The design and synthesis of novel spirocyclic heterocyclic sulfone ROMK inhibitors as diuretics.

A spirocyclic class of ROMK inhibitors was developed containing a structurally diverse heterocyclic sulfone moiety and spirocyclic core starting from lead 1. These compounds not only displayed exquisite ROMK potency but significantly improved selectivity over hERG. The lead compounds were found to have favorable pharmacokinetic properties and displayed robust diuretic, natriuretic and blood pressure lowering effects in spontaneously hypertensive rats.

Improvement of hERG-ROMK index of spirocyclic ROMK inhibitors through scaffold optimization and incorporation of novel pharmacophores.

SAR in the previously described spirocyclic ROMK inhibitor series was further evolved from lead 4 by modification of the spirocyclic core and identification of novel right-side pharmacophores. In this process, it was discovered that the spiropyrrolidinone core with the carbonyl group α to the spirocenter was preferred for potent ROMK activity. Efforts aimed at decreasing hERG affinity within the series led to the discovery of multiple novel right-hand pharmacophores including 3-methoxythiadiazole, 2-methoxypyrimidine, and pyridazinone. The most promising candidate is pyridazinone analog 32 that showed an improved functional hERG/ROMK potency ratio and preclinical PK profile. In vivo evaluation of 32 demonstrated blood pressure lowering effects in the spontaneously hypertensive rat model.

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

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.

Differentiation of ROMK potency from hERG potency in the phenacetyl piperazine series through heterocycle incorporation.

Following the discovery of small molecule acyl piperazine ROMK inhibitors and their initial preclinical validation as a novel diuretic agent, our group set out to discover new ROMK inhibitors with reduced risk for QT effects, suitable for further pharmacological experiments in additional species. Several strategies for decreasing hERG affinity while maintaining ROMK inhibition were investigated and are described herein. The most promising candidate, derived from the newly discovered 4-N-heteroaryl acetyl series, improved functional hERG/ROMK ratio by >10× over the previous lead. In vivo evaluation demonstrated comparable diuretic effects in rat with no detectable QT effects at the doses evaluated in an in vivo dog model.

Discovery of a potent and selective ROMK inhibitor with improved pharmacokinetic properties based on an octahydropyrazino[2,1-c][1,4]oxazine scaffold.

Following the discovery of small molecule acyl piperazine ROMK inhibitors, the acyl octahydropyrazino[2,1-c][1,4]oxazine series was identified. This series displays improved ROMK/hERG selectivity, and as a consequence, the resulting ROMK inhibitors do not evoke QTc prolongation in an in vivo cardiovascular dog model. Further efforts in this series led to the discovery of analogs with improved pharmacokinetic profiles. This new series also retained comparable ROMK potency compared to earlier leads.

Evaluation of cynomolgus monkeys for the identification of endogenous biomarkers for hepatic transporter inhibition and as a translatable model to predict pharmacokinetic interactions with statins in humans.

Inhibition of hepatic transporters such as organic anion transporting polypeptides (OATPs) 1B can cause drug-drug interactions (DDIs). Determining the impact of perpetrator drugs on the plasma exposure of endogenous substrates for OATP1B could be valuable to assess the risk for DDIs early in drug development. As OATP1B orthologs are well conserved between human and monkey, we assessed in cynomolgus monkeys the endogenous OATP1B substrates that are potentially suitable to assess DDI risk in humans. The effect of rifampin (RIF), a potent inhibitor for OATP1B, on plasma exposure of endogenous substrates of hepatic transporters was measured. From the 18 biomarkers tested, RIF (18 mg/kg, oral) caused significant elevation of plasma unconjugated and conjugated bilirubin, which may be attributed to inhibition of cOATP1B1 and cOATP1B3 based on in vitro to in vivo extrapolation analysis. To further evaluate whether cynomolgus monkeys are a suitable translational model to study OATP1B-mediated DDIs, we determined the inhibitory effect of RIF on in vitro transport and pharmacokinetics of rosuvastatin (RSV) and atorvastatin (ATV). RIF strongly inhibited the uptake of RSV and ATV by cOATP1B1 and cOATP1B3 in vitro. In agreement with clinical observations, RIF (18 mg/kg, oral) significantly decreased plasma clearance and increased the area under the plasma concentration curve (AUC) of intravenously administered RSV by 2.8- and 2.7-fold, and increased the AUC and maximum plasma concentration of orally administered RSV by 6- and 10.3-fold, respectively. In contrast to clinical findings, RIF did not significantly increase plasma exposure of either intravenous or orally administered ATV, indicating species differences in the rate-limiting elimination pathways.

