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Research ArticleCardiovascular

The Renal Outer Medullary Potassium Channel Inhibitor, MK-7145, Lowers Blood Pressure, and Manifests Features of Bartter’s Syndrome Type II Phenotype

Caryn Hampton, Xiaoyan Zhou, Birgit T. Priest, Lee-Yuh Pai, John P. Felix, Brande Thomas-Fowlkes, Jessica Liu, Martin Kohler, Jianying Xiao, Aaron Corona, Olga Price, Charles Gill, Kashmira Shah, Cordelia Rasa, Vince Tong, Karen Owens, James Ormes, Haifeng Tang, Sophie Roy, Kathleen A. Sullivan, Joseph M. Metzger, Magdalena Alonso-Galicia, Gregory J. Kaczorowski, Alexander Pasternak and Maria L. Garcia
Journal of Pharmacology and Experimental Therapeutics October 2016, 359 (1) 194-206; DOI: https://doi.org/10.1124/jpet.116.235150

Abstract

The renal outer medullary potassium (ROMK) channel, located at the apical surface of epithelial cells in the thick ascending loop of Henle and cortical collecting duct, contributes to salt reabsorption and potassium secretion, and represents a target for the development of new mechanism of action diuretics. This idea is supported by the phenotype of antenatal Bartter’s syndrome type II associated with loss-of-function mutations in the human ROMK channel, as well as, by cardiovascular studies of heterozygous carriers of channel mutations associated with type II Bartter's syndrome. Although the pharmacology of ROMK channels is still being developed, channel inhibitors have been identified and shown to cause natriuresis and diuresis, in the absence of any significant kaliuresis, on acute oral dosing to rats or dogs. Improvements in potency and selectivity have led to the discovery of MK-7145 [5,5′-((1R,1′R)-piperazine-1,4-diylbis(1-hydroxyethane-2,1-diyl))bis(4-methylisobenzofuran-1(3H)-one)], a potential clinical development candidate. In spontaneously hypertensive rats, oral dosing of MK-7145 causes dose-dependent lowering of blood pressure that is maintained during the entire treatment period, and that displays additive/synergistic effects when administered in combination with hydrochlorothiazide or candesartan, respectively. Acute or chronic oral administration of MK-7145 to normotensive dogs led to dose-dependent diuresis and natriuresis, without any significant urinary potassium losses or changes in plasma electrolyte levels. Elevations in bicarbonate and aldosterone were found after 6 days of dosing. These data indicate that pharmacological inhibition of ROMK has potential as a new mechanism for the treatment of hypertension and/or congestive heart failure. In addition, Bartter’s syndrome type II features are manifested on exposure to ROMK inhibitors.

Introduction

Hypertension and congestive heart failure are medical conditions of different etiologies that affect many individuals. Antihypertensive drugs with different mechanisms of action exist, with diuretics being among the most widely prescribed medications, alone or in combination with other drugs. Resistant hypertension, defined as blood pressure above normal values despite adherence to a regimen of at least three optimally dosed antihypertensive drugs, one of which is a diuretic, can affect up to 14% of patients undergoing treatment (Rossignol et al., 2015). Recommendations for treating resistant hypertension include the testing of new antihypertensive drugs in this patient population. Congestive heart failure is one of the most common causes for hospitalizations in individuals >65 years of age, with 93% of these patients presenting symptoms of dyspnea, and 70% manifesting peripheral edema. As a consequence, most hospitalized patients are treated with loop diuretics to diminish volume overload and congestion (Felker et al., 2011). There are, however, issues associated with the use of diuretics, such as those related to the development of diuretic resistance, and reflex neurohormonal stimulation of the sympathetic nervous system and the renin–angiotensin–aldosterone system. Given the above, there is an unmet clinical need for developing new mechanism of action drugs with which to treat hypertension and/or heart failure.

The kidney plays an important role in long-term regulation of blood pressure by modulating net renal salt and water reabsorption (Lifton et al., 2001). At the thick ascending loop of Henle (TALH), ∼30% salt reabsorption occurs through the luminal Na+/K+/2Cl− cotransporter, the target of loop diuretics. In the distal convoluted tubule, the Na+/Cl− cotransporter, the clinical target of thiazide-class diuretics, is responsible for ∼7% salt reabsorption. Final regulation of reabsorption occurs in the cortical collecting duct (CCD) through amiloride-sensitive epithelial sodium channels. Because of tight coupling between sodium reabsorption and potassium secretion at the CCD, the use of loop or thiazide diuretics is clinically associated with hypokalemia.

Bartter’s syndrome type II is an autosomal-recessive disorder caused by loss-of-function mutations in the renal outer medullary potassium (ROMK) channel (Simon et al., 1996). The disease is characterized by renal salt wasting and polyuria-associated low blood pressure, mild hypokalemia, metabolic alkalosis, hypercalciuria, and elevated plasma renin and aldosterone levels. Importantly, heterozygous carriers of channel mutations associated with type II Bartter’s syndrome have reduced blood pressure and decreased risk of developing hypertension by 60 years of age (Ji et al., 2008), suggesting that ROMK represents a target for developing channel inhibitors as novel treatment of hypertension and/or heart failure.

ROMK, the KCNJ1 gene product, belongs to the inwardly rectifying family of potassium (Kir) channels (Nichols and Lopatin, 1997), and is expressed at the apical membrane of epithelial cells lining the TALH and CCD (Palmer et al., 1997; Xu et al., 1997; Lu et al., 2002). At the TALH, ROMK recycles potassium across the apical membrane, which is critical for proper function of the Na+/K+/2Cl− cotransporter. At the CCD, ROMK contributes to potassium secretion that is tightly coupled to sodium reabsorption through the amiloride-sensitive sodium channel. The presence of ROMK channels at both TALH and CCD suggests that selective channel inhibitors would display diuretic/natriuretic efficacy that is equivalent or superior to that of other diuretics, with potential benefit for attenuating hypokalemia associated with the use of loop or thiazide diuretics.

The development of selective ROMK inhibitors is in progress. Two independent groups have reported identification of small molecule Kir1.1 inhibitors (Bhave et al., 2010; Tang et al., 2012; Walsh et al., 2015). Compound A, 5-(2-(4-(2-(4-(1H-tetrazol-1-yl)phenyl)acetyl)piperazin-1-yl)ethyl)isobenzofuran-1(3H)-one (Tang et al., 2013), was shown to display diuresis/natriuresis in normotensive rats and dogs, with no significant kaliuresis, and no changes in plasma electrolyte levels (Garcia et al., 2014). Despite its 100-fold selectivity over the human ether-a go-go related gene (hERG) channel, compound A caused corrected QT (QTc) prolongation when evaluated in a cardiovascular dog model, suggesting that further improvements in selectivity would be needed to identify an appropriate clinical development candidate. Medicinal chemistry efforts directed to achieve these goals led to the discovery of MK-7145 [5,5′-((1R,1′R)-piperazine-1,4-diylbis(1-hydroxyethane-2,1-diyl))bis(4-methylisobenzofuran-1(3H)-one)] (Tang et al., 2016) as a potential clinical candidate.

In the present study, MK-7145 has been evaluated in preclinical in vivo studies. In spontaneously hypertensive rats (SHRs), oral dosing of MK-7145 causes dose-dependent lowering of blood pressure that is maintained during the treatment period, and that displays an additive/synergistic effect when administered in combination with hydrochlorothiazide (HCTZ) or candesartan, respectively. In conscious dogs, short-term or long-term oral MK-7145 administration led to dose-dependent diuresis and natriuresis, without any significant urinary potassium losses or changes in plasma electrolyte levels. These data indicate that pharmacological inhibition of ROMK has potential as a new mechanism for the treatment of hypertension and/or congestive heart failure.

Materials and Methods

Materials.

