Pharmacodynamic and pharmacokinetic evaluation of clopidogrel and ...

Pharmacodynamic and pharmacokinetic evaluation of clopidogrel and the carboxylic acid metabolite SR 26334 in healthy dogs Benjamin M. Brainard, VMD; Stephanie A. Kleine, BS; Mark G. Papich, DVM, MS; Steven C. Budsberg, DVM, MS

Objective—To determine pharmacodynamic and pharmacokinetic properties of clopidogrel and the metabolite SR 26334 in dogs. Animals—9 mixed-breed dogs. Procedures—8 dogs received clopidogrel (mean ± SD 1.13 ± 0.17 mg/kg, PO, q 24 h) for 3 days; 5 of these dogs subsequently received a lower dose of clopidogrel (0.5 ± 0.18 mg/kg, PO, q 24 h) for 3 days. Later, 5 dogs received clopidogrel (1.09 ± 0.12 mg/kg, PO, q 24 h) for 5 days. Blood samples were collected for optical platelet aggregometry, citrated native and platelet mapping thrombelastography (TEG), and measurement of plasma drug concentrations. Impedance aggregometry was performed on samples from 3 dogs in each 3-day treatment group. Results—ADP-induced platelet aggregation decreased (mean ± SD 93 ± 6% and 80 ± 22% of baseline values, respectively) after 72 hours in dogs in both 3-day treatment groups; duration of effect ranged from ≥ 3 to > 7 days. Platelet mapping TEG and impedance aggregometry yielded similar results. Citrated native TEG was not different among groups. Clopidogrel was not detected in any samples; in dogs given 1.13 ± 0.17 mg/kg, maximum concentration of SR 26334 (mean ± SD, 0.206 ± 0.2 µg/mL) was detected 1 hour after administration. Conclusions and Clinical Relevance—Clopidogrel inhibited ADP-induced platelet aggregation in healthy dogs and may be a viable antiplatelet agent for use in dogs. Impact for Human Medicine—Pharmacodynamic effects of clopidogrel in dogs were similar to effects reported in humans; clopidogrel may be useful in studies involving dogs used to investigate human disease. (Am J Vet Res 2010;71:822–830)

C

ritically ill dogs are at risk for thromboembolic disease.1–4 The most severe manifestations are pulmonary and aortic thromboembolism, both of which are associated with severe illness and death.4–6 Diseases as diverse as infection with parvovirus, immune-mediated hemolytic anemia, atrial fibrillation, and neoplasia have been associated with hypercoagulability or the formation of arterial or venous thrombi.4–6 Dogs of some breeds, such as the Soft Coated Wheaten Terrier, may inherit glomerulonephropathies that predispose them to urinary protein loss and subsequent hypercoagulability.7 After thrombus formation, treatment

ASA CD CYP HD HPLC LD OPA PRP TEG WBA

Received March 17, 2009. Accepted June 23, 2009. From the Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, University of Georgia, Athens, GA 30602 (Brainard, Kleine, Budsberg); and the Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606 (Papich). Supported by the Morris Animal Foundation (MAF D07CA-303) and the University of Georgia Clinical Research Committee. Presented in abstract form at the American College of Veterinary Internal Medicine Forum, San Antonio, Tex, June 2008. Address correspondence to Dr. Brainard ([email protected]).

with thrombolytic agents (eg, streptokinase) carries a substantial risk of hemorrhage or metabolic derangements caused by reperfusion.8 Consequently, it is preferable to prophylactically treat dogs that have known or suspected hypercoagulability to decrease the chance of thromboembolic complications. This is frequently accomplished by administration of anticoagulant (eg, heparin or warfarin) or antiplatelet (eg, aspirin) medication.3 Whereas oral antiplatelet drug protocols have been extensively evaluated in humans, there are currently no drugs approved for this indication in compan-

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Abbreviations

Acetylsalicylic acid Chronic-dose Cytochrome P450 High-dose High-pressure liquid chromatography Low-dose Optical aggregometry Platelet-rich plasma Thrombelastography Whole blood aggregation

