Target Cell Activation of a Structurally Novel NOD-Like Receptor Pyrin Domain-Containing Protein 3 Inhibitor NT-0796 Enhances Potency.

The NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome is a central regulator of innate immunity, essential for processing and release of interleukin-1ß and pyroptotic cell death. As endogenous NLRP3 activating triggers are hallmarks of many human chronic inflammatory diseases, inhibition of NLRP3 has emerged as a therapeutic target. Here we identify NDT-19795 as a novel carboxylic acid-containing NLRP3 activation inhibitor in both human and mouse monocytes and macrophages. Remarkably, conversion of the carboxylate to an isopropyl-ester (NT-0796) greatly enhances NLRP3 inhibitory potency in human monocytes. This increase is attributed to the ester-containing pharmacophore being more cell-penetrant than the acid species and, once internalized, the ester being metabolized to NDT-19795 by carboxylesterase-1 (CES-1). Mouse macrophages do not express CES-1, and NT-0796 is ineffective in these cells. Mice also contain plasma esterase (Ces1c) activity which is absent in humans. To create a more human-like model, we generated a mouse line in which the genome was modified, removing Ces1c and replacing this segment of DNA with the human CES-1 gene driven by a mononuclear phagocyte-specific promoter. We show human CES-1 presence in monocytes/macrophages increases the ability of NT-0796 to inhibit NLRP3 activation both in vitro and in vivo. As NLRP3 is widely expressed by monocytes/macrophages, the co-existence of CES-1 in these same cells affords a unique opportunity to direct ester-containing NLRP3 inhibitors precisely to target cells of interest. Profiling NT-0796 in mice humanized with respect to CES-1 biology enables critical modeling of the pharmacokinetics and pharmacodynamics of this novel therapeutic candidate. SIGNIFICANCE STATEMENT: Inhibition of NLRP3 represents a desirable therapeutic strategy for the treatment of multiple human disorders. In this study pharmacological properties of a structurally-novel, ester-containing NLRP3 inhibitor NT-0796 are characterized. To study pharmacodynamics of NT-0796 in vivo, a mouse line was engineered possessing more human-like traits with respect to carboxylesterase biology. In the context of these hCES-1 mice, NT-0796 serves as a more effective inhibitor of NLRP3 activation than the corresponding acid, highlighting the full translational potential of the ester strategy.

In vitro Growth Pattern of Primary Human Osteoblasts on Calcium Phosphate- and Polymethylmethacrylate-Based Bone Cement.

BACKGROUND/PURPOSE: Polymethylmethacrylate (PMMA) and calcium phosphate (Ca-P) cements are widely used for arthroplasty surgery and augmentation of bone defects. However, aseptic implant loosening in absence of wear-induced osteolysis indicates an unfavourable interaction between the cement surface and human osteoblasts. Our underlying hypothesis is that cement surfaces directly modify cell viability, proliferation rate, and cell differentiation. METHODS: To test this hypothesis, we examined primary human osteoblasts harvested from six individuals. These cells were pooled and subsequently seeded directly on cement pellets prepared from Palacos® R, Palacos® R+G, and Norian® Drillable cements. After incubation for 24 and 72 h, cell viability, proliferation rate, apoptosis rate, and cell differentiation were analysed. RESULTS: Upon cultivation of human osteoblasts on cement surfaces, we observed a significantly reduced cell viability and DNA content compared to the control. Analysis of the apoptosis rate revealed an increase for cells on Palacos R and Norian Drillable, but a significant decrease on Palacos R+G compared to the control. Regarding osteogenic differentiation, significantly lower values of alkaline phosphatase enzyme activity were identified for all cement surfaces after 24 and 72 h compared to cultivation on tissue culture plastic, serving as control. CONCLUSIONS: In summary, these data suggest a limited biocompatibility of both PMMA and Ca-P cements, necessitating further research to reduce unfavourable cell-cement interactions and consequently extend implant survival.

The ester-containing prodrug NT-0796 enhances delivery of the NLRP3 inflammasome inhibitor NDT-19795 to monocytic cells expressing carboxylesterase-1.

NT-0796 is an ester prodrug which is metabolized by carboxylesterase-1 (CES1) to yield the carboxylic acid NDT-19795, an inhibitor of the NLR family pyrin domain-containing protein 3 (NLRP3) inflammasome. When applied to human monocytes/macrophages which express CES1, however, NT-0796 is much more potent at inhibiting NLRP3 inflammasome activation than is NDT-19795. Comparison of the binding of NDT-19795 and NT-0796 in a cell-based NLRP3 target engagement assay confirms that NDT-19795 is the active species. Moreover, microsomes expressing CES1 efficiently convert NT-0796 to NDT-19795, confirming CES1-dependent activation. To understand the basis for the enhanced potency of the ester prodrug species in human monocytes, we analyzed the accumulation and de-esterification of NT-0796 in cultured cells. Our studies reveal NT-0796 rapidly accumulates in cells, achieving estimated cellular concentrations above those applied to the medium, with concomitant metabolism to NDT-19795 in CES1-expressing cells. Using cells lacking CES1 or a poorly hydrolysable NT-0796 analog demonstrated that de-esterification is not required for NT-0796 to achieve high cellular levels. As a result of a dynamic equilibrium whereby NDT-19795 formed intracellularly is subsequently released to the medium, concentrations of NT-0796 sufficient to inhibit NLRP3 can be completely metabolized to NDT-19795 resulting in a transient pharmacodynamic response. In contrast, when NDT-19795 is applied directly to cells, observed cell-associated levels are below those present in the medium and remain stable over time. Dynamics observed within the context of a closed tissue culture system highlight the utility of NT-0796 as a vehicle for delivering the NDT-19795 acid payload to CES1 expressing cells.

