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Novel P2Y12 adenosine diphosphate receptor antagonists for inhibition of platelet aggregation (II): Pharmacodynamic and pharmacokinetic characterization
Thrombosis Research (2008) 122, 533—540
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Novel P2Y12 adenosine diphosphate receptor antagonists for inhibition of platelet aggregation (II): Pharmacodynamic and pharmacokinetic characterization Joseph M. Post, Serene Alexander, Yi-Xin Wang ⁎, Jon Vincelette, Ron Vergona, Lorraine Kent, Judi Bryant, Mark E. Sullivan, William P. Dole, John Morser, Babu Subramanyam Berlex Biosciences, Richmond, CA 94804 Received 2 December 2007; received in revised form 20 April 2008; accepted 23 April 2008 Available online 9 June 2008
KEYWORDS BX 667; P2Y12 receptor; Platelet inhibitor
Abstract Antiplatelet drugs are used to prevent aberrant platelet activation in pathophysiologic conditions such as myocardial infarction and ischemic stroke. The key role that ADP plays in this process has led to the development of antiplatelet drugs that target the P2Y12 receptor. The aim of this study was to characterize the pharmacodynamic (PD) and pharmacokinetic (PK) properties of the novel P2Y12 receptor antagonists, BX 667 and BX 048. BX 667 blocks ADP-induced platelet aggregation in human, dog and rat blood (IC50 = 97, 317 and 3000 nM respectively). BX 667 had nominal effects on collagen-induced aggregation and weakly inhibited arachidonic acid-induced aggregation. BX 667 has an active metabolite, BX 048, that also potently inhibits ADP-induced aggregation (IC50 = 290 nM) in human blood. BX 667 was shown to have high oral bioavailability in both dog and rat unlike BX 048. Administration of BX 667 resulted in a rapid and sustained inhibition of platelet aggregation where the extent and duration of platelet inhibition was directly proportional to circulating plasma levels. This report describes the PK/PD properties of BX 667 showing that it has the properties required for a potential antiplatelet therapeutic agent. © 2008 Elsevier Ltd. All rights reserved.
Introduction ⁎ Corresponding author. Arete Therapeutics, Inc., 3912 Trust Way, Hayward, CA 94545. Tel.: +1 510 300 1868; fax: +1 510 785 7061. E-mail address: [email protected] (Y.-X. Wang).
Adenosine-5'-diphospate (ADP) activates platelets and is known to play an important role in haemostasis
0049-3848/$ - see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2008.04.009
534 and thrombosis [1,2] through interacting with P2Y1 and P2Y12 [3–7]. Recognition of the therapeutic benefits of preventing thrombotic complications have led to the development of P2Y12 receptor antagonists such as ticlopidine and clopidogrel which are used both for prevention and treatment of cardiovascular diseases such as atherosclerosis, myocardial infarction and cerebral vascular diseases [8–11]. Clopidogrel is an orally available irreversible P2Y12 platelet antagonist that has been reported to be safer than ticlopidine [8]. Although, clopidogrel is an effective antithrombotic, it requires hepatic activation which delays inhibition of platelet aggregation and its irreversible binding to P2Y12 receptors may prolong or exacerbate bleeding [8,12–14]. We have focused our efforts on the discovery and development of a P2Y12 receptor antagonist with anti-platelet properties that will provide an improved alternative to currently available P2Y12 receptor inhibitors. These efforts led to the discovery of the selective P2Y12 receptor antagonists, BX 667 and BX 048. In part I of the companion paper, we describe the molecular pharmacology of these compounds. BX 667 is a potent and orally active P2Y12 receptor antagonist which unlike clopidogrel, does not require hepatic activation and is reversible. In this paper we determine the pharmacokinetic (PK) and pharmacodynamic (PD) properties of BX 667 and BX 048. Metabolism of BX 667 results in the transition of the parent ester to its corresponding acid, BX 048, which is also a potent antagonist of P2Y12 receptors. The in vivo activity of BX 667 could be explained by the sum of the PD activity of the parental compound and its active metabolite. Methods Chemicals BX 667, “(S)-4-({4-[1-(Ethoxycarbonyl)-1-methylethoxy]-7methyl-2-quinolyl} Carbamoyl)-5-[4-(ethoxycarbonyl) piperazin-1-yl]-5-oxopentanoic acid”, and BX 048, “(S)-4-({[4-(1Carboxy-1-methylethoxy)-7-methylquinolin-2-yl]carbonyl} amino)-5-[4-(ethoxycarbonyl) piperazin-1-yl]-5-oxopentanoic acid”, were used in this study. The structures of the 2 compounds were published in the previous paper [14].
Whole-blood platelet aggregation Platelet aggregation was assessed in heparinized (10 U/mL) blood diluted 1:1 with 0.9% saline induced with either 5 μM ADP, 3 μg/mL collagen (Type 1, Chrono-Log Corp, Havertown PA) or 300 μM arachidonic acid and incubated at 37 oC. Experiments were performed using human, male beagle dog or male SpragueDawley rat blood. BX 667 or BX 048 was added to individual cuvettes at half-log concentrations ranging from 0.01 to 30 µM. The cuvettes were then transferred to a whole-blood platelet aggregometer (ChronoLog, Havertown, PA), which measures
J.M. Post et al. impedance to quantify platelet aggregation. BX 667 or BX 048 were added 4 minutes prior to addition of agonist. Previous experiments in our laboratory have shown that use of these concentrations of ADP, collagen, and arachidonic acid produce a near maximal (≈ IC90) aggregatory response by experiments in which whole blood was challenged with different concentrations of the agonists, platelet aggregation determined, and the concentration necessary for a 90% aggregation response was determined. The extent of platelet aggregation was calculated by measuring the area under the curve (AUC) for 15 minutes in the presence or absence of drug. Inhibition of platelet aggregation was expressed as a % inhibition with respect to controls (i.e. no drug added). IC50 values were calculated by averaging the concentrations from individual experiments necessary to inhibit agonist-induced responses by 50%.
In vitro stability and metabolism Due to species differences in systemic esterase activity, the stability of the parent ester, BX 667, was evaluated in whole blood, hepatic microsomes and intestinal (S9) fractions of human, rat and dog. For evaluation in blood, BX 667 was incubated in heparinized whole blood at concentrations of 10 or 25 μM at 37 °C. A 0.5 mL aliquot was removed at 0, 15 and 30 minutes post incubation and plasma was immediately prepared. The resulting samples were denatured by addition of cold acetonitrile or methanol (1:4) containing an internal reference standard for quantification. The denatured proteins were removed by centrifugation and BX 667 was quantified utilizing LC/MS-MS. The hepatic microsomal stability studies were conducted utilizing commercially available microsomes (Gentest Corp. Woburn, MA). Rat, dog and human hepatic microsomes of known P450 content and protein concentration were incubated with BX 667 and BX 048 at 37 °C at a substrate concentration of 10 μM. Incubations were carried out in the presence and absence of a NADPH generating cofactor system (8 mM glucose-6phosphate, 4 mM MgCl2, 1 IU/mL glucose-6-phosphage dehydrogenase, 0.5 mM NADP+ in 100 mM sodium phosphate buffer). Following a specified incubation period, samples were quenched with ice cold methanol, precipitated proteins were removed by centrifugation and the remaining drug concentrations were determined by LC/MS/MS. Testing of BX 667 hydrolysis to BX 048 in intestinal jejunal S9 fractions from human, dog and rat was performed by Quintiles (Kansas City, MO). BX 667 was incubated in triplicate with rat, dog and human jejunal S9, respectively, for 0, 5, 15 and 30 minutes at concentrations of 1 or 10 μM. Following incubation, acetonitrile containing 0.5 μM of an internal standard was added to terminate the reactions. Incubations were performed at 37 °C, in a 96-well plate on a Hamilton Microlab 2200 Liquid Handler. BX 667 and BX 048 concentrations were determined using ESI LC/MS.