Pharmacologic inhibition of the renal outer medullary potassium channel causes diuresis and natriuresis in the absence of kaliuresis.

The renal outer medullary potassium (ROMK) channel, which is located at the apical membrane of epithelial cells lining the thick ascending loop of Henle and cortical collecting duct, plays an important role in kidney physiology by regulating salt reabsorption. Loss-of-function mutations in the human ROMK channel are associated with antenatal type II Bartter's syndrome, an autosomal recessive life-threatening salt-wasting disorder with mild hypokalemia. Similar observations have been reported from studies with ROMK knockout mice and rats. It is noteworthy that heterozygous carriers of Kir1.1 mutations associated with antenatal Bartter's syndrome have reduced blood pressure and a decreased risk of developing hypertension by age 60. Although selective ROMK inhibitors would be expected to represent a new class of diuretics, this hypothesis has not been pharmacologically tested. Compound A [5-(2-(4-(2-(4-(1H-tetrazol-1-yl)phenyl)acetyl)piperazin-1-yl)ethyl)isobenzofuran-1(3H)-one)], a potent ROMK inhibitor with appropriate selectivity and characteristics for in vivo testing, has been identified. Compound A accesses the channel through the cytoplasmic side and binds to residues lining the pore within the transmembrane region below the selectivity filter. In normotensive rats and dogs, short-term oral administration of compound A caused concentration-dependent diuresis and natriuresis that were comparable to hydrochlorothiazide. Unlike hydrochlorothiazide, however, compound A did not cause any significant urinary potassium losses or changes in plasma electrolyte levels. These data indicate that pharmacologic inhibition of ROMK has the potential for affording diuretic/natriuretic efficacy similar to that of clinically used diuretics but without the dose-limiting hypokalemia associated with the use of loop and thiazide-like diuretics.

Discovery of a novel sub-class of ROMK channel inhibitors typified by 5-(2-(4-(2-(4-(1H-Tetrazol-1-yl)phenyl)acetyl)piperazin-1-yl)ethyl)isobenzofuran-1(3H)-one.

A sub-class of distinct small molecule ROMK inhibitors were developed from the original lead 1. Medicinal chemistry endeavors led to novel ROMK inhibitors with good ROMK functional potency and improved hERG selectivity. Two of the described ROMK inhibitors were characterized for the first in vivo proof-of-concept biology studies, and results from an acute rat diuresis model confirmed the hypothesis that ROMK inhibitors represent new mechanism diuretic and natriuretic agents.

The role of the therapeutic alliance in the regulation of emotion in psychosis: an attachment perspective.

OBJECTIVES: This article aims to explore the value of attachment theory as a framework for understanding the ways in which the staff-patient relationship is associated with different methods of regulating emotion in individuals with a diagnosis of psychosis. METHOD: Patient participants with a diagnosis of schizophrenia, schizoaffective disorder or psychosis not otherwise specified and mental health workers were recruited from 24-hour rehabilitation teams. Patient participants completed questionnaires assessing emotion regulation, attachment, therapeutic alliance and symptomatology. The therapeutic alliance was also assessed from mental workers' perspective. RESULTS: Insecure attachment was significantly associated with greater difficulties in regulating emotions. A strong therapeutic alliance was associated with fewer difficulties in regulating emotions. CONCLUSIONS: Attachment is a useful theoretical construct for understanding psychosis, with evidence for a link between a positive staff-patient relationship and enhanced emotion regulation. Clinical implications of these findings are discussed. KEY PRACTITIONER MESSAGE: The study supports a link between attachment organisation and psychosis, characterised by difficulties in emotion regulation. A positive patient-key worker relationship may facilitate the development of emotion regulation in this client group. A key implication of these findings is the training of MDT staff in the implementation of attachment-informed interventions.

