Determination of finerenone - a novel, selective, nonsteroidal mineralocorticoid receptor antagonist - in human plasma by high-performance liquid chromatography-tandem mass spectrometry and its application to a pharmacokinetic study in venous and capillary human plasma.

A straightforward and rapid high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay allowing the sensitive and selective quantitation of finerenone (BAY 94-8862) in lithium heparin human plasma is described. Finerenone is a novel, selective, nonsteroidal mineralocorticoid receptor antagonist that is in phase III clinical trials for the treatment of chronic kidney disease. Finerenone quantitation is performed after addition of its stable isotope-labelled internal standard (ISTD) by protein precipitation with acidified acetonitrile followed by HPLC-MS/MS separation and detection. The determination of finerenone concentrations was validated for a plasma volume of 0.100 mL and subsequently also for a lower plasma volume of 0.010 mL, collected e.g. in paediatric studies. The analytical range was from 0.100 µg/L (lower limit of quantification) to 200 µg/L (upper limit of quantification). Inter-day accuracy was 99.7-105.0% for the plasma volume of 0.100 mL and 101.1-104.5% for the plasma volume of 0.010 mL. Inter-day precision was ≤ 7.0%, independent of the extracted plasma volume. A moderate, concentration-independent matrix effect on ionisation was observed for both finerenone and its ISTD of 0.535-0.617, which is fully compensated by the ISTD (ISTD-normalised matrix factors were 0.98-1.03). The assay was successfully applied with both validated plasma volumes to a clinical phase I study in which the pharmacokinetics of 20 mg finerenone were compared in capillary plasma (0.010 mL) and venous plasma (0.100 mL) in a concentration range from the lower limit of quantification to 310 µg/L (capillary plasma) and 252 µg/L (venous plasma). The area under the plasma concentration versus time curve was similar in both matrices, while maximum concentrations were 37% higher in capillary plasma. In conclusion, capillary sampling should not bias pharmacokinetic exposure estimates compared with venous plasma values, if limited to sampling times in the distribution and elimination phases of finerenone.

Pharmacokinetic interactions of esaxerenone with amlodipine and digoxin in healthy Japanese subjects.

BACKGROUND: To investigate the effects of coadministration of esaxerenone with amlodipine on the pharmacokinetics (PK) of each drug, and of esaxerenone on the PK of digoxin. METHODS: In three open-label, single-sequence, crossover studies, healthy Japanese males received single oral doses of esaxerenone 2.5 mg (Days 1, 15), with amlodipine 10 mg/day (Days 8-18) (Study 1, N = 24); single doses of amlodipine 2.5 mg (Days 1, 21), with esaxerenone 5 mg/day (Days 8-25) (Study 2; N = 20); or digoxin 0.25 mg/day (Days 1-15) with esaxerenone 5 mg/day (Days 11-15) (Study 3; N = 20). PK parameters and safety were assessed. RESULTS: Study 1: esaxerenone peak plasma concentration (Cmax) and time to Cmax were unaltered by amlodipine coadministration, but mean half-life was slightly prolonged from 18.5 to 20.9 h. Geometric least-squares mean (GLSM) ratios for Cmax, area under the plasma concentration-time curve (AUC) from zero to last measurable concentration and from zero to infinity for esaxerenone + amlodipine versus esaxerenone were 0.958, 1.154, and 1.173, respectively. Study 2: corresponding GLSM ratios for amlodipine + esaxerenone versus amlodipine were 1.099, 1.185, and 1.214. Study 3: esaxerenone did not markedly alter digoxin PK. GLSM ratios for Cmax, trough plasma concentration, and AUC during a dosing interval for digoxin versus esaxerenone + digoxin were 1.130, 1.088, and 1.072, respectively. CONCLUSIONS: No drug-drug interactions are expected during combination therapy with esaxerenone and either amlodipine or digoxin, based on a lack of any clinically relevant PK changes. TRIAL REGISTRATION: Studies 1 and 2: JapicCTI-163379 (registered on 20 September 2016); Study 3: JapicCTI-163443 (registered on 24 November 2016).

The human adrenal gland as a drug metabolizer: First in-vivo evidence for the conversion of steroidal drugs.

