Study design, endpoints and safety
EPATH is a single-center, double-blind, placebo-controlled, randomized, parallel group trial. Patients with PHPT diagnosed according to the report of the 3rd International Workshop on Diagnosis of PHPT will be screened for inclusion into the study[14]. Patients diagnosed with symptomatic/asymptomatic PHPT will be consented and screened for eligibility to participate in the EPATH trial. Overall 110 patients will be randomized to receive eplerenone or placebo in a 1:1 ratio (55 patients will receive eplerenone and 55 patients will receive placebo).
The design, conduction and reporting of the EPATH study adheres to the recommendations of the CONSORT Statement (http://www.consort-statement.org/). The study will be performed in accordance with the Good Clinical Practice (GCP) guidelines and the Declaration of Helsinki. The study has been approved (N° 24–032 ex 11/12; EudraCT number: 2011-005683-21) by the Ethics Committee of the Medical University of Graz, Austria.
The primary endpoint of the study is to evaluate the efficacy of eplerenone relative to placebo to reduce PTH(1–84) concentration, measured by two different laboratory methods, in patients with PHPT. Secondary endpoints are the mean changes from baseline after 8-weeks with eplerenone versus placebo, in the following measurements: (1) 24-hour systolic and diastolic ambulatory blood pressure (ABP) levels; (2) biomakers of CVD; (3) biomarkers of bone metabolism: osteocalcin, β-crosslaps, bone alkaline phosphatase and tartrate-resistant acid phosphatase; (4) 24-h urinary albumin/protien excretion; and (5) echocardiographic parameters related to systolic and diastolic function as well as cardiac dimensions (i.e. left ventricular ejection fraction, E/E´ ratio, left ventricular end-diastolic/systolic pressure/volume/index and LV posterior wall thickness).
We decided to evaluate the effect of MR-blocker eplerenone on iPTH(1–84) - the primary outcome – and on CV and bone health – the secondary outcomes - after 8 weeks because (1) preliminary data from the Graz Endocrine Causes of Hypertension (GECOH) study suggest that MR-blockade with eplerenone might exert “rapid” effects on iPTH(1–84) levels[15]; (2) eplerenone has been shown to significantly lower BP after a few days of treatment, indicating that this dose may be sufficient to impact on iPTH(1–84) levels[16]; and (3) in order to minimize the drop-out number due to eplerenone’s higher selectivity for the MR, which yields a superior tolerability profile in terms of sexual side effects, compared to spironolactone. In addition, findings from previous studies suggested that eplerenone appears to produce more consistent inhibition of some of the nongenomic effects of aldosterone than spironolactone[17]. Finally, it has been speculated that the extended half-life of the active metabolites of spironolactone increase the risk of hyperkalemia and its associated complications[18]. Conversely, the relatively short half-life of eplerenone and lack of inactive metabolites may decrease the risk of hyperkalemia.
Considering the higher prevalence of arterial hypertension and cardiovascular disease (CVD) in PHPT patients, which is suggested to result in part from a reciprocal interaction between aldosterone and PTH, MR-blockade might be a promising therapeutic strategy to decrease risk of the development and progression of CVD even in normotensive patients without materially increasing the risk of hypotensive side effects[19, 20]. In case of severe side effects related to hypotension, the study drug however will be discontinued.
The study schema and visit schedule are illustrated in flow chart in Figure1. The preliminary screening for study eligibility is based on medical records performed by the study staff at or before the baseline visit. A written informed consent will be provided by each eligible participant, who will be randomly assigned in a double blind fashion to receive eplerenone 25 mg once daily (given in the morning) or a matching placebo. If no adverse effects of eplerenone treatment with 25 mg once daily will be observed after 4 weeks, the dose will be up-titrated by protocol to the target dose of 50 mg once daily. This dose titration strategy was chosen to minimise the risk of adverse effects, in particular hyperkalemia. At week 1 and each subsequent clinic visit, the serum potassium and creatinie level will be checked. Overall, the study consists of an 8-week, double-blind, randomized (active) treatment period.
Patients will be requested to be fasting and abstain from tea, coffee and smoking for overnight at least 12 hours before applying at the study outpatient. Intake of any drug should be avoided in the morning before blood collection. Investigations will be done in the morning (8:00 to 12:00). Extreme care will be taken to avoid haemolysis and prolonged stasis. Laboratory surveillance of serum electrolytes (potassium, sodium, magnesium and calcium) and renal function (serum creatinine and estimated glomerular filtration rate (GFR) according to the MDRD formula) is required at each study visit. Immediate suspension of the study drug eplerenone will be disposed for any measured serum potassium ≥ 5.5 mmol/L, and a down-titration of the study drug will be initiated for any measured serum potassium ≥ 5.0 mmol/L. Furthermore, the study drug will be discontinued in case of a rise of serum creatinine ≥ 3.0 mg/dL or GFR < 30 mL/min (or downtitration between GFR 30-49 mL/min), severe anaphylactic reactions following or intolerance due to eplerenone intake, requirement for an open-label use of eplerenone or potassium sparing diuretics, or formal withdrawal of the consent.
