J. vet. Pharmacol. Therap. 38, 65--73. doi: 10.1111/jvp.12154.

Aldosterone breakthrough with benazepril in furosemide-activated renin-angiotensin-aldosterone system in normal dogs A. C. LANTIS* , 1 M. K. AMES*

,2

C. E. ATKINS*

Lantis, A. C., Ames, M. K., Atkins, C. E., DeFrancesco, T. C., Keene, B. W., Werre, S. R. Aldosterone breakthrough with benazepril in furosemide-activated renin-angiotensin-aldosterone system in normal dogs. J. vet. Pharmacol. Therap. 38, 65–73.

T. C. DEFRANCESCO* B. W. KEENE* & S. R. WERRE* , † *Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA; †Virginia Maryland Regional College of Veterinary Medicine, Blacksburg, VA, USA

Pilot studies in our laboratory revealed that furosemide-induced renin-angiotensin-aldosterone system (RAAS) activation was not attenuated by the subsequent co-administration of benazepril. This study was designed to evaluate the effect of benazepril on angiotensin-converting enzyme (ACE) activity and furosemide-induced circulating RAAS activation. Our hypothesis was that benazepril suppression of ACE activity would not suppress furosemideinduced circulating RAAS activation, indicated by urinary aldosterone concentration. Ten healthy hound dogs were used in this study. The effect of furosemide (2 mg/kg p.o., q12h; Group F; n = 5) and furosemide plus benazepril (1 mg/kg p.o., q24h; Group FB; n = 5) on circulating RAAS was determined by plasma ACE activity, 4–6 h posttreatment, and urinary aldosterone to creatinine ratio (UAldo:C) on days 1, 2, 1, 3, and 7. There was a significant increase in the average UAldo:C (lg/g) after the administration of furosemide (Group F baseline [average of days 1 and 2] UAldo: C = 0.41, SD 0.15; day 1 UAldo:C = 1.1, SD 0.56; day 3 UAldo:C = 0.85, SD 0.50; day 7 UAldo:C = 1.1, SD 0.80, P < 0.05). Benazepril suppressed ACE activity (U/L) in Group FB (Group FB baseline ACE = 16.4, SD 4.2; day 1 ACE = 3.5, SD 1.4; day 3 ACE = 1.6, SD 1.3; day 7 ACE = 1.4, SD 1.4, P < 0.05) but did not significantly reduce aldosterone excretion (Group FB baseline UAldo:C = 0.35, SD 0.16; day 1 UAldo:C = 0.79, SD 0.39; day 3 UAldo:C 0.92, SD 0.48, day 7 UAldo:C = 0.99, SD 0.48, P < 0.05). Benazepril decreased plasma ACE activity but did not prevent furosemide-induced RAAS activation, indicating aldosterone breakthrough (escape). This is particularly noteworthy in that breakthrough is observed at the time of initiation of RAAS suppression, as opposed to developing after months of therapy. (Paper received 8 January 2014; accepted for publication 28 June 2014) Dr Clarke E. Atkins, Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27607, USA. E-mail: [email protected] 1 Present address: Veterinary Emergency and Referral Group, Brooklyn, NY 11201, USA 2 Present address: Department of Clinical Sciences, College of Veterinary Medicine Colorado State University Fort Collins, CO 80523, USA This study was performed at the Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA.

INTRODUCTION Renin-angiotensin-aldosterone system (RAAS) activation attends the administration of both loop diuretics (Vander & Carlson, 1969; Lovern et al., 2001; Gardner et al., 2007; Hori et al., © 2014 John Wiley & Sons Ltd

2007; Sayer et al., 2009) and vasodilating (afterload reducing) drugs such as amlodipine (Atkins et al., 2007) and hydralazine (H€ aggstr€ om et al., 1996). Loop diuretics are utilized in the setting of congestive heart failure (CHF) to relieve clinical signs associated with pulmonary and systemic venous congestion due 65