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ion animals, and few reports are available to establish reliable guidelines. In the authors’ experience, many owners are unable or unwilling to administer injectable anticoagulant drugs (eg, heparin) to their pets, so oral formulations are preferred for long-term treatment of a hypercoagulable state in animals. The most commonly used drug for chronic treatment of affected animals has been aspirin (ie, ASA), which inhibits platelet cyclooxygenase and decreases platelet production of thromboxane.3 In general, platelet aggregation is sustained by the release of thromboxane A2 from activated platelets; however, this is not the case in all dogs.9,10 Acetylsalicylic acid does not reliably decrease canine platelet aggregation,10 and consequently, administration of ASA alone may be suboptimal for prophylaxis against thromboembolism in some dogs. Adverse effects of ASA can also include gastrointestinal bleeding, which may be exacerbated in patients with altered platelet function.11 The combination of corticosteroids and ASA may increase the risk of gastrointestinal complications. For these reasons, oral administration of a drug that inhibits ADPinduced canine platelet aggregation (eg, clopidogrel) may result in safer and more reliable inhibition of platelet aggregation in dogs. Clopidogrel has been safely administered to cats,12 rabbits,13 and calves,14 but there are limited data available on its effects in dogs.15 Clopidogrel is a prodrug; it is not active in the administered form and must be converted to an active metabolite. In humans, it is primarily metabolized by 2 main pathways, 1 that leads to the formation of the inactive carboxylic acid metabolite SR 26334 and 1 that leads to the active metabolite (a thiol derivative) via formation of the 2-oxo-clopidogrel intermediate.16 The antiplatelet effects of clopidogrel are attributed to this active metabolite.17 In humans, conversion to the active metabolite is via activities of CYP enzymes, including CYP2C19, CYP3A4, CYP1A2, and CYP2B6.18 Some drug treatments are reported to inhibit CYP2C19.18,19 Although dogs have CYP2C19, CYP3A4, CYP1A2, and CYP2B6 activities,20 it is not known whether the metabolic pathway for clopidogrel activation in dogs is the same as that in humans. Concentrations of the parent drug and active metabolite are usually undetectable in plasma; therefore, clopidogrel pharmacokinetics have been assessed via measurement of the concentration of SR 26334 as a surrogate marker.21 In humans, SR 26334 represents 85% of the circulating clopidogrel metabolites.21 We hypothesized that it would be possible to measure concentrations of SR 26334 in canine plasma and to determine an association between the pharmacokinetic activity of this metabolite and the reported antiplatelet effects in dogs. We also sought to detect and measure plasma concentrations of the clopidogrel parent compound, although it is rapidly metabolized in humans. The objective of the study reported here was to provide a thorough assessment of the pharmacodynamics and pharmacokinetics of clopidogrel administered PO to healthy dogs and to determine the relationship between plasma concentrations of clopidogrel or SR26334 and platelet aggregation. AJVR, Vol 71, No. 7, July 2010

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Materials and Methods Animals—Eight adult mixed-breed dogs (5 males and 3 females) were randomly selected from a research colony and assigned to the HD group. Subsequently, 5 of these 8 dogs (3 males and 2 females) were randomly selected for assignment to the LD group. The CD group also consisted of 5 randomly selected dogs (3 males and 2 females); 4 of these had been part of the HD group, and 1 had not previously received clopidogrel. Mean ± SD weight was 30 ± 6.4 kg, 28.5 ± 5.9 kg, and 27.1 ± 4.6 kg for dogs in the HD, LD, and CD groups, respectively; no dog had received any medication for ≥ 14 days prior to the study. Dogs were considered healthy on the basis of results of physical examination, serum biochemical analysis, and a CBC. During periods of drug administration, the dogs received 2 physical examinations daily and were monitored closely for adverse effects (eg, petechiae, hemorrhage, melena, or bruising). Food was withheld for 12 hours prior to collection of all blood samples for OPA. All procedures were approved by the University of Georgia Institutional Animal Care and Use Committee. Experimental design and dose determination— Clopidogrela was initially evaluated in short-term experiments at 2 doses. The 8 dogs in the HD group received (mean ± SD) 1.13 ± 0.17 mg of clopidogrel/kg PO every 24 hours for 3 days. This initial dose was derived from the human dose of 75 mg/d. Statistical power analysis for the subsequent experiments was based on the results from the HD experiment, and only 5 dogs were assigned to experimental groups thereafter. After a 2-week washout period during which no medications were administered, 5 of the dogs from the HD group were randomly assigned to the LD group by a technician who was unaware of the results of the HD experiment. Dogs in the LD group received (mean ± SD) 0.50 ± 0.18 mg of clopidogrel/kg PO every 24 hours for 3 days. Experiments for the CD group were performed after a 5-month washout period; 5 dogs were randomly assigned to this group by an individual who was unaware of the results of the HD and LD studies. Four of these dogs had been part of the HD group, and 1 had not previously received clopidogrel. The dose used in the CD experiment (mean ± SD; 1.09 ± 0.12 mg/kg, PO, q 24 h) was determined to be effective on the basis of results of the HD and LD experiments and was administered for 5 days. Drug preparation—Investigators were aware of the dose of clopidogrel administered to each dog. For the HD and CD groups, 75-mg tablets were divided into doses on the basis of tablet weight (each 75-mg tablet was weighed and divided into individual portions for each dog to provide the required dose). For the LD group, tablets (containing 75 mg of clopidogrel) were weighed and ground to a powder. The powder was weighed and aliquoted; the calculated dose for each dog was transferred into empty gelatin capsulesb for administration. Clopidogrel administration was designated as time 0. Sample collection for the HD and LD groups— Blood samples were collected from a jugular vein into 12-mL syringesc that contained no additives by use of