Animal studies All animal experimental procedures were approved by the Institutional Animal Care and Use Committee and were in accordance with the Guide for the Care and Use of Laboratory Animals (NIH). For dog studies, male beagles were fasted for 18 hours and instrumented with indwelling jugular catheters on the day of the experiment for taking blood samples. Dogs were allowed water ad libitum and were housed individually. BX 667 or BX 048 was administered via oral gavage (PO) in a vehicle consisting of 100% propylene glycol or in gelatin capsules as a solid dose form or intravenousely (IV).
PK/PD of a platelet inhibitor
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For each time-point, 2.7 mL of blood was collected in a 3 cc syringe and placed in a culture tube containing 10 units of heparin in 300 µL PBS. Blood samples were immediately tested for platelet aggregation. In selected studies, plasma drug levels were also measured. For drug level determinations, the remaining blood was centrifuged (1000 x g for 10 min at 4 °C) to obtain plasma. Plasma samples were then frozen (−20 °C) until assayed for drug levels. For rat PK studies, fasted male Sprague-Dawley rats were used. BX 667 or BX 048 (2 mg/kg) was administered in 10%DMSO/ 90% saline by oral gavage or intravenously. Blood samples were drawn at pre-designated time points via an indwelling jugular cannula. Plasma levels of BX 667 and BX 048 were quantified utilizing LC/MS/MS methods described below. For ex vivo platelet aggregation studies in rat, inhibition of ADP-induced platelet aggregation was monitored following oral gavage administration in male Sprague-Dawley rats. Blood samples were taken from the jugular vein of cannulated rats at 5 minutes before dosing to determine control platelet aggregation and at 15, 60, 120 and 180 minutes after dosing.
Determination of plasma concentration Samples and control plasma were thawed at room temperature. BX 290 was used as an internal standard. The range of the calibration curve was 0.01 µM–30 µM. Following the preparation of calibration standards and quality control samples, 50 µL standards and experimental samples were pipetted into cluster tubes. The plasma proteins were precipitated by addition of 200 μL cold acetonitrile containing an internal reference standard for quantification. The precipitated proteins were removed by centrifugation at 1000 ×g for 20 minutes at ambient temperature. Approximately 80 µL of the supernatant was placed into HPLC vial inserts for analysis. Plasma levels of BX 667 and BX 048 were measured by LC/MS/MS method. Pharmacokinetic parameters were calculated using (WinNonlin): Cmax: maximum concentration attained; tmax: time to maximum concentration; t1/2: half-life of compound elimination; AUCinf; area under the concentration time curve with the last concentration extrapolated based on the elimination rate; Cltotal: total clearance; F: % oral bioavailability.
Results In vitro platelet aggregation BX 667 and BX 048 potently inhibited ADP-induced platelet aggregation in human, dog and rat whole blood (Fig. 1). The IC50 values for BX 667, which represents the concentration at which 50% inhibition of ADP-induced platelet aggregation occurred were 97 ± 39 (n = 10), 317 ± 86 (n = 7), and 3000 ± 620 (n = 4) nM in human, dog and rat blood, respectively. ADPinduced aggregation was inhibited nearly 100% at 1 μM in human and dog. In rat maximal inhibition was achieved at 10 μM. For the primary metabolite, BX 048, the IC50 values for inhibition of ADP-induced aggregation were 290 ± 63 (n = 10), 230 ± 60 (n = 4), and 3600 ± 96 (n = 4) nM in human, dog and rat blood respectively. Concentrations required for maximal inhibition were similar to those observed with BX 667. The potency of BX 667 and BX 048 was found to be species dependent with the rank order of potency: human N dog N rat. Since other agents than ADP can induce platelet aggregation, BX 667 and BX 048 were tested against platelet aggregation induced by arachidonic acid and collagen. The compounds had
Figure 1 The in vitro effects of BX 667 and BX 048 on ADPinduced platelet aggregation in whole blood from human (top), dog (middle) and rat (bottom). BX 667 and BX 048 inhibited ADP (5 µM) —induced platelet aggregation in a concentration dependent manner. Data points represent the mean± SE. nominal effects on platelet aggregation induced by 3 μg/mL collagen (Fig. 2). However, BX 667 and BX 048 were found to inhibit platelet aggregation induced with 300 μM arachidonic acid in a concentration dependent manner albeit with a lower potency than ADP-induced aggregation. The IC50 values of BX 667 and BX 048 for arachidonic acid induced aggregation were 8.5 µM and 15 µM respectively.
In vitro stability and metabolism The stability of BX 667 at concentrations of 10 or 25 μM was evaluated in human, dog and rat blood. BX 667 was nearly completely converted to its primary metabolite BX 048 in rat
536
J.M. Post et al. Table 1 Metabolic stability of BX 667 in human, dog and rat jejunal S9 (n = 3), expressed as percent of remaining compound at the specified time points Species
Time (minutes)
% ± SD
Human
5 15 30 5 15 30 5 15 30
89.2 ± 5.0 91.9 ± 3.4 80.8 ± 2.1 79.8 ± 14.5 78.1 ± 4.2 81.7 ± 14.4 65.7 ± 10.3 32.5 ± 3.9 20.8 ± 1.4
Dog
Rat
* p b 0.05 vs. human and dog samples at corresponding time points.
found to be stable in microsomes from human, dog and rat with less than 10% conversion observed following 3 hours of incubation (data not shown). BX 667 and BX 048 were found to be metabolized to the same extent in either the presence or absence of NADPH which suggests that metabolic conversion is mediated by non-specific esterases rather than NADPHdependent cytochrome P450 enzymes. The potential for presystemic metabolism of BX 667 was evaluated in jejunal S9 fractions from human, dog and rat. BX 667 had relatively minimal metabolism in human and dog jejunal S9 fractions (Table 1) suggesting that intestinal Figure 2 Effect of BX 667 and BX 048 on platelet aggregation induced with 3 µg/mL collagen (top) or 300 µM arachidonic acid (bottom) in human blood. Datapoints represent the mean ± SE of 4 experiments. blood (N 99%) by 30 minutes. In contrast, BX 667 was relatively stable in human and dog blood with only 1–2% conversion to BX 048 after 30 minutes. The in vitro metabolic stability of BX 667 and BX 048 was also evaluated in hepatic microsomes from human, dog and rat. Metabolism of BX 667 to BX 048 occurred rapidly in rat relative to human and dog. The time-course for BX 667 metabolism in microsomes is illustrated in Fig. 3. The metabolite, BX 048, was
Figure 3 Time-course of BX 667 metabolism in hepatic microsomes from human, dog and rat.
