Effect of standard‐dose and high‐dose pimobendan on select indices of renal and cardiac function in dogs with American College of Veterinary Internal Medicine stage B2 myxomatous mitral valve disease

Abstract Background Pimobendan might have favorable effects on renal function but this has not been well‐studied in dogs with myxomatous mitral valve disease (MMVD). Objectives Determine the effects of standard‐dose (SD_pimo) and high‐dose pimobendan (HD_pimo) on glomerular filtration rate (GFR) and cardiac size and function in dogs with preclinical MMVD. Animals Thirty nonazotemic dogs with stage B2 MMVD. Methods Prospective, randomized, double‐blinded, placebo‐controlled clinical study. Dogs had an echocardiographic examination, assessment of GFR (iohexol clearance), N‐terminal probrain natriuretic peptide (NT‐proBNP), and quality of life (QOL) score at baseline and 7 to 10 days after placebo (n = 6), SD_pimo 0.2 to 0.3 mg/kg q12 (n = 12), or HD_pimo 0.5 to 0.6 mg/kg q12h (n = 12). Results No significant differences in GFR or QOL scores were detected between groups (P ≥.07). After HD_pimo, the mean [SD] percent change of NT_proBNP (−46.1 [20.2]%), left atrial volume (LAV; −27.1 [16.9]%), left ventricular end‐diastolic volume (EDV; −21.8 [15.0]%), and end‐systolic volume (ESV; −55.0 [20.7]%) were significantly different (P ≤.004) from placebo (0.5 [19.9]%, 1.3 [15.6]%, −0.2 [8.2]%, −7.3 [35.6]%, respectively) but not the percent change after SD_pimo (−36.6 [16.1]%, −22.7 [14.9]%, −16.7 [12.5]%, −41.6 [14.8]%, respectively; P > .05). After SD_pimo, percent change of NT_proBNP, LAV, EDV, and ESV were significantly different from placebo (P < .05). Conclusions and Clinical Importance Results suggest that pimobendan (SD_pimo or HD_pimo) might not affect renal function in nonazotemic dogs with stage B2 MMVD. High‐dose pimobendan did not demonstrate advantages over SD_pimo within the constraints of our study.

B2 MMVD. High-dose pimobendan did not demonstrate advantages over SD_pimo within the constraints of our study. Pimobendan is an inodilator drug recommended for dogs with American College of Veterinary Internal Medicine (ACVIM) stage B2, C, and D MMVD. 1 In contrast to other drugs used in the management of MMVD (vasodilators, diuretics), 7,8 pimobendan does not activate the renin angiotensin aldosterone system (RAAS). [9][10][11] In preclinical studies of healthy dogs, pimobendan increases cardiac output, 12 renal blood flow, 13 and offsets furosemide-induced prerenal azotemia. 11 Pimobendan might have favorable effects on renal function and increase glomerular filtration rate (GFR), particularly at high doses. 11 However, 1 study in healthy dogs 14 and another in dogs with experimentally-induced mitral valve regurgitation 13 suggest that pimobendan does not have an important effect on GFR. Off-label escalation of pimobendan dose, frequency, or both has become a clinical strategy for dogs with MMVD and refractory CHF, azotemia, or both. 1,15,16 Although the benefits of standard-dose pimobendan (SD_pimo) on cardiac function are well-documented in dogs with MMVD, [17][18][19] to our knowledge, the effect of SD_pimo and particularly high-dose pimobendan (HD_pimo) on renal and cardiac function have not been well-studied.
The primary objective of this study was to determine the effect of SD_pimo (0.2-0.3 mg/kg PO q12) and HD_pimo (0.5-0.6 mg/kg PO q12) on glomerular filtration rate (GFR) in dogs with ACVIM stage B2 MMVD. A secondary objective was to determine the effect of SD_pimo and HD_pimo on echocardiographic indices of cardiac size and function. We hypothesized that SD_pimo and HD_pimo would improve renal and cardiac function.

| MATERIALS AND METHODS
All study procedures were approved by the Institutional Animal Care and Use Committee at the University of California, Davis (protocol #: 20226). All dog owners gave written, informed consent prior to enrollment.

| Study design
This was a single-site, prospective, randomized, double-blinded, placebo-controlled clinical study. Before inclusion (at the study screening visit), the case history (including diet and medications) was reviewed for each dog. Physical examination, systolic arterial blood pressure, echocardiographic examination, and thoracic radiography were performed. The screening echocardiographic examination was reviewed by 2 primary study investigators (JLK, LCV). Packed cell volume with total solids, a serum biochemistry with creatinine, blood urea nitrogen (BUN), electrolyte concentrations, and urine specific gravity were assessed. Once enrollment eligibility was confirmed, owners were scheduled to drop off their dogs within 5-days of the study screening visit. Owners were instructed to not alter their dog's diet throughout the study period and to fast their dogs 12-hours prior to each study visit. Free access to water was permitted.
At baseline and 7 to 10 days after, each dog underwent a physical examination, an echocardiographic examination, systolic arterial blood pressure assessment, and phlebotomy for assessment of packed cell volume and total solids, serum creatinine, BUN, electrolytes, and symmetric dimethylarginine (SDMA), plasma N-terminal probrain natriuretic peptide (NT-proBNP), and GFR by plasma clearance of iohexol Randomization, drug preparation, and distribution were carried out by an independent veterinary pharmacist and technician not involved in the study and unaware of any patient information other than group assignment. Each treatment group (SD_pimo and HD_pimo) used a 2:1 allocation ratio relative to placebo. Thus, each treatment group had twice as many dogs as placebo group. The placebo group was primarily included to account for day-to-day variability of the echocardiographic and renal function variables. Study investigators and owners were blinded to the group assignment. Each owner was instructed to administer the final treatment <1 hour prior to the final study visit, which was verified verbally. All diagnostics including the echocardiographic examination and phlebotomy were performed 1 to 3 hours posttreatment.

