In cats with cardiomyopathy, thromboembolic complications account for approximately 9% to 38% of deaths, justifying the investigation of antithrombotic drugs as preventives in high-risk patients.1,2 Possible antithrombotic approaches include inhibition of platelet function, which can be achieved by antagonism of specific platelet cell surface receptors, or interference with intracellular signaling pathways. Although the former provides high specificity, inhibition of platelet signal transduction might exert broader effects and suppress platelet activation independent of the initial stimulus.3
In human medicine, there is recent and growing interest in the use of phosphodiesterase (PDE) inhibitors as antithrombotic agents.3–6 Phosphodiesterase enzymes catalyze the hydrolytic inactivation of cyclic adenosine monophosphate and cyclic guanosine monophosphate, intracellular second messengers that inhibit platelet activation. Thus, PDE inhibition favors reduced platelet aggregation. The PDE3-inhibitors dipyridamole, cilostazol, and milrinone have demonstrable antiplatelet activity when administered to human patients.6–11 These drugs exert their antiplatelet effects not only through PDE inhibition but also by reducing the reuptake of adenosine, an important platelet function inhibitor.3–5,9,11
Pimobendan is a positive inotrope and vasodilator that has been shown to delay the onset of congestive heart failure (CHF) in dogs affected by myxomatous mitral valve disease or dilated cardiomyopathy and to prolong survival time in dogs with CHF secondary to these conditions.12–14 Recently, there has been interest in the use of pimobendan for the treatment of CHF in cats affected by cardiomyopathy.15,16
In addition to its calcium-sensitizing effects, pimobendan is an inhibitor of PDE3. A limited number of studies have evaluated the in vitro effects of pimobendan on human or animal platelets.7,17–19 In 1, pimobendan, as well as its active metabolite, O-desmethylpimobendan (ODMP), reduced the in vitro aggregation of human platelets in a dose-dependent manner.18 Pimobendan also reduced aggregation of canine platelets; however, this effect was only demonstrated at in vitro pimobendan concentrations that were 1000-fold greater than those considered achievable with clinically recommended oral dosages.19 Only 1 investigation of pimobendan’s effects on feline platelets has been reported.20 In that in vitro study, concentration-dependent inhibition of platelet aggregation was demonstrated;20 however, the concentration necessary to achieve this effect was higher than plasma concentrations subsequently reported in pharmacokinetic studies in cats.20–22 Nonetheless, pimobendan’s potential antiplatelet effects continue to be considered in clinical settings, highlighting the need for in vivo investigation.23,24
To date, there are no published reports characterizing pimobendan’s effects on platelet function when given orally to cats at clinically relevant dosages. If oral pimobendan therapy produces demonstrable antiplatelet effects, additional benefits could be derived from its usage. Further, because heart disease is frequently complicated by arterial thromboembolism (ATE) in cats, and affected patients are often treated with antiplatelet and/or anticoagulant agents, increased risk of bleeding could be of concern if pimobendan also inhibits platelet function in cats.
The goal of this study was to evaluate the effects of pimobendan, administered orally at 2 dosages, on platelet function in healthy adult cats. We hypothesized that compared with no treatment, oral administration of twice daily pimobendan would not be associated with statistically significant changes in platelet function in healthy cats.
Methods
Cats
Six sexually intact (n = 3 female), healthy, purpose-bred, adult domestic shorthair cats, aged 1.1 to 1.4 years, with mean ± SD body weight of 4.603 ± 0.489 kg, were used for this study. Before the start of the study, each cat’s general and cardiac health was confirmed by normal findings of physical examination, complete blood count, serum biochemical profile, urinalysis, and echocardiography. Cats were vaccinated against common viral diseases and tested negative for FeLV antigen and antibodies against FIV. Cats had access to water at all times and were fed a commercially available adult feline ration twice daily. Ambient temperature was maintained between 20 and 22 °C, inclusive. All research activities were approved by the Institutional Animal Care and Use Committee of the University of Georgia (AUP A2018-12-017-A3).
Pimobendan administration
Each cat was administered chewable pimobendan (Vetmedin) orally at a dosage of 0.625 mg/cat twice daily (low-dose) for 15 doses (treatment days 0–7), followed by 1.25 mg/cat twice daily (high-dose) for 15 doses (treatment days 7–14), with no washout period between. A 1-week washout period was then allowed. Each cat was observed to ingest the full dose at each administration. Evaluated dosages were based on those previously advocated for the treatment of CHF in cats and used in studies describing the pharmacokinetics of pimobendan in healthy adult cats.21,22 On sampling days, food was withheld for approximately 12 hours before drug administration (or blood sampling for baseline timepoints) and was provided after blood sampling.