Figure 4 The kinetics following intravenous and oral administration of 2 mg/kg BX 667 (top) or BX 048 (bottom) in conscious rats (n = 3).
PK/PD of a platelet inhibitor
537
Table 2 Pharmacokinetic parameters following intravenous or oral administration of BX 667 and BX 048 at 2 mg/kg in rat (n = 3/dose group) BX 667 Cmax (µM) tmax (min) t½ (β) (h) AUCinf (µg·h/mL) Cltotal (mL/min/kg) F (%)
BX 048
i.v.
p.o.
i.v.
p.o.
33 1 2 12 3
1.9 60 3 6.9
56 1 1.4 23 1.4
0.18 30 4.5 0.42
57
2
Pharmacokinetic parameters were calculated using (WinNonlin): Cmax: maximum concentration attained; tmax: time to maximum concentration; t1/2: half-life of compound elimination; AUCinf; area under the concentration time curve with the last concentration extrapolated based on the elimination rate; Cltotal: total clearance; F: % oral bioavailability.
metabolism should have a minor impact on BX 667 bioavailability in higher species but in rat intestinal S9 fractions was metabolized 80 percent at 30 minutes.
Pharmacokinetics Pharmacokinetic experiments with BX 667 and BX 048 were performed in dog and rat to assess oral bioavailability. BX 667 is converted in vivo from the ester to its corresponding acid, BX 048, which is also a potent inhibitor of P2Y12 receptors (see above). Therefore, pharmacokinetic parameters for BX 667 were calculated by measuring both the parent compound and its metabolite BX 048 in dog studies. In the rat, due to rapid and extensive esterase activity, BX 667 was rapidly converted to BX 048 and therefore it was only possible to calculate the pharmacokinetic parameters of the active metabolite, BX 048.
Pharmacokinetics in rats The plasma concentrations of BX 048 following IV or PO administration of 2 mg/kg BX 667 or BX 048 in rat are illustrated in Fig. 4. Oral administration of BX 667 in rat led to rapid and well-maintained circulating levels of BX 048 with a peak concentration of approximately 2 μM at 60 minutes. Based on the circulating concentrations of BX 048 in the rat, the oral availability of BX 667 was deduced to be 57%. This finding supports the suitability of BX 667 for oral administration. In contrast, the oral availability of BX 048 was determined to be 2%. The pharmacokinetic parameters of BX 667 and BX 048 in rat are shown in Table 2.
Pharmacokinetics in dogs Oral administration of 10 mg/kg of BX 667 in dog resulted in rapid absorption and well-maintained circulating levels of BX 667 and its active metabolite BX 048 (Fig. 5, top). BX 667 was rapidly absorbed and reached a cumulative peak circulating concentration of approximately 14 μM after 60 minutes. Due
Table 3 Pharmacokinetic parameters for BX 667 and BX 048 following intravenous (2 mg/kg) or oral (10 mg/kg) administration of BX 667 or BX 048 in dogs (n = 3 per dose group) Compound
BX 667
Route
Figure 5 The pharmacokinetic profile of BX 667 and its metabolite BX 048 in conscious dogs (n = 3): Circulating plasma levels of BX 667 and BX 048 following PO (10 mg/kg) dose of BX 667 (top) and IV/PO kinetics of BX 667 itself following a IV (2 mg/ kg) and PO (10 mg/kg) dose (bottom). The insertion is the area under the curve (AUC) calculated from the data in the top panel.
IV
BX 048 PO
IV
PO
Plasma Conc.
BX 667
BX 048
BX 667
BX 048
BX 048
BX 048
Cmax (µM) tmax (min) t½ (β) (h) AUCinf (µg·h/mL) Cltotal (mL/min/kg) F (%)
35 1 0.21 2
5.2 15 7 4.2
4.3 15 3 3.3
10 60 6.9 16
8 12
6 4.1
17
N/A⁎
2.7 62
7
Cmax: maximum concentration attained; tmax; time to maximum concentration; t1/2; half- life of compound elimination; AUCinf; area under the concentration time curve with the last concentration extrapolated based on the elimination rate; Cltotal; total clearance; F; % oral bioavailability.
538
J.M. Post et al.
to hepatic or enteric esterase activity, the circulating level of BX 048 following IV or PO administration was substantially higher than the parent (BX 667) as illustrated by the greater AUC for BX 048 relative to BX 667 (Fig. 5, top insertion). The sum of the plasma circulating levels of BX 667 and BX 048 was 280 nM at 12 hours. The magnitude and duration of the circulating levels of BX 667 and BX 048 relative to their in vitro potencies suggests that platelet inhibition will be maintained for several hours. The plasma concentration time courses of BX 667 following IV (2 mg/kg) or PO (10 mg/kg) administration in dog are shown in Fig. 5 (bottom) and the PK parameters are shown in Table 3. Because BX 667 and its metabolite BX 048 are both potent inhibitors of platelet aggregation in blood, the aggregate of their circulating concentrations are responsible for their pharmacodynamic activity. Therefore, the %F was calculated using the sum of the AUC values for BX 667 and BX 048 after iv/po dosing of BX 667. The F was 62% suggesting that BX 667 is a suitable candidate for oral administration. The bioavailability of BX 048 was also calculated after IV/ PO dosing (Table 3) and as predicted by the data in rats, the oral bioavailability of this compound was much lower (F = 7%).
Pharmacodynamics Pharmacodynamics in rats Oral administration of doses of 1, 3 and 10 mg/kg BX 667 resulted in a rapid and dose-dependent inhibition of platelet aggregation (Fig. 6). The extent of inhibition was dosedependent and maintained at relatively constant levels throughout the 3 hour study. The average percent inhibition over the 3 hour time course following 1, 10 and 30 mg/kg dosing was 36 ± 15, 49 ± 24 and 73 ± 13 respectively. The PK profile in rat (Fig. 4, top) shows that the plasma level of BX 048 was near steady state between 1 and 3 hours following PO
Figure 7 Effect of oral administration of BX 667 (1, 3, 10 mg/kg) on ADP (5 µM)-induced platelet aggregation in conscious dogs (top). Data points represent the mean ± SEM. Platelet inhibition at 3 and 10 mg/kg was significantly greater than that observed at 1 mg/kg (p b 0.005). Correlation of the percent inhibition of ADP-induced platelet aggregation to the sum of the circulating plasma levels of BX 667 and BX 048 in dog (bottom). Each point represents one measurement from one animal from Fig. 5, top panel.
administration, which is consistent with the observed steadystate inhibition of platelet aggregation.
Figure 6 Effect of BX 667 on ADP (5 µM)-induced platelet aggregation after oral administration in conscious rats. Data points represent the mean ± SEM. Platelet inhibition was significant at all doses relative to vehicle control over the time course (p b 0.01). Platelet inhibition at 30 mg/kg was greater than at 1 mg/kg (p b 0.001) or 10 mg/kg (p b 0.02).