| Blood sample collection and analysis
All blood samples were collected by jugular venipuncture. Analysis of serum creatinine, BUN, electrolytes, as well as packed cell volume, and total solids was performed and analyzed immediately through our inhouse diagnostic laboratory. An attempt was made to collect urine (free catch or cystocentesis) from all dogs and, if collected, urine specific gravity was determined. Samples for SDMA were sent to IDEXX for immediate analysis. Blood samples for iohexol concentrations and GFR analysis, as well as NT-pro-BNP concentrations were collected in lithium heparin tubes and ethylenediaminetetraacetic acid (EDTA) tubes, respectively.
These were centrifuged at 2000g for 15 minutes within 20 minutes of collection, and plasma was separated, aliquoted into cryotubes containing 300 μL of each sample, and stored in at À80 C for future analysis. 21 Samples for NT-proBNP were sent in batch to IDEXX laboratories for analysis once all samples were obtained.

| Glomerular filtration rate measurements and calculations
After placement of an intravenous cephalic catheter, an intravenous injection of iohexol (Omnipaque concentration of 300 mg I/mL) at a dose of 1 mL/kg was administered as a bolus. Blood samples (2 mL) for iohexol concentration were collected at 2-, 3-, and 4-hours after iohexol administration. All blood samples for GFR analysis were collected in lithium heparin tubes. Once all samples were obtained, plasma iohexol concentrations were measured in batch using a highperformance liquid chromatography-tandem mass spectrometry  Tukey-Kramer post hoc test (or nonparametric equivalent). Statistical significance was set at P < .05.

| RESULTS
Thirty-one dogs with ACVIM stage B2 MMVD were enrolled in this study. One dog was withdrawn from the study at its owner's request due to perceived behavior changes of aggression toward the owner and ataxia after receiving several doses of the study medication (later determined to be HD_pimo). Thus, 30 dogs completed the study. Study population characteristics are summarized in Table 1. There were no statistically significant differences in clinical, clinicopathological, or echocardiographic variables, or owner perceived quality of life (FETCH) scores among the groups, aside from dogs enrolled in the HD_pimo group weighed slightly but significantly more (P = .05) than dogs enrolled in the SD_pimo group. One dog in the placebo group, and 3 each in the SD_pimo and HD_pimo groups had a baseline GFR <2 mL/kg/min.
Results of GFR, NT-proBNP, FETCH score and echocardiographic indices of cardiac size and function before and 7 to 10 days after treatment are summarized in Table 2. No statistically significant differences in GFR or FETCH scores were identified within the SD_pimo or HD_pimo groups. However, NT-proBNP and echocardiographic indices of cardiac size (LAV and EDV) exhibited statistically significant decreases relative to baseline within the SD_pimo and HD_pimo groups. Echocardiographic indices of systolic function (ESV and EF) were significantly changed (decreased for ESV and increased for EF) relative to baseline within the SD_pimo and HD_pimo groups. Regarding the among group comparisons in percent change in NT-proBNP, LAV, EDV, and ESV, and EF, there were no statistically significant differences in the percent change after standard-dose pimobendan compared to after high-dose pimobendan. When percent changes were compared among groups, the only statistically significant differences identified were between the placebo group and groups receiving pimobendan.
No adverse effects, adverse clinical events or clinical signs were reported in any of the dogs that completed the study. However, as previously mentioned, 1 dog that received HD_pimo withdrew from the study due to owner perceived aggressive behavior and ataxia.
Hydration status based on physical examination and total solids remained static at each examination. One dog in the SD_pimo group developed a mild anemia, which was noted at the second study visit.
The PCV decreased from 50% to 32% (hematocrit = 27.6%). This anemia was self-limiting and resolved in 72-hours, which was verified with a complete blood count at the dog's primary veterinarian. To our knowledge, the effects of HD_pimo on cardiac and renal function have not been well-studied in dogs with cardiovascular disease. One study evaluated SD_pimo and HD_pimo in dogs with experimentallyinduced MR and found that pimobendan reduced LA pressure in a dosedependent manner. 16 Another study found that HD_pimo did not suppress or potentiate furosemide-induced renin angiotensin aldosterone system activation in healthy dogs. 11 These studies have prompted support for increasing pimobendan dose, frequency (q8h administration), or both for dogs with refractory CHF. 1,15 Additionally, some clinicians prescribe HD_pimo for the potential added benefit that pimobendan might improve renal function in the setting of furosemide-induced prerenal azotemia, 11 and because renal dysfunction is common in dogs with advanced stages of MMVD. 3 However, cross-over study designs present numerous additional challenges, particularly with client-owned dogs, and recruiting more dogs would have increased study cost. Our assessment of renal function in this study does not represent the gold standard assessment of GFR in dogs. Renal clearance of inulin presents many challenges including assay availability, 24-hour urine collection, and frequent urinary catheterizations. 32 We opted to utilize plasma clearance of iohexol as detected by high-performance liquid chromatography, which has been previously validated for measurement of GFR in dogs. 30,32,33 In conclusion, our study was unable to demonstratable an effect of SD_pimo or HD_pimo on renal function in dogs with ACVIM stage B2 MMVD. High-dose pimobendan (0.5-0.6 mg/kg q12h) did not demonstrate advantages over SD_pimo in terms of cardiac function as assessed by selected echocardiographic indices of cardiac size and systolic function.