66 A. C. Lantis et al.

to avid sodium and water retention. Loop diuretics are not generally recommended for long-term use in the absence of concurrent RAAS suppression, as chronic RAAS activation harms cardiac, renal, and vascular tissues in experimental animal models (Wang et al., 1992; Fullerton & Funder, 1994; Rocha et al., 2000) and human beings (Weber, 2001; Beygui et al., 2006). An ACVIM consensus panel on canine mitral valvular disease has recently indicated that chronic pharmacologic management of heart failure in dogs, due to valvular degeneration, should include furosemide, pimobendan, and an angiotensinconverting enzyme (ACE) inhibitor (Atkins et al., 2009). ACE inhibitors (ACEIs) utilized in veterinary medicine blunt plasma ACE activity >50% when administered as directed and the benefits of ACE inhibition have been demonstrated in multiple clinical trials in dogs with CHF due to chronic degenerative valvular disease and dilated cardiomyopathy (The COVE Study Group, 1995; Hamlin & Nakayama, 1998; Ettinger et al., 1999; The BENCH Study Group, 1999; Amberger et al., 2004). While it is clear that ACEIs are beneficial in the setting of CHF, aldosterone secretion does not appear to be sufficiently suppressed in some dogs with CHF treated with an ACEI (H€ aggstr€ om et al., 1996). Chronic exposure to high concentrations of aldosterone results in excessive sodium retention and resultant expansion of extracellular volume (Weber, 2001). It also contributes to endothelial dysfunction in rats (Rocha et al., 2000) and is independently associated with renal (Rocha et al., 2000) and cardiac remodeling in experimental dog models (Fullerton & Funder, 1994; Brilla et al., 1995; Suzuki et al., 2002) and human patients with CHF (Pitt et al., 1999, 2003), inhibits myocardial norepinephrine uptake, diminishes heart rate variability in people with CHF (Weber, 2001), and decreases baroreceptor sensitivity in dogs (Wang et al., 1992). Increasing evidence links aldosterone excess and/or chronic activation of mineralocorticoid receptors to the development and progression of various cardiovascular disease processes in humans (Pitt et al., 1999, 2003; Suzuki et al., 2002). Both direct and indirect evidence of the harmful effects of RAAS activation are evident in veterinary cardiac patients (The COVE Study Group, 1995; Hamlin & Nakayama, 1998; Ettinger et al., 1999; The BENCH Study Group, 1999; Amberger et al., 2004; Bernay et al., 2010; Hezzell et al., 2010). In a prospective veterinary study by Hezzell et al. (2010) urinary aldosterone concentrations were found to be negatively associated with survival (P = 0.005) in 54 dogs with mitral valve disease. A recent double-blind, field study in 190 dogs demonstrated a 69% reduction in risk of cardiac morbidity and mortality in dogs with chronic degenerative mitral valve disease that were treated with spironolactone in addition to an ACEI with furosemide  digoxin when compared to furosemide, an ACEI  digoxin alone (Bernay et al., 2010). Previous studies in cats (MacDonald et al., 2006), experimental models of heart failure in dogs (Cataliotti et al., 2002), and dogs with naturally occurring chronic degenerative valve disease (H€ aggstr€ om et al., 1996; Hezzell et al., 2010) have revealed persistent aldosterone secretion despite ACEI therapy. Pilot studies in our laboratory revealed that

furosemide-induced RAAS activation of 10 days duration was not attenuated by the subsequent co-administration of a 7-day course of benazepril to the furosemide regimen. Potential explanations for this finding include the phenomenon of aldosterone breakthrough (escape) or possibly failure of ACEI-induced suppression of ACE activity. This study was designed to confirm the findings of our pilot study and expand our knowledge regarding ACE activity. We sought to evaluate benazepril’s effect on furosemide-induced RAAS activation by way of evaluation of plasma ACE activity and urinary aldosterone excretion in dogs receiving both benazepril and furosemide vs. dogs receiving only furosemide. Our hypothesis, based on pilot data, was that benazepril suppression of ACE activity would not suppress furosemide-induced circulating RAAS activation. MATERIALS AND METHODS Animals This prospective study was designed to examine the time course and magnitude of the response of the RAAS to the administration of furosemide (Salix; Intervet Canada, Whitby, ON, Canada) alone (2 mg/kg p.o. [SD 0.068] q12h) as compared to that associated with the co-administration of furosemide (2 mg/kg p.o. [SD 0.083] q12h) and benazepril (Fortekor; Novartis, Eschborn, Germany) (1 mg/kg p.o. [SD 0.047] q24h) over 7 days. This study was approved by the North Carolina State University College of Veterinary Medicine’s Institutional Animal Care and Use Committee. Ten mature (>1 year of age) hound dogs (four female and six male) were enrolled in the study, randomized between groups. Each dog was determined to be healthy by history, physical examination, and analysis of a minimum database, consisting of systemic blood pressure, complete blood count, serum chemistry profile, and urinalysis. These tests were performed on baselineday 0, prior to study day -1. The mean body weight of the dogs was 21.18 (SD 8.942; range 7.5–34) kg. The dogs were housed in an Association for Assessment and Accreditation of Laboratory Animal Care International – approved facility with controlled light/dark cycles and received a standard diet (Iams ProActive HealthTM; MinichunksTM, Iams, Mason, OH, USA). Each dog served as its own control for linear variables and was randomly assigned to one of two treatment groups, with the control evaluations preceding the treatment phases. Comparisons were also made between treatment groups. Group F (control group; n = 5, two male and three female) received furosemide (2 mg/kg p.o., q12h at 7 AM and 7 PM) for 7 days. Group FB (n = 5, four male and one female) received furosemide (2 mg/kg p.o., q12h at 7 AM and 7 PM), and benazepril (1 mg/kg p.o., q24h at 7 AM) for 7 days. To measure the time course and magnitude of the response of the RAAS (and hematologic and hemodynamic changes) to the medications, body weight, systolic blood pressure, an abbreviated serum chemistry panel, plasma ACE activity, and twice daily samples for urinary aldosterone:creatinine ratio (UAldo:C) were measured on days 1, 2, 1, 3, and 7. © 2014 John Wiley & Sons Ltd