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a 19-gauge butterfly catheterd when possible. Samples (9 mL) for OPA were collected into a 12-mL syringe containing 1 mL of 3.2% sodium citratee for a final citrate-to-blood ratio of 1:9. Samples for some pharmacokinetic time points were collected by direct cephalic venipuncture. Blood samples were collected for serum biochemical analysis and CBC immediately prior to oral administration of clopidogrel (ie, baseline, time = 0) and at 96 hours (time = 96) after initial administration for all experiments. For HD and LD groups, baseline blood samples were used for OPA,f citrated native TEG,g platelet mapping TEG,g and pharmacokinetic analysis. Blood samples were collected 20, 40, 60, 120, and 180 minutes after initial clopidogrel administration for platelet mapping and pharmacokinetic analysis. In 3 dogs from each group, samples collected at these intervals were also used for WBAf (ie, impedance) analysis. Additionally, blood samples from each group were collected daily, immediately prior to oral administration of clopidogrel, for 4 days (24, 48, 72, and 96 hours after initial clopidogrel administration) for OPA, citrated native TEG, and pharmacokinetic analysis. Sample collection for the CD group—Blood samples were collected from dogs in the CD group on days 1, 3, and 6 and then once daily until day 10 or until platelet function returned to OPA values similar to baseline values from the HD and LD study. Additional samples were obtained from 3 dogs on day 13 because platelet function had not returned to baseline values by day 10. Blood samples were collected via jugular venipuncture as described for dogs in the HD and LD experiments. Samples were evaluated for citrated native TEG, platelet mapping TEG, and OPA at each time point. OPA—The PRP was prepared from 20 mL of citrated blood. The blood was centrifuged twice at room temperature (ie, 20°C) and 540 X g for 10 minutes. The PRP was removed between centrifugations. The remaining blood was then centrifuged at 1,500 X g for 10 minutes to prepare platelet-poor plasma. The PRP was diluted as necessary with platelet-poor plasma to obtain a final platelet count between 250,000 and 300,000 platelets/µL. Platelet counts on PRP were performed by use of an impedance technique.h The PRP was allowed to equilibrate at room temperature for 30 minutes and then 500 µL of the sample was added to a glass cuvette containing a magnetic stir bar. The sample was warmed to 37°C and stirred at 1,200 revolutions/min. An optical aggregometerf was used to evaluate aggregation in response to ADPi (10 or 15µM) and collageni (10 or 15 µg/mL) as agonists.22 The agonist concentration that yielded 50% to 80% aggregation in the baseline samples was used for subsequent aggregation of samples from the same dog. TEG—Each blood sample was transferred into a 1.8-mL tubej containing 3.2% sodium citrate to yield a 1:9 citrate-to-blood ratio. The sample was allowed to equilibrate at room temperature for 30 minutes prior to analysis. A TEG cupk containing 20 µL of 0.2M CaCl2k was warmed to 37°C, and 340 µL of citrated blood was added to the cup to initiate analysis. Continuous measurements relating to the viscoelastic properties of the sample as clot formation progressed were transduced 824

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and transferred to a computer. The data were translated into a thrombelastogram via softwarel that calculated and displayed several variables: reaction time (ie, R), time to reach a standard clot firmness (ie, K), maximum clot strength (ie, maximum amplitude), and speed of clot formation (ie, α-angle).2 Platelet mapping TEG—Each blood sample was transferred into a 4-mL tubej containing lithium heparin and allowed to equilibrate at room temperature for 30 minutes prior to analysis; 360 µL of heparinized blood was added to a warmed (37°C) TEG cup containing 10 µL of a proprietary mixturem of a reptilase-based substance and activated factor XIII23 with 10 µL of ADPk for a final concentration of 2µM ADP. The sample was was added to the TEG, partially aspirated back into the pipette tip 3 times to mix the contents, and then replaced in the cup to start the test. The data were transduced to the computer as described for citrated native TEG analysis. WBA—Each blood sample was transferred into a 1.8-mL tubej containing 3.2% sodium citrate to yield a 1:9 citrate-to-blood ratio. The sample was allowed to equilibrate at room temperature for 30 minutes prior to analysis; 500 µL of citrated blood was added to a plastic cuvette containing a magnetic stir bar and 500 µL of 0.9% NaCl solution.n The cuvette was warmed to 37°C and stirred at 1,200 revolutions/min. Aggregation was measured by use of an impedance probe and aggregometer.f Aggregation was induced with ADPi at final concentrations of 10 or 15µM to achieve a total impedance of 8 to 12 Ω on the baseline sample. The concentration of agonist that yielded the best baseline tracing was used to activate subsequent samples from the same dog. Pharmacokinetic analysis—An HPLC assay was developed for clopidogrel and SR 26334; the method used was developed in the laboratory of 1 of the authors (MGP). Clopidogrel and the SR 26334 pure reference standardso were dissolved in pure methanole to make a 1 mg/mL stock solution. Dilutions of the stock solution were made in distilled water to provide fortifying solutions for blank (control) plasma used for quality control and calibration. The stock solution was stored at 4°C in a tightly sealed dark vial. Clopidogrel and SR 26334 solutions were added to blank (control) plasma to create 9 calibration standards (range, 0 to 10 µg/mL). The mobile phase for HPLC analysis consisted of 0.01M potassium phosphate buffere (75%) and acetonitrile (25%).e The pH of the mobile phase (3.0) was adjusted with 85% phosphoric acid.e Fresh mobile phase was prepared, filtered, and degassed each day. The HPLC system consisted of a quaternary solvent delivery systemp at a flow rate of 1 mL/min, an autosampler,q and a UV detector set at a wavelength of 220 nm.r The chromatograms were integrated with a computer program.s The column was a reverse-phase 4.6 mm X 15 cm C8 columnt kept at a constant temperature of 40°C. All plasma samples, calibration samples, and control plasma samples were prepared identically. Solidphase extraction cartridgesu were conditioned with 1 mL of methanol followed by 1 mL of 0.01M acetate buffer (pH, 4.2). Each plasma sample was added to a conditioned cartridge, followed by a wash step of 1 mL AJVR, Vol 71, No. 7, July 2010