MK-7145 was synthesized at Merck Research Laboratories (Rahway, NJ), as described previously (Tang et al., 2016). The FluxOR Thallium Detection Kit was obtained from Invitrogen (Carlsbad, CA). Probenecid and ouabain were obtained from Sigma-Aldrich (St. Louis, MO). 86RbCl was obtained from PerkinElmer (Waltham, MA). The pCI-neo vector and the FuGENE 6 transfection reagent were from Promega (Madison, WI). Amphotericin B was from Sigma-Aldrich. HCTZ was purchased from MP Biomedicals, LLC (Solon, OH). Candesartan cilexetil was purchased from Sequioa Research Products (Oxford, UK). All other reagents were obtained from commercial sources and were of the highest purity commercially available.

Cells.

All tissue culture reagents were obtained from Invitrogen. All Kir1.1 constructs represent the ROMK1 splice variant of Kir1.1. HEK293 cell lines stably transfected with human Kir1.1 (hKir1.1), rat Kir1.1 (rKir1.1), human Kir2.1 (hKir2.1), human Kir4.1 (hKir4.1), or human Kir7.1 (hKir7.1); CHO cell lines stably expressing hKir1.1 or human Kir2.3 (hKir2.3); and MDCKII-Flp cell line stably expressing rKir1.1 were acquired as previously described (Felix et al., 2006, 2012; Garcia et al., 2014). An HEK293 cell line stably expressing dog Kir1.1 (dKir1.1) was constructed by transfecting cells with dKir1.1-pCIneo expression plasmid using FuGENE 6. Stable pools of HEK293 cells expressing dKir1.1 were prepared by geneticin (G418) selection (1000 μg/ml) and were analyzed for functional expression of dKir1.1 in a 86Rb+ efflux assay. Individual stable cell lines were generated by limiting dilution under continuous selection with G418. HEK293 cells were grown in minimum essential medium Alpha medium, 10% fetal bovine serum (FBS), 500 µg/ml G418, 1× penicillin/streptomycin/glutamine and 1× minimum essential medium nonessential amino acids. CHO cells were grown in Iscove's modified Dulbecco's medium supplemented with HT Supplement Solution with 10% heat-inactivated FBS, 500 mg/ml G418, and 1% penicillin/streptomycin. The MDCK-rKir1.1 cell line was grown in Dulbecco’s modified Eagle medium with GlutaMAX, supplemented with penicillin/streptomycin, 200 μg/ml hygromicin B, and 10% FBS. Procedures for handling TsA-201 cells and their transfection with FuGENE 6 have been described previously (Felix et al., 2006). All cell lines were maintained at 37°C in a 10% CO2 atmosphere.

Kir1.1 Thallium Flux Assay.

Permeation of thallium through open hKir1.1 channels was monitored as previously described (Felix et al., 2012; Garcia et al., 2014). Briefly, HEK293 cells stably transfected with hKir1.1 were plated at approximately 20,000 cells/well on black-wall, clear-bottom, 384-well poly-d-lysine–coated plates (Becton Dickinson, Franklin Lakes, NJ) in 50 µl of growth medium, and incubated overnight (16–20 hours) at 37°C in a 10% CO2 atmosphere. Cell growth medium was removed, and cells were then incubated with 0.025 ml of a solution containing FluxOR dye loading reagent, prepared according to the manufacturer instructions in Hanks’ balanced salt solution containing 1.26 mM CaCl2 and 0.49 mM MgCl2 (Life Technologies, Carlsbad, CA), pH adjusted to 7.4 by the addition of NaOH. After incubation in the dark for 90 minutes at ambient temperature (22–24°C), cells were washed once with 0.04 ml of Hanks’ balanced salt solution buffer solution, and incubated in the dark for 30 minutes at ambient temperature (22–24°C) with 0.025 ml of FluxOR assay solution containing 2.5 mM probenecid, and 300 μM ouabain, in the absence or presence of test compound. At the end of the 30-minute incubation period, the plate was placed in a FLIPRTETRA instrument (Molecular Devices, Sunnyvale, CA) and illuminated at 490 nm, and fluorescence emission was recorded at 525 nm. After an 80-second baseline reading, 0.00625 ml of a 5× solution containing 7.5 mM thallium sulfate, 0.75 mM K2SO4, prepared in the FluxOR chloride-free buffer was added, and fluorescence emission was recorded for an additional 8–9 minutes, with an exposure time of 0.4 second and a read interval of 10 seconds. The relative change in fluorescence was calculated as previously described (Felix et al., 2012).

Kir1.1 86Rb+ Flux Assays.

The ability of 86Rb+ to permeate through Kir1.1 channels was evaluated as previously described (Felix et al., 2012; Garcia et al., 2014). Briefly, HEK293 cells stably expressing rKir1.1 or dKir1.1 were seeded at 120,000 cells/well in 96-well plates (BioCoat; Becton Dickinson), in complete growth medium containing 1.5 μCi/ml 86Rb+, and incubated in 10% CO2 at 37°C overnight. On the day of the assay, the 86Rb+-containing medium was removed, and the cells were washed 1× with Low K assay buffer containing the following (in mM): 126.9 NaCl, 4.6 KCl, 2 CaCl2, 1 MgCl2, and 10 HEPES/NaOH, pH 7.4. High K assay buffer (100 μl) containing (in mM) 121.5 NaCl, 10 KCl, 2 CaCl2, 1 MgCl2, and 10 HEPES/NaOH, pH 7.4, with or without test compound was added, and cells were incubated at ambient temperature (22–24°C) for 30 minutes. Radioactivity associated with the assay buffer and cells was determined in a Packard TopCount Counter (GMI, Ramsey, MN). The amount of radioactivity in the assay buffer (percentage efflux) was normalized to the total radioactivity content of the assay buffer and cells. For experiments in which serum was included, compounds were prepared in high K assay buffer, in the absence or presence of 10, 30, or 100% of the corresponding serum. All other steps were carried out as described above.

Electrophysiological Assay.

Block of hKir1.1 (CHO-hKir1.1) currents was studied by whole-cell voltage clamp using the IonWorks Quattro automated electrophysiology platform (Molecular Devices), as previously described (Felix et al., 2012). Briefly, cells suspended in 150 mM NaCl, 10 mM KCl, 2.7 mM CaCl2, 0.5 mM MgCl2, and 5 mM Na-HEPES, pH 7.4 were placed in the IonWorks instrument. The intracellular solution consisted of 80 mM K gluconate, 40 mM KCl, 20 mM KF, 3.2 mM MgCl2, 3 mM EGTA, and 5 mM K-HEPES, pH 7.4. Electrical access to the cytoplasm was achieved by perforation for 4 minutes in the presence of 0.13 mg/ml amphotericin B. Voltage protocols and current recordings were performed using the IonWorks Quattro software/hardware system in population patch-clamp mode. The test pulse, consisting of a 100-millisecond step to 0 mV from a holding potential of −70 mV, followed by a 100-millisecond voltage ramp from −70 to +70 mV, was applied before and after incubation with MK-7145 for 6 minutes. Current amplitudes were measured using the IonWorks software, and the extent of block was assessed during the voltage step to 0 mV. Block of rKir1.1, dKir1.1, and rhesus Kir1.1 by MK-7145 was examined by manual whole-cell voltage clamp, as described previously (Felix et al., 2006; Garcia et al., 2014). Briefly, experiments were performed at room temperature, using an EPC-9 amplifier and Pulse software (HEKA Electronics, Lamprecht, Germany). Data were acquired at 10 kHz and filtered at 2.9 kHz. The bath solution consisted of 120 mM NaCl, 40 mM KCl, 2.7 mM CaCl2, 0.5 mM MgCl2, and 5 mM Na-HEPES, pH 7.4. The internal (pipet) solution contained 130 mM KCl, 5 mM NaCl, 2 mM MgCl2, 5 mM EGTA, 0.2 mM MgATP, and 5 mM Na-HEPES, pH 7.4. Kir1.1 currents were recorded during 100-millisecond voltage ramps from −100 to +100 mV at 10-second intervals and the current corresponding to a membrane potential of −100 mV was measured. To evaluate the quality of the recording, a bath solution containing a low concentration of K+ (156 mM NaCl, 4 mM KCl, 2.7 mM CaCl2, 0.5 mM MgCl2, and 5 mM Na-HEPES, pH 7.4) was used at the beginning of each recording, and the shift in the reversal potential was used to assess the contribution of nonselective leak currents.