Blood sample collection and processing
On the mornings of treatment days 0, 7, and 14, and again after a final 1-week washout period (study day 21; posttreatment baseline), blood was sampled for platelet function testing and plasma drug concentrations. Sampling was performed immediately before the first dose of pimobendan (day 0; pretreatment baseline), 1 hour after the morning dose of pimobendan (i.e., at the time of expected peak drug levels21,22 on days 7 and 14), or at approximately 9 am (day 21; posttreatment baseline). Pre- and posttreatment baseline samples were obtained to evaluate the effect of time and repeated venipuncture on tested variables.
At each time point, approximately 6 mL of blood was obtained using a 21-gauge butterfly needle for cephalic venipuncture of gently restrained cats. Before collection, cats were sedated with a combination of intramuscular ketamine (2–10 mg/kg), midazolam (0.2–0.5 mg/kg), and alfaxalone (0–2.25 mg/kg) at variable doses based on individual cat demeanor. Approximately 3.6 mL of blood were immediately transferred to collection tubes containing sodium citrate (BD Vacutainer; Becton Dickinson) at a 1:9 final citrate:blood ratio, which was gently inverted 5 times and held at room temperature (23 °C) for platelet function testing. An additional 2 mL of blood was transferred to a collection tube containing lithium heparin, which was gently inverted 3 times, placed immediately on ice, and centrifuged within 20 minutes of collection at 2 °C to 4 °C and 2,000 X g for 10 minutes. Plasma was separated and stored at −80 °C within 1 hour of collection, until later analysis of drug concentrations.
Platelet testing
Whole blood aggregometry (Chronolog 700; Chronolog Corp) using an impedance-based method, with collagen and adenosine diphosphate (ADP) as agonists (Chronolog Corp) was performed as previously described within 2 hours of collection.25 Briefly, citrated whole blood, diluted 1:1 with isotonic saline, was placed in 2 aggregometer cuvettes and warmed to 37 °C. Samples were stirred at 1,200 rpm and a stable baseline was established before induction of aggregation by adding 5 µM ADP or 5 µg/mL collagen. Aggregation curves were recorded for 7 minutes after agonist addition. The amplitude (Ω) of the aggregometry curve after 7 minutes, as well as the rate of aggregation (Ω/min), were recorded for each sample.
Platelet function was also assessed by use of a platelet function analyzer (PFA-100, Siemens) as previously described.26,27 Briefly, citrated whole blood was transferred into a disposable test cartridge containing a collagen- and ADP- coated membrane. The cartridge was then inserted into the instrument and time to aperture occlusion (closure time), indicative of platelet plug formation, was recorded. A duplicate assessment of PFA closure time was not performed due to low sample volume.
Plasma drug analytical methods
Plasma samples were shipped overnight on dry ice and stored at −80 °C until batch analysis. Pimobendan and ODMP concentrations were quantified using ultra-high performance liquid chromatography with tandem mass spectrometry at Auburn University’s Specialized Pharmaceutical and Experimental Center for Translational Research and Analysis. In brief, reference standards for pimobendan, ODMP, as well as internal standard, 13C,2H3-pimobendan were obtained from Alsachim (Illkirch Graffenstaden, Bas-Rhin, France). Reagent-grade formic acid was obtained from Sigma-Aldrich (Sigma-Aldrich; Merck KGaA), and MS-grade acetonitrile and water were obtained from VWR (Avantor delivered by VWR).
Analysis was performed using an Agilent 1290 Infinity II LC ultra-high performance liquid chromatography (UHPLC) system coupled to an Agilent 6460 triple quadrupole (QQQ) mass spectrometer (MS/MS). Samples were introduced into the LC-MS/MS system using electrospray ionization in positive-ion mode for detection. Data processing was performed using the Agilent MassHunter software package (MassHunter Qualitative Analysis B.06.00; Agilent).
Stock solutions of pimobendan, ODMP, and 13C,2H3-pimobendan were prepared at 1 mg/mL and 1 µg/mL. The 1 mg/mL stock solutions were prepared in DMSO (100%) and diluted in 1:7 (V/V) DMSO:H2O and stored at −20 °C. Calibration solutions were prepared in plasma at a concentration range from 400 to 0.049 ng/mL. Separately prepared quality control (QC) samples were prepared (n = 3) at levels of 0.7831, 25, and 400 ng/mL.