Characterization of multidrug resistance 1a/P-glycoprotein knockout rats generated by zinc finger nucleases.

The development of zinc finger nuclease (ZFN) technology has enabled the genetic engineering of the rat genome. The ability to manipulate the rat genome has great promise to augment the utility of rats for biological and pharmacological studies. A Wistar Hannover rat model lacking the multidrug resistance protein Mdr1a P-glycoprotein (P-gp) was generated using a rat Mdr1a-specific ZFN. Mdr1a was completely absent in tissues, including brain and small intestine, of the knockout rat. Pharmacokinetic studies with the Mdr1a P-gp substrates loperamide, indinavir, and talinolol indicated that Mdr1a was functionally inactive in the blood-brain barrier and intestine in Mdr1a(-/-) rats. To identify possible compensatory mechanisms in Mdr1a(-/-) rats, the expression levels of drug-metabolizing enzyme and transporter-related genes were compared in brain, liver, kidney, and intestine of male and female Mdr1a(-/-) and control rats. In general, alterations in gene expression of these genes in Mdr1a(-/-) rats seemed to be modest, with more changes in female than in male rats. Taken together, our studies demonstrate that the ZFN-generated Mdr1a(-/-) rat will be a valuable tool for central nervous system drug target validation and determining the role of P-gp in drug absorption and disposition.

Design and synthesis of a new class of malonyl-CoA decarboxylase inhibitors with anti-obesity and anti-diabetic activities.

A new series of thiazole-substituted 1,1,1,3,3,3-hexafluoro-2-propanols were prepared and evaluated as malonyl-CoA decarboxylase (MCD) inhibitors. Key analogs caused dose-dependent decreases in food intake and body weight in obese mice. Acute treatment with these compounds also led to a drop in elevated blood glucose in a murine model of type II diabetes.

Medical Countermeasures for Children in Radiation and Nuclear Disasters: Current Capabilities and Key Gaps.

OBJECTIVE: Despite children's unique vulnerability, clinical guidance and resources are lacking around the use of radiation medical countermeasures (MCMs) available commercially and in the Strategic National Stockpile to support immediate dispensing to pediatric populations. To better understand the current capabilities and shortfalls, a literature review and gap analysis were performed. METHODS: A comprehensive review of the medical literature, Food and Drug Administration (FDA)-approved labeling, FDA summary reviews, medical references, and educational resources related to pediatric radiation MCMs was performed from May 2016 to February 2017. RESULTS: Fifteen gaps related to the use of radiation MCMs in children were identified. The need to address these gaps was prioritized based upon the potential to decrease morbidity and mortality, improve clinical management, strengthen caregiver education, and increase the relevant evidence base. CONCLUSIONS: Key gaps exist in information to support the safe and successful use of MCMs in children during radiation emergencies; failure to address these gaps could have negative consequences for families and communities. There is a clear need for pediatric-specific guidance to ensure clinicians can appropriately identify, triage, and treat children who have been exposed to radiation, and for resources to ensure accurate communication about the safety and utility of radiation MCMs for children. (Disaster Med Public Health Preparedness. 2019;13:639-646).

Extending the Reach of Pediatric Emergency Preparedness: A Virtual Tabletop Exercise Targeting Children's Needs.

OBJECTIVES: Virtual tabletop exercises (VTTXs) simulate disaster scenarios to help participants improve their emergency-planning capacity. The objectives of our study were to (1) evaluate the effectiveness of a VTTX in improving preparedness capabilities specific to children's needs among pediatricians and public health practitioners, (2) document follow-up actions, and (3) identify exercise strengths and weaknesses. METHODS: In February 2017, we conducted and evaluated a VTTX facilitated via videoconferencing among 26 pediatricians and public health practitioners from 4 states. Using a mixed-methods design, we assessed participants' knowledge and confidence to fulfill targeted federal preparedness capabilities immediately before and after the exercise. We also evaluated the degree to which participants made progress on actions through surveys 1 month (n = 14) and 6 months (n = 14) after the exercise. RESULTS: Participants reported a greater ability to identify their state's pediatric emergency preparedness strengths and weaknesses after the exercise (16 of 18) compared with before the exercise (10 of 18). We also observed increases in (1) knowledge of and confidence in performing most pediatric emergency preparedness capabilities and (2) most dimensions of interprofessional collaboration. From 1 month to 6 months after the exercise, participants (n = 14) self-reported making progress in increasing awareness for potential preparedness partners and in conducting similar pediatric exercises (from 4-7 for both). CONCLUSIONS: Participants viewed the VTTX positively and indicated increased pediatric emergency preparedness knowledge and confidence. Addressing barriers to improving local pediatric emergency preparedness-particularly long term-is an important target for future tabletop exercises.