The metabolism of drugs in mammals is attributed mainly to the liver and its cytochromes P450 localized in the endoplasmic reticulum. Here, we demonstrate for the first time in humans that there is no strict subdivision between P450 s involved in exogenous and endogenous metabolism. We determined the widely used mineralocorticoid receptor antagonist spironolactone, its active metabolite canrenone and their metabolites in the adrenal venous blood of treated patients with gas chromatography-mass spectrometry. 11- and 18-hydroxylated metabolites of canrenone were found in the efferent right and left adrenal veins, indicating that they were produced by the adrenal mitochondrial cytochromes P450 CYP11B1 and CYP11B2. Thus, the adrenal has to be considered as a new organ for drug metabolism. In future, application of drugs may need further investigations concerning side effects due to interactions with adrenal enzymes.

Pharmacokinetics of the Novel, Selective, Non-steroidal Mineralocorticoid Receptor Antagonist Finerenone in Healthy Volunteers: Results from an Absolute Bioavailability Study and Drug-Drug Interaction Studies In Vitro and In Vivo.

BACKGROUND AND OBJECTIVES: Finerenone is a selective, non-steroidal mineralocorticoid receptor antagonist. In vivo and in vitro studies were performed to assess absolute bioavailability of finerenone, the effect of metabolic enzyme inhibitors on the pharmacokinetics of finerenone and its metabolites, the quantitative contribution of the involved enzymes cytochrome P450 (CYP) 3A4 and CYP2C8 and the relevance of gut wall versus liver metabolism. METHODS: The pharmacokinetics, safety and tolerability of finerenone (1.25-10 mg orally or 0.25-1.0 mg intravenously) were evaluated in healthy male volunteers in four crossover studies. Absolute bioavailability was assessed in volunteers receiving finerenone orally and by intravenous infusion (n = 15) and the effects of erythromycin (n = 15), verapamil (n = 13) and gemfibrozil (n = 16) on finerenone pharmacokinetics were investigated. Finerenone was also incubated with cryopreserved human hepatocytes in vitro in the presence of erythromycin, verapamil or gemfibrozil. RESULTS: Finerenone absolute bioavailability was 43.5% due to first-pass metabolism in the gut wall and liver. The geometric mean AUC0-∞ ratios of finerenone (drug + inhibitor/drug alone) were 3.48, 2.70 and 1.10 with erythromycin, verapamil and gemfibrozil, respectively. The contribution ratio of CYP3A4 to the metabolic clearance of finerenone derived from these values was 0.88-0.89 and was consistent with estimations based on in vitro data, with the remaining metabolic clearance due to CYP2C8 involvement. CONCLUSION: Finerenone is predominantly metabolized by CYP3A4 in the gut wall and liver. Increases in systemic exposure upon concomitant administration of inhibitors of this isoenzyme are predictable and consistent with in vitro data. Inhibition of CYP2C8, the second involved metabolic enzyme, has no relevant effect on finerenone in vivo.

Population Pharmacokinetics of LY2623091 in Patients With Hypertension and Chronic Kidney Disease.

LY2623091 is a selective, orally active, nonsteroidal, competitive mineralocorticoid receptor antagonist that blocks the actions of aldosterone and other mineralocorticoid receptor ligands at the receptor level. The aim of this work was to explore and establish a population pharmacokinetic model, quantify the degree of interindividual variability, and identify significant disease-, patient-, and study-specific covariates that alter the disposition of LY2623091. The data included concentrations from 294 healthy subjects and patients with hypertension and/or chronic kidney disease (CKD), sampled in 5 phase 1 and 2 studies. The pharmacokinetics of LY2623091 was well described by a 2-compartment model with first-order absorption and elimination. Formulation (on oral bioavailability) as well as weight and age (both on apparent central volume of distribution) were found to be significant covariates. The relative bioavailability of the capsule formulation was 68.4% compared to that of the solution. Hypertension and CKD status were not significant covariates. The pharmacokinetic model suggests that given the same dose, patients with hypertension and/or CKD would receive a similar exposure compared to subjects without these disease conditions.