Pharmacodynamics in dogs Oral administration of doses of 1, 3 and 10 mg/kg BX 667 led to a rapid and dose-dependent inhibition of platelet aggregation (Fig. 7). The extent of inhibition was maximal (≈100%) at 30 minutes following 3 and 10 mg/kg administration. Platelet inhibition gradually declined with time, consistent with the observed time course for elimination of BX 667 and BX 048 (Fig. 5). Inhibition of ADP-induced platelet aggregation was well maintained for several hours (Fig. 7, top). A high correlation was found between the extent of inhibition of ADP-induced platelet aggregation and the sum of the circulating levels of BX 667 and BX 048 (r2 = 0.72; Fig. 7, bottom). The ex vivo potency of BX 667 to inhibit ADP-induced
PK/PD of a platelet inhibitor platelet aggregation (IC50 ≈ 450 nM) was also consistent with in vitro potency measurements (IC50 ≈ 317 nM) in dog.
Discussion Whole blood platelet aggregometry was used in the present study to evaluate the pharmacodynamic activity of BX 667 and BX 048. The use of this method minimizes manipulation of the blood samples and allows for the monitoring of platelet activity in a biological matrix which reflects the in vivo pharmacological activity of platelet inhibitors [15]. In vitro platelet aggregation experiments indicate that BX 667 and BX 048 potently inhibit ADP-induced platelet aggregation in human, dog and rat blood. An important pharmacological property of BX 667 is that both the parent compound and its metabolite, BX 048, are highly potent and selective inhibitors of P2Y12 receptors and that the rank order potency is the same in whole blood as it is in binding studies and aggregation experiments on washed platelets (see accompanying manuscript). Therefore, the extent of inhibition of platelet aggregation is dependent on the combined circulating levels of BX 667 and BX 048. The in vitro potencies of BX 667 and BX 048 were species dependent with the greatest potency observed in human relative to dog and rat. It has to be point out the in vitro potency in the whole blood assay (Fig. 1) is different from that in the washed platelets (Fig. 2 of part I of the companion paper) in the human samples between the 2 compounds, this may be due to the presence of enzymes in the blood, such as hydrolase that could convert BX667 to BX048. In addition, the different protein binding for the 2 compounds may also impact on the potency. In vivo, modulators of platelet function provide the appropriate balance for maintaining hemostasis without causing either excessive thromboembolic events such as stroke or making platelets so unresponsive that hemorrhagic risk or bleeding is increased [16]. Therefore, in addition to ADP, it was of interest to test the effects of BX 667 and BX 048 with the platelet activators, collagen and arachidonic acid, to evaluate any potential cross-talk that may exist. Collagen is an important thrombogenic component of the subendothelial matrix and induces platelet aggregation using a multistep process involving the integrin α2β1 and glycoprotein VI (GPVI) [16,17]. Collagen is able to amplify its response by stimulating release of ADP and thromboxane A2 (TxA2) from platelets [17,18]. Additional platelet activators such as thrombin via proteaseactivated receptors (PAR1) and arachidonic acid are also able to stimulate platelet aggregation. BX 667
539 and BX 048 had nominal effects on collagen-induced platelet aggregation suggesting that P2Y12 receptors play only a minor role in collagen-induced platelet activation. In transgenic studies with platelets derived from P2Y1 –deficient mice, collagen-induced aggregation was greatly attenuated [19]. Together, these results suggest that P2Y1 receptors play a greater role in collagen-induced aggregations than P2Y12 receptors. In contrast to collagen, BX 667 and BX 048 were able to inhibit arachidonic acid induced aggregation albeit at concentrations much greater than those needed to inhibit ADP-induced platelet aggregation. This is consistent with results from studies with the P2Y12 receptor inhibitor, clopidogrel, where it was observed that although clopidogrel does not significantly alter collagen-induced aggregation [20]; inhibition of P2Y12 can inhibit thromboxane A2 mediated platelet effects [21]. These results indicate that although BX 667 and BX 048 may provide an anti-thrombotic benefit by inhibition of ADP-mediated platelet aggregation, other platelet activators such as collagen may continue to assist in the maintenance of hemostatic function when the subendothelial matrix or vessel wall is compromised (e.g. during surgery). Oral administration of BX 667 in dog and rat leads to a rapid and dose-dependent inhibition of platelet aggregation in contrast to clopidogrel which requires hepatic activation and has a delayed antithrombotic effect. Furthermore, the extent of inhibition of platelet activity correlated well with the circulating plasma levels of BX 667 and BX 048. The potency calculated from the in vivo experimental determination of the PD for BX 667 was consistent with the in vitro potency measurements (See accompanying manuscript). The observation that BX 667 reversibly inhibits P2Y12 receptors also distinguishes it from irreversible platelet inhibitors such as clopidogrel or aspirin. The direct relationship between PK and PD will assist with dose selection and response monitoring. BX 667 was highly bioavailable in dog and rat following oral administration and as expected its acid metabolite, BX 048, was poorly bioavailable. In rat, BX 667 was rapidly metabolized in blood, hepatic microsomes and intestinal fractions. In human and dog, BX 667 was relatively stable in both blood and intestinal fractions. BX 667 was gradually metabolized by an NADPH-independent process in hepatic microsomes suggesting that it is a poor P-450 substrate. The overall PK and pharmacodynamic profiles of BX 667 indicate its suitability as an orally available therapeutic candidate. These results suggest that complete inhibition of ADP-induced platelet
540 aggregation would occur at circulating levels of 1 μM in humans. It is anticipated that because BX 667 is a reversible ADP-receptor inhibitor as demonstrated in the companion paper (ref), it will be easier to predict the extent and duration of platelet inhibition compared to irreversible inhibitors providing a greater safety margin if adverse events such as bleeding occur or if greater control over hemostasis is desired. Recently, the efficacy and safety of BX 667 in comparison to clopidogrel in various preclinical animal models of thrombosis were described [14]. BX 667 demonstrated superior efficacy relative to bleeding risk when these agents were administered either alone or in combination with aspirin. In the studies reported here and in the accompanying paper (ref) we show that BX 667 is a reversible antagonist of platelet aggregation and as such its drug-target residence time will be lower than that of an irreversible antagonist such as clopidogrel. Drug-target residence time affects both the efficacy and potential side effects of a drug [22]. In the case of P2Y12 receptor antagonists, efficacy and bleeding are both due to the blockade of the receptor. Thus while a long drugtarget residence time improves efficacy, it also inevitably increases bleeding. These findings suggest that BX 667 as a reversible P2Y12 receptor antagonist may provide a wider therapeutic index relative to irreversible platelet inhibitors because of its lower drug-target residence time. In summary, the present report characterizes the pharmacodynamic and pharmacokinetic properties of the novel P2 Y12 receptor inhibitor BX 667 and its active metabolite BX 048. BX 667 is an orally available, reversible P2Y12 receptor inhibitor that potently inhibits ADP-induced aggregation. Inhibition of platelet aggregation is rapid in onset and the duration and extent of platelet inhibition is directly proportional to the plasma concentration of BX 667 and BX 048. The direct PK/PD relationship should assist in dose selection to accurately predict the extent and duration of activity in order to provide optimal therapeutic benefit.
