Evaluation of oral telmisartan administration as a suppression test for diagnosis of primary hyperaldosteronism in cats

Abstract Background Development of a telmisartan‐based suppression test may facilitate the diagnosis of primary hyperaldosteronism (PHA) in cats, which remains difficult today. Objectives To develop a telmisartan suppression test (TST) that is safe, and able to suppress aldosterone secretion in healthy cats but not in cats with PHA. Animals Ten healthy cats and 6 cats with PHA. Methods Prospective study using a placebo‐controlled crossover design to investigate a TST in healthy cats, and evaluation of TST in cats with PHA. Plasma aldosterone concentration, potassium concentration, and systolic blood pressure (SBP) were measured before (T0), and 1 hour (T1) and 1.5 hours after (T1.5) PO administration of 1 mg/kg of telmisartan, 2 mg/kg of telmisartan or placebo. Results Median age in healthy cats was 3 years old (range, 1‐7). In healthy cats, a telmisartan dose of 2 mg/kg significantly decreased aldosterone concentration at T1 and T1.5 compared with T0. Placebo had no significant effect on aldosterone concentration. In cats diagnosed with PHA, a 2‐mg/kg dose of telmisartan did not induce any significant change in aldosterone concentration at T1 or T1.5 compared with T0. No adverse effects of telmisartan (e.g., hyperkalemia, systemic hypotension) were observed in any cats. Conclusions and Clinical Importance The oral TST shows promise as a diagnostic test for the diagnosis of PHA in cats.

ARR, aldosterone-to-renin ratio; AVR, aldosterone variation rate; FST, fludrocortisone suppression test; PAC, plasma aldosterone concentration; PHA, primary hyperaldosteronism; PRA, plasma renin activity; RAAS, renin-angiotensine-aldosterone system; SBP, systolic blood pressure. can lead to hypokalemia or systemic hypertension or both and has been associated with progressive renal disease in cats. 2 Cases are being reported more frequently, and understanding of the pathogenesis and clinical manifestations of the disease has improved. 1,3 However, diagnosis of PHA in cats remains challenging. 1 The aldosterone-to-renin ratio (ARR) currently is considered the gold standard measurement for PHA screening, 1 but assays for plasma renin activity (PRA) are not widely available, and stringent pre-analytical conditions make its evaluation difficult. Moreover, evidence that PRA is suppressed in all cats with PHA is lacking. Currently, suspicion of PHA is based on a combination of suggestive clinical signs, an increased plasma aldosterone concentration (PAC) in the presence of otherwise unexplained hypokalemia, and visualization of an adrenal mass on ultrasound examination or computed tomography scan. However, in the case of bilateral adrenocortical hyperplasia, there may be minimal or no detectable change on ultrasonography, making PHA diagnosis even more challenging. For these reasons, a reliable and accessible confirmatory test is needed.
In human medicine, the 4 commonly used confirmatory tests for PHA are the fludrocortisone suppression test (FST), the saline infusion test, the captopril challenge test and the oral sodium loading test. 4 The FST has already been evaluated in cats and a lack of aldosterone suppression was observed in 6/9 cats with PHA. 5,6 However, there are concerns that fludrocortisone could be detrimental to cats with PHA that have severe hypokalemia or severe hypertension or both. In elderly humans, who are considered to be a vulnerable population, an alternative suppression test that uses the selective angiotensin 2 type 1 receptor (AT1R) antagonist losartan has been demonstrated to be safe and accurate to confirm PHA. 7 This test is quick, with only 1 blood sample required 2 hours after PO administration of losartan. Telmisartan is the only AT1R antagonist authorized for use in cats. It has proven to be an effective and safe antiproteinuric and antihypertensive drug at the dosage of 1 to 2 mg/kg PO q24h, respectively. 7,8 Given that angiotensin II (and hyperkalemia) are the most powerful stimuli for adrenocortical aldosterone secretion, we hypothesized that AT1R blockade would lead to a significant reduction in PAC in healthy cats but not in cats with PHA that have autonomous secretion of aldosterone.
Our aim was to evaluate the ability of PO telmisartan to suppress PAC in healthy cats, and to identify the dosage and time corresponding to optimal aldosterone suppression. The potential of a telmisartan suppression test to discriminate cats with PHA from healthy cats also was investigated.

| Cats
We enrolled 10 healthy student-owned cats. Eligibility criteria were age ≥6 months, an unremarkable history and physical examination, plasma concentrations of creatinine, sodium, and potassium within reference intervals, urine specific gravity >1.030, and systolic blood pressure (SBP) ≤160 mm Hg.
Six client-owned cats admitted to Veterinary Teaching Hospital of the National Veterinary School of Alfort and diagnosed with PHA also were enrolled between September 2016 and September 2018.
The diagnosis of PHA was based on increased PAC (>540 pmol/L) associated with hypokalemia in the presence of an adrenal mass or increased ARR (>3.8 Â 10 À9 ). 8 Exclusion criteria were concurrent treatment expected to affect the renined angiotensin aldosterone system (e.g., angiotensin converting enzyme inhibitor, angiotensin receptor blocker, aldosterone receptor antagonist, calcium channel blocker, diuretic) within the week before enrollment. In PHA cats that were hypertensive, the telmisartan suppression test was performed as soon as PHA was suspected and adequate medications were initiated after the test was completed.