Aldosterone breakthrough with benazepril 67

Systolic blood pressure evaluation After allowing the dogs to acclimate, Doppler systolic blood pressure (MModel 811-B; Parks Medical Electronics, Inc., Aloha, OR, USA; coccygeal artery, with cuff width approximating 40% of the circumference of the tail base) was determined by taking the average of three consecutive measurements within 10% of each another. ACE activity Serum samples for the determination of plasma ACE activity (U/L) were collected 6 h after benazepril administration. The ACE REA kit (ACE REA Kit 01-RK-ACD; ALPCO Diagnostics, Salem, NH, USA) was used and ACE activity was calculated according to the manufacturer’s instructions. The principle of this assay is that ACE mediates the cleavage of a synthetic substrate, 3 H-hippuryl-glycyl-glycine, into 3 H-hippuric acid and the glycylglycine dipeptide. After acidification with hydrochloric acid, the titrated hippuric acid is separated from unreacted substrate by extraction with scintillation cocktail and measured in a beta counter. Briefly, scintillation vials (Fisher Scientific, Suwanee, GA, USA) were labeled in duplicate for the standards and serum samples. Frozen canine serum samples ( 80 °C) were thawed on ice. After vortexing, 10 lL of each standard and sample was pipetted into the corresponding vial. Hundred microliters of the titrated substrate was added to each vial and mixed well by vortexing, before being incubated in a water bath at 37 °C for 1 h. Then, 50 lL of 1 N hydrochloric acid was added and each was vortexed again. A 1.5-mL aliquot of this scintillation cocktail was added and the vials were thoroughly vortexed, and allowed to stand at room temperature for 1 h before being quantitating radioactivity using a Perkin Elmer Tri-Carb 2900 TR Beta Counter (PerkinElmer Life and Analytical Sciences, Shelton, CT, USA) for 5 min each. Urinary aldosterone:creatinine ratio Five milliliters of urine was collected from each dog in the morning (voided or by cystocentesis, 1–2 h after the administration of medications) and evening (8–10 h after the administration of morning medications) for UAldo:C determination. Each sample was frozen at 70 °C within an hour of collection. Later, equal aliquots of each subject’s morning and evening urine samples were thawed, equal aliquots were mixed and refrozen, and submitted for the determination of UAldo:C (Esoterix Laboratories, Calabasas, CA, USA), as previously described (Atkins et al., 2007; Gardner et al., 2007). Statistical analysis Blood pressure, body weight, urine creatinine, urine aldosterone, urine aldosterone:creatinine ratio, plasma ACE activity, serum electrolyte concentrations (phosphorus, magnesium, sodium, potassium, calcium, and chloride), serum urea © 2014 John Wiley & Sons Ltd

nitrogen, creatinine, protein and bicarbonate concentration, and packed cell volume were measured at four time points (baseline, and study days 1, 3, and 7) following an approximate Gaussian distribution. Accordingly, these outcomes were analyzed using mixed model ANOVA to assess the effect of time point within treatment, the effect of treatment grouping at baseline and at each time point during the administration of medications. The linear model included treatment, time, and treatment*time, as fixed effects, while dog identification constituted the random effect. For each model, residual plots were inspected to verify model adequacy (i.e., that the errors followed a normal distribution with constant variance). Statistical significance was set at a = 0.05. All analyses were performed using SAS version 9.2 (SAS Institute, Cary, NC, USA).