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REGULAR ARTICLE
Novel P2Y12 adenosine diphosphate receptor antagonists for inhibition of platelet aggregation (II): Pharmacodynamic and pharmacokinetic characterization Joseph M. Post, Serene Alexander, Yi-Xin Wang ⁎, Jon Vincelette, Ron Vergona, Lorraine Kent, Judi Bryant, Mark E. Sullivan, William P. Dole, John Morser, Babu Subramanyam Berlex Biosciences, Richmond, CA 94804 Received 2 December 2007; received in revised form 20 April 2008; accepted 23 April 2008 Available online 9 June 2008
KEYWORDS BX 667; P2Y12 receptor; Platelet inhibitor
Abstract Antiplatelet drugs are used to prevent aberrant platelet activation in pathophysiologic conditions such as myocardial infarction and ischemic stroke. The key role that ADP plays in this process has led to the development of antiplatelet drugs that target the P2Y12 receptor. The aim of this study was to characterize the pharmacodynamic (PD) and pharmacokinetic (PK) properties of the novel P2Y12 receptor antagonists, BX 667 and BX 048. BX 667 blocks ADP-induced platelet aggregation in human, dog and rat blood (IC50 = 97, 317 and 3000 nM respectively). BX 667 had nominal effects on collagen-induced aggregation and weakly inhibited arachidonic acid-induced aggregation. BX 667 has an active metabolite, BX 048, that also potently inhibits ADP-induced aggregation (IC50 = 290 nM) in human blood. BX 667 was shown to have high oral bioavailability in both dog and rat unlike BX 048. Administration of BX 667 resulted in a rapid and sustained inhibition of platelet aggregation where the extent and duration of platelet inhibition was directly proportional to circulating plasma levels. This report describes the PK/PD properties of BX 667 showing that it has the properties required for a potential antiplatelet therapeutic agent. © 2008 Elsevier Ltd. All rights reserved.
Introduction ⁎ Corresponding author. Arete Therapeutics, Inc., 3912 Trust Way, Hayward, CA 94545. Tel.: +1 510 300 1868; fax: +1 510 785 7061. E-mail address: [email protected] (Y.-X. Wang).
Adenosine-5'-diphospate (ADP) activates platelets and is known to play an important role in haemostasis
0049-3848/$ - see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2008.04.009
534 and thrombosis [1,2] through interacting with P2Y1 and P2Y12 [3–7]. Recognition of the therapeutic benefits of preventing thrombotic complications have led to the development of P2Y12 receptor antagonists such as ticlopidine and clopidogrel which are used both for prevention and treatment of cardiovascular diseases such as atherosclerosis, myocardial infarction and cerebral vascular diseases [8–11]. Clopidogrel is an orally available irreversible P2Y12 platelet antagonist that has been reported to be safer than ticlopidine [8]. Although, clopidogrel is an effective antithrombotic, it requires hepatic activation which delays inhibition of platelet aggregation and its irreversible binding to P2Y12 receptors may prolong or exacerbate bleeding [8,12–14]. We have focused our efforts on the discovery and development of a P2Y12 receptor antagonist with anti-platelet properties that will provide an improved alternative to currently available P2Y12 receptor inhibitors. These efforts led to the discovery of the selective P2Y12 receptor antagonists, BX 667 and BX 048. In part I of the companion paper, we describe the molecular pharmacology of these compounds. BX 667 is a potent and orally active P2Y12 receptor antagonist which unlike clopidogrel, does not require hepatic activation and is reversible. In this paper we determine the pharmacokinetic (PK) and pharmacodynamic (PD) properties of BX 667 and BX 048. Metabolism of BX 667 results in the transition of the parent ester to its corresponding acid, BX 048, which is also a potent antagonist of P2Y12 receptors. The in vivo activity of BX 667 could be explained by the sum of the PD activity of the parental compound and its active metabolite. Methods Chemicals BX 667, “(S)-4-({4-[1-(Ethoxycarbonyl)-1-methylethoxy]-7methyl-2-quinolyl} Carbamoyl)-5-[4-(ethoxycarbonyl) piperazin-1-yl]-5-oxopentanoic acid”, and BX 048, “(S)-4-({[4-(1Carboxy-1-methylethoxy)-7-methylquinolin-2-yl]carbonyl} amino)-5-[4-(ethoxycarbonyl) piperazin-1-yl]-5-oxopentanoic acid”, were used in this study. The structures of the 2 compounds were published in the previous paper [14].
Whole-blood platelet aggregation Platelet aggregation was assessed in heparinized (10 U/mL) blood diluted 1:1 with 0.9% saline induced with either 5 μM ADP, 3 μg/mL collagen (Type 1, Chrono-Log Corp, Havertown PA) or 300 μM arachidonic acid and incubated at 37 oC. Experiments were performed using human, male beagle dog or male SpragueDawley rat blood. BX 667 or BX 048 was added to individual cuvettes at half-log concentrations ranging from 0.01 to 30 µM. The cuvettes were then transferred to a whole-blood platelet aggregometer (ChronoLog, Havertown, PA), which measures
J.M. Post et al. impedance to quantify platelet aggregation. BX 667 or BX 048 were added 4 minutes prior to addition of agonist. Previous experiments in our laboratory have shown that use of these concentrations of ADP, collagen, and arachidonic acid produce a near maximal (≈ IC90) aggregatory response by experiments in which whole blood was challenged with different concentrations of the agonists, platelet aggregation determined, and the concentration necessary for a 90% aggregation response was determined. The extent of platelet aggregation was calculated by measuring the area under the curve (AUC) for 15 minutes in the presence or absence of drug. Inhibition of platelet aggregation was expressed as a % inhibition with respect to controls (i.e. no drug added). IC50 values were calculated by averaging the concentrations from individual experiments necessary to inhibit agonist-induced responses by 50%.
In vitro stability and metabolism Due to species differences in systemic esterase activity, the stability of the parent ester, BX 667, was evaluated in whole blood, hepatic microsomes and intestinal (S9) fractions of human, rat and dog. For evaluation in blood, BX 667 was incubated in heparinized whole blood at concentrations of 10 or 25 μM at 37 °C. A 0.5 mL aliquot was removed at 0, 15 and 30 minutes post incubation and plasma was immediately prepared. The resulting samples were denatured by addition of cold acetonitrile or methanol (1:4) containing an internal reference standard for quantification. The denatured proteins were removed by centrifugation and BX 667 was quantified utilizing LC/MS-MS. The hepatic microsomal stability studies were conducted utilizing commercially available microsomes (Gentest Corp. Woburn, MA). Rat, dog and human hepatic microsomes of known P450 content and protein concentration were incubated with BX 667 and BX 048 at 37 °C at a substrate concentration of 10 μM. Incubations were carried out in the presence and absence of a NADPH generating cofactor system (8 mM glucose-6phosphate, 4 mM MgCl2, 1 IU/mL glucose-6-phosphage dehydrogenase, 0.5 mM NADP+ in 100 mM sodium phosphate buffer). Following a specified incubation period, samples were quenched with ice cold methanol, precipitated proteins were removed by centrifugation and the remaining drug concentrations were determined by LC/MS/MS. Testing of BX 667 hydrolysis to BX 048 in intestinal jejunal S9 fractions from human, dog and rat was performed by Quintiles (Kansas City, MO). BX 667 was incubated in triplicate with rat, dog and human jejunal S9, respectively, for 0, 5, 15 and 30 minutes at concentrations of 1 or 10 μM. Following incubation, acetonitrile containing 0.5 μM of an internal standard was added to terminate the reactions. Incubations were performed at 37 °C, in a 96-well plate on a Hamilton Microlab 2200 Liquid Handler. BX 667 and BX 048 concentrations were determined using ESI LC/MS.