Discovery of N-{(1S,2S)-2-(3-cyanophenyl)- 3-[4-(2-[18F]fluoroethoxy)phenyl]-1-methylpropyl}- 2-methyl-2-[(5-methylpyridin-2-yl)oxy]propanamide, a cannabinoid-1 receptor positron emission tomography tracer suitable for clinical use.

The discovery of a structurally distinct cannabinoid-1 receptor (CB1R) positron emission tomography tracer is described. Starting from an acyclic amide CB1R inverse agonist (1) as the lead compound, an efficient route to introduce 18F to the molecule was developed. Further optimization focused on reducing the lipophilicity and increasing the CB1R affinity. These efforts led to the identification of [18F]-16 that exhibited good brain uptake and an excellent signal-to-noise ratio in rhesus monkeys.

Differential anti-thrombotic benefit and bleeding risk profiles of antagonists of protease-activated receptor 1 and 4 in Cynomolgus Macaques.

Platelet activation plays a crucial role in hemostasis and thrombosis. Thrombin, the most potent stimulus of platelet activation, mediates platelet activation via the protease activated receptors (PARs). The platelet PAR repertoire in mediating thrombin's action differs across species. Only nonhuman primate (NHP) platelet activation is known to be similar to humans, mediated by PAR1 and PAR4, hence limiting translational in vivo studies of PAR's role in thrombosis and hemostasis to NHPs. Earlier studies have demonstrated a range of distinct in vitro activities of PAR1 and 4 in platelet activation yet the implications of these events in vivo is unclear. The objective of this study is to investigate and compare the roles of PAR1 and PAR4 in hemostasis and thrombosis in a relevant animal species. NHP models for pharmacokinetic, ex vivo platelet aggregation responses, FeCI3 injury-mediated arterial thrombosis and template bleeding were developed in Cynomolgus Macaques. Potent and selective small molecule antagonists of PAR1 and PAR4 were characterized in an array of in vitro assays, and subsequently examined head-to-head in the NHP models. Treatment of NHPs with antagonists of PAR1 or PAR4 both resulted in strong inhibition of ex vivo platelet aggregation. At doses that led to similar inhibition of platelet aggregation, animals treated with the PAR4 antagonist showed similar levels of anti-thrombotic efficacy, but longer bleeding times, compared to animals treated with the PAR1 antagonist. These findings suggest that PAR1 antagonism will likely produce a larger therapeutic index (ie. a larger anti-thrombotic efficacy over bleeding risk margin) than PAR4 antagonism.

Discovery of MK-7145, an Oral Small Molecule ROMK Inhibitor for the Treatment of Hypertension and Heart Failure.

ROMK, the renal outer medullary potassium channel, is involved in potassium recycling at the thick ascending loop of Henle and potassium secretion at the cortical collecting duct in the kidney nephron. Because of this dual site of action, selective inhibitors of ROMK are expected to represent a new class of diuretics/natriuretics with superior efficacy and reduced urinary loss of potassium compared to standard-of-care loop and thiazide diuretics. Following our earlier work, this communication will detail subsequent medicinal chemistry endeavors to further improve lead selectivity against the hERG channel and preclinical pharmacokinetic properties. Pharmacological assessment of highlighted inhibitors will be described, including pharmacodynamic studies in both an acute rat diuresis/natriuresis model and a subchronic blood pressure model in spontaneous hypertensive rats. These proof-of-biology studies established for the first time that the human and rodent genetics accurately predict the in vivo pharmacology of ROMK inhibitors and supported identification of the first small molecule ROMK inhibitor clinical candidate, MK-7145.

