and 2.4 μm for clopidogrel AM. Both AMs also inhibited platelet aggregation induced by 2-MeS-ADP (0.1 μm), a stable analog of ADP, with IC50 values of 1.2 μm and 1.4 μm, respectively (data not shown). We further examined the effects of both AMs on washed human platelet aggregation induced by ADP (10 μm) or 2-MeS-ADP (0.1 μm). Both prasugrel AM and clopidogrel AM inhibited the ADP-induced platelet aggregation in a concentration-related manner (Fig. 6). The IC50 values were 0.30 μm for prasugrel AM and 0.30 μm for clopidogrel AM. Both AMs also inhibited human platelet aggregation induced by 2-MeS-ADP, with IC50 values of 0.40 μm and 0.45 μm, respectively (data not shown).

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Figure 5.  Effects of prasugrel and clopidogrel active metabolites (AMs) on ADP-induced rat platelet aggregation. The in vitro effects of prasugrel AM and clopidogrel AM on ADP-induced (10 μm) platelet aggregation were measured using washed rat platelets. Results are expressed as mean ± SEM (= 6). *< 0.05, **< 0.01 vs. control (0 μm).

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image

Figure 6.  Effects of prasugrel and clopidogrel active metabolites (AMs) on ADP-induced human platelet aggregation. The in vitro effects of prasugrel AM and clopidogrel AM on ADP-induced (10 μm) platelet aggregation were measured using washed human platelets. Results are expressed as mean ± SEM (= 5). *< 0.05, **< 0.01 vs. control (0 μm).

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Effects of prasugrel AM and clopidogrel AM on ADP-induced decrease in platelet cAMP level

cAMP levels were markedly increased (about tenfold) 3 min after the addition of PGE1 (10 μm) to washed rat platelets, indicating the activation of adenylyl cyclase (Fig. 7). The cAMP levels in prasugrel AM-treated platelets (0.3–3 μm) at 0 and 3 min were not statistically different from those in control platelets (no AM) (Fig. 7A). The addition of ADP (10 μm) to platelets resulted in a reduction of cAMP levels between 3 and 6 min. Pretreatment with prasugrel AM (0.3–3 μm) attenuated this ADP-induced reduction in cAMP levels in a concentration-related manner. In comparison to control platelets, statistical significance was observed at 1 and 3 μm. Clopidogrel AM (0.3–3 μm) also reduced the ADP-induced decrease in cAMP levels in a concentration-related manner, and this neutralizing effect also reached statistical significance in comparison to control platelets at 1 and 3 μm (Fig. 7B). These results indicate that both AMs can antagonize the Gi-linked P2Y12 receptors, and that the in vitro antagonistic activities of the mixture of the (R,S)-isomer and (S,R)-isomer of prasugrel AM and the (R,S)-isomer of clopidogrel AM are approximately equipotent.

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Figure 7.  Effects of prasugrel and clopidogrel active metabolites (AMs) on ADP-induced cAMP reduction in PGE1-stimulated platelets. The effects of prasugrel AM (A) and clopidogrel AM (B) on ADP-induced (10 μm) cAMP decreases in PGE1-stimulated (10 μm) rat platelets. The cAMP levels in prasugrel AM-treated (0.3–3 μm) platelets at 0 and 3 min were not statistically significant from those in control platelets (no AM). Results are expressed as mean ± SEM (= 6). **< 0.01 vs. control (0 μm).

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

Ex vivo effect of prasugrel on platelet aggregation

We have previously reported that prasugrel demonstrates more potent ex vivo antiplatelet activity than clopidogrel in rats and humans [10–13,23]. The present study confirmed an approximately tenfold more potent effect of prasugrel over clopidogrel on ex vivo platelet aggregation in rats. Our previous results suggested that this potent in vivo antiplatelet and antithrombotic activity of prasugrel is mediated by the AM [10,24], by analogy to other thienopyridines [20]. However, the precise mechanism providing prasugrel’s more potent activities has not been elucidated. In the present study, we have tried to answer two fundamental questions regarding the basis of prasugrel’s increased potency, namely: (i) comparison of the in vivo production of each agent’s AM after oral administration; and (ii) direct comparison in vitro of the effects of prasugrel and clopidogrel AMs on platelet P2Y12-mediated activities, including ADP-induced aggregation and cAMP inhibition.