Animal studies All animal experimental procedures were approved by the Institutional Animal Care and Use Committee and were in accordance with the Guide for the Care and Use of Laboratory Animals (NIH). For dog studies, male beagles were fasted for 18 hours and instrumented with indwelling jugular catheters on the day of the experiment for taking blood samples. Dogs were allowed water ad libitum and were housed individually. BX 667 or BX 048 was administered via oral gavage (PO) in a vehicle consisting of 100% propylene glycol or in gelatin capsules as a solid dose form or intravenousely (IV).
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For each time-point, 2.7 mL of blood was collected in a 3 cc syringe and placed in a culture tube containing 10 units of heparin in 300 µL PBS. Blood samples were immediately tested for platelet aggregation. In selected studies, plasma drug levels were also measured. For drug level determinations, the remaining blood was centrifuged (1000 x g for 10 min at 4 °C) to obtain plasma. Plasma samples were then frozen (−20 °C) until assayed for drug levels. For rat PK studies, fasted male Sprague-Dawley rats were used. BX 667 or BX 048 (2 mg/kg) was administered in 10%DMSO/ 90% saline by oral gavage or intravenously. Blood samples were drawn at pre-designated time points via an indwelling jugular cannula. Plasma levels of BX 667 and BX 048 were quantified utilizing LC/MS/MS methods described below. For ex vivo platelet aggregation studies in rat, inhibition of ADP-induced platelet aggregation was monitored following oral gavage administration in male Sprague-Dawley rats. Blood samples were taken from the jugular vein of cannulated rats at 5 minutes before dosing to determine control platelet aggregation and at 15, 60, 120 and 180 minutes after dosing.
Determination of plasma concentration Samples and control plasma were thawed at room temperature. BX 290 was used as an internal standard. The range of the calibration curve was 0.01 µM–30 µM. Following the preparation of calibration standards and quality control samples, 50 µL standards and experimental samples were pipetted into cluster tubes. The plasma proteins were precipitated by addition of 200 μL cold acetonitrile containing an internal reference standard for quantification. The precipitated proteins were removed by centrifugation at 1000 ×g for 20 minutes at ambient temperature. Approximately 80 µL of the supernatant was placed into HPLC vial inserts for analysis. Plasma levels of BX 667 and BX 048 were measured by LC/MS/MS method. Pharmacokinetic parameters were calculated using (WinNonlin): Cmax: maximum concentration attained; tmax: time to maximum concentration; t1/2: half-life of compound elimination; AUCinf; area under the concentration time curve with the last concentration extrapolated based on the elimination rate; Cltotal: total clearance; F: % oral bioavailability.
Results In vitro platelet aggregation BX 667 and BX 048 potently inhibited ADP-induced platelet aggregation in human, dog and rat whole blood (Fig. 1). The IC50 values for BX 667, which represents the concentration at which 50% inhibition of ADP-induced platelet aggregation occurred were 97 ± 39 (n = 10), 317 ± 86 (n = 7), and 3000 ± 620 (n = 4) nM in human, dog and rat blood, respectively. ADPinduced aggregation was inhibited nearly 100% at 1 μM in human and dog. In rat maximal inhibition was achieved at 10 μM. For the primary metabolite, BX 048, the IC50 values for inhibition of ADP-induced aggregation were 290 ± 63 (n = 10), 230 ± 60 (n = 4), and 3600 ± 96 (n = 4) nM in human, dog and rat blood respectively. Concentrations required for maximal inhibition were similar to those observed with BX 667. The potency of BX 667 and BX 048 was found to be species dependent with the rank order of potency: human N dog N rat. Since other agents than ADP can induce platelet aggregation, BX 667 and BX 048 were tested against platelet aggregation induced by arachidonic acid and collagen. The compounds had
Figure 1 The in vitro effects of BX 667 and BX 048 on ADPinduced platelet aggregation in whole blood from human (top), dog (middle) and rat (bottom). BX 667 and BX 048 inhibited ADP (5 µM) —induced platelet aggregation in a concentration dependent manner. Data points represent the mean± SE. nominal effects on platelet aggregation induced by 3 μg/mL collagen (Fig. 2). However, BX 667 and BX 048 were found to inhibit platelet aggregation induced with 300 μM arachidonic acid in a concentration dependent manner albeit with a lower potency than ADP-induced aggregation. The IC50 values of BX 667 and BX 048 for arachidonic acid induced aggregation were 8.5 µM and 15 µM respectively.
In vitro stability and metabolism The stability of BX 667 at concentrations of 10 or 25 μM was evaluated in human, dog and rat blood. BX 667 was nearly completely converted to its primary metabolite BX 048 in rat
536
J.M. Post et al. Table 1 Metabolic stability of BX 667 in human, dog and rat jejunal S9 (n = 3), expressed as percent of remaining compound at the specified time points Species
Time (minutes)
% ± SD
Human
5 15 30 5 15 30 5 15 30
89.2 ± 5.0 91.9 ± 3.4 80.8 ± 2.1 79.8 ± 14.5 78.1 ± 4.2 81.7 ± 14.4 65.7 ± 10.3 32.5 ± 3.9 20.8 ± 1.4
Dog
Rat
* p b 0.05 vs. human and dog samples at corresponding time points.
found to be stable in microsomes from human, dog and rat with less than 10% conversion observed following 3 hours of incubation (data not shown). BX 667 and BX 048 were found to be metabolized to the same extent in either the presence or absence of NADPH which suggests that metabolic conversion is mediated by non-specific esterases rather than NADPHdependent cytochrome P450 enzymes. The potential for presystemic metabolism of BX 667 was evaluated in jejunal S9 fractions from human, dog and rat. BX 667 had relatively minimal metabolism in human and dog jejunal S9 fractions (Table 1) suggesting that intestinal Figure 2 Effect of BX 667 and BX 048 on platelet aggregation induced with 3 µg/mL collagen (top) or 300 µM arachidonic acid (bottom) in human blood. Datapoints represent the mean ± SE of 4 experiments. blood (N 99%) by 30 minutes. In contrast, BX 667 was relatively stable in human and dog blood with only 1–2% conversion to BX 048 after 30 minutes. The in vitro metabolic stability of BX 667 and BX 048 was also evaluated in hepatic microsomes from human, dog and rat. Metabolism of BX 667 to BX 048 occurred rapidly in rat relative to human and dog. The time-course for BX 667 metabolism in microsomes is illustrated in Fig. 3. The metabolite, BX 048, was
Figure 3 Time-course of BX 667 metabolism in hepatic microsomes from human, dog and rat.