Signal Enhancement in the HPLC-ESI-MS/MS analysis of spironolactone and its metabolites using HFIP and NH4F as eluent additives.

This paper describes an LC-MS/MS method to determine the concentration of spironolactone and its metabolites 7-alpha-methylthiospironolactone and canrenone in blood plasma samples. The resulting assay is simple (using protein precipitation for sample preparation) and sensitive (the lower limit of quantification is close to 0.5 ng/ml) while requiring only 50 µl of plasma, making it especially suitable for analyzing samples obtained from pediatric and neonatal patients where sample sizes are limited. The sensitivity is achieved by using ammonium fluoride as an eluent additive, which in our case amplifies the signal from our analytes in the plasma solution on average about 70 times. The method is fully validated according to the European Medicines Agency's guideline and used for the measurement of pediatric patients' samples in clinical trials for evaluating oral spironolactone's and its metabolites' pharmacokinetics in children up to 2 years of age.

Population Pharmacokinetics of Eplerenone in Japanese Patients With Chronic Heart Failure.

To characterize eplerenone pharmacokinetics (PK) in Japanese chronic heart failure (CHF) patients and to estimate the impact of factors that may affect eplerenone PK, population pharmacokinetic (PPK) analysis was conducted. In addition, PK of Japanese CHF and Western CHF patients from a previous clinical pharmacology study were compared in the analysis. Eplerenone PK was characterized by a 1-compartment PPK model with first-order absorption and lag time in Japanese CHF patients. The population mean of apparent oral clearance (CL/F) in Japanese CHF patients was estimated as 5.31 L/h, which was similar to the mean CL/F for Western CHF patients. In the full model approach, creatinine clearance (CLcr) on CL/F and body weight on apparent central volume of distribution (Vc/F) were selected as factors that may affect PK. The effect of CLcr on CL/F predicted that CL/F would be decreased by 25% when CLcr was decreased from 80 mL/min to 50 mL/min. The effect of body weight on Vc/F predicted that Vc/F would be decreased by 18% when body weight was decreased from 80 kg to 60 kg. Distribution of individual CL/F estimates for Japanese CHF patients overlapped CL/F observed values for Western CHF patients, and CL/F values for Western CHF patients were contained within the distribution of CL/F estimates for Japanese CHF patients. No obvious difference between Japanese and Western subjects was detected even in the updated model by adding the data obtained from Western CHF patients and Western healthy adults to the model constructed with data from Japanese CHF patients.

Drospirenone intake alters plasmatic steroid levels and cyp17a1 expression in gonads of juvenile sea bass.

Drospirenone (DRO) is one of the most widely used progestins in contraceptive treatments and hormone replacement therapies. The pharmacokinetics and potential toxicological effects of DRO were investigated in juvenile sea bass (Dicentrarchus labrax) exposed through the diet (0.01-10 µg DRO/g) for up to 31 days. DRO was detected in the blood (4-27 ng/mL) of fish exposed to the highest concentration, with no significant bioaccumulation over time and no alteration of hepatic metabolizing enzymes, namely, CYP1A and CYP3A-catalysed activities and UDP-glucuronyltransferase (UGT). Pregnenolone (P5), progesterone (P4), 17α-hydroxyprogesterone (17P4), 17α-hydroxypregnenolone (17P5), androstenedione (AD) and testosterone (T) were determined in plasma and gene expression of cyp17a1, cyp19a1a and cyp11ß analysed by qRT-PCR in gonads. The significant increase in plasmatic levels of 17P5, 17P4 and AD detected after 31 days exposure to 10 ng DRO/g together with the increased expression of cyp17a1 in females evidence the ability of DRO to alter steroid synthesis at low intake concentrations (7 ng DRO/day). However, the potential consequences of this steroid shift for female reproduction remain to be investigated.

ENVIRONMENTALLY FRIENDLY LC/MS DETERMINATION OF EPLERENONE IN HUMAN PLASMA.