To minimise the risk of adverse events (e.g. hyperkalemia) patients with an eGFR ≤ 50 mL/min and/or a serum potassium > 5 mmol/L will be excluded from enrollment into the EPATH Study. Furthermore, the dose of 25 mg eplerenone once daily is comparably low and will be titrated to 50 mg once daily, if no adverse effects have been observed and if well tolerated.
If women of child-bearing age participate in the EPATH study, pregnancy will be ruled out at the beginning of the active participation before study enrolment. Further pregnancy tests will be performed monthly in our outpatients (spot urine test), and additionally as a self-test at home (after thorough education and instruction to perform test self-tests and after notification per telephone). Female participants of child-bearing age will be instructed to perform contraception throughout the active study period.
Because MR-blocker treatment is currently recommended in all patients with persisting symptoms (NYHA class II–IV) and an EF <35%, despite treatment with an ACE inhibitor (or an ARB) and a beta-blocker, special care was taken to avoid serious side effects such as hyperkalemia in PHPT patients by (1) excluding those participants from study participation, who are at higher risk of developing hyperkalemia; (2) a dose titration strategy; (3) monitoring of serum electrolytes and renal function at each study visit[21].
Randomization will be done with assistance of the Institute of Medical Informatics, Statistics and Documentation of the Medical University of Graz (http://www.randomizer.at). The study staff and participants will be blinded to treatment assignment for the duration of the study.
Study medication of either eplerenone (Pfizer Corporation Austria, Vienna, Austria) or placebo will be packed in numbered pill boxes (identical in appearance and packages) according to both a computer generated randomization list and to international standards, respectively. This will be done by qualified staff at the pharmacy of the Medical University of Graz, which has extensive experience with placebo controlled trials.
Setting
Eligible study participants will be recruited from the outpatient clinic of the Department of Internal Medicine, Division of Endocrinology and Metabolism and Division of Cardiology, Medical University of Graz, Austria. All baseline and follow-up visits will be conducted at the Department of Internal Medicine, Division of Endocrinology and Metabolism and Division of Cardiology and at the Center of Medical Research of the Medical University of Graz, Austria.
Inclusion and exclusion criteria
Eligible patients will be men and women of at least 18 years of age with PHPT, who (1) do not meet criteria for surgical treatment; or (2) in whom surgery is recommended, but not performed because of patient and/or physician preference; or (3) perceived medical contraindications[22].
Exclusion criteria include (1) 25(OH)D levels <20 ng/dl (50 nmol/liter); (2) estimated GFR (according to the MDRD formula) ≤ 50 ml/min; (3) serum potassium > 5.0 mEq/L (mmol/L) at baseline or > 5.5 mEq/L (mmol/L) during active study period; (4) side effects related to hypotension; (5) pregnancy or lactating women; (6) drug intake as part of another clinical study 4 weeks before enrolment into the EPATH study and/or during the active study period; (7) any disease with an estimated life expectancy below 1 year; (8) chemotherapy or radiation therapy during the study; (9) intolerance to eplerenone or any ingredient occurring in eplerenone; (10) severe acute or chronic liver diseases (Child-Pugh Class C); and (11) concurrent intake of potassium sparing drugs, e.g. diuretics (amiloride and triamterene) or CPY3A4-inhibitors and ongoing potassium supplementation.
Measurements
Anthropometry
Weight and height will be measured wearing no shoes and light clothes and waist and hip ratio will be determined according to published recommendations.
Laboratory measurements
All blood samples will be centrifuged within 1 hour after sampling and will be measured at latest 4 hours after blood collection. Before analysis or freezing all samples will be kept at room temperature, except of the samples for determination of iPTH(1–84) (and plasma aldosterone), which will be kept at 4° Celsius. Remaining samples for long-term storage (whole blood, serum, plasma, urine and saliva samples) will be kept at −70° to 80° Celsius at the Biobank Institute of the Medical University of Graz.
Measurement of iPTH(1–84) (pg/mL) will be performed by electrochemiluminiscence immunoassay “ECLIA” (Elecsys immunoassay analyzer, Cobas®, Roche Diagnostics GmbH, Mannheim, Germany). The Elecsys assay for determining intact PTH employs a sandwich test principle in which a biotinylated monoclonal antibody reacts with the N-terminal fragment (1–37) and a monoclonal antibody labeled with a ruthenium complex reacts with the C-terminal fragment (38–84). The antibodies used in this assay are reactive with epitopes in the amino acid regions 26–32 and 37–42. Blood for iPTH(1–84) determination will be collected with standard EDTA plasma tubes and centrifuged within 5 to 10 minutes.
[Conversion factors of iPTH(1–84) are: pg/mL × 0.106 = pmol/L and pmol/L × 9.43 = pg/mL, respectively. Measuring range of the assay: 1.20 (=lower detection limit) to 5000 pg/mL (=maximum of the master curve); Reference range: 15–65 pg/mL, interassay coefficient of variation 3.0-6.5% for PTH 26.7-261 pg/mL).