| Study design
The initial study in healthy cats was a randomized, placebo-controlled, crossover study. At study baseline, all cats had a physical examination and SBP determination, plasma creatinine, sodium and potassium concentrations were measured, and urinalysis was performed.
For the telmisartan suppression test, healthy cats were given telmisartan solution PO (Semintra, 4 mg/mL, Boehringer Ingelheim) at a dose of 1 mg/kg, 2 mg/kg or the equivalent volume of the 2 mg/kg dose of tap water as a placebo. Blood samples were taken before (T0), and 1 hour (T1) and 1.5 hours (T1.5) after telmisartan or placebo administration for the measurement of plasma aldosterone and potassium concentrations. Systolic blood pressure (SBP) was measured at the same time, before blood sampling, to evaluate the safety of the test. The telmisartan dosage was chosen based on previous studies evaluating the antiproteinuric and antihypertensive efficacy and safety of telmisartan. 9,10 A 1-week washout period was applied between each test.
Based on results in healthy cats, an uncontrolled follow-on study of the best-performing protocol for the suppression test was conducted on cats with PHA using only telmisartan at a dosage of 2 mg/kg.
The study protocol was approved by the scientific ethics committee of the National Veterinary School of Alfort (Comerc 2017-09-06) and all owners consented to their cats' participation in the study.

| Sample collection and analysis
Blood samples were collected by jugular venipuncture into heparin-coated tubes for the measurement of plasma sodium, potassium, and aldosterone concentrations. Sodium and potassium concentrations were measured using an electrode-based analyzer (Smart 30 Vet, I-Sens, France). Blood also was collected into ice-chilled EDTA-coated tubes and then centrifuged at 4 C for 8 minutes at 3000g. Plasma was stored at À20 C and À80 C pending measurement of aldosterone and PRA, respectively.
Aldosterone concentration was determined using a validated radioimmunoassay (RIAZENco Aldosterone, ZenTech) at the laboratory of the veterinary school of Lyon (Vetagrosup, Lyon, France). The aldosterone variation rate (AVR) was calculated at T1 and T1.5 using the for- The lowest value of AVR (i.e., the highest aldosterone inhibition) that occurred at either T1 or T1.5 then was determined. Plasma renin activity was measured at the Department of Nephrology of the University Medical Center, Utrecht, the Netherlands as previously described. 8 In brief, plasma was incubated at pH 6.0 for 1 hour at 37 C in the presence of inhibitors of angiotensinases and angiotensin I converting enzyme. Samples then were deproteinized with acetone and ammonia and centrifuged. The supernatants were evaporated and redissolved in assay buffer, and angiotensin I was measured by radioimmunoassay. Plasma ARR was calculated as the ratio of PAC (pmol/L) to PRA (fmol/L/s).

| Cats with PHA
F I G U R E 1 Plasma aldosterone concentration before (T0), 1 hour (T1) and 1.5 hours (T1.5) after oral administration of 1 mg/kg (A, n = 9), 2 mg/kg (B, n = 10) of telmisartan and placebo (C, n = 7) in healthy cats. For each plot, the box represents the interquartile (25th-75th percentiles) range, the solid horizontal line within the box represents the median. Each filled circle represents the value for an individual cat. Asterisk (*) indicates significant differences.
F I G U R E 2 Plasma aldosterone concentration before (T0), 1 hour (T1) and 1.5 hours (T1.5) after oral administration of 2 mg/kg of telmisartan in cats with primary hyperaldosteronism (n = 6, see Figure 1 for legend).

| DISCUSSION
Administration of telmisartan to healthy cats in our study was associated with significant suppression of PAC, whereas no significant effect was associated with placebo administration. A telmisartan dose of 2 mg/kg appeared to be more efficient at decreasing aldosterone concentration (AVR was À40% at T1 and À47% at T1.5) than a 1-mg/kg dose (AVR was À24% at T1 and À22% at T1.5). The decrease in PAC was also larger with the higher telmisartan dose, although not significantly. We did not identify any absolute value of PAC that could be used to accurately discriminate between cats with and without aldosterone suppression. As far as the relative variation of aldosterone concentration from T0 (ie, AVR) is concerned, it was lower than À33% in all healthy cats at T1 or T1.5 or both when a 2 mg/kg telmisartan dose was chosen. For these reasons, the dosage of 2 mg/kg of telmisartan was chosen to evaluate the telmisartan suppression test in cats with PHA. It could be hypothesized that aldosterone suppression with telmisartan is dose-dependent, and that a higher telmisartan dosage would be better at discriminating between healthy cats and cats with PHA.
We chose to measure the PAC at T1 and T1.5 because pharmacokinetic studies show that the maximum serum concentration of telmisartan is reached 30 minutes after PO administration (unpublished data). Assuming a plasma half-life of aldosterone of approximately 15 to 20 minutes (as in humans), 12 the direct effect of telmisartan on aldosterone concentration is expected to be detectable between 1 and 1.5 hours after PO administration. In our population of healthy cats, aldosterone suppression occurred at T1 or T1.5 or both. Therefore, both sampling times were retained for further evaluation of the telmisartan suppression test in the 6 cats with PHA. It is possible that suppression might be higher at timepoints beyond 1.5 hours after telmisartan dosing, and this possibility needs to be investigated. Because our population of healthy cats was young, we also need to confirm the ability of telmisartan to suppress aldosterone secretion in elderly healthy cats.
Our study confirms the safety of telmisartan when administered to healthy cats. No significant change in plasma potassium concentration was noted in either dose group. Moreover, the anticipated change in potassium concentration secondary to telmisartan administration would constitute hyperkalemia, which should not worsen the F I G U R E 3 Aldosterone variation rate 1 hour (T1) and 1.5 hours (T1.5) after oral administration of 1 mg/kg or 2 mg/kg of telmisartan or placebo in healthy cats, and of 2 mg/kg of telmisartan in cats with primary hyperaldosteronism (PHA Low-renin hypertension also has been observed in cats, 14