Discovery of Clinical Candidate NT-0796, a Brain-Penetrant and Highly Potent NLRP3 Inflammasome Inhibitor for Neuroinflammatory Disorders.

The NLRP3 inflammasome is a component of the innate immune system involved in the production of proinflammatory cytokines. Neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, multiple sclerosis, and amyotrophic lateral sclerosis, have been shown to have a component driven by NLRP3 inflammasome activation. Diseases such as these with large unmet medical needs have resulted in an interest in inhibiting the NLRP3 inflammasome as a potential pharmacological treatment, but to date, no marketed drugs specifically targeting NLRP3 have been approved. Furthermore, the requirement for CNS-penetrant molecules adds additional complexity to the search for NLRP3 inflammasome inhibitors suitable for clinical investigation of neuroinflammatory disorders. We designed a series of ester-substituted carbamate compounds as selective NLRP3 inflammasome inhibitors, leading to NT-0796, an isopropyl ester that undergoes intracellular conversion to NDT-19795, the carboxylic acid active species. NT-0796 was shown to be a potent and selective NLRP3 inflammasome inhibitor with demonstrated in vivo brain penetration.

Discovery and Optimization of Triazolopyrimidinone Derivatives as Selective NLRP3 Inflammasome Inhibitors.

The NLRP3 inflammasome is a multiprotein complex that facilitates activation and release of the proinflammatory cytokines interleukin-1ß (IL-1ß) and IL-18 in response to infection or endogenous stimuli. It can be inappropriately activated by a range of danger signals resulting in chronic, low-grade inflammation underlying a multitude of diseases, such as Alzheimer's disease, Parkinson's disease, osteoarthritis, and gout. The discovery of potent and specific NLRP3 inhibitors could reduce the burden of several common morbidities. In this study, we identified a weakly potent triazolopyrimidone hit (1) following an in silico modeling exercise. This was optimized to furnish potent and selective small molecule NLRP3 inflammasome inhibitors. Compounds such as NDT-30805 could be useful tool molecules for a scaffold-hopping or pharmacophore generation project or used as leads toward the development of clinical candidates.

Discovery of MAP855, an Efficacious and Selective MEK1/2 Inhibitor with an ATP-Competitive Mode of Action.

Mutations in MEK1/2 have been described as a resistance mechanism to BRAF/MEK inhibitor treatment. We report the discovery of a novel ATP-competitive MEK1/2 inhibitor with efficacy in wildtype (WT) and mutant MEK12 models. Starting from a HTS hit, we obtained selective, cellularly active compounds that showed equipotent inhibition of WT MEK1/2 and a panel of MEK1/2 mutant cell lines. Using a structure-based approach, the optimization addressed the liabilities by systematic analysis of molecular matched pairs (MMPs) and ligand conformation. Addition of only three heavy atoms to early tool compound 6 removed Cyp3A4 liabilities and increased the cellular potency by 100-fold, while reducing log P by 5 units. Profiling of MAP855, compound 30, in pharmacokinetic-pharmacodynamic and efficacy studies in BRAF-mutant models showed comparable efficacy to clinical MEK1/2 inhibitors. Compound 30 is a novel highly potent and selective MEK1/2 kinase inhibitor with equipotent inhibition of WT and mutant MEK1/2, whose drug-like properties allow further investigation in the mutant MEK setting upon BRAF/MEK therapy.

Indazole derivatives as novel bradykinin B1 receptor antagonists.

A new class of indazole-derived bradykinin B(1) antagonists and their structure-activity relationships (SAR) is reported. A number of compounds were found to have low-nanomolar affinity for the human B(1) receptor and possess acceptable P-gp and pharmacokinetics properties.

Discovery and expanded SAR of 4,4-disubstituted quinazolin-2-ones as potent T-type calcium channel antagonists.

The discovery and synthesis of 4,4-disubstituted quinazolinones as T-type calcium channel antagonists is reported. Based on lead compounds 2 and 3, a focused SAR campaign driven by the optimization of potency, metabolic stability, and pharmacokinetic profile identified 45 as a potent T-type Ca(2+) channel antagonist with minimized PXR activation. In vivo, 45 suppressed seizure frequency in a rat model of absence epilepsy and showed significant alterations of sleep architecture after oral dosing to rats as measured by EEG.

Amiloride derived inhibitors of acid-sensing ion channel-3 (ASIC3).