Figure 4 The kinetics following intravenous and oral administration of 2 mg/kg BX 667 (top) or BX 048 (bottom) in conscious rats (n = 3).
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Table 2 Pharmacokinetic parameters following intravenous or oral administration of BX 667 and BX 048 at 2 mg/kg in rat (n = 3/dose group) BX 667 Cmax (µM) tmax (min) t½ (β) (h) AUCinf (µg·h/mL) Cltotal (mL/min/kg) F (%)
BX 048
i.v.
p.o.
i.v.
p.o.
33 1 2 12 3
1.9 60 3 6.9
56 1 1.4 23 1.4
0.18 30 4.5 0.42
57
2
Pharmacokinetic parameters were calculated using (WinNonlin): Cmax: maximum concentration attained; tmax: time to maximum concentration; t1/2: half-life of compound elimination; AUCinf; area under the concentration time curve with the last concentration extrapolated based on the elimination rate; Cltotal: total clearance; F: % oral bioavailability.
metabolism should have a minor impact on BX 667 bioavailability in higher species but in rat intestinal S9 fractions was metabolized 80 percent at 30 minutes.
Pharmacokinetics Pharmacokinetic experiments with BX 667 and BX 048 were performed in dog and rat to assess oral bioavailability. BX 667 is converted in vivo from the ester to its corresponding acid, BX 048, which is also a potent inhibitor of P2Y12 receptors (see above). Therefore, pharmacokinetic parameters for BX 667 were calculated by measuring both the parent compound and its metabolite BX 048 in dog studies. In the rat, due to rapid and extensive esterase activity, BX 667 was rapidly converted to BX 048 and therefore it was only possible to calculate the pharmacokinetic parameters of the active metabolite, BX 048.
Pharmacokinetics in rats The plasma concentrations of BX 048 following IV or PO administration of 2 mg/kg BX 667 or BX 048 in rat are illustrated in Fig. 4. Oral administration of BX 667 in rat led to rapid and well-maintained circulating levels of BX 048 with a peak concentration of approximately 2 μM at 60 minutes. Based on the circulating concentrations of BX 048 in the rat, the oral availability of BX 667 was deduced to be 57%. This finding supports the suitability of BX 667 for oral administration. In contrast, the oral availability of BX 048 was determined to be 2%. The pharmacokinetic parameters of BX 667 and BX 048 in rat are shown in Table 2.
Pharmacokinetics in dogs Oral administration of 10 mg/kg of BX 667 in dog resulted in rapid absorption and well-maintained circulating levels of BX 667 and its active metabolite BX 048 (Fig. 5, top). BX 667 was rapidly absorbed and reached a cumulative peak circulating concentration of approximately 14 μM after 60 minutes. Due
Table 3 Pharmacokinetic parameters for BX 667 and BX 048 following intravenous (2 mg/kg) or oral (10 mg/kg) administration of BX 667 or BX 048 in dogs (n = 3 per dose group) Compound
BX 667
Route
Figure 5 The pharmacokinetic profile of BX 667 and its metabolite BX 048 in conscious dogs (n = 3): Circulating plasma levels of BX 667 and BX 048 following PO (10 mg/kg) dose of BX 667 (top) and IV/PO kinetics of BX 667 itself following a IV (2 mg/ kg) and PO (10 mg/kg) dose (bottom). The insertion is the area under the curve (AUC) calculated from the data in the top panel.
IV
BX 048 PO
IV
PO
Plasma Conc.
BX 667
BX 048
BX 667
BX 048
BX 048
BX 048
Cmax (µM) tmax (min) t½ (β) (h) AUCinf (µg·h/mL) Cltotal (mL/min/kg) F (%)
35 1 0.21 2
5.2 15 7 4.2
4.3 15 3 3.3
10 60 6.9 16
8 12
6 4.1
17
N/A⁎
2.7 62
7
Cmax: maximum concentration attained; tmax; time to maximum concentration; t1/2; half- life of compound elimination; AUCinf; area under the concentration time curve with the last concentration extrapolated based on the elimination rate; Cltotal; total clearance; F; % oral bioavailability.
538
J.M. Post et al.
to hepatic or enteric esterase activity, the circulating level of BX 048 following IV or PO administration was substantially higher than the parent (BX 667) as illustrated by the greater AUC for BX 048 relative to BX 667 (Fig. 5, top insertion). The sum of the plasma circulating levels of BX 667 and BX 048 was 280 nM at 12 hours. The magnitude and duration of the circulating levels of BX 667 and BX 048 relative to their in vitro potencies suggests that platelet inhibition will be maintained for several hours. The plasma concentration time courses of BX 667 following IV (2 mg/kg) or PO (10 mg/kg) administration in dog are shown in Fig. 5 (bottom) and the PK parameters are shown in Table 3. Because BX 667 and its metabolite BX 048 are both potent inhibitors of platelet aggregation in blood, the aggregate of their circulating concentrations are responsible for their pharmacodynamic activity. Therefore, the %F was calculated using the sum of the AUC values for BX 667 and BX 048 after iv/po dosing of BX 667. The F was 62% suggesting that BX 667 is a suitable candidate for oral administration. The bioavailability of BX 048 was also calculated after IV/ PO dosing (Table 3) and as predicted by the data in rats, the oral bioavailability of this compound was much lower (F = 7%).
Pharmacodynamics Pharmacodynamics in rats Oral administration of doses of 1, 3 and 10 mg/kg BX 667 resulted in a rapid and dose-dependent inhibition of platelet aggregation (Fig. 6). The extent of inhibition was dosedependent and maintained at relatively constant levels throughout the 3 hour study. The average percent inhibition over the 3 hour time course following 1, 10 and 30 mg/kg dosing was 36 ± 15, 49 ± 24 and 73 ± 13 respectively. The PK profile in rat (Fig. 4, top) shows that the plasma level of BX 048 was near steady state between 1 and 3 hours following PO
Figure 7 Effect of oral administration of BX 667 (1, 3, 10 mg/kg) on ADP (5 µM)-induced platelet aggregation in conscious dogs (top). Data points represent the mean ± SEM. Platelet inhibition at 3 and 10 mg/kg was significantly greater than that observed at 1 mg/kg (p b 0.005). Correlation of the percent inhibition of ADP-induced platelet aggregation to the sum of the circulating plasma levels of BX 667 and BX 048 in dog (bottom). Each point represents one measurement from one animal from Fig. 5, top panel.
administration, which is consistent with the observed steadystate inhibition of platelet aggregation.
Figure 6 Effect of BX 667 on ADP (5 µM)-induced platelet aggregation after oral administration in conscious rats. Data points represent the mean ± SEM. Platelet inhibition was significant at all doses relative to vehicle control over the time course (p b 0.01). Platelet inhibition at 30 mg/kg was greater than at 1 mg/kg (p b 0.001) or 10 mg/kg (p b 0.02).