Calibration, QC, and test samples (200 μL each) were deproteinated with the addition of 400 μL acetonitrile followed by vortexing for 20 seconds and centrifugation (13,000 X g, 4 °C). Before deproteinization, a 10 μL volume of internal standard was added to all standards, QC, and test samples to achieve a concentration of 40 ng/mL. After centrifugation, 580 µL of supernatant was transferred to an HPLC vial, and 200 µL of H2O was added to each to aid in separation and peak shape.
A 2 uL sample was injected into UHPLC-MS/MS system via autosampler and separation was carried out on an Agilent Zorbax SB C18 column (2.1 X 50 mm, 1.8 µm) maintained at 40 °C. Gradient elution was performed according to the following timing: 95% to 60% mobile phase A (H2O + 0.1% (V/V) formic acid) and 5% to 40% mobile phase B (ACN + 0.1% (V/V) formic acid) from 0 to 0.7 minutes, at a flow rate of 0.6 mL/min. Retention times of ODMP, pimobendan, and pimobendan-13C were 1.047, 1.166, and 1.164, respectively with a total run time of 2.2 minutes. During assay development, blank samples were injected throughout the analysis of standard and actual samples to ensure chromatographic response returned to baseline before subsequent injections. During the analysis of all samples, blank samples were also used after standards and in conjunction with QC samples to monitor the performance of the assay.
Quantitation was performed using multiple reaction monitoring (MRM) modes to monitor parent to product-ion (m/z) transitions for pimobendan, ODMP, and 13C,2H3-pimobendan. Both a quantifying and qualifying ion were selected for each analyte. MS/MS conditions and MRM transitions monitored are listed elsewhere (Supplementary Material S1). The standard curve was linear with concentrations within 10% of their nominal values, and QC samples were within 10% of their nominal values with <15% coefficient of variation. No evidence of carry-over, alterations in baseline, or changes in pump pressure were observed during the analysis.
Column washing was carried out before and after the run of each sample set. This was done using a mixture of 70% H2O, 15% methanol, and 15% ACN for at least 10 column volumes, 100% MeOH for at least 10 column volumes, and 100% ACN for at least 10 column volumes. Blank plasma and calibration standards were run before and after the sample runs and showed no signs of carryover.
Statistical methods
The number of cats studied was found to be adequate to detect a 35% prolongation of the PFA closure time,27 targeting power of 0.8, and α of 0.05 (SigmaStat 4.0; Systat Software Inc). Aggregometry and PFA data were analyzed using commercially available software (GraphPad Prism 8.0; GraphPad Software LLC). Friedman tests were used to test for differences in aggregometry and PFA data among treatments. For statistically significant comparisons (plasma drug and ODMP concentrations), pairwise comparisons were performed using Dunn’s test for multiple comparisons. Differences with a P-value < .05 were considered significant. Data are presented as median (range).
Results
Five of 6 cats accepted pimobendan readily, with the remaining cat requiring occasional manual administration of the tablet. Blood samples of adequate volume were successfully collected from all cats at all timepoints. Median (range) administered doses of ketamine, midazolam, and alfaxolone were 3 mg/kg (2–10 mg/kg), 0.2 mg/kg (0.2–0.5 mg/kg), and 1 mg/kg (0–2.25 mg/kg), respectively. The median (range) time from administration of the oral dose of pimobendan to blood collection was 62.5 minutes (61–75 minutes) and 68.5 minutes (65–70 minutes) for low-dose and high-dose pimobendan, respectively. Platelet function testing was performed within 2 hours of collection for all samples.
Median (range) plasma pimobendan concentrations were 0.296 ng/mL (0.103–1.77 ng/mL), 15.1 ng/mL (6.89–20.2 ng/mL), 32.8 ng/mL (23.3–44.8 ng/mL), and 0.188 ng/mL (0.090–1.23 ng/mL) for samples obtained at pretreatment baseline, from cats receiving low-dose pimobendan, from cats receiving high-dose pimobendan, and at posttreatment baseline, respectively. Median (range) plasma ODMP concentrations were 0.0263 ng/mL (0.000–0.584 ng/mL), 4.03 ng/mL (2.45–7.26 ng/mL), 11.6 ng/mL (7.45–14.6 ng/mL), and 0.498 ng/mL (0.351–1.03 ng/mL) for samples obtained at pretreatment baseline, from cats receiving low-dose pimobendan, from cats receiving high-dose pimobendan, and at posttreatment baseline, respectively.