PTH will be further determined using the electrochemiluminiscence LIAISON N-tact PTH immunoassay (DiaSorin Inc., Stillwater, MN, USA).
The measurements of further parameters (25(OH)D, osteocalcin, β-CrossLaps, bone alkaline phosphatase, tartrate-resistant acid phosphatase, osteoprotegerin, NT-pro-BNP) have been previously described in detail[15, 23].
All other laboratory measurements will be performed according to current routine laboratory methods at the Medical University of Graz.
Measurement of omics-based biomarkers will be performed by Liquid chromatography–mass spectrometry at the Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz[24].
Genetic investigations
Genetic analyses (e.g. PCR) will be performed in order to investigate the impact of e.g. polymorphisms of the calcium-sensing receptor or of the aldosterone synthase (CYP11B2) on PTH- and aldosterone secretion. Genome-wide association analyses will be performed - within the limits of a hypothesis testing strategy - in order to examine novel genes/loci implicated in the regulation of aldosterone and PTH synthesis in concert with larger studies. Genetic testing will be further initiated in suspicion of familial form of hyperparathyroidism. Emphasis will be placed upon the genes associated with familial hypocalciuric hypercalcemia (FHH), multiple endocrine neoplasia syndrome type I (MEN1), MEN2, and the hyperparathyroidism jaw-tumor syndrome.
Echocardiography
Comprehensive echocardiographic analysis of cardiac function and dimensions will be performed by experienced physicians on echocardiographic device (GE Vivid 3® Pro Echocardiography System, United Kingdom) according to current guidelines of the American Society of Echocardiography. Tissue Doppler indices will be recorded at the septal and lateral base of the mitral annulus.
24-hour systolic/diastolic ambulatory blood pressure monitoring (ABPM)
Our stuff will be carefully trained to ensure the accuracy of the ABPM measurement. The ABPM measurements will be performed in accordance to the established protocols of the European Society of Hypertension and the American Association for Medical Instrumentation. Measurements of 24-hour ABP will be performed by a validated ABP monitor (Spacelabs 90217; Spacelabs Healthcare GmbH) and mean 24-hour systolic and diastolic ABP will be recorded. A minimum of 14 readings during the day and 7 readings overnight will be required for valid 24-hour ABPM.
Questionnaires
The validated Short Form 36 questionnaire (SF-36)12 will be used as a general estimate for qualtity of life. The Kansas City Cardiomyopathy Questionnaire is a 23-item, self-administered instrument, which will be used to quantify physical function, symptoms (frequency, severity and recent change), social function, self-efficacy and knowledge, and quality of life.
Statistical methods
Sample size calculation
Sample size calculation for the primary endpoint iPTH(1–84) is based on (1) an observed effects of eplerenone treatment (and adrenalectomy) on iPTH(1–84) levels in a previously published study[25]; (2) the proven significant association between (elevated) PTH levels on risk of fatal cardiovascular events in humans[3]; and (3) data derived from patients, which have been managed at the outpatient clinic of the Division of Endocrinology and Metabolism at the Medical University of Graz, with primary hyperparathyroidism.
Based on the observational data from the GECOH study, MR-blockade (by eplerenone and spironolactone) in patients with primary aldosteronism was associated with an approximate 10.0 pg/mL decrease in PTH levels after a mean follow-up of 12 months[25]. However, two patients at short-term follow-up of 4 weeks revealed a similar decrease of PTH levels with MR-blockade compared to long-term follow-up, indicating a rapid effect of MR-blockade on PTH levels. We are, however, aware that due to limited evidence regarding the effects of eplerenone in patients with PHPT, final conclusions cannot be drawn.
We further evaluated whether an increase of 10.0 pg/mL PTH is translated into a significant higher risk of CV mortality. In the LURIC study comprising 3.300 patients referred to coronary angiography the increment of 10 pg/mL PTH was significantly related to a 4% [Hazard Ratio (95% CI): 1.04 (1.03-1.06) p < 0.001] higher risk of cardiovascular death[3]. We assumed an effect size of 10.0 pg/mL, assuring sufficient power to decrease PTH by eplerenone 25/50 mg once daily. Thus, for a two-sided alternative-hypothesis with an α of 0.05 and a power (1-β) of 80% we calculated a sample size of 51 study participants per group. To compensate for drop-outs during the study (estimated to around 10%) we plan to enrol 55 patients per group.
Outcomes
Outcome variables will be tested for normal distribution by use of the Kolmogorov-Smirnov test and by further descriptive statistics. Normally distributed continuous variables will be given as mean [with standard deviation (SD)], variables with skewed distribution as median with interquartile range, and categorical variables as percentages. For parametric procedures all skewed distributed continuous parameters will be logarithmically transformed (log10). Analysis of covariance, adjusted for the respective baseline values, will be used to analyze the continuous primary and secondary outcome variables.
All statistical tests to assess the treatment difference between eplerenone and placebo will be performed using a 2-sided hypothesis test at 5% significance level. Data will be analysed using SPSS 17.0 statistical package (SPSS, Inc., Chicago, IL, USA).