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Results

of the acetate buffer. The drug was eluted with 1 mL of methanol and collected in clean glass tubes. Solutions in the tubes were evaporated at 40°C for 25 to 35 minutes. Each product was then reconstituted with 200 µL of mobile phase and vortexed. Fifty microliters of each solution was then injected into the HPLC system. Retention time for the peak of interest was 4.0 to 4.5 minutes. A fresh set of calibration and blank samples was prepared each day. All calibration curves were linear with an R2 value of 0.99 or higher. Limit of quantification for clopidogrel and SR 26334 in canine plasma was 0.05 µg/mL, which was determined from the lowest point on a linear calibration curve that yielded an appropriate signal-to-noise ratio. Laboratory personnel used guidelines published by the United States Pharmacopeia.24 Plasma drug concentrations were plotted on linear and semilogarithmic graphs for analysis. Analysis of curves and pharmacokinetic modeling was performed by use of a commercial pharmacokinetic software program.v

Pharmacodynamics of clopidogrel—All dogs tolerated the drug well for the duration of the study; no evidence of petechiae, bruising, or hemorrhage was observed in any dog. There was no evidence of hematoma formation at venipuncture sites in any dog in any group. The mean ± SD dose of clopidogrel administered during the short-term experiments was 1.13 ± 0.17 mg/ kg for the HD group and 0.50 ± 0.18 mg/kg for the LD group. Dogs in the CD group received a mean dose of 1.09 ± 0.12 mg/kg. Platelet mapping results indicated a rapid inhibitory effect on ADP-induced clot strength (Table 1). Maximum amplitudes were significantly decreased from baseline values beginning 60 minutes after clopidogrel administration in samples from the HD group and were 11.5% of baseline values 180 minutes after administration. For dogs in the LD group, only the 180-minute maximum amplitude was significantly (P < 0.001) decreased from baseline values. No significant difference was observed in any monitored variables (R, K, α angle, and maximum amplitude) for citrated native TEG from dogs in the HD or LD groups after clopidogrel administration (data not shown). The results for ADP-induced WBA closely resembled the results for platelet mapping TEG; a significant decrease in impedance was observed within 60 minutes after clopidogrel administration. The mean ± SD impedance of WBA for the 3 HD-group dogs in which this variable was monitored differed significantly (P = 0.04) at 60 minutes from the baseline value of 6.5 ± 1.0 Ω, decreasing to 1.0 ± 2.0 Ω. Values at baseline and

Statistical analysis—Statistical analysis was performed by use of commercial software.w Data were evaluated for normality by use of a Kolmogorov-Smirnov test. Parametric data between days were compared via a 1-way ANOVA for repeated measures with the HolmSidak test for pairwise multiple comparisons. For nonparametric data, a 1-way repeated-measures ANOVA on ranks was performed, with a Tukey test for pairwise comparisons. Parametric or nonparametric data between the start and end of a dose regimen (eg, CBC data) were evaluated by use of a paired Student t test or a Wilcoxon signed rank test, respectively.

Table 1—Mean ± SD maximum amplitude (ie, a representation of maximum clot strength) derived from platelet mapping analysis of heparinized whole blood samples from healthy adult dogs that received clopidogrel (1.13 ± 0.17 mg/kg, PO, q 24 h [HD group], or 0.5 ± 0.18 mg/kg, PO, q 24 h [LD group]) for 3 days. Variable Maximum amplitude (mm)

Baseline 15.1

20

HD group 40

60

LD group

120

180

Baseline

20

40

60

180

24.5  17.7 19.8  18.2 12.7  4.4* 10.7  3.6* 4.4  0.2* 31.8  15.9 23.5  15.2 15.8  5.6 16.3  8.9 7.0  3.9*

The HD group consisted of 8 dogs (5 males and 3 females); 5 of these 8 dogs (3 males and 2 females) were randomly selected for the LD group after a washout period of 2 weeks. Administration of the initial dose of clopidogrel was designated as time 0. Baseline measurements were determined in blood samples obtained from dogs immediately prior to the initial administration of clopidogrel, and times indicate the interval (number of minutes) after initial administration. *Amplitudes differ significantly (P  0.001) from the baseline value within that group.

Table 2—Mean ± SD percentage of platelet aggregation (measured by use of OPA after incubation in vitro with ADP or collagen as agonist) in plasma samples obtained from healthy adult dogs that received clopidogrel. Variable

Baseline

Aggregation 82.6  17.0 after ADP (%) Aggregation 80.9  15.0 after collagen (%)

HD group

LD group

24

48

72

Baseline

24

48

72

6.2  6.0*

2.4  3.9*

6.0  7.3*

72.2  26.0

23  25*

12.8  16.0*

15.0  16.6*

20.5  15.0*

9.3  5.0*

17.8  25.0*

71.4  19.9

34.0  30.0

27.2  32.0

29.0  26.5

Agonist concentrations were chosen for individual dogs to result in a baseline aggregation between 50% and 90%. Samples were obtained immediately prior to administration of the initial dose of clopidogrel (baseline) or immediately prior to administration of the daily dose of clopidogrel at 24, 48, and 72 hours. At 48 and 72 hours, platelets in samples from all dogs in the HD group and 4 of 5 dogs in the LD group disaggregated after initial response to ADP; maximum aggregation values were used for comparisons, despite the disaggregation. See Table 1 for remainder of key. AJVR, Vol 71, No. 7, July 2010