Eplerenone (EPL), a selective aldosterone receptor antagonist, is indicated in the treatment of chronic heart failure and hypertension. It is hard to find a green assay among a few published methods for its determination in human plasma or serum. Following a liquid-liquid extraction with methyl t-butyl ether, eplerenone and isotope labelled eplerenone - used as an internal standard - were separated from the endogenous compounds on an Atlantis dCl8 column (150 x 3 mm, 3.0 [im). An isocratic elution with the mobile phase consisting of methanol and ammonium acetate (3 : 2, v/v) was used. A single quadrupole mass spectrometer was operated in positive electrospray ionization using the selected ion monitoring mode. The method is more environmentally-friendly than the previously reported assays. Acetonitrile in the mobile phase was replaced with methanol which is a removable solvent. Plasma sample volume was reduced to 250 pL which significantly decreased waste volume. Chlorinated solvents used previously for liquid-liquid extraction were eliminated and the safety of the laboratory staff was increased by eliminating diethyl ether. The method is characterized by a short analysis time, simple sample preparation and reduction of waste volume, which are important advantages when analyzing large numbers of samples. The method was validated according to international regulatory guidelines and may be applied to human pharmacokinetic studies following a single 25 or 50 mg oral dose.

Chronic antagonism of the mineralocorticoid receptor ameliorates hypertension and end organ damage in a rodent model of salt-sensitive hypertension.

We investigated the effects of chronic mineralocorticoid receptor blockade with eplerenone on the development and progression of hypertension and end organ damage in Dahl salt-sensitive rats. Eplerenone significantly attenuated the progressive rise in systolic blood pressure (SBP) (204 ± 3 vs. 179±3 mmHg, p < 0.05), reduced proteinuria (605.5 ± 29.6 vs. 479.7 ± 26.1 mg/24h, p < 0.05), improved injury scores of glomeruli, tubules, renal interstitium, and vasculature in Dahl salt-sensitive rats fed a high-salt diet. These results demonstrate that mineralocorticoid receptor antagonism provides target organ protection and attenuates the development of elevated blood pressure (BP) in a model of salt-sensitive hypertension.

Abbott ARCHITECT clinical chemistry and immunoassay systems: digoxin assays are free of interferences from spironolactone, potassium canrenoate, and their common metabolite canrenone.

Spironolactone, which is metabolized to canrenone, is often used in combination with digoxin. Potassium canrenoate is a similar drug that is also metabolized to canrenone. As a result of reported interference of spironolactone, potassium canrenoate, and their common metabolite canrenone with digoxin immunoassays, we investigated potential interference of these compounds with two relatively new digoxin assays for application on ARCHITECT clinical chemistry platforms (cDig, particle-enhanced turbidimetric inhibition immunoassay) and ARCHITECT immunoassay platforms (iDig, chemiluminescent microparticle immunoassay), both from Abbott Diagnostics. When aliquots of drug-free serum pool were supplemented with various amounts of spironolactone, potassium canrenoate, and canrenone, no apparent digoxin concentration was observed using cDig assay on ARCHITECT c4000, c8000, and c16000 or iDig assay on i1000SR and i2000SR analyzers. In addition, we observed no false increase in serum digoxin value when aliquots of a digoxin pool were further supplemented with various amounts of spironolactone, potassium canrenoate, or canrenone. We conclude that both the cDig and iDig assays on the ARCHITECT analyzers are free from interferences by spironolactone, potassium canrenoate, and canrenone.

Effect of spironolactone, potassium canrenoate, and their common metabolite canrenone on Dimension Vista Digoxin Assay.

Spironolactone, a potassium sparing diuretic metabolized to canrenone, is often used with digoxin to treat various conditions including congestive heart failure. Potassium canrenoate is a similar drug that is also metabolized to canrenone. Due to reported interference of spironolactone, potassium canrenoate, and their common metabolite canrenone with digoxin immunoassays, we investigated potential interference of these compounds with Dimension Vista Digoxin immunoassay using Flex reagent cartridge. Aliquots of a drug-free serum pool were supplemented with various amounts of spironolactone, potassium canrenoate, or canrenone and apparent digoxin values were measured using Dimension Vista digoxin assay, we observed none-detected value except when aliquots were supplemented with higher amounts of spironolactone or canrenone. Similarly, when aliquots of a serum digoxin pool (prepared by pooling specimens from patients receiving digoxin) where further supplemented with various amounts of spironolactone, potassium canrenoate, or canrenone, we observed moderately falsely elevated digoxin values only in specimens containing higher amounts of spironolactone or canrenone. We conclude that spironolactone and canrenone but not potassium canrenoate may cause modest interference with Dimension Vista digoxin assay but such interferences may not be clinically significant except with very high amounts of canrenone.