Profile of plasma AM levels

In parallel with the measurement of ex vivo platelet aggregation, we measured the plasma concentrations of prasugrel and clopidogrel AMs following oral dosing. In rats treated with clopidogrel at 10 times the dose of clopidogrel (i.e. up to 30 mg/kg−1 clopidogrel), the pharmacokinetic parameters for clopidogrel AM did not reach the levels observed for prasugrel AM, strongly suggesting that the increased potency of prasugrel reflects more efficient conversion to its AM. Previous reports have shown that clopidogrel undergoes rapid hydrolysis at its methyl ester moiety [position (b) in Fig. 1] to form an inactive carboxylic acid metabolite, which is the most abundant metabolite, estimated to comprise up to 85% of the total clopidrogel metabolites in human plasma [25]. In contrast, hydrolysis of the acetate ester moiety of prasugrel [position (a) in Fig. 1] leads to a thiolactone metabolite, which is the immediate precursor of prasugrel AM, thereafter, requiring just a single cytochrome P450 oxidation step [26]. In contrast, clopidogrel is converted to its AM in a two-step cytochrome P450 oxidation process. The first step is conversion of clopidogrel to its 2-oxo metabolite, and the second step is conversion of the 2-oxo metabolite to the AM [27]. These differences in metabolic pathways to the AMs most likely account for significantly higher systemic exposure, as reflected in the Cmax and AUC0–6 h values, to the AM of prasugrel than to clopidogrel AM. Recently, more efficient AM production for prasugrel has been observed in healthy volunteers [14]. Taken together with results of the ex vivo platelet inhibitory effects, this suggests that the more efficient production of prasugrel AM plays a major role in its more potent in vivo/ex vivo antiplatelet activity and preclinical antithrombotic effects as compared to clopidogrel.

Comparison of in vitro antiplatelet activity of prasugrel AM and clopidogrel AM

The in vitro antiplatelet effects of prasugrel AM were compared to those of clopidogrel AM, using rat and human platelets. To our knowledge, this is the first report of a direct comparison of these AMs with regard to their antiplatelet activities. The aggregation studies clearly showed that prasugrel AM, a mixture of the (R,S)-isomer and the (S,R)-isomer, has almost identical antiplatelet activities to the (R,S)-isomer of clopidogrel AM. In addition, the similar antiplatelet activities of both AMs were independently confirmed in a cAMP-based assay system reflecting a more specific test of P2Y12 antagonism, utilizing a well-known property of this Gi-linked receptor. It is of note that the two AMs showed similar effects on ADP-induced (10 μm) reductions in PGE1-stimulated rat platelet cAMP levels. Our previous study showed that the most potent isomer of prasugrel AM is the (R,S)-form [19], and, theoretically, a mixture of the (R,S)-isomer and the (S,R)-isomer of prasugrel AM contains 50% of the (R,S)-isomer; thus, these results suggest that the (R,S)-isomer of prasugrel AM has at least 2-fold more potent activity than the (R,S)-isomer of clopidogrel AM. Further delineation of the contributions of individual isomers to the observed platelet inhibition following oral dosing will require a detailed examination of the in vivo formation of each of the prasugel AM and clopidogrel AM isomers.

Correlation between plasma AM and antiplatelet activity

Several previous reports suggested that thienopyridine antiplatelet agents such as prasugrel and clopidogrel demonstrate irreversible and cumulative antiplatelet effects based on disulfide bond formation between the thiol group of the AM and P2Y12 receptors [10,28,29]. In the present study, discrete plasma AM concentrations after single oral administration of prasugrel and clopidogrel to rats did not significantly correlate with inhibition of platelet aggregation induced by ADP at the same time point (= 0.21 and = 0.60 for prasugrel, and = −0.42 and = 0.22 for clopidogrel). In other words, the antiplatelet effects of prasugrel and clopidogrel were maintained for extended periods of time despite the disappearance of each AM from the plasma. Thus, the lack of a relationship between plasma AM concentration and antiplatelet activity reflects the irreversible binding of AMs to P2Y12, with the resulting effect probably lasting for the life of the platelet (i.e. 7–10 days). In contrast, as shown in Fig. 4, a very good relationship between total plasma AM production (AUC0–t) and inhibition of platelet aggregation was observed in both prasugrel-treated rats (= 0.96, < 0.0001) and clopidogrel-treated rats (= 0.92, < 0.0001). This high correlation between AUC0–t and inhibition of platelet aggregation serves as additional evidence supporting the view that the in vivo antiplatelet activities of prasugrel and clopidogrel largely reflect their AM levels.

In summary, these studies confirm that prasugrel has at least tenfold greater in vivo/ex vivo antiplatelet activity than clopidogrel, and that this reflects more efficient generation of its AM. It is also possible that small differences in the activity of the AM may contribute to the overall potency differences.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

We thank M. Hasegawa, Y. Yokouchi, M. Takahashi, K. Kawabata, N. Suzuki and T. Shimoji for their expert technical assistance, and T. Kimura, T. Ikeda, M. Kazui, T. Isobe and Ken-ichi Otsuguro for their helpful discussions.

Disclosure of Conflict of Interests

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

All authors are employees of Darichi-Sankyo or Eli Lilly and Company.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References
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