Other Ion Channel Assays.

All procedures for evaluation of hKir2.1 (HEK-hKir2.1), hKir.2.3 (CHO-hKir2.3), hKir4.1 (HEK-Kir4.1), hKir7.1 (HEK-Kir7.1), hERG (CHO-hERG), the human voltage-gated sodium channel Nav1.5 (HEK-Nav1.5), and the human L-type calcium channel Cav1.2 (HEK-hCav1.2) have been described previously (Felix et al., 2004, 2012; Abbadie et al., 2010; Schmalhofer et al., 2010; Garcia et al., 2014).

Animal Studies.

All protocols for animal experiments were approved by the Institutional Animal Care and Use Committee of Merck Research Laboratories (Rahway, NJ, and West Point, PA) and were carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the U.S. National Institutes of Health.

Blood Pressure and Renal Excretory Function Studies in Conscious Rats.

Male SHRs 11–23 months of age were purchased from Taconic Biosciences (Hudson, NY) and were housed in temperature- and humidity-controlled rooms with a 12-hour light-dark cycle, and with access to rodent chow (catalog #7012; Teklad Diets, Madison, WI) and water ad libitum. Animals were implanted with a TA11PA-C40 telemetry device (Data Sciences International, St. Paul, MN) under either isoflurane or ketamine/xylazine/acepromazine or ketamine/domitor anesthesia. The telemetry unit catheter was inserted into the descending aorta via the femoral artery, and the telemetry device was implanted subcutaneously in the area between the caudal edge of the ribcage and the most cranial extension of the range of motion of the knee. Animals received postoperative analgesia, subcutaneous buprenorphine (0.1 ml/kg body weight), to alleviate pain. Animals were allowed to recover from surgery for 14 days before use in any experiments. Conscious, freely moving animals were housed in metabolism cages (Laboratory Products, Seaford, DE), and were used for blood pressure and renal function studies. After the acclimation period, animals were assigned to treatment groups based on the 24- to 48-hour average systolic blood pressure (SBP) determined before initiation of the experiment. Baseline control values were obtained on the day before treatment. Unless otherwise stated, animals were trained to receive a daily 5 ml/kg dose by oral gavage of either vehicle (0.5% methylcellulose in deionized water) or compound treatment of specified periods of time. Blood pressure and heart rate were recorded continuously for 10 seconds, every 10 minutes, using a computer-driven data acquisition system (Dataquest A.R.T. version 4; Data Sciences International). Data were pooled as hourly averages, and changes in blood pressure were calculated by subtracting from all values the corresponding control baseline. To assess renal excretory function, urine samples were collected daily, at 0–4 and 4–24 hours after dosing, during the baseline control period, and on each day of treatment while blood pressure was recorded. Samples were collected at room temperature, and urine volume was recorded for each animal. Samples were then subjected to centrifugation at 3000 rpm for 10 minutes, portioned in aliquots, and stored at −80°C until analyzed. When needed, blood samples were obtained by jugular vein puncture to determine compound plasma exposure levels.

Renal Excretory Function Studies in Conscious Dogs.

Female mongrel dogs were used as previously described (Garcia et al., 2014). Briefly, animals were sling restrained for the duration of the study, and Foley and percutaneous catheters were inserted for the collection of urine and blood samples, respectively. Saline was infused for 30 minutes, followed by a stabilization period in which creatinine and para-aminohippurate were infused for glomerular filtration rate (GFR) and effective renal plasma flow (eRPF) measurements. Dogs were assigned to receive either vehicle (Imwitor742:Tween80) (1:1, v:v) or compound treatment, once a day for specified periods of time, by oral gavage (feeding tube) at a dose volume of 1 ml/kg. On study days 1 and 7, dogs received vehicle or compound treatment, immediately after the collection of control blood and urine samples (two 30-minute collection periods). Six additional blood and urine collections (30 minutes for each period) up to 180 minutes were obtained. Urine volume was recorded for each collection period. Urine and blood samples were then subjected to centrifugation, aliquoted, and frozen at −20°C until analyzed. Dogs were continually observed while in sling restraint. Upon completion of the study, the Foley catheter was gently removed, and dogs were returned to their home cages.

Renal Excretory Function Studies in Conscious Nonhuman Primates.

Adult male rhesus macaques fasted for 16 hours were dosed with either vehicle (0.5% methylcellulose in deionized water) or 3 mg/kg MK-7145 via orogastric intubation and gavage, and were given access to water 2.5 hours after dosing. Animals were transferred to restraint chairs and received an oral dose of saline (5 ml/kg) followed by either vehicle or MK-7145 at 1 ml/kg via orogastric intubation and gavage. Urine samples were collected for 0–4 hours post-treatment, subjected to centrifugation, aliquoted, and frozen at −70°C until analyzed. Animals were returned to cages after the 4-hour collection and provided with free access to food.

Blood and Urinary Electrolyte Analysis.

Blood and urine electrolytes were measured from freshly thawed samples by a Modular Chemistry System (Roche Diagnostics, Indianapolis, IN). Creatinine was measured using an enzymatic method on an automated P Module Clinical Chemistry Analyzer (Roche Diagnostics). Aldosterone levels were determined using the aldosterone canine ELISA kit KA2286 (Abnova, Walnut CA), according to manufacturer instructions.

Plasma and Urine MK-7145 Level Analysis.

Plasma and urine concentrations of MK-7145 were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an API 5000 LC-MS/MS mass spectrometer (Applied Biosystems/MDS Sciex, Foster City, CA) operated in positive ion atmospheric pressure chemical ionization mode with multiple-reaction monitoring. Plasma was prepared for analysis by protein precipitation with a 6-fold volume of acetonitrile/methanol (2:1)/0.1% formic acid as described previously (Garcia et al., 2014). Extracts were subjected to chromatography using a Kinetex 1.7 μm PFP 50 × 2.1 mm column (Phenomenex, Torrance, CA) and eluted at 0.75 ml/min using a linear gradient of acetonitrile.

Statistics.

IC50 values for inhibition were determined according to the Hill equation from concentration–response curves by a nonlinear regression analysis, where all parameters were left unconstrained. Data are presented as the mean ± S.D. or S.E.M. of n experiments. Statistical analysis for comparisons of groups was conducted using one-way or two-way analysis of variance followed by Tukey’s post hoc test using Prism (version 6.04; GraphPad, La Jolla, CA). A P value <0.05 is considered to be statistically significant.

Results

MK-7145 Inhibits Kir1.1 Channels.