RESULTS There were no significant abnormalities detected on baseline physical examination, complete blood counts, serum chemistry analyses, urinalyses, or blood pressure evaluations. Those changes after the administration of furosemide alone or the combination of furosemide and benazepril that were not significant are presented only in Tables 1 & 2, while significant changes are reported in both the text and Table 2. The administration of furosemide and benazepril resulted in an increase in serum albumin concentration at day 3 (P < 0.01; Table 2). Serum albumin concentration declined at day 7 as compared to baseline in Group F and Group FB (P < 0.01; Table 2). The administration of furosemide alone and the combination of furosemide and benazepril resulted in an initial increase in phosphorus concentration at day 1 (P < 0.01; Table 2), which subsequently returned toward baseline values at days 3 and 7. Serum phosphorus concentrations were not significantly different between Groups F and FB at any sampling point (Table 2). Furosemide and benazepril administration resulted in an initial decline in serum sodium concentration (day 1, P < 0.002; Table 2), that subsequently returned toward baseline values at

Table 1. Mean (SD) values for packed cell volume (PCV), systolic blood pressure, and body weight are displayed for all treatment situations for dogs receiving furosemide (2 mg/kg q12h) and the combination of benazepril (1 mg/kg q24h) and furosemide (2 mg/kg q12h) Day 0 (baseline)

Day 1

PCV (%) Reference interval: 43–62% Group F 50 (5) 53 Group FB 53 (5) 55 BW (kg) Group F 24 (8) 24 Group FB 23 (5) 22 SBP (mmHg) Reference interval: 120–160 mmHg Group F 139 (23) 140 Group FB 127 (14) 132

Day 3

Day 7

(5) (6)

53 (5) 53 (4)

53 (5) 55 (6)

(8) (11)

24 (8) 22 (11)

23 (8) 22 (11)

(24) (15)

133 (13) 135 (17)

146 (13) 132 (21)

68 A. C. Lantis et al. Table 2. Mean (SD) values for serum chemistry parameters are displayed for all treatment situations for dogs receiving furosemide (2 mg/kg q12h) and the combination of benazepril (1 mg/kg q24h) and furosemide (2 mg/kg q12h) Day 0 (baseline)

Day 1

Albumin (g/dL) Reference interval: 2.8–3.7 Group F 3.9 (0.1) 3.9 Group FB 3.7 (0.1) 3.9 Phosphorus (mg/dL) Reference interval: 2–6.7 Group F 4.0 (0.4) 4.6 Group FB 4.0 (0.6) 4.5 Sodium (mEq/L) Reference interval: 147–154 Group F 145.5 (1.2) 140.6 Group FB 143.6 (0.9) 141.0 Chloride (mEq/L) Reference interval: 104–117 Group F 110.7 (0.91) 106.6 Group FB 111.9 (1.52) 108.0 Calcium (mg/dL) Reference interval: 9.4–10.7 Group F 11.0 (0.1)† 10.5 Group FB 10.5 (0.5)† 10.2 Potassium (mEq/L) Reference interval: 3.9–5.2 Group F 4.6 (0.20) 4.3 Group FB 4.5 (0.48) 4.3 Bicarbonate (mEg/L) Reference interval: 18–25.8 Group F 22.2 (1.3) 22.8 Group FB 19.9 (1.1) 21.8 SUN (mg/dL) Reference interval: 9–30 Group F 19.7 (8.3) 20.6 Group FB 13.8 (3.8) 15.4 Creatinine (mg/dL) Reference interval: 0.7–1.3 Group F 0.9 (0.1) 0.9 Group FB 0.9 (0.1) 0.9

Day 3

Day 7

(0.2) (0.2)

4.0 (0.2) 3.9 (0.1)*

3.5 (0.1)* 3.5 (0.1)*

(0.6)* (0.7)*

4.3 (0.6) 3.9 (0.7)

4.0 (0.4) 4.0 (0.5)

(0.9)* (1.0)*

143.2 (0.4)* 142.4 (0.5)

144.0 (0.9) 143.4 (1.7)

(1.52)* 106.8 (1.92)* 108.6 (2.07)* (1.58)* 107.2 (1.64)* 108.0 (1.41)*

(0.1)* (0.3)

10.7 (0.1)† 10.0 (0.7)*,†

10.4 (0.2)* 9.9 (0.2)*

(0.19) (0.52)

4.1 (0.29)* 4.3 (0.68)

4.2 (0.37) 4.2 (0.38)

(1.9) (2.3)