Data for PFA and aggregometry parameters were obtained for all cats (Table 1). No significant difference in PFA closure time, amplitude of the aggregometry curve in response to ADP or collagen, or rate of aggregation in response to ADP or collagen, were found among any of the treatment conditions.
Whole blood platelet function and platelet aggregation data in 6 healthy cats treated with pimobendan orally for 15 doses at 0.625 mg/cat twice daily (low-dose) or 1.25 mg/cat twice daily (high-dose), or untreated (pretreatment and posttreatment baselines).
| ADP agonist (5 μM) | Collagen agonist (5 µg/mL) | ||||
|---|---|---|---|---|---|
| Treatment | PFA closure time (s) | Amplitude of aggregation (Ω) | Rate of aggregation (Ω/min) | Amplitude of aggregation (Ω) | Rate of aggregation (Ω/min) |
| Pretreatment baseline | 81.5 (58–138) | 18 (0–27) | 10.5 (0–17) | 25.5 (23–30) | 22 (17–28) |
| Low-dose pimobendan | 70 (59–83) | 19 (6–24)* | 12 (3–18)* | 25 (17–30) | 23.5 (17–30) |
| High-dose pimobendan | 62 (51–83) | 20 (2–23) | 12 (2–20) | 27.5 (24–30) | 24 (18–26) |
| Posttreatment baseline | 78.5 (56–86) | 13.5 (0–20) | 13 (0–18) | 28 (22–30) | 23 (16–27) |
Blood samples were obtained 1 hour postdose for pimobendan treatment periods. Platelet aggregation was induced with ADP or collagen. No significant differences in any analyzed parameter were found among any of the treatment conditions. Data are reported as median (range). PFA = Platelet function analyzer-100®. ADP = Adenosine diphosphate.
Data only available for 5 cats.
While all samples demonstrated measurable aggregation in response to 5 µg/mL collagen agonist, those obtained from 2 cats demonstrated minimal or no whole blood aggregation in response to 5 µM ADP across treatment conditions. For these cats, whole blood aggregometry was repeated at some time points using higher ADP agonist concentrations (10 µM, 20 µM, or both), which resulted in a modest improvement of aggregation response when compared with results obtained with 5 µM ADP (Supplementary Table S1). Assessment of whole blood aggregometry in response to 10 μM and 20 μM ADP was not possible for all time points due to inadequate sample volume.
Discussion
This study did not demonstrate a significant effect of 2 clinically relevant oral doses of pimobendan, when administered to healthy cats, on platelet aggregation or PFA-100® closure time. The authors of a previous report found dose-dependent inhibition of platelet aggregation when pimobendan was added in vitro to feline platelet-rich plasma.20 In that report, the half-maximal effective concentration (EC50) of pimobendan was 0.92 μg/mL, and pimobendan concentration of 2 μg/mL essentially abolished platelet aggregation.20 In the present study, at the time of blood sampling for platelet function testing, the median plasma drug concentration of pimobendan after high-dose oral dosing was 32.8 ng/mL, a value similar to the reported maximum plasma drug concentrations in 2 pharmacokinetic studies of healthy cats.21,22 As such, the average plasma drug concentration after oral administration of 1.25 mg pimobendan (i.e., the highest dose tested here) in cats is more than 25-fold lower than the EC50 concentration that demonstrated inhibition of platelet aggregation in the previous report.20 Similarly, in another in vitro study, pimobendan reduced aggregation of canine platelets only at drug concentrations that were 1,000-fold greater than clinically achievable concentrations.19 Although demonstrable antiplatelet effects of other PDE inhibitors are reported,6–11 the concentration of pimobendan required to achieve platelet inhibition is likely greater than that achievable by clinically relevant oral doses in veterinary species. While our results are not unexpected, the present study was necessary to show the absence of an in vivo antiplatelet effect in cats.
Our study evaluated the effects of pimobendan alone, which does not necessarily mimic the clinical use of this drug in cats with CHF. In these patients, a combination of drugs, often including antiplatelet and/or anticoagulant agents, is common. Although no antiplatelet effects of pimobendan were identified in the current study, it is unknown if the combination of pimobendan with other agents, such as clopidogrel, would confer additional antiplatelet effect over clopidogrel alone, especially as the 2 drugs would act on complementary but different pathways for platelet activation. Further studies are warranted to investigate the effect of cardiac drug combinations on feline platelet function.