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at 60 minutes did not differ significantly (P = 0.12) for the dogs in the LD group. Results obtained at 120 and 180 minutes after clopidogrel administration were not significantly different from the results obtained at 60 minutes in dogs from the HD and LD groups. Analysis of the results of OPA testing revealed a profound inhibition of ADP-induced platelet aggregation 24 hours after the initial administration of clopidogrel in dogs in the HD group (Table 2). For all dogs in the HD group, ADP-induced platelet aggregation tracings for the 48- and 72-hour time points after initial clopidogrel administration were characterized by an initial aggregation response, which was then followed by disaggregation (Figure 1). For this group of dogs, the greatest downward deflection (ie, the maximum aggregation) was analyzed, even though this likely represented an initial platelet reaction to the addition of agonist. Inhibition of ADP-induced platelet aggregation in dogs of the HD group was not significantly different between samples obtained at 24, 48, and 72 hours after initial clopidogrel administration, and percentage of aggregation at all of these time points was significantly (P < 0.001) decreased, compared with baseline aggregation values. In dogs in the LD group, the magnitude

Figure 1—Representative tracings from OPA analysis for percentage of ADP-induced platelet aggregation in PRP from a healthy mixed-breed dog after administration of clopidogrel (mean ± SD; 1.13 ± 0.17 mg/kg, PO, q 24 h) for 3 days (HD group). Administration of the initial dose of clopidogrel was designated as time 0. Values < 0 indicate the effect of the initial platelet shape change. Tracings represent results for samples obtained before daily clopidogrel administration (baseline [blue line], 24 hours [black line], 48 hours [red line], and 72 hours [green line]).

of ADP-induced platelet aggregation was significantly (P < 0.001) decreased from the baseline values at all time points. In contrast with the HD group, disaggregation was not detected in all dogs from the LD group; samples from 4 of 5 dogs at 48 and 72 hours after initial administration of clopidogrel disaggregated after an initial response to the ADP, with the tracing slowly returning to zero. Peak aggregation was also recorded for samples from the LD group, even though disaggregation was observed. The percentage of collagen-induced platelet aggregation measured by use of OPA also decreased after clopidogrel administration in dogs in the HD and LD groups. Compared with baseline aggregation values, the percentage of aggregation in dogs from the HD group was significantly (P < 0.010, P < 0.009, and P < 0.013, respectively) decreased at 24, 48, and 72 hours after initial administration of clopidogrel; there were no significant differences among aggregation values at these 3 time points (Table 2). Collagen-induced aggregation did not differ significantly among samples from dogs in the LD group at any time point, including the baseline value. Disaggregation was not observed during collagen-induced aggregation experiments. No clinically relevant changes from baseline values were identified during serum biochemical analysis of samples obtained 96 hours after initial administration of clopidogrel in dogs in the HD group, and no values were outside of laboratory reference ranges before or after the experiment. There was a significant (P = 0.003) decrease in alkaline phosphatase activity (from a mean ± SD of 50 ± 27 U/L to 47 ± 26 U/L [reference range, 13 to 122 U/L]) as well as a slight but significant (P = 0.006) increase in total calcium concentration (from a mean ± SD of 10.33 ± 0.34 mg/dL to 10.68 ± 0.28 mg/dL [reference range, 9.3 to 11.4 mg/dL]) in samples from these dogs. Complete blood counts in the HD group similarly had no clinically relevant changes and few significant changes; platelet counts decreased significantly (P = 0.039) from a mean ± SD of 317,000 ± 62,000 platelets/µL to 289,000 ± 47,000 platelets/µL (reference range, 235,000 to 694,000 platelets/µL), and a slight but significant (P < 0.01) increase in circulating monocytes (from a mean ± SD of 1,182 ± 650 cells/µL to 2,250 ± 880 cells/µL [reference range, 100 to 1,400 cells/µL]) was detected. No significant differences from baseline values were detected in CBC variables or serum biochemical results

Table 3—Mean ± SD percentage of platelet aggregation (measured by use of OPA after incubation in vitro with the platelet receptor agonist ADP [15µM]) in plasma obtained from 5 healthy adult dogs that received clopidogrel (1.09 ± 0.12 mg/kg, PO, q 24 h) for 5 days (CD group).* Variable Aggregation after ADP (%)

3

6

4.0  6.0 5.2  5.5

Time (d)

7

8

9

10

13

8.8  6.3 36.0  13.3† 39.2  24.0† 44.8  13.9† 44.4  17.2†

Platelets in samples from all dogs in the group disaggregated after the initial response to ADP on days 3, 6, and 7; the maximum aggregation values prior to disaggregation were used for comparison. On days when clopidogrel was administered, samples were obtained immediately prior to drug administration. *The CD group consisted of 5 dogs (3 males and 2 females); 4 of these dogs had received (mean ± SD) 1.13 ± 0.17 mg of clopidogrel/kg PO once daily for 3 days  5 months prior to this experiment. †Values were significantly (P  0.005) different from values determined on day 3. See Table 1 for remainder of key. 826