Previous efforts had led to the identification of compound A (Tang et al., 2013), a Kir1.1 inhibitor with in vivo properties in rats and dogs consistent with pharmacological modulation of ROMK channels (Garcia et al., 2014). However, despite its ∼100-fold selectivity for ROMK versus hERG, compound A was found to cause QTc prolongation when evaluated in a cardiovascular dog model (Tang et al., 2016). The search for potent Kir1.1 inhibitors with better selectivity over hERG has led to the discovery of MK-7145 (Tang et al., 2016) (Fig. 1). In HEK cells stably expressing hKir1.1 channels, MK-7145 inhibits Kir1.1-mediated thallium flux with an IC50 value (mean ± S.E.M.) of 6.6 ± 0.6 nM (n = 9) (Fig. 2A). In MDCK cells stably expressing rKir1.1, thallium flux is inhibited by MK-7145 with an IC50 value (mean ± S.E.M.) of 6.3 ± 1.7 nM (n = 4) (data not shown). In functional cell-based assays that measure the ability of 86Rb+ to permeate through rat or dog Kir1.1 channels, MK-7145 inhibits these channels with IC50 values (mean ± S.D.) of 32.5 ± 1.6 nM (n = 2) and 11 ± 3 nM (n = 3), respectively, in the absence of serum, and 46.2 ± 2.6 nM (n = 2) and 23.4 ± 9.2 nM (n = 3), respectively, in the presence of 100% rat (Fig. 2B) or dog serum (Fig. 2C).

Fig. 1.
Fig. 1.

Structure of MK-7145 [5,5′-((1R,1′R)-piperazine-1,4-diylbis(1-hydroxyethane-2,1-diyl))bis(4-methylisobenzofuran-1(3H)-one)].

Fig. 2.
Fig. 2.

MK-7145 is a potent and selective inhibitor of Kir1.1 channels. (A) HEK293 cells stably expressing hKir1.1 (●), hKir2.1 (■), hKir4.1 (▲), or hKir7.1 (□) channels were preloaded with the FluxOR reagent, and incubated in the absence or presence of increasing concentrations of MK-7145, as described under Materials and Methods. Upon recording the emission of the dye for 90 seconds, a thallium sulfate/potassium sulfate solution was added, and fluorescence was monitored for an additional 510 seconds. Data were analyzed by the Hill equation where all parameters were left unconstrained. MK-7145 inhibits hKir1.1 with an IC50 value of 4.9 nM and a Hill coefficient of 1.1. In marked contrast, MK-7145 does not have any significant effect on hKir2.1 at concentrations of up to 100 μM or on hKir4.1, or hKir7.1 at concentrations of up to 10 μM. Data shown are the mean ± S.D. (n = 4); however, error bars may be smaller than the symbol size. HEK-rKir1.1 (B) or HEK-dKir1.1 (C) cells were incubated with 86Rb+ overnight, as indicated in Materials and Methods. On the day of the experiment, the medium was removed, and cells were placed into assay medium containing 0% (●), 10% (△), 30% (♦), or 100% (□) rat (B), or dog (C) serum, in the absence or presence of increasing concentrations of MK-7145, and incubated at room temperature for 30 minutes. The amount of 86Rb+ efflux was calculated as indicated in Materials and Methods. Data were analyzed by the Hill equation where all parameters were left unconstrained. The data shown are average ± S.D. (n = 2–4). IC50 values were as follows (in nM): (B) 32.5 (●), 33.5 (△), 35.5 (♦), 46.3 (□); (C) 8 (●), 11.3 (△), 10.2 (♦), and 23.4 (□).

Inhibition of recombinant hKir1.1 channels by MK-7145 was also examined by whole-cell voltage-clamp recordings. Using the automated IonWorks Quattro platform, CHO-hKir1.1 currents recorded at 0 mV in the presence of 10 mM extracellular potassium were inhibited in a concentration-dependent manner by MK-7145 with an IC50 value (mean ± S.E.M.) of 10 ± 1.3 nM (n = 6) (Fig. 3A). In manual electrophysiological recordings of HEK-rKir1.1, HEK-dKir1.1, or rhesus Kir1.1 channels transiently transfected into TsA-201 cells, MK-7145 caused concentration-dependent inhibition of Kir1.1 currents (Fig. 3B). The estimated IC50 values of 4.9, 8.8, and 8.6 nM, for rat, rhesus, or dog Kir1.1, respectively, indicate little species differences. In summary, these results are consistent with MK-7145 being a potent Kir1.1 inhibitor with the potential for displaying a high unbound fraction in serum. In vitro plasma binding studies carried out at MK-7145 concentrations of 1 and 10 μM confirmed that the compound has similar plasma unbound fractions in rat (57–65%), dog (62–63%), rhesus monkey (67–69%), and human (29–37%) (n = 3 for each determination).

Fig. 3.
Fig. 3.

Inhibition of Kir1.1 channels by MK-7145. (A) Human Kir1.1 channels stably expressed in CHO cells were examined by whole-cell voltage clamp using the IonWorks Quattro automated platform, as described in Materials and Methods. Currents were recorded at 0 mV in 10 mM extracellular potassium, in the absence or presence of increasing concentrations of MK-7145. Inhibition was plotted as a function of MK-7145 concentration, where the solid line represents the results of fitting data to the Hill equation, yielding an IC50 of 9 nM and a Hill coefficient of 1.2. Data are shown as the mean ± S.D. (n = 4). (B) Rat or dog Kir1.1 channels stably expressed in HEK293 cells or rhesus Kir1.1 channels transiently transfected into TsA-201 cells were analyzed using standard manual whole-cell voltage-clamp protocols. Currents corresponding to membrane potential of −100 mV were measured, and the observed inhibition is presented as a function of MK-7145 concentration. One to three cells were tested at each concentration, and error bars reflect the S.D. of the mean. The estimated IC50 values determined from fitting the Hill equation to the data points were 4.9, 8.8, and 8.6 nM for rat, rhesus, or dog Kir1.1, respectively.

The selectivity of MK-7145 for other related Kir channels, such as cardiac Kir2.1, or renal Kir2.3, Kir4.1, and Kir7.1, was determined in functional assays. In the absence of serum, MK-7145 had no significant effect on hKir2.1 (Fig. 2A) or hKir2.3 (data not shown) at concentrations of up to 100 μM; or on hKir4.1 and hKir7.1 (Fig. 2A) channels at concentrations of up to 10 μM. Consistent with the thallium flux data, MK-7145, when tested at 30 μM, did not cause any significant inhibition in electrophysiological recordings of HEK cells expressing either Kir4.1 or Kir7.1 channels (data not shown).

In a binding assay that measures the interaction of [35S]MK-499 with membranes derived from HEK cells stably expressing the hERG channel, MK-7145 displays an IC50 (mean ± S.E.M.) of 23.6 ± 2.6 μM (n = 7), a value similar to the IC50 determined in electrophysiological recordings of hERG channels, 28.7 ± 6.1 μM (n = 4). In functional assays, MK-7145 at concentrations of up to 30 μM had no significant effect (<5% inhibition) on the voltage-gated sodium channel hNav1.5 or the voltage-gated calcium channel hCav1.2. As previously reported (Tang et al., 2016), MK-7145 did not display any treatment-related effects on heart rate, mean arterial pressure, PR, QRS, or QT/QTc intervals at plasma concentrations of up to 11.6 μM when administered intravenously to anesthetized and vagotomized dogs. Moreover, in a panel of 166 enzyme and radioligand binding assays performed at MSD Pharma Services (King of Prussia, PA), MK-7145, when tested at 10 μM, only inhibited the human serotonin transporter, somatostatin receptor type 1, and acetyl cholinesterase with IC50 values of 0.12, 2.63, and 9.94 μM, respectively.

Overall, MK-7145 is a more potent and selective Kir1.1 inhibitor than compound A and, unlike compound A, does not display cardiovascular liabilities. The pharmacokinetic properties of MK-7145 in rats, dogs, and rhesus (Tang et al., 2016) indicate that the compound has good oral bioavailability with moderate clearance rates, making it suitable for in vivo evaluation.

MK-7145 Elicits Blood Pressure Lowering in SHRs.

In a previous short-term study, compound A was shown to cause natriuresis/diuresis, in the absence of kaliuresis, when orally dosed to conscious, volume-loaded rats (Garcia et al., 2014). Under an identical paradigm, MK-7145 displays a similar profile, although the magnitude of its diuretic and natriuretic activity (9.4- and 8.5-fold over vehicle, respectively) is larger than that of compound A (4.3- and 3.8-fold over vehicle, respectively), and the pharmacological effects occur at a lower compound dose (Tang et al., 2016). Similar to compound A, kaliuresis was not significantly affected by MK-7145 (data not shown).