This study had several limitations. Samples obtained from 2 cats demonstrated minimal or no response to ADP agonist (5, 10, or 20 µM) for whole blood aggregometry, although these cats had PFA-100 closure times that were similar to those observed for the other cats in the study. Other reports of feline whole blood platelet aggregation have demonstrated absent or very low aggregation responses to 20 μM ADP, though this finding was attributed to sampling or handling problems leading to platelet clumping.28 In the authors’ experience, platelet aggregation using ADP as an agonist is less predictable and robust, particularly when tested at relatively low concentrations, than aggregation initiated by collagen. We consider that potential errors in sample collection or handling may have led to reduced platelet aggregation results in these cats; however, reduced aggregometry with ADP agonist was noted in samples obtained from the same 2 cats at all timepoints, and these cats showed robust aggregation responses to collagen, indicating a possible inherent insensitivity to ADP. Moreover, blood sampling in these 2 cats was not considered more difficult than sampling from other cats in the study. Consistent with the manufacturer’s guidelines, we used citrate anticoagulant for impedance aggregometry. Multiple ex vivo primate and canine studies using a different impedance aggregometer (Multiplate® Analyzer) found hirudin or heparin anticoagulants resulted in increased aggregation compared with citrate in ADP-activated samples.29–34 To the authors’ knowledge, there is no such study investigating different anticoagulants in cats nor a study investigating different anticoagulants for the impedance aggregometer we used. Citrate might not have been the optimal anticoagulant to detect platelet aggregation in ADP-activated samples from these 2 cats.
Cats in the present study were sedated to minimize platelet activation secondary to the release of tissue factors from traumatic venipuncture. In previous studies assessing feline platelet function, cats have been sedated with ketamine, acepromazine, benzodiazepines, opioids, or some combination of these.25,26,35,36 While we do not suspect that sedative drugs had a major influence on our results, these drugs may have influenced feline platelet function. However, as the sedation protocol was standardized among timepoints for each cat, any effect should have been similar across treatments.
Low plasma concentrations of pimobendan and ODMP were detected in most cats before drug exposure at the pretreatment baseline (day 0), and at the posttreatment baseline (day 21), when the drug should have been fully cleared based on the results of 2 pharmacokinetic studies of healthy cats.21,22 Background signal leading to reduced test specificity is considered the most likely explanation for this finding.
In the present study, we chose 2 established methods for the assessment of platelet activation. A previous report found a difference in platelet aggregation response using the Plateletworks assay, but not whole blood aggregometry, between clopidogrel- and placebo-treated cats, highlighting differences in the sensitivity of these tests.28 More sensitive methods, such as flow cytometry for platelet activation (P-selectin expression) and optical aggregometry,37 might have allowed the detection of subtle antiplatelet effects of pimobendan treatment.
While our study utilized healthy purpose-bred adult cats, previous work suggests that platelets from cats with HCM might have altered function.35,38,39 In 1 study, biomarkers of platelet activation were increased in platelets obtained from cats with HCM when compared with those obtained from healthy cats.38 Results of a previous report suggested that platelets from cats with HCM are more sensitive to agonist stimulation, while another failed to identify a significant difference in PFA closure time between healthy cats and cats with HCM.35,39 Therefore, our findings, generated in healthy cats, might not accurately represent the platelet response in cats with HCM receiving oral pimobendan.
Using whole blood aggregometry and PFA, we did not detect a measurable antiplatelet effect of pimobendan when administered orally to healthy adult cats at 2 clinically relevant dosages. Prospective studies are warranted to further investigate the effect of pimobendan on platelet function in cats with heart disease in which the drug is administered alone or alongside other antiplatelet medications.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org.
Acknowledgments
The authors thank Anna-Claire Bullington, Lydia Moss, Robert Miller, Kathleen Hoover, Samantha Salmon, Jackson Sanders, and Clinton Lynn for assistance with blood collection and sample processing.
Disclosures
The authors have nothing to disclose. No AI-assisted technologies were used in the generation of this manuscript.
Funding
Funding was provided by ACVIM Resident Research Grant. The funding source did not have any involvement in study design, data analysis and interpretation, or writing and publication of the manuscript.
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