Interference between eplerenone and digoxin in fluorescence polarization immunoassay, microparticle enzyme immunoassay, and affinity column-mediated immunoassay.

Digitalis-like immunoreactive substances have crossreactivity with antidigoxin antibodies and the interference between digoxin and spironolactone/canrenone has been reported. The structure of eplerenone is similar to that of spironolactone/canrenone. Therefore, we hypothesized that eplerenone might also interfere with the measurement of digoxin by immunoassay. We performed three types of assays (fluorescence polarization immunoassay [FPIA], microparticle enzyme immunoassay [MEIA], and affinity column-mediated immunoassay [ACMIA]) to determine crossreactions between eplerenone and antidigoxin antibodies. Furthermore, we used FPIA, MEIA, and ACMIA to measure the apparent digoxin concentration in mixed solutions of eplerenone (1-100 µg/mL) and digoxin (1-3 ng/mL). In the crossreaction tests, eplerenone was detected as digoxin by FPIA and ACMIA. By FPIA, a known concentration of 1 µg/mL of eplerenone was measured as 0.33 ± 0.11 ng/mL of digoxin (crossreaction rate, 0.03%). By ACMIA, a known concentration of 10 µg/mL of eplerenone was measured as 0.13 ± 0.05 ng/mL of digoxin (crossreaction rate, 0.001%). No crossreaction between eplerenone and digoxin was determined by MEIA. In the interference of eplerenone coadministered with digoxin, the apparent concentration of digoxin was increased in FPIA, but decreased in MEIA and ACMIA. The results suggest that eplerenone crossreacts with antidigoxin antibodies in FPIA, MEIA, and ACMIA, but that the interference of eplerenone might be smaller than that of spironolactone/canrenone.

Clinical repercussions of analytical interferences due to aldosterone antagonists in digoxin immunoassays: an assessment.

Analytical interferences in digoxin immunoassays constitute a well-known problem, with repercussions for therapeutic drug monitoring. Clinically effective doses of spironolactone and potassium canrenoate cross-react in several digoxin immunoassays, producing falsely elevated or lowered concentrations. This study evaluates the interferences caused by these drugs in the microparticle enzyme immunoassay (MEIA III) in comparison with another 3 immunoassays used for digoxin therapeutic drug monitoring: MEIA II, fluorescence polarization immunoassay, and enzyme multiplied immunoassay. The potential clinical implications of assay discrepancies in patient care are also assessed. To evaluate assay performance, in vitro specimens and real patient samples were measured using the 4 assays. Five serum pools were spiked with digoxin to achieve concentrations of 1 and 2.25 ng/mL digoxin and measured with the immunoassays before and after supplementation. Real samples from patients receiving digoxin (n = 39), digoxin and spironolactone (n = 35), or digoxin and potassium canrenoate (n = 4) were also quantified. The influence of ultrafiltration was evaluated in 3 pools from 29 additional patients. The implications of assay discrepancies for dose recommendations were also evaluated. In general, the results obtained for the in vitro and in vivo approaches coincided, confirming statistically significant differences in the assays regardless of the type of sample. MEIA III showed positive interference against the well-known negative interference attributed to MEIA II. According to Bland-Altman analysis, it is not possible to assume the interchangeability of the immunoassays evaluated. Thus, individual patients must be monitored with the same technique even in the absence of potential interferences. Discordant digoxin dose recommendations were estimated in 31% of patients not treated with interfering drugs and in 43% of cotreated patients. From a clinical perspective, analytical interferences in digoxin immunoassays are a real and frequent problem, which seems even more important in view of the lower therapeutic range now recommended.

Effect of spironolactone, potassium canrenoate and their common metabolite canrenone on serum digoxin measurement by digoxin III, a new digoxin immunoassay.