22.6 (2.7) 21.8 (1.3)

23.4 (1.7) 23.8 (1.9)*

(2.2) (6.7)

17.4 (5.7) 13.8 (5.4)

18.0 (6.0) 16.6 (5.8)

(0.1) (0.2)

0.9 (0.2) 0.9 (0.2)

1.0 (0.2) 1.0 (0.1)

Baseline values are those determined prior to drug administration. See text for detailed description. *P < 0.05 within group compared to baseline. † P < 0.05 between treatment groups.

days 3 and 7. The administration of furosemide alone resulted in a decrease in serum sodium concentration at days 1 and 3 (P < 0.005; Table 2). Serum sodium concentration was not significantly different between Groups F and FB at any sampling point (Table 2). The administration of furosemide alone (P < 0.01) and the combination of furosemide and benazepril (P < 0.0001) resulted in a decrease in serum chloride concentrations at days 1, 3, and 7 (Table 2). There was no significant difference between the two groups’ serum chloride concentrations at any time point (Table 2). Serum calcium concentration declined with the administration of furosemide alone at days 1 and 7 and the combination of furosemide and benazepril at days 3 and 7 (P < 0.05; Table 2). Serum calcium concentration

was significantly different between Groups F and FB at baseline and day 3 (P < 0.05; Table 2). Serum potassium concentration declined significantly at day 3 with the administration of furosemide alone (P < 0.05; Table 2). Serum bicarbonate concentration was increased with the administration of furosemide and benazepril at day 7 (P < 0.01; Table 2), but not with furosemide alone. The changes in serum chemistry values in both F and FB, which did not deviate outside reference range, were clinically irrelevant, and compatible with known effects of furosemide administration to dogs in both groups. With the exception of day 3, plasma ACE activity after the administration of furosemide alone was not significantly different than that observed at baseline (Group F ACE activity [U/L]: baseline ACE = 15.2, SD 2.7; day 1 ACE = 15.6, SD 6.6; day 3 ACE = 12.5, SD 3.4; day 7 ACE = 15.0, SD 4.4, P > 0.05; Fig. 1a). Contrarily, plasma ACE activity was significantly blunted in the group receiving furosemide and benazepril (Group FB ACE activity [U/L]: baseline ACE = 16.4, SD 4.2; day 1 ACE = 3.5, SD 1.4; day 3 ACE = 1.6, SD 1.3; day 7 ACE = 1.4, SD 1.4, P < 0.05; Fig. 1b). On day 3, compared to baseline, there was an 18% reduction in ACE activity in dogs of Group F, as compared to 90% suppression from baseline when benazepril was added to the furosemide treatment (Fig. 1a,b). Furthermore, the day 3 mean ACE activity in the F group was significantly (P = 0.0026) greater than in the FB group (12.5 vs. 1.6 U/L) and there was no significant difference between ACE activity at baseline and that found on day 3 in the F Group. There was a statistically significant difference in the plasma ACE activity at days 1, 3, and 7 between Groups F and FB (P < 0.01; Fig. 1a). Furosemide administration was associated with an approximate threefold increase in the UAldo:C (lg/g); Group F baseline UAldo:C = 0.41, SD 0.15; day 1 UAldo:C = 1.1, SD 0.56, P < 0.005; day 3 UAldo:C = 0.85, SD 0.50, P < 0.05; day 7 UAldo:C = 1.1, SD 0.80, P < 0.02; Fig. 2). Concurrent furosemide and benazepril administration was associated with a similar significant increase in urinary UAldo:C (Group FB baseline UAldo:C = 0.35, SD 0.16; day 1 UAldo:C = 0.79, SD 0.39, P < 0.05; day 3 UAldo:C 0.92, SD 0.48, P < 0.01; day 7 UAldo:C = 0.99, SD 0.48, P < 0.02; Fig. 2). There was no significant difference in the UAldo:C on days 1, 3, and 7 between Groups F and FB (P > 0.05; Fig. 2).