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96 hours after initial administration of clopidogrel for dogs in the LD group. The assessment of platelet function in dogs in the CD group after 5 days of clopidogrel administration revealed a potent and long-lasting inhibition of ADPinduced platelet aggregation. This variable was more profoundly affected in some dogs than in others, with the return to baseline values delayed up to 7 days after the final dose of drug was administered (Table 3). Analysis of the results of platelet mapping TEG revealed a slow return to baseline values for most dogs in the CD group; at 5 days after discontinuation of clopidogrel (ie, day 10), samples from some dogs still had significantly decreased responses to ADP (Figure 2). When evaluated as a group, maximum amplitudes were significantly (P < 0.001) lower than baseline values on days 3, 6, 7, and 8, but the differences were not significant (P = 0.31, P = 0.12, and P = 0.114, respectively) on days 9, 10, and 13. Complications in obtaining baseline aggregation values for dogs in the CD group were encountered because of a malfunctioning centrifuge, which resulted in production of inadequate PRP for the experiment. All dogs had been previously evaluated for adequate platelet function and had normal platelet aggregation responses to ADP. Because the purpose of the CD group was to evaluate the return of platelet function and because it was impossible to collect additional blood samples for PRP after clopidogrel administration, samples from these dogs were only evaluated for a return to normal ADP-induced aggregation variables by use of a high concentration of ADP (15µM). The ADP-induced platelet aggregation was minimal, and it was characterized by disaggregation in all dogs on days 3, 6, and 7; beginning on day 8, a gradual return to normal aggregation was detected (Table 3). There was no significant difference in the percentage of ADP-induced platelet aggregation among days 3, 6, and 7; however, on days 8 through 10 and 13, a significantly (P < 0.005 for all) greater per-

Figure 2—Maximum amplitudes (ie, a representation of maximum clot strength) obtained via platelet mapping TEG of heparinized whole blood samples for 5 healthy adult dogs (3 males and 2 females) that received clopidogrel (mean 6 SD, 1.09 6 0.12 mg/ kg, PO, q 24 h) for 5 days (CD group). Four of these 5 dogs had received 1.13 6 0.17 mg of clopidogrel/kg PO once daily for 3 days > 5 months prior to this experiment. Samples were obtained immediately prior to administration of the daily dose of clopidogrel on days when the drug was given. Values were significantly (P < 0.001) different from baseline (day 0) on days 3, 6, 7, and 8. Each symbol represents results for 1 dog. AJVR, Vol 71, No. 7, July 2010

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centage of aggregation was detected, compared with the percentage aggregation on day 3 in response to the same dose of ADP. On day 13, ADP-induced aggregation (mean ± SD, 44.4 ± 17.2%) was still less than would be expected for untreated dogs (65% to 85%, based on baseline values for HD and LD dogs), which may have indicated some residual effects of the drug. Similar to the results for dogs from HD and LD groups that received clopidogrel for the short term, no significant change was detected in any variables of the citrated native TEG analysis after clopidogrel treatment in dogs from the CD group (data not shown). All results of serum biochemical analysis were within the reference ranges for dogs in the CD group prior to administration of clopidogrel, and these tests were not repeated at the completion of the experiment. Initial results of CBCs for this group were also within reference ranges for the laboratory, except for 2 dogs that had mild thrombocytopenia (179,000 and 194,000 platelets/µL, respectively). At the completion of treatment (day 5), CBC results indicated a significant (P = 0.018) increase in mean platelet count (from a mean

Figure 3—Mean ± SD plasma concentrations of the inactive carboxylic acid metabolite (SR 26334) of clopidogrel in samples obtained from healthy adult dogs that received clopidogrel (mean 6 SD; 1.13 6 0.17 mg/kg, PO, q 24 h [HD group; white circles], or 0.50 6 0.18 mg/kg, PO, q 24 h [LD group; black squares]) for 3 days. Samples were obtained at various time points during the first 24 hours after clopidogrel administration (A) as well as at 24-hour intervals for up to 96 hours after initial clopidogrel administration (B). The HD group consisted of 8 dogs (5 males and 3 females); 5 of these 8 dogs (3 males and 2 females) were randomly selected for the LD group after a washout period of 2 weeks. When repeat doses were administered at 24, 48, and 72 hours, the samples were obtained immediately prior to drug administration.