To further explore the hemodynamic consequences of ROMK inhibition by MK-7145, blood pressure, and kidney excretory function were assessed in SHRs after oral dosing, once a day, for 4 days with 0.3, 1, 3, 10, or 30 mg/kg compound. In these experiments, HCTZ was also included as a reference diuretic at a dose of 25 mg/kg, giving approximately maximal efficacy of that compound in this animal model. Figure 4 illustrates the results of these experiments. MK-7145 caused a dose-dependent decrease in systolic (Fig. 4A) and diastolic blood pressure (data not shown). Maximal lowering of SBP, ∼20 mm Hg, was observed by day 2 at both the 10 and 30 mg/kg MK-7145 doses. The extent of blood pressure lowering by 3 mg/kg MK-7145, ∼12 mm Hg, was similar to that achieved by 25 mg/kg HCTZ. Blood pressure lowering was maintained through the last day of treatment in all dosing groups. Once-a-day dosing was sufficient to sustain maximum decrease in blood pressure, as indicated by the data in Fig. 4B. At the time of dosing, blood pressure was maintained at the minimum level established by the previous dose and did not change significantly during the 24 hours after subsequent doses. Changes in heart rate were not observed in any of the treatment groups (data not shown).

Fig. 4.
Fig. 4.

MK-7145 lowers blood pressure. SHRs were orally dosed, once a day, for 4 days with vehicle (0.5% methylcellulose) (●), or 0.3 (○), 1 (△), 3 (◊), 10 (∇), or 30 (□) mg/kg MK-7145, or 25 mg/kg (■) HCTZ, and were housed in metabolic cages for blood pressure and renal function studies. (A) MK-7145 caused a dose-dependent decrease in mean SBP. (B) Average hourly SBP at day 3 of treatment with vehicle (●); 3 (◊) or 10 (∇) mg/kg MK-7145; or 25 mg/kg (■) HCTZ. At the time of dosing, blood pressure was already at the minimum level established by the previous dose and did not change significantly during the 24 hours upon dosing. (C) Correlation between blood pressure lowering on day 3 and diuresis for 0–24 hours on day 1. (D) Correlation between blood pressure lowering on day 3 and natriuresis for 0–24 hours on day 1. Data are presented as the mean ± S.E.M. and represent changes from baseline values obtained prior to treatment administration (n = 6 for vehicle group; n = 6-7 for MK-7145 and HCTZ treatment groups). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus vehicle.

Diuresis was significantly larger in MK-7145-dosed SHRs than in those animals receiving vehicle or HCTZ, showing little dose dependence over the 0–4 hours after dosing (data not shown), but a clear dose dependence over the 24-hour period (Fig. 5A). Increases in diuresis occurred on the first day of MK-7145 dosing and were maintained throughout the entire treatment. HCTZ caused an increase in diuresis when measured over 4 hours after dosing (data not shown), but there were no differences from vehicle on the 24-hour postdosing interval on any day of treatment (Fig. 5A). The extent of 24-hour diuresis after MK-7145 dosing correlated well with the magnitude of blood pressure lowering (Fig. 4C).

Fig. 5.
Fig. 5.

Renal excretory function studies in conscious rats. SHRs were orally dosed, once a day, for 4 days with vehicle (0.5% methylcellulose) (●);0.3 (○), 1 (△), 3 (◊), 10 (∇), or 30 (□) mg/kg MK-7145; or 25 mg/kg (■) HCTZ; and were housed in metabolic cages for blood pressure and renal function studies. (A) Urine output 0–24 hours after dosing. (B) Urinary sodium excretion 0–24 hours after dosing. (C) Urinary potassium excretion 0–24 hours after dosing. (D) Urinary calcium excretion 0–24 hours after dosing. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus vehicle.

Dose-dependent increases in urinary sodium excretion were present in the first 4 hours after dosing on all days of the study, but there were only significant increases during the 24 hours postdosing on day 1 (Fig. 5B). The absence of increased urinary sodium excretion after day 1 is a consequence of reduced sodium excretion during the 4- to 24-hour postdosing period (data not shown). It is possible that this period of compensatory sodium retention reflects normal homeostatic control mechanisms for animals that are not volume expanded. There was a good correlation between the extent of 24-hour natriuresis on day 1 and the extent of blood pressure lowering on day 3 (Fig. 4D). Although a similar extent of kaliuresis (∼2-fold over baseline) was observed during the first 4 hours after dosing in both MK-7145 and HCTZ groups, kaliuresis was not different from vehicle-treated animals when assessed over 24 hours (Fig. 5C). Urine samples were also analyzed to determine the rate of calcium excretion after MK-7145 treatment. There was an increase in the calcium excretion rate relative to vehicle for all MK-7145 groups over the 24-hour period after dosing (Fig. 5D) that was maintained through the entire treatment, consistent with inhibition of ROMK channels at the TALH. In marked contrast, an opposite effect was observed in HCTZ-treated animals, a result that is also consistent with the mechanism of action of this drug. There was no significant effect on food intake and body weight (mean average values for the different groups ranged between 420 and 445 g) for any of the treatment groups (data not shown). Increases in water intake occurred for all MK-7145 treatment groups (Fig. 1; Supplemental Data).

In a parallel PK study, concentrations of MK-7145 were determined in plasma and urine samples. For clarity, only those values corresponding to day 3 at doses of 3 and 10 mg/kg are presented (Table 1). Despite low plasma exposures at trough, 24-hour blood pressure–lowering efficacy is maintained (Fig. 4, A and B). However, urinary levels of MK-7145 are significantly higher than plasma levels, suggesting that concentrations of compound in the tubules may drive pharmacological efficacy. Because of the relatively low protein-binding characteristics of MK-7145 (>50% free fraction in rat serum), it is likely that filtration represents a significant component of the renal elimination of the compound, although other mechanisms such as tubular secretion could also play a role in contributing to the accumulation of the compound in the tubular fluid.

View this table:
TABLE 1

Plasma and urine concentrations (μM) of MK-7145 on day 3 after daily oral administration to SHRs

A 14-day study in SHRs using two doses of MK-7145, 1 and 3 mg/kg, given once a day, was carried out to determine the extent of blood pressure lowering during prolonged days of treatment. There was significant and sustained blood pressure–lowering effects throughout the 2-week treatment of the two MK-7145 dosing groups (Fig. 2; Supplemental Data). In addition, increases in 24 hour urine volume over vehicle were maintained throughout the study (data not shown), and there were no significant changes in urinary sodium, potassium, or chloride excretion over the 24-hour period after 14 days of dosing (Table 1; Supplemental Data).

MK-7145 Blood Pressure Lowering Efficacy in Combination with Candesartan or HCTZ.

To achieve target blood pressure, many patients require combination therapies with antihypertensive drugs having different mechanisms of action. Thiazide-like diuretics and angiotensin receptor blockers, such as candesartan, are widely used, alone or in combination, for blood pressure control (Sood et al., 2010). To determine the potential additive or synergistic effects of candesartan with MK-7145 in SHRs, MK-7145 was dosed once daily for 7 days at 10 mg/kg. HCTZ, at 25 mg/kg, was used in these experiments as a positive control. Subsequently, all treatment and vehicle groups received a submaximal dose of candesartan (0.1 mg/kg) via oral gavage, in addition to their corresponding treatment of 2 additional days. This dose of candesartan was selected because it elicits submaximal blood pressure lowering in SHRs (data not shown). Afterward, treatments were ended, and all SHR groups were monitored for blood pressure recovery for 3 consecutive days. As illustrated in Fig. 6A, MK-7145 caused a reduction in SBP of ∼20 mm Hg by day 2, which was sustained for the duration of the 7-day treatment. The magnitude of HCTZ blood pressure lowering was ∼10-13 mm Hg. The addition of candesartan lowered blood pressure in vehicle-treated animals by ∼14 and 18 mm Hg after days 1 and 2 of treatment, respectively. In HCTZ-treated animals, the addition of candesartan provided an additional reduction of 14–22 mm Hg in SBP, whereas in the MK-7145 treatment group, a further 20–29 mm Hg in blood pressure lowering was observed when dosing in combination with candesartan. Although the extent of blood pressure lowering caused by HCTZ and candesartan is additive, there appears to be a synergistic effect for the MK-7145 and candesartan combination (Table 2; Supplemental Data). Three days after treatment ended, blood pressure returned to baseline values in all groups, although reversibility was not complete within the course of the experiment.