Spironolactone and potassium canrenoate (aldosterone antagonist diuretics) are often used with digoxin in clinical practice. It has been well documented in the literature that spironolactone, potassium canrenoate, and their common metabolite canrenone cross-react with several digoxin immunoassays at concentrations expected after therapeutic usage of these drugs and falsely elevate or lower serum digoxin concentrations. Recently, Abbott Laboratories marketed a new Digoxin III immunoassay for application on the AxSYM analyzer. We studied the potential interference of these compounds with this new digoxin assay. The Tina-quant assay was used as the reference method because spironolactone, potassium canrenoate, and canrenone do not interfere with serum digoxin measurement using this assay. Aliquots of drug-free serum were supplemented with therapeutic and above therapeutic concentrations of spironolactone, canrenone, and potassium canrenoate, and apparent digoxin concentrations were measured using the Digoxin III assay and Tina-quant assay. Significant apparent digoxin concentrations were observed when the Digoxin III digoxin assay was used, but no apparent digoxin levels was observed using the Tina-quant assay. When serum pools prepared from patients receiving digoxin were further supplemented with these compounds in concentrations expected in sera of patients receiving these medications, falsely elevated digoxin levels were observed using Digoxin III assay, but no statistically significant change was observed using the Tina-quant assay. We conclude that spironolactone, potassium canrenoate, and their common metabolite canrenone interfere with the serum digoxin measurements using the new Digoxin III assay.

Pharmacokinetics and pharmacodynamics of drospirenone-estradiol combination hormone therapy product coadministered with hydrochlorothiazide in hypertensive postmenopausal women.

The effects of combination hormone therapy of drospirenone (DRSP), a novel progestin with antialdosterone properties, and 17beta-estradiol (E2) on hydrochlorothiazide (HCTZ) pharmacokinetics/pharmacodynamics versus placebo were investigated in a double-blind, placebo-controlled, crossover study. Thirty-six postmenopausal women with stage 1 hypertension maintained on 25 mg of HCTZ once daily were randomized to receive either 3 mg of DRSP/1 mg of E2 or placebo once daily for 4 weeks. Plasma HCTZ, serum DRSP, E2, potassium, aldosterone, and plasma renin activity were determined at baseline and after 4 weeks. Results showed that the combination of DRSP/E2 plus 25 mg of HCTZ is safe and well tolerated in hypertensive postmenopausal women. The pharmacokinetics of HCTZ were not affected by coadministration of DRSP/E2. The geometric mean ratios and 90% confidence intervals ([HCTZ + DRSP/E2]/[HCTZ + placebo]) for HCTZ (a) area under the serum/plasma concentration-time curve from 0 to 24 hours and (b) maximum plasma concentration were 101 (90.7, 112) and 103 (92.8, 115), respectively. In the HCTZ + DRSP/E2 group, serum potassium, aldosterone, and plasma renin activity all increased in a manner marginally consistent with a beneficial antialdosterone effect, counteracting the HCTZ-induced potassium loss and lowering both systolic and diastolic blood pressure. No dose adjustment is required when DRSP/E2 is added to antihypertensive therapy with HCTZ in hypertensive postmenopausal women.

Effect of drospirenone on serum potassium and drospirenone pharmacokinetics in women with normal or impaired renal function.

This open-label study investigated whether drospirenone, a novel progestin with antimineralocorticoid properties, increases the risk for hyperkalemia in subjects with renal impairment. Women with normal renal function and mild or moderate renal impairment were given drospirenone 3 mg/d for 14 days. Mean serum potassium concentrations did not significantly change during steady-state drospirenone treatment. No difference in drospirenone effects on serum potassium concentrations in subjects with renal insufficiency versus subjects with normal renal function was found. Steady-state pharmacokinetics was similar in subjects with normal renal function and mild renal impairment, whereas, due to results from 1 subject, drospirenone exposure was slightly higher in the moderate renal impairment group. Given the reported tolerability of drospirenone, this small increase in serum drospirenone is not expected to be clinically significant. In conclusion, this study demonstrated that drospirenone has no significant effect on serum potassium levels in patients with mild to moderate renal insufficiency.