DISCUSSION The results of this study demonstrate RAAS activation, evident in a significant increase in urinary aldosterone excretion, in normal dogs receiving furosemide at 2 mg/kg bid. Group F (furosemide alone) had a two- to threefold increase in urinary aldosterone excretion, which was maintained over the course of 7 days (Fig. 2). The administration of furosemide in combination with benazepril (Group FB) resulted in a similar increase in UAldo:C that was maintained over the course of the study period, despite significant reduction in plasma ACE activity (Fig. 1). Plasma ACE activity, 4–6 h postpill, was blunted by >65% © 2014 John Wiley & Sons Ltd

Aldosterone breakthrough with benazepril 69

(a)

(b)

Fig. 1. (a) Group F (n = 5 dogs) – plasma angiotensin-converting enzyme (ACE) activity (U/L) at baseline (average of days 1 and 2; preF) and 4–6 h after the administration of furosemide (2 mg/kg q12h) on days 1, 3, and 7. (b) Group FB (n = 5 dogs) – plasma ACE activity (U/L) at baseline (average of days 1 and 2; pre-FB) and 4–6 h after the administration of the combination of furosemide (2 mg/kg q12h) and benazepril (1 mg/kg q24h) on days 1, 3, and 7. The figure demonstrates the mean and standard error of the mean (bars). ACE activity is essentially unchanged in the furosemide group (with the exception of a minor, but significant fall of 18% on day 3 vs. 90% on day 3 in the FB group), whereas ACE activity is significantly attenuated by the combination of furosemide and benazepril at all time points. *P ≤ 0.01 within group compared to baseline; **P ≤ 0.01 between treatment groups, †P < 0.05 between treatment day and baseline. F, furosemide; FB, furosemide and benazepril.

(range 66–100%) in these normal dogs, receiving benazepril in combination with furosemide. Maximum RAAS suppression is known to occur 1–3 h posttreatment (Hamlin & Nakayama, 1998). Furosemide administered alone did not result in a significant change in plasma ACE activity, with the exception of a mild, but significant and unexplained, fall on day 3. It is noteworthy that the day 3 decrease from baseline in Group F was only 18%, compared to 90% on day 3 in the FB group. In addition, the differences in ACE activity between groups on day 3 were substantially different with mean values of 12.5 U/L in Group F and 1.6 U/L in Group FB (P = 0.0026). The differences in serum electrolyte concentrations over time with treatment (Tables 1 & 2) are likely reflective of the © 2014 John Wiley & Sons Ltd

administration of furosemide in both groups. Previous studies have documented significant decreases in serum potassium, magnesium, sodium, chloride, calcium, and bicarbonate concentrations during furosemide administration (Penman et al., 1972; Cobb & Michell, 1992; Lovern et al., 2001). While changes in the values within each group were statistically significant, all values remained within reference ranges or were only slightly outside the reference ranges and remained clinically unimportant throughout the study. Decreased renal perfusion pressure, diminished delivery of sodium chloride to the macula densa, and beta-adrenergic stimulation of the juxtaglomerular apparatus result in renin release (Vander & Carlson, 1969; Osborn et al., 1983; MartinezMaldonado et al., 1990). Furosemide can directly stimulate renin release by inhibiting NKCC2-dependent NaCl transport across macula densa cells, as demonstrated by studies in dogs, rats, and mice (Vander & Carlson, 1969; Martinez-Maldonado et al., 1990; Castrop et al., 2005). Furosemide-induced RAAS activation was expected in Group F as RAAS activation after the administration of furosemide has been well documented in experimental studies in normal dogs and in canine patients with CHF (Lovern et al., 2001; Gardner et al., 2007; Hori et al., 2007; Sayer et al., 2009). There are several potential explanations for persistent aldosterone excretion in group FB (aldosterone breakthrough) despite reduced plasma ACE activity in these dogs receiving both benazepril and furosemide. Potential explanations include timing of sample collection, drug dosage, high plasma levels of angiotensin II (non-ACE enzymes cleaving angiotensin I to angiotensin II), hyperkalemia, hyponatremia, increased corticotrophin serum concentration, and decreased atrial natriuretic hormone (Staessen et al., 1981; Willard et al., 1987; Lovern et al., 2001; Sp€ at & Hunyady, 2004; Rossi, 2006; Bomback & Klemmer, 2007; Hori et al., 2010). Persistent aldosterone secretion, despite presumed reduction in plasma ACE activity and production of angiotensin II, its major secretagogue, has been referred to as ‘aldosterone escape’ or ‘aldosterone breakthrough’ (MacFadyen et al., 1999). Aldosterone breakthrough has been defined in the human literature as any increase in serum aldosterone concentration that exceeds a baseline value after initiation of RAAS-blocking therapy (Bomback & Klemmer, 2007). There appears to be no consensus among investigators, regarding the exact definition and time course of aldosterone breakthrough. Some authors describe it as a serum level exceeding baseline (pre-ACEI and/or angiotensin II receptor blocker [ARB] therapy) value 6–12 months after initiation of RAAS-blocking therapy (Bomback & Klemmer, 2007), while others have used this term when aldosterone concentrations become elevated to a certain level 4–6 weeks after initiation of an ACEI (Staessen et al., 1981; Cleland et al., 1984). Several studies have revealed the potential for aldosterone breakthrough in veterinary patients (H€ aggstr€ om et al., 1996; Cataliotti et al., 2002; MacDonald et al., 2006). Cats with hypertrophic cardiomyopathy receiving ramipril (0.5 mg/kg q24h) had a 97% suppression of plasma ACE activity at 3, 6, 9, and 12 months, however, plasma aldosterone concentration