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± SD of 235,000 ± 56,000 platelets/µL to 289,000 ± 38,000 platelets/µL); mean platelet volume was also significantly (P = 0.001) increased (from a mean ± SD of 7.1 ± 0.86 fL to 10.6 ± 0.73 fL) in dogs from the CD group. Pharmacokinetics of clopidogrel—Analysis of the plasma concentration-versus-time profiles for SR 26334 revealed a significant (P = 0.012) difference between the samples from dogs in the HD and LD groups; samples from the HD group had a higher peak (Figure 3). No detectable concentration of clopidogrel was found in any samples, and a pharmacokinetic analysis could not be performed. For SR 26334, there was no defined terminal halflife that could be derived from these concentrations. Without a defined terminal half-life, determinations such as area under the curve and variables derived from the area under the curve could not be calculated reliably. Other pharmacokinetic values, such as maximum plasma concentration and time to maximum plasma concentration, could be derived directly from a graph of the data (Figure 3). In samples from dogs in the HD group, the plasma concentration of SR 26334 peaked 60 minutes after initial administration of clopidogrel (mean ± SD, 0.206 ± 0.2 µg/ mL). In samples from dogs in the LD group, the concentration of SR 26334 peaked 120 minutes after initial administration (mean ± SD, 0.086 ± 0.1 µg/mL). The highest detected concentration of SR 26334 in dogs from the HD group was 0.705 µg/mL; this same dog had the highest detected concentration of SR 26334 in the LD group (0.276 µg/mL). After initial administration of clopidogrel, only 2 dogs had detectable plasma concentrations of SR 26334 at 24 hours, and only 3 dogs had detectable concentrations at 48 hours; all of these were in the HD group. The 24- and 48-hour samples were obtained prior to administration of the next dose of clopidogrel, and analysis revealed mean ± SD trough concentrations of 0.045 ± 0.016 µg/mL and 0.065 ± 0.014 µg/mL, respectively. However, some dogs had concentrations below the limit of quantification (ie, 0.05 µg/mL), which may have biased the data (ie, they were counted as 0 in the analysis). In the CD group, only 1 dog had detectable concentrations of SR 26334; these were identified on days 3 and 6, although the concentrations were below the limit of quantification (0.05 and 0.03 µg/mL, respectively). Discussion At the doses investigated in the study reported here, oral administration of clopidogrel caused rapid and effective inhibition of canine platelet aggregation responses to ADP and collagen. The effect for ADP was most profound, which indicated that the drug may exert an effect at the canine P2Y12 ADP receptor. Collagen is a more general activator of platelet aggregation, with an effect transduced via both GP VI (ie, glycoprotein 6) and integrin α2β1 on platelets.25 Platelet activation by collagen results in the release of ADP from dense granules of platelets, which magnifies the aggregation response. In dogs in the study reported here, inhibition of the ADP receptor effect may have resulted in a decrease in the magnitude of aggregation when collagen was used as an agonist.26 828

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The overall coagulability of the blood determined via whole blood TEG analysis was not significantly affected by administration of clopidogrel at the doses administered in the present study. Platelet activation and aggregation contributed to the final TEG tracing; however, the multiple mechanisms available for platelet activation, combined with the interactions among other procoagulant factors and the platelets, likely overwhelmed the effect of ADP receptor inhibition. In humans, a native TEG tracing of samples obtained from a patient after clopidogrel administration may display a shallow α-angle (which indicates slower clot formation) or prolonged R time (indicating a slower time to initial fibrin formation), but frequently does not reveal a difference and is not specific for single receptor platelet inhibition.27 By contrast, when global platelet function is disabled via administration of other drugs such as cytochalasin-D or abciximab, profound effects are detected on whole blood TEG tracings.28 Platelet activation via ADP is through at least 2 separate G-protein–coupled receptors, P2Y1 and P2Y12.29 The P2Y12 receptor is antagonized by a metabolite of clopidogrel; however, the P2Y1 receptor is theoretically still able to react with ADP. Interaction at the P2Y1 receptor is likely the reason for the initial shape change and minor aggregation of platelets detected in samples from most dogs prior to disaggregation.30 In some dogs (which may have variants of the P2Y12 receptor), clopidogrel activity may be absent or diminished, similar to responses reported in humans with polymorphisms in this receptor.31 The individual variation in results, even in this small subset of mixed-breed dogs, indicates that clopidogrel will have varying effects in different animals, perhaps because of polymorphisms in ADP receptors. The doses used in the present study were approximate, and this may have resulted in some of the observed variability. Variation in aggregation was more pronounced at lower doses, which may have been the result of unresponsiveness of platelets or of an insufficient number of affected platelets in circulation. In humans that had an exaggerated response to ADP-induced platelet aggregation, a lower degree of ADP-induced aggregation was detected for a given dose of clopidogrel.32 A report33 in the veterinary literature has described dogs that were less responsive to the inhibitory effects of NSAIDs on platelet function than other dogs, and all dogs should not be expected to have the same response to platelet-inhibiting drugs. Monitoring with platelet aggregometry or TEG platelet mapping is recommended for verification and quantification of drug effect. The inhibitory effect of clopidogrel on platelets appears to be irreversible. In humans treated once daily for 7 days with 75 mg of clopidogrel, platelet activity (measured by P-selectin expression in response to ADP activation) returned to expected values at 7 days after discontinuation of the drug.34 This appears to be similar to the effects of clopidogrel in dogs in the present study; platelet function in many dogs returned to baseline values approximately 8 days after cessation of drug treatment, although some individual variability was detected (Figure 2; Table 3). The mean ± SD life span of a canine platelet has been reported as 6.0 ± 1.1 AJVR, Vol 71, No. 7, July 2010