Fig. 6.
Fig. 6.

MK-7145 efficacy in combination with candesartan or HCTZ. (A) SHRs were dosed once daily, for 7 days, with vehicle (●), 10 mg/kg MK-7145 (∇), or 25 mg/kg HCTZ (■). Subsequently, all groups received 0.1 mg/kg candesartan, in addition to their corresponding treatment of 2 days. Afterward, treatments were ended, and all SHR groups were monitored for 3 consecutive days. The combination HCTZ/candesartan caused an additive effect on blood pressure lowering, whereas the result of combining MK-7145/candesartan appears to be synergistic. Three days after treatment ended, blood pressure returned to baseline values in all groups, although reversibility was not complete within the course of the experiment. Data are presented as the mean ± S.E.M. and represent changes from baseline values obtained prior to treatment administration (n = 6 for vehicle group; n = 7 for MK-7145 and HCTZ treatment groups). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus vehicle. (B) SHRs were dosed once daily for 4 days with 1 or 10 mg/kg MK-7145; 10 or 25 mg/kg HCTZ; or 1 and 10, 10 and 10, or 10 and 25 mg/kg MK-7145 and HCTZ, respectively. Data are presented as the mean ± S.E.M. and represent changes from baseline values obtained prior to treatment administration (n = 6 and 12 for 1 and 10 mg/kg MK-7145; 12 and 6 for 10 and 25 mg/kg HCTZ; 6, 11 and 6 for 1 and 10, 10 and 10, or 10 and 25 mg/kg MK-7145 and HCTZ, respectively). *P < 0.05, ***P < 0.001

View this table:
TABLE 2

Coadministration of HCTZ diminishes MK-7145 calciuretic response

To determine the blood pressure–lowering effects of MK-7145 in combination with HCTZ, SHRs were treated once daily for 4 days with vehicle, MK-7145 at 1 or 10 mg/kg, HCTZ at 10 or 25 mg/kg, or combinations of MK-7145 and HCTZ. Results from these experiments after 4 days of dosing are presented in Fig. 6B. At concentrations of either 1 or 10 mg/kg MK-7145, which, when administered alone, provide an intermediate or maximal blood pressure–lowering effect, in combination with HCTZ caused additive reductions in SBP. These data are consistent with the contribution of two independent mechanisms to blood pressure regulation. In addition to blood pressure, urinary calcium levels were determined for the 24 hours period after dosing at day 4. As expected, MK-7145 treatment alone led to an increase in urinary calcium levels, whereas the opposite effect was found in HCTZ-treated animals. When dosed in combination with HCTZ, there was a significant attenuation on the levels of urinary calcium compared with MK-7145 treatment alone (Table 2).

MK-7145 Therapy Manifests Features of the Bartter’s Syndrome Type II Phenotype in Conscious Dogs.

Single-dose oral administration of MK-7145 at 0.1, 0.3, 1, or 3 mg/kg increased urine output in conscious, euvolemic mongrel dogs, with maximal diuretic effect (10-fold over vehicle) observed at the 1–3 mg/kg dose (Fig. 7A). Similarly, MK-7145 led to dose- and time-dependent increases in urinary Na+ excretion (Fig. 7B), and a maximal natriuretic effect was also seen at the 1–3 mg/kg dose. Consistent with previous studies (Garcia et al., 2014), oral HCTZ dosed at 10 mg/kg also increased urine flow (3.9-fold), urinary Na+ excretion (5.2-fold), and urinary K+ excretion (1.7-fold) (data not shown). The effects of single MK-7145 dosing on diuresis, natriuresis, and kaliuresis (1.2-fold; data not shown) are similar to those previously reported for compound A (Garcia et al., 2014), although they occur at lower MK-7145 doses. Similarly, short-term dosing of 3 mg/kg MK-7145 to nonhuman primates caused a 1.6-fold increase in urine output over a baseline of 209 ± 60 ml/4 hours for vehicle-treated animals, and a 7.7-fold increase in urinary Na+ excretion from a baseline value of 2.45 ± 0.54 mmol/4 hours recorded in vehicle-treated animals (n = 7 per treatment group).

Fig. 7.
Fig. 7.

Renal excretory function studies in conscious dogs. Female dogs (n = 3–6 per treatment) were dosed by oral gavage with either vehicle (Imwitor742:Tween80 [1:1, v:v]) (●), or 0.1 mg/kg (◧), 0.3 mg/kg (○), 1 mg/kg (△), or 3 mg/kg (◊) MK-7145. Urine samples were collected before and after oral dosing at 30-minute intervals up to 3 hours postdosing, and analyzed for volume (A), or sodium (B) content. Data (mean ± S.E.M.) are presented as absolute values obtained after vehicle or MK-7145 dosing. The zero time point (BL) represents the average of two 30-minute baseline periods collected before vehicle or MK-7145 administration. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus vehicle.

To determine the effects of MK-7145 in mongrel dogs after extended treatment, MK-7145 was dosed, once a day at 0.3 mg/kg, for 7 days, and blood and urine samples were collected on days 1 and 7 for analysis. The 0.3 mg/kg dose of MK-7145 was selected for these studies because of its slightly larger effects on diuresis (6.7-fold) and natriuresis (8.0-fold) compared with HCTZ (3.9- and 5.2-fold, respectively) in the single-dosing experiments. As illustrated in Fig. 8, the extent of diuresis (Fig. 8A) and natriuresis (Fig. 8B) due to MK-7145 appears to be similar regardless of the day of treatment. Kaliuresis (Fig. 8C), on the other hand, was not present on day 1, although it was noted at day 7 after MK-7145 dosing. In parallel experiments, 10 mg/kg HCTZ was dosed once a day for 7 days to conscious dogs. Compared with day 1, the extent of diuresis, natriuresis, and kaliuresis at day 7 was diminished (data not shown). Interestingly, plasma potassium levels were not significantly altered by 7 days of MK-7145 treatment, both at baseline and 180 minutes postdosing, whereas a marked decrease in plasma potassium was observed at day 7 in HCTZ-treated dogs (Fig. 8D). GFR, determined by calculating creatinine clearance, and eRPF, determined by para-aminohippurate clearance were not significantly affected at either day 1 or day 7 in MK-7145–treated or HCTZ-treated dogs, and were maintained at ∼60 and 150 ml/min, respectively, throughout the study in all groups (data not shown). Compared with vehicle, 7-day dosing with MK-7145 had no effect on plasma glucose, calcium, magnesium, sodium, chloride, uric acid, or blood urea nitrogen levels (data not shown). However, at baseline and 180 minutes postdosing bicarbonate levels were found to be elevated after 7 days of dosing with MK-7145 (data not shown).

Fig. 8.
Fig. 8.