70 A. C. Lantis et al.

*

*

1.6

*

1.5 1.4

* *

*

1.3 1.2 UAldo:C (µg/g)

1.1

F

1.0

F B

0.9

F B

F

F B

Day 3 - F+B

Day 7 - F

Day 7 - F+B

F

0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0

Pre-F

Pre-F+B

Day 1 - F

Day 1 - F+B

Day 3 - F

Fig. 2. The UAldo:C (lg/g) before (pre-F and pre-FB, average of baseline days 1 and 2) and after the administration of furosemide (2 mg/kg q12h) and furosemide and benazepril (1 mg/kg q24h) at days 1, 3, 7. The figure demonstrates the mean and the standard error of the mean (bars). Compared to baseline values (pre-F and pre-FB), there is activation of circulating renin-angiotensin-aldosterone system (RAAS) with furosemide administration alone (P < 0.05) and with the combination of furosemide and benazepril (P < 0.05). Significance (*) was reached or was approached (P-value provided) at each individual time point. There is no difference between the two groups at any sampling date (P > 0.05). F, furosemide; FB, furosemide and benazepril.

was not different in cats treated with ramipril, compared to those receiving placebo (MacDonald et al., 2006). A pacinginduced CHF model in dogs receiving an intravenous bolus of fosinoprilat (1 lmol/kg) and intravenous furosemide (40 mg administered over 20 min) resulted in a significant increase in plasma aldosterone concentration at 30, 60, 90, and 150 min (Cataliotti et al., 2002). We addressed the timing of UAldo:C sample collection by sampling twice daily (one AM urine sample taken 2 h after the medications were administered and one PM urine sample collected 8–10 h after the administration of the medications were combined in equal amounts for analysis). Benazepril successfully suppressed plasma ACE levels by 60% 4–6 h postpill in the dogs in this study. This was not likely the nadir of ACE activity, as benazepril (0.5 mg/kg p.o., every 24 h) has been shown to suppress plasma ACE activity by 80% 1.5 h after dosing, 70% at 3 h, 50% at 6 h, and 25% at 8 h postpill in dogs (Hamlin & Nakayama, 1998). Benazepril (2 mg/kg) presumably blocked angiotensin II formation and aldosterone secretion (as measured by serum concentration of aldosterone) in a study evaluating the effects of benazepril in a canine remnant kidney model of chronic renal failure (Mishina & Watanabe, 2008). The discrepancy in the attenuation of aldosterone secretion between the present study and the aforementioned chronic renal failure study may be due to the difference in benazepril dosage or, more likely, study design (furosemide-induced activation of RAAS vs. canine remnant kidney model). The dosage of benazepril used in the present study (1 mg/kg p.o., q24h) is a clinically relevant dosage, an approximation of which has been utilized in multiple canine clinical trials and is known to be beneficial in the management of canine heart failure, due to dilated cardiomyopathy and mitral valve disease (Kitagawa et al., 1997; The BENCH Study Group, 1999; O’Grady et al., 2009).