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days35; because aggregation responses are an average of the activity of many platelets (some affected and some not), the return to aggregation activity similar to baseline values likely indicates the contribution of new, unaffected platelets. Because plasma drug concentrations decrease quickly in dogs, newly released platelets are not exposed to the residual parent drug or measured metabolite in the plasma. All dogs in the study reported here retained minimal aggregation responses to ADP for at least 3 days after the final dose of drug; this may imply that every-other-day administration is a feasible option after an initial period of daily drug administration. Analysis of the plasma concentration profiles also suggested that an intermittent dosing schedule may be effective. The SR 26334 metabolite of clopidogrel was detectable, even at 72 hours after the initial dose (Figure 3), although some dogs had concentrations that were below the limit of quantification, and the pharmacokinetics in dogs between the active metabolite (which is inherently unstable) and SR 26334 likely differ.17 The dosing regimen of clopidogrel requires further evaluation before it can be recommended for administration to dogs in a clinical setting, especially because cessation of treatment may result in transient platelet hyperactivity.36 No adverse reactions referable to platelet dysfunction were identified in the healthy dogs in the study reported here. At the doses tested, clopidogrel appears to be a safe drug for use in dogs for the purpose of decreasing platelet aggregation. In particular, clopidogrel may be a safe alternative to NSAIDs for prophylaxis against thromboembolism in dogs concurrently receiving corticosteroids because it avoids the adverse drug interactions associated with the coadminstration of corticosteroids and NSAIDs.37 Many animals with immune-mediated hemolytic anemia are hypercoagulable and treated with high doses of corticosteroids. Although presumed platelet inhibition by the use of LD (0.5 mg/kg, PO, q 24 h) aspirin appears to confer a survival advantage in dogs with this disease,3 the combination of aspirin and corticosteroids may result in serious adverse effects. Clopidogrel is a prodrug that requires hepatic conversion to an active molecule to exert its effects. The active metabolite is the thiol derivative R-130964, a molecule that requires 2 enzymatic conversions from the inactive prodrug form.18 The inactive carboxylic acid metabolite SR 26334 is reliably generated in humans who take clopidogrel at a dose of approximately 1 mg/kg PO every 24 hours (75 mg/person/d), resulting in maximum plasma concentrations > 3 µg/mL.38 In the study reported here, dogs treated with a similar dose produced SR 26334 at much lower concentrations. The metabolic pathway for clopidogrel activity has been defined in humans16; specific CYP enzymes (esterases) have been identified that convert the parent drug to the active metabolite, and other enzymes have been identified that convert clopidogrel to SR 26334. Although these enzymes are also found in dogs,20 the metabolic pathway for clopiogrel in dogs has not been defined. Because antiplatelet effects were observed in the present study despite extremely low measured concentrations of SR 26334, it is possible that dogs produce an active metabolite but have a different pathway for inactive metabolites. For example, the dogs in the present AJVR, Vol 71, No. 7, July 2010

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study may have preferentially produced higher concentrations of another metabolite that was not assayed. Alternatively, these dogs may have rapid clearance of SR 26334 once it is produced. Without a study of the parent drug and metabolites administered to dogs IV, these patterns of metabolism cannot be identified for this species. The pharmacodynamic profile established in the study reported here indicated that most dogs had significant inhibition of platelet function within 3 hours after the initial PO administration of clopidogrel. This peak coincided with the highest production of SR 26334 as well, which indicated a time of maximum drug absorption. Inhibition of aggregation lasted at least 24 hours and was not associated with plasma concentrations of clopidogrel or SR 26334. The lack of detectable concentrations of clopidogrel in any samples was thought to be attributable to extensive first-pass metabolism, but it may also have been attributable to degradation of the drug in the gastrointestinal tract. Although the present study provides data on the effectiveness of clopidogrel in healthy dogs, it does not necessarily provide information on the use of clopidogrel in dogs that are critically ill or receiving other drugs. Because clopidogrel is not available as an injectable solution for IV use, the patient population is necessarily limited to those that can tolerate orally administered medications. Hepatic CYP inhibition by drugs such as rifampin or cimetidine may change the pharmacodynamics of clopidogrel in dogsx and is also likely to affect its pharmacokinetics. These are relevant pharmacological interactions that will define the future use of clopidogrel in veterinary medicine. a.

Plavix, clopidogrel, 75 mg, Bristol-Meyers Squibb, Princeton, NJ. b. Gelatin capsules, size 0, Eli Lilly, Indianapolis, Ind. c. Monoject syringe, Tyco Healthcare, Mansfield, Mass. d. 19 gauge, 3/4-inch Transiva winged infusion set, Abbott Laboratories, North Chicago, Ill. e. Sigma-Aldrich Co, St Louis, Mo. f. Model 700 Whole Blood/Optical Lumi-Aggregometer, Chronolog Corp, Havertown, Pa. g. TEG 5000, Haemoscope Corp, Niles, Ill. h. Heska, Loveland, Colo. i. Chrono-log Corp, Havertown, Pa. j. Vacutainer, Becton-Dickinson, Franklin Lakes, NJ. k. Haemoscope Corp, Niles, Ill. l. TEG Analytical Software, version 4.2.95, Haemoscope Corp, Niles, Ill. m. Activator F, Haemoscope Corp, Niles, Ill. n. 0.9% saline for irrigation, Abbott Laboratories, North Chicago, Ill. o. Clopidogrel and SR 26334 pharmaceutical standards for HPLC analysis, generously provided by Sanofi-Aventis, Paris, France. p. Agilent Technologies, Wilmington, Del. q. 1100 Series autosampler, Agilent Technologies, Wilmington, Del. r. 1100 Series variable wavelength detector, Agilent Technologies, Wilmington, Del. s. 1100 Series Chemstation software, Agilent Technologies, Wilmington, Del. t. Zorbax Rx-C8, MAC-MOD Analytical Inc, Chadds Ford, Pa. u. Bond Elut CNE, 3-mL cartridges, Varian Inc, Palo Alto, Calif. v. WinNonlin, version 5.0.1, Pharsight Corp, Mountain View, Calif. w. SigmaStat, version 3.5, Systat Software, Chicago, Ill. x. Goodwin JC, Hogan DF, Green HW. Altered hepatic metabolism of clopidogrel in dogs with inducers and inhibitors of hepatic

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