MK-7145 dosing in conscious dogs. Female dogs (n = 3 per treatment) were dosed by oral gavage, once a day for 7 days, with either vehicle (Imwitor742:Tween80 [1:1, v:v]) (●, ○), 0.3 mg/kg MK-7145 (▲, △), or 10 mg/kg HCTZ (□). Urine samples were collected before and after oral dosing at 30-minute intervals up to 3 hours postdosing, and analyzed for volume (A), sodium (B), and potassium (C) content at days 1 and 7. The zero time point (BL) represents the average of two 30-minute baseline clearance periods collected prior to administration of vehicle or test compound. Data (mean ± S.E.M.) are presented as absolute values obtained after vehicle or MK-7145 dosing on day 1 (●, ▲) or day 7 (○, △). (D) Plasma potassium levels were not significantly altered by MK-7145 (△), whereas a marked decrease was observed at day 7 in HCTZ-treated (□) versus vehicle-treated (○) dogs. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus vehicle.

Inhibition of ROMK at the TALH should attenuate the transepithelial potential that provides the driving force for paracellular uptake of ions such as calcium and magnesium. This is a consequence of diminished chloride reabsorption and potassium recycling at the TALH. Consistent with this notion, MK-7145 caused an increase in chloride, calcium, and magnesium excretion of similar magnitude when monitored on days 1 and 7 for the first 180 minutes after dosing (Fig. 9, A–C).

Fig. 9.
Fig. 9.

Chloride, calcium, and magnesium excretion after MK-7145 dosing in conscious dogs. Female dogs (n = 3 per treatment) were dosed by oral gavage, once a day for 7 days, with either vehicle (Imwitor742:Tween80 [1:1, v:v]) (●, ○), or 0.3 mg/kg MK-7145 (▲, △). Urine samples were collected before and after oral dosing at 30-minute intervals up to 3 hours postdosing, and analyzed for chloride (A), calcium (B), and magnesium (C) content at days 1 and 7. Data (mean ± S.E.M.) are presented as absolute values obtained after vehicle or MK-7145 dosing on day 1 (●, ▲) or day 7 (○, △). The zero time point (BL) represents the average of two 30-minute baseline periods collected prior to the administration of vehicle or MK-7145. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus vehicle.

Loss of salt and water by the kidney should activate the renin–angiotensin–aldosterone system, and high renin and aldosterone blood levels are characteristics of individuals with Bartter’s syndrome. On the seventh day of MK-7145 dosing, baseline and 180-minute postdosing levels of serum aldosterone were found to be significantly elevated over vehicle-treated animals (Fig. 3; Supplemental Data).

MK-7145 levels upon oral dosing in mongrel dogs were found to be significantly higher in urine than in plasma, and compound did not seem to accumulate in either compartment with days of dosing (Table 3). Similar to SHRs, high MK-7145 levels in the tubular fluid could be an important factor in the efficacy of this compound.

View this table:
TABLE 3

Plasma and urine concentrations of MK-7145 after oral administration to mongrel dogs

Discussion

This study presents, for the first time, a thorough characterization of the in vivo properties of MK-7145, a potent and selective Kir1.1 inhibitor, which is a potential clinical development candidate. MK-7145 displays pronounced diuretic and natriuretic activity in normotensive and SHRs, and these features translate into dose-dependent, sustained blood pressure lowering in SHRs upon once-a-day oral dosing, for up to 14 days. The magnitude of blood pressure lowering by maximally efficacious doses of MK-7145 appears to be synergistic when combined with submaximal doses of the angiotensin II receptor blocker candesartan, whereas an additive effect was found when MK-7145 was tested in combination with HCTZ, providing evidence for the contribution of independent mechanisms to control blood pressure. MK-7145 levels are significantly higher in urine than in plasma compartments, suggesting that concentrations of compound in the tubular fluid are related to the sustained efficacy of MK-7145 for lowering blood pressure 24 hours after dosing. It is important to note that MK-7145 does not display any significant activity on other targets that could influence blood pressure and that the effects on pressure levels observed with this compound appear to be solely the consequence of its ROMK inhibitory activity. In conscious mongrel dogs, 7 days oral dosing with submaximal doses of MK-7145 features natriuresis and diuresis, as monitored during the first 180 minutes after treatment, that display similar characteristics on days 1 and 7. In addition, the excretion of chloride, calcium, and magnesium is also increased as expected from the inhibition of ROMK at the TALH, and increases in bicarbonate and aldosterone plasma levels were observed after 7 days of dosing with MK-7145. Importantly, no significant effects on plasma potassium or other electrolyte levels were observed in these experiments. These data, taken together, suggest that selective ROMK inhibitors represent novel diuretics for the treatment of hypertension and/or congestive heart failure and that features of Bartter’s syndrome type II phenotype, due to recessive loss of ROMK function, are manifested upon treatment with MK-7145.

Hypertension is a complex, multifactorial disorder that often requires combination therapy to achieve target blood pressure levels. Within the diuretic class, thiazides, such as HCTZ, are the most widely used as first-line therapy to treat uncomplicated hypertension, or as add-on therapy to drugs with other mechanisms of action, such as angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, and calcium channel blockers (Sood et al., 2010). In addition, resistant hypertension can affect up to 14% of patients treated for hypertension, and its prevalence is twice as high in patients with chronic kidney disease (Rossignol et al., 2015). Thus, there is a need for developing antihypertensive agents with new mechanisms of action that could be used alone or in combination with other drugs for achieving better blood pressure control. Congestive heart failure is one of the most common chronic conditions and the most common cause of hospital admissions in patients older than 65 years of age (Felker et al., 2011). Loop diuretics are commonly used to treat these patients, and they clearly improve hemodynamics and symptoms of the disease. However, many studies have not been able to demonstrate a benefit on mortality from the use of loop diuretics, in part because of the development of diuretic resistance, which may require the use of a second type of thiazide-like diuretic, and because their therapeutic benefit may be limited by adverse effects including electrolyte imbalances and neurohormonal activation. New treatments for heart failure should overcome some of the current limitations associated with the use of loop diuretics. In this respect, features of MK-7145, such as minimal disturbances in potassium balance, direct access to its target from the tubular fluid, long duration of action, its synergistic or additive effect with an angiotensin II receptor blocker, such as candesartan, or HCTZ, respectively, and lack of effects on GFR and eRPF could represent an advantage for treating this disease.

ROMK channels are present in two different regions of the nephron (Xu et al., 1997). The inhibition of ROMK at the TALH should provide natriuresis/diuresis similar to that caused by inhibitors of the Na+/K+/2Cl− cotransporter, such as furosemide or bumetanide. In addition, the inhibition of ROMK at the CCD, where it participates in potassium secretion, may ameliorate the hypokalemia resulting from the use of loop and thiazide diuretics. These assumptions are consistent with recent data showing that the major diuretic target site of compound A is the TALH, and that the compound prevents the kaliuretic response caused by either bumetanide or HCTZ as a consequence of its inhibitory actions at both TALH and CCD (Kharade et al., 2015). The results presented in this study with MK-7145 also support these expectations of a ROMK inhibitor, and further illustrate constant diuretic and blood pressure effects in SHRs, without evidence for the development of diuretic resistance during the course of the experiments. Lack of diuretic resistance occurred despite the fact that ROMK inhibitors are expected to activate the renin–angiotensin system, as indicated by the elevation in plasma aldosterone levels after 7 days of treatment in dogs, which could attenuate the extent of natriuresis/diuresis upon long-term dosing. However, 24-hour sodium excretion was increased only on day 1 of MK-7145 dosing in SHRs, and returned to control values on subsequent dosing days, reflecting normal homeostatic control mechanisms for animals that are not volume expanded. A much longer chronic treatment with MK-7145 may be needed to determine the extent of diuretic/natriuretic resistance that may develop with time, which could limit the utility of this mechanism as a monotherapy agent. In any event, the possibility of combining MK-7145 with drugs having other mechanisms of action may overcome the issues associated with the activation of the renin–angiotensin system and provide an additional therapeutic benefit.