Increasing angiotensin II levels have been noted in human patients receiving an ACEI for the treatment of chronic heart failure, and is typically attributed to an increased ACE activity (Jorde et al., 2000; Tang et al., 2002). Angiotensin II concentrations were not measured in the present study. Angiotensin II formation, in the presence of adequate ACE activity suppression, may reflect the existence of alternative enzymes (i.e., chymase, and others) for the formation of angiotensin II, a potential explanation for aldosterone breakthrough (Swedberg et al., 1990; Urata et al., 1990; Borghi et al., 1993; Lee et al., 1999; MacFadyen et al., 1999; Dell’Italia, 2002; Yamane et al., 2008). This study involved a small number of dogs and the duration of the study was relatively brief, when compared to the duration of therapy (months, to even years) for most veterinary patients with CHF. We did not evaluate RAAS activation, using a placebo-treated control group. We evaluated the effects of furosemide and the combination of furosemide and benazepril in healthy dogs and extrapolation to dogs with CHF is not necessarily applicable. Several pieces of information that would have made interpretation of the data more complete include assessment of tissue RAAS; more extensive pharmacokinetic data; and serum angiotensin II, renin, and norepinephrine concentrations. Therapeutic options to treat or prevent aldosterone breakthrough include the use of drugs which blunt the RAAS via different mechanisms. Drugs with this potential include ARBs, such as losartan, telmisartan, and valsartan, and direct renin inhibitors (DRIs), such as aliskiren. Aldosterone breakthrough is known to occur with ARBs when used in the management of CHF in people (Bomback & Klemmer, 2007). To the authors’ knowledge, the occurrence and prevalence of aldosterone breakthrough in dogs with heart failure treated with ARBs and/or DRIs has not been studied. A recent study in human patients with mild to moderate proteinuric renal disease and systemic © 2014 John Wiley & Sons Ltd

Aldosterone breakthrough with benazepril 71

hypertension evaluated the prevalence of aldosterone breakthrough in people treated for 9 months with an ARB (valsartan) alone, a DRI (aliskiren) alone, or the two in combination (Pitt et al., 2003). Aldosterone breakthrough occurred in all three groups (defined as a sustained increase in 24-h urine aldosterone secretion over the study period) and the prevalence of aldosterone breakthrough was not significantly different among these three treatment groups and ranged from 25% to 30%. This study was small (33 people divided among three groups), as it was terminated early, when a concurrent study evaluating aliskiren in diabetic patients found an increased risk of nonfatal stroke in patients receiving this drug. The mineralocorticoid receptor blockers (spironolactone and eplerenone) provide sequential RAAS blockade and have been shown in multiple trials to improve survival and quality of life in human and canine patients with heart failure, both severe and relatively mild (Pitt et al., 1999, 2003; Bomback et al., 2012; Krum et al., 2013). The accepted explanation for the added benefit of mineralocorticoid receptor blockade is that RAAS blockade with an ACEI or ARB was incomplete (breakthrough) in some patients. In conclusion, we have shown a nearly threefold increase in urinary aldosterone secretion, as indicated by the urine UAldo: C, with furosemide monotherapy. This was maintained for the 7 days of the study. We also found an expected >60% reduction in plasma ACE activity 4–6 h posttreatment in the furosemide and benazepril-treated group (FB), without a concomitant, consistent decrease in mean urinary aldosterone excretion. The benefits of ACE inhibition have been demonstrated in the laboratory (Wang et al., 1992; Fullerton & Funder, 1994; Kitagawa et al., 1997; Rocha et al., 2000) and in multiple clinical trials in dogs with CHF, due to chronic degenerative valvular disease and dilated cardiomyopathy (The COVE Study Group, 1995; Hamlin & Nakayama, 1998; Ettinger et al., 1999; The BENCH Study Group, 1999; Amberger et al., 2004). The discrepancy between the data of this study, which might suggest that benazepril was not effective, and the proven beneficial effect of ACE-I in dogs with CHF is of interest. We postulate that the clinical benefit may be the result of benazepril’s effect on tissue RAAS (not evaluated clinically), in addition to or rather than, circulating RAAS, evaluated here, with subsequent reduction in the formation of angiotensin II; prevention of the degradation of bradykinin; and/or additional, noncardiac survival benefits (i.e., the beneficial effects in dogs with concurrent chronic renal disease or hypertension, for example; Tenhu¨ndfeld et al., 2009; Bomback et al., 2012). Furthermore, there are likely patients that respond initially, giving overall study population benefit during the time of RAAS suppression, and importantly, current data indicate that approximately 50% of human patients never experience aldosterone breakthrough. While the results of this study must be confirmed in clinical patients, given the present data and data from dogs with CHF (Bernay et al., 2010), it appears that standard CHF therapy (Atkins et al., 2009) should be accompanied by an aldosterone antagonist in dogs (Bernay et al., 2010). © 2014 John Wiley & Sons Ltd

ACKNOWLEDGMENTS The authors thank Ms. Allison Klein for technical assistance. This work was funded by Novartis Animal Health. Pilot studies funded through the Jane Lewis Seaks Endowment.

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Aldosterone breakthrough with benazepril in furosemide-activated renin-angiotensin-aldosterone system in normal dogs.

Pilot studies in our laboratory revealed that furosemide-induced renin-angiotensin-aldosterone system (RAAS) activation was not attenuated by the subs...
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