Discovery of a Potent and Selective ROMK Inhibitor with Pharmacokinetic Properties Suitable for Preclinical Evaluation.

A new subseries of ROMK inhibitors exemplified by 28 has been developed from the initial screening hit 1. The excellent selectivity for ROMK inhibition over related ion channels and pharmacokinetic properties across preclinical species support further preclinical evaluation of 28 as a new mechanism diuretic. Robust pharmacodynamic effects in both SD rats and dogs have been demonstrated.

Discovery of Selective Small Molecule ROMK Inhibitors as Potential New Mechanism Diuretics.

The renal outer medullary potassium channel (ROMK or Kir1.1) is a putative drug target for a novel class of diuretics that could be used for the treatment of hypertension and edematous states such as heart failure. An internal high-throughput screening campaign identified 1,4-bis(4-nitrophenethyl)piperazine (5) as a potent ROMK inhibitor. It is worth noting that this compound was identified as a minor impurity in a screening hit that was responsible for all of the initially observed ROMK activity. Structure-activity studies resulted in analogues with improved rat pharmacokinetic properties and selectivity over the hERG channel, providing tool compounds that can be used for in vivo pharmacological assessment. The featured ROMK inhibitors were also selective against other members of the inward rectifier family of potassium channels.

Chemical genetics define the roles of p38alpha and p38beta in acute and chronic inflammation.

The p38 MAP kinase signal transduction pathway is an important regulator of proinflammatory cytokine production and inflammation. Defining the roles of the various p38 family members, specifically p38alpha and p38beta, in these processes has been difficult. Here we use a chemical genetics approach using knock-in mice in which either p38alpha or p38beta kinase has been rendered resistant to the effects of specific inhibitors along with p38beta knock-out mice to dissect the biological function of these specific kinase isoforms. Mice harboring a T106M mutation in p38alpha are resistant to pharmacological inhibition of LPS-induced TNF production and collagen antibody-induced arthritis, indicating that p38beta activity is not required for acute or chronic inflammatory responses. LPS-induced TNF production, however, is still completely sensitive to p38 inhibitors in mice with a T106M point mutation in p38beta. Similarly, p38beta knock-out mice respond normally to inflammatory stimuli. These results demonstrate conclusively that specific inhibition of the p38alpha isoform is necessary and sufficient for anti-inflammatory efficacy in vivo.

Enhanced external counterpulsation treatment improves arterial wall properties and wave reflection characteristics in patients with refractory angina.

OBJECTIVES: To determine if arterial properties and wave reflection characteristics are favorably altered after enhanced external counterpulsation (EECP) treatment in patients with refractory angina. BACKGROUND: Early return of reflected waves from the lower body, resulting from increased arterial stiffness, augments central aortic pressure and increases left ventricular (LV) afterload and myocardial oxygen demand. EECP acutely enhances coronary perfusion (supply) and reduces LV afterload (demand). However, the mechanisms responsible for the sustained beneficial effects of EECP treatment are unclear. METHODS: Radial artery pressure waveforms were recorded by applanation tonometry and central aortic pressure waveforms generated using a mathematical transfer function in 20 patients with stable refractory angina. Data were collected before and after 34 1-h EECP sessions. Augmentation index (AI(a)) and timing of the reflected pressure wave were calculated from the aortic waveform. RESULTS: EECP treatment caused a decline in AI(a) and an increase in reflected wave travel time. These modifications in wave reflection characteristics caused a decrease in aortic systolic pressure and wasted LV pressure energy. The average number of angina episodes and Canadian Cardiovascular Society (CCS) class, both decreased in concordance with the physiologic changes due to EECP treatment. CONCLUSIONS: EECP treatment reduces arterial stiffness and improves wave reflection characteristics in patients with refractory angina. These changes decrease LV afterload and myocardial oxygen demand and reduce the number of angina episodes, therefore enabling patients to participate in continuous exercise programs which in turn may provide long-term benefits and sustained improved quality of life.