Pharmacodynamics in dogs Oral administration of doses of 1, 3 and 10 mg/kg BX 667 led to a rapid and dose-dependent inhibition of platelet aggregation (Fig. 7). The extent of inhibition was maximal (≈100%) at 30 minutes following 3 and 10 mg/kg administration. Platelet inhibition gradually declined with time, consistent with the observed time course for elimination of BX 667 and BX 048 (Fig. 5). Inhibition of ADP-induced platelet aggregation was well maintained for several hours (Fig. 7, top). A high correlation was found between the extent of inhibition of ADP-induced platelet aggregation and the sum of the circulating levels of BX 667 and BX 048 (r2 = 0.72; Fig. 7, bottom). The ex vivo potency of BX 667 to inhibit ADP-induced
PK/PD of a platelet inhibitor platelet aggregation (IC50 ≈ 450 nM) was also consistent with in vitro potency measurements (IC50 ≈ 317 nM) in dog.
Discussion Whole blood platelet aggregometry was used in the present study to evaluate the pharmacodynamic activity of BX 667 and BX 048. The use of this method minimizes manipulation of the blood samples and allows for the monitoring of platelet activity in a biological matrix which reflects the in vivo pharmacological activity of platelet inhibitors [15]. In vitro platelet aggregation experiments indicate that BX 667 and BX 048 potently inhibit ADP-induced platelet aggregation in human, dog and rat blood. An important pharmacological property of BX 667 is that both the parent compound and its metabolite, BX 048, are highly potent and selective inhibitors of P2Y12 receptors and that the rank order potency is the same in whole blood as it is in binding studies and aggregation experiments on washed platelets (see accompanying manuscript). Therefore, the extent of inhibition of platelet aggregation is dependent on the combined circulating levels of BX 667 and BX 048. The in vitro potencies of BX 667 and BX 048 were species dependent with the greatest potency observed in human relative to dog and rat. It has to be point out the in vitro potency in the whole blood assay (Fig. 1) is different from that in the washed platelets (Fig. 2 of part I of the companion paper) in the human samples between the 2 compounds, this may be due to the presence of enzymes in the blood, such as hydrolase that could convert BX667 to BX048. In addition, the different protein binding for the 2 compounds may also impact on the potency. In vivo, modulators of platelet function provide the appropriate balance for maintaining hemostasis without causing either excessive thromboembolic events such as stroke or making platelets so unresponsive that hemorrhagic risk or bleeding is increased [16]. Therefore, in addition to ADP, it was of interest to test the effects of BX 667 and BX 048 with the platelet activators, collagen and arachidonic acid, to evaluate any potential cross-talk that may exist. Collagen is an important thrombogenic component of the subendothelial matrix and induces platelet aggregation using a multistep process involving the integrin α2β1 and glycoprotein VI (GPVI) [16,17]. Collagen is able to amplify its response by stimulating release of ADP and thromboxane A2 (TxA2) from platelets [17,18]. Additional platelet activators such as thrombin via proteaseactivated receptors (PAR1) and arachidonic acid are also able to stimulate platelet aggregation. BX 667
539 and BX 048 had nominal effects on collagen-induced platelet aggregation suggesting that P2Y12 receptors play only a minor role in collagen-induced platelet activation. In transgenic studies with platelets derived from P2Y1 –deficient mice, collagen-induced aggregation was greatly attenuated [19]. Together, these results suggest that P2Y1 receptors play a greater role in collagen-induced aggregations than P2Y12 receptors. In contrast to collagen, BX 667 and BX 048 were able to inhibit arachidonic acid induced aggregation albeit at concentrations much greater than those needed to inhibit ADP-induced platelet aggregation. This is consistent with results from studies with the P2Y12 receptor inhibitor, clopidogrel, where it was observed that although clopidogrel does not significantly alter collagen-induced aggregation [20]; inhibition of P2Y12 can inhibit thromboxane A2 mediated platelet effects [21]. These results indicate that although BX 667 and BX 048 may provide an anti-thrombotic benefit by inhibition of ADP-mediated platelet aggregation, other platelet activators such as collagen may continue to assist in the maintenance of hemostatic function when the subendothelial matrix or vessel wall is compromised (e.g. during surgery). Oral administration of BX 667 in dog and rat leads to a rapid and dose-dependent inhibition of platelet aggregation in contrast to clopidogrel which requires hepatic activation and has a delayed antithrombotic effect. Furthermore, the extent of inhibition of platelet activity correlated well with the circulating plasma levels of BX 667 and BX 048. The potency calculated from the in vivo experimental determination of the PD for BX 667 was consistent with the in vitro potency measurements (See accompanying manuscript). The observation that BX 667 reversibly inhibits P2Y12 receptors also distinguishes it from irreversible platelet inhibitors such as clopidogrel or aspirin. The direct relationship between PK and PD will assist with dose selection and response monitoring. BX 667 was highly bioavailable in dog and rat following oral administration and as expected its acid metabolite, BX 048, was poorly bioavailable. In rat, BX 667 was rapidly metabolized in blood, hepatic microsomes and intestinal fractions. In human and dog, BX 667 was relatively stable in both blood and intestinal fractions. BX 667 was gradually metabolized by an NADPH-independent process in hepatic microsomes suggesting that it is a poor P-450 substrate. The overall PK and pharmacodynamic profiles of BX 667 indicate its suitability as an orally available therapeutic candidate. These results suggest that complete inhibition of ADP-induced platelet
540 aggregation would occur at circulating levels of 1 μM in humans. It is anticipated that because BX 667 is a reversible ADP-receptor inhibitor as demonstrated in the companion paper (ref), it will be easier to predict the extent and duration of platelet inhibition compared to irreversible inhibitors providing a greater safety margin if adverse events such as bleeding occur or if greater control over hemostasis is desired. Recently, the efficacy and safety of BX 667 in comparison to clopidogrel in various preclinical animal models of thrombosis were described [14]. BX 667 demonstrated superior efficacy relative to bleeding risk when these agents were administered either alone or in combination with aspirin. In the studies reported here and in the accompanying paper (ref) we show that BX 667 is a reversible antagonist of platelet aggregation and as such its drug-target residence time will be lower than that of an irreversible antagonist such as clopidogrel. Drug-target residence time affects both the efficacy and potential side effects of a drug [22]. In the case of P2Y12 receptor antagonists, efficacy and bleeding are both due to the blockade of the receptor. Thus while a long drugtarget residence time improves efficacy, it also inevitably increases bleeding. These findings suggest that BX 667 as a reversible P2Y12 receptor antagonist may provide a wider therapeutic index relative to irreversible platelet inhibitors because of its lower drug-target residence time. In summary, the present report characterizes the pharmacodynamic and pharmacokinetic properties of the novel P2 Y12 receptor inhibitor BX 667 and its active metabolite BX 048. BX 667 is an orally available, reversible P2Y12 receptor inhibitor that potently inhibits ADP-induced aggregation. Inhibition of platelet aggregation is rapid in onset and the duration and extent of platelet inhibition is directly proportional to the plasma concentration of BX 667 and BX 048. The direct PK/PD relationship should assist in dose selection to accurately predict the extent and duration of activity in order to provide optimal therapeutic benefit.
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