Three different ROMK isoforms are present in human and rat kidney (Dong et al., 2016). These isoforms differ at their cytoplasmic amino acid terminus and are differentially expressed in the nephron. Although ROMK knockout mice and rats lacking the three isoforms recapitulate the phenotype of type II Bartter’s syndrome (Lu et al., 2002; Lorenz et al., 2002; Zhou et al., 2013), unique functions of the ROMK1 isoform have only been recently elucidated (Dong et al., 2016). ROMK1 knockout did not produce the Bartter’s phenotype, and there was no functional coupling between ROMK1 and the Na+/K+/2Cl− cotransporter at the TALH. It appears that ROMK1 contributes to potassium secretion in the CCD in response to a high potassium intake. Although selective modulation of specific ROMK isoforms could lead to unique pharmacological consequences, compound A and related ROMK inhibitors display identical potency on the three human ROMK isoforms (data not shown), and therefore they should result in the complete inhibition of all ROMK channels present in the nephron.

Bartter’s syndrome type II phenotype is characterized by salt wasting, polyuria, calcium loss, and secondary hyperreninemia and hyperaldosteronemia that lead to hypokalemia and metabolic alkalosis (Reinalter et al., 2004). As expected, some of these features are manifested in mongrel dogs upon 7 days of treatment with MK-7145. Unlike the significant hypokalemia observed after HCTZ treatment, there were, however, no indications of changes in plasma potassium levels in dogs dosed for 7 days with 0.3 mg/kg MK-7145. Interestingly, the 0.3 mg/kg MK-7145 dose used in these studies is at least as efficacious on diuresis and natriuresis as HCTZ, although it represents a submaximal efficacious dose of the compound. Although it is not possible to determine the extent of target engagement that was achieved in these experiments, it is unlikely that 100% of channels would have been inhibited. In this sense, it is important to note that heterozygous carriers of ROMK mutations associated with type II Bartter’s syndrome, identified in the Framingham Heart Study, had a positive cardiovascular outcome (Ji et al., 2008), and that heterozygous ROMK rats display normal plasma potassium levels and are associated with reduced blood pressure when challenged with a high-salt diet (Zhou et al., 2013). In addition, ROMK-deficient mice did not develop hypokalemia despite exhibiting higher potassium excretion than wild-type animals (Lorenz et al., 2002; Lu et al., 2002). It is possible that different outcomes in plasma potassium levels in mongrel dogs would have occurred at maximal efficacious doses of MK-7145. Ultimately, however, clinical trials monitoring safety and efficacy of MK-7145 will determine the extent to which the results from preclinical species translate to humans. In particular, it would be important to compare the efficacy of MK-7145 and loop diuretics for diuretic resistance, and in patients with kidney failure.

The search for Kir1.1 inhibitors has identified a limited number of compounds with appropriate potency, selectivity, and physical–chemical and pharmacokinetic properties to be used in proof of concept studies (Lewis et al., 2009; Bhave et al., 2010; Tang et al., 2012, 2013, 2016; Garcia and Kaczorowski, 2014; Garcia et al., 2014; Walsh et al., 2015, 2016). MK-7145 represents the most potent and selective Kir1.1 inhibitor disclosed to date. Results from the in vivo evaluation of MK-7145 provide convincing evidence for the development of this, or similar compounds, for the treatment of hypertension and/or congestive heart failure.

Acknowledgments

The authors thank Timothy Bailey, Richard Brochu, Randal Bugianesi, Rodolfo Haedo, and Melba Hernandez for expert technical assistance; Xiaolan Shen, Keiana Dunn and the SALAR team, and Carol Ann Keohane and the Pharmacology team for assistance with animal studies; and Drs. Mike Forrest, Sandy G. Mills, Emma Parmee, Andrew Swensen, and Lihu Yang for important discussions during the course of this work.

Authorship Contributions

Participated in research design: Hampton, Zhou, Priest, Pai, Kohler, Tong, Ormes, Roy, Sullivan, Metzger, Alonso-Galicia, Kaczorowski, Pasternak, Garcia

Conducted experiments: Hampton, Zhou, Priest, Pai, Felix, Thomas-Fowlkes, Liu, Xiao, Corona, Price, Gill, Shah, Rasa, Owens, Alonso-Galicia

Contributed new reagents or analytic tools: Tang, Pasternak

Performed data analysis: Hampton, Zhou, Priest, Felix, Thomas-Fowlkes, Liu, Kohler, Owens, Alonso-Galicia, Garcia

Wrote or contributed to the writing of the manuscript: Hampton, Zhou, Priest, Kaczorowski, Pasternak, Garcia

Footnotes

    • Received May 17, 2016.
    • Accepted July 14, 2016.

  • ↵1 Current affiliation: Lilly Research Laboratories, Eli Lilly & Co., Indianapolis, Indiana

  • ↵2 Current affiliation: Consultant, General Biologicals Corp., Westfield, New Jersey

  • ↵3 Current affiliation: Evotec, Princeton, New Jersey

  • ↵4 Current affiliation: Bristol-Myers-Squibb, Princeton, New Jersey

  • ↵5 Current affiliation: University of Texas-Rio Grande Valley, Edinburg, Texas

  • ↵6 Current affiliation: Stemcentrx, South San Francisco, California

  • ↵7 Current affiliation: Sanofi-Genzyme, Framingham, Massachusetts

  • ↵8 Current affiliation: 3409 Park Place, Springfield, New Jersey

  • ↵9 Current affiliation: Bayer HealthCare LLC, San Francisco, California

  • ↵10 Current affiliation: Kanalis Consulting, L.L.C., Edison, New Jersey

  • C.H. and X.Z. contributed equally to this work.

  • dx.doi.org/10.1124/jpet.116.235150.

  • ↵Embedded ImageThis article has supplemental material available at jpet.aspetjournals.org.

Abbreviations

CCD
cortical collecting duct
CHO
Chinese hamster ovary
eRPF
effective renal plasma flow
FBS
fetal bovine serum
GFR
glomerular filtration rate
G418
geneticin
HCTZ
hydrochlorothiazide
HEK293
human embryonic kidney 293 cell line
hERG
human ether-a-go-go related gene
Kir
inwardly rectifying potassium channel
LC-MS/MS
liquid chromatography-tandem mass spectrometry
MDCK
Madin-Darby canine kidney epithelial
MK-499
[(+)-N-[1′-(6-cyano-1,2,3,4-tetrahydro-2(R)-naphthalenyl)-3,4-dihydro-4(R)-hydroxyspiro(2H-1-benzopyran-2,4′-piperidin)-6-yl)methanesulfonamide
MK-7145
[5,5′-((1R,1′R)-piperazine-1,4-diylbis(1-hydroxyethane-2,1-diyl))bis(4-methylisobenzofuran-1(3H)-one)]
QTc
corrected QT
ROMK
renal outer medullary potassium channel
SBP
systolic blood pressure
SHR
spontaneously hypertensive rat
TALH
thick ascending loop of Henle

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Research ArticleCardiovascular

ROMK Inhibitors and Bartter’s Syndrome Type II Phenotype

Caryn Hampton, Xiaoyan Zhou, Birgit T. Priest, Lee-Yuh Pai, John P. Felix, Brande Thomas-Fowlkes, Jessica Liu, Martin Kohler, Jianying Xiao, Aaron Corona, Olga Price, Charles Gill, Kashmira Shah, Cordelia Rasa, Vince Tong, Karen Owens, James Ormes, Haifeng Tang, Sophie Roy, Kathleen A. Sullivan, Joseph M. Metzger, Magdalena Alonso-Galicia, Gregory J. Kaczorowski, Alexander Pasternak and Maria L. Garcia
Journal of Pharmacology and Experimental Therapeutics October 1, 2016, 359 (1) 194-206; DOI: https://doi.org/10.1124/jpet.116.235150
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