A series of amiloride derivatives modified at the 5-position of the pyrazine ring were evaluated as inhibitors of acid-sensing ion channel-3 (ASIC3), a novel target for the treatment of chronic pain.

The Discovery of LML134, a Histamine H3 Receptor Inverse Agonist for the Clinical Treatment of Excessive Sleep Disorders.

Histamine H3 receptor (H3R) inverse agonists that have been in clinical trials for the treatment of excessive sleep disorders, have been plagued with insomnia as a mechanism-based side effect. We focused on the identification of compounds that achieve high receptor occupancy within a short time, followed by rapid disengagement from the receptor, a target profile that could provide therapeutic benefits without the undesired side effect of insomnia. This article describes the optimization work that led to the discovery of 1-(1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)piperidin-4-yl 4-cyclobutylpiperazine-1-carboxylate (18 b, LML134).

CYP2C75-involved autoinduction of metabolism in rhesus monkeys of methyl 3-chloro-3'-fluoro-4'-{(1R)-1-[({1-[(trifluoroacetyl)amino]cyclopropyl}carbonyl)amino]ethyl}-1,1'-biphenyl-2-carboxylate (MK-0686), a bradykinin B1 receptor antagonist.

After oral treatment (once daily) for 4 weeks with the potent bradykinin B(1) receptor antagonist methyl 3-chloro-3'-fluoro-4'-{(1R)-1-[({1-[(trifluoroacetyl)amino]cyclopropyl}carbonyl)-amino]ethyl}-1,1'-biphenyl-2-carboxylate (MK-0686), rhesus monkeys (Macaca mulatta) exhibited significantly reduced systemic exposure of the compound in a dose-dependent manner, suggesting an occurrence of autoinduction of MK-0686 metabolism. This possibility is supported by two observations. 1) MK-0686 was primarily eliminated via biotransformation in rhesus monkeys, with oxidation on the chlorophenyl ring as one of the major metabolic pathways. This reaction led to appreciable formation of a dihydrodiol (M11) and a hydroxyl (M13) product in rhesus liver microsomes supplemented with NADPH. 2) The formation rate of these two metabolites determined in liver microsomes from MK-0686-treated groups was > or = 2-fold greater than the value for a control group. Studies with recombinant rhesus P450s and monoclonal antibodies against human P450 enzymes suggested that CYP2C75 played an important role in the formation of M11 and M13. The induction of this enzyme by MK-0686 was further confirmed by a concentration-dependent increase of its mRNA in rhesus hepatocytes, and, more convincingly, the enhanced CYP2C proteins and catalytic activities toward CYP2C75 probe substrates in liver microsomes from MK-0686-treated animals. Furthermore, a good correlation was observed between the rates of M11 and M13 formation and hydroxylase activities toward probe substrates determined in a panel of liver microsomal preparations from control and MK-0686-treated animals. Therefore, MK-0686, both a substrate and inducer for CYP2C75, caused autoinduction of its own metabolism in rhesus monkeys by increasing the expression of this enzyme.

A new class of bradykinin B1 receptor antagonists with high oral bioavailability and minimal PXR activity.

The design and synthesis of a novel class of human bradykinin B1 antagonists featuring difluoroethyl ether and isoxazole carboxamide moieties are disclosed. Compound 7g displayed excellent pharmacokinetic properties, efficient ex vivo receptor occupancy, and low potential for P450 induction via PXR activation.

Bradykinin B1 receptor antagonists: an alpha-hydroxy amide with an improved metabolism profile.

A series of carbo- and heterocyclic alpha-hydroxy amide-derived bradykinin B1 antagonists was prepared and evaluated. A 4,4-difluorocyclohexyl alpha-hydroxy amide was incorporated along with a 2-methyl tetrazole in lieu of an oxadiazole to afford a suitable compound with good pharmacokinetic properties, CNS penetration, and clearance by multiple metabolic pathways.

Alpha-hydroxy amides as a novel class of bradykinin B1 selective antagonists.

Antagonism of the bradykinin B(1) receptor represents a potential treatment for chronic pain and inflammation. Novel antagonists incorporating alpha-hydroxy amides were designed that display low-nanomolar affinity for the human bradykinin B(1) receptor and good bioavailability in the rat and dog. In addition, these functionally active compounds show high passive permeability and low susceptibility to phosphoglycoprotein mediated efflux, predictive of good CNS exposure.

Aerococcus urinae Mitral Valve Endocarditis-Related Stroke: A Case Report and Literature Review.

Background. Aerococcus urinae is a rare causative pathogen of infective endocarditis that results in a high risk of embolic events. The mortality rate for A urinae endocarditis is high. Old age and underlying urologic conditions are the best-known risk factors for infection. Case Description. We report the clinical course of the disease in a 49-year-old man who presented symptoms of a urinary tract infection. A few days later, transthoracic echocardiography showed a conspicuous mitral valve with myxomatous alterations. Following the detection of a cerebral embolism with associated stroke symptoms, as well as at the beginning of cardiac failure, the emergency indication for the surgical treatment of mitral valve endocarditis was given. On the second day following the operation, circulatory collapse rapidly developed. Following an unsuccessful attempt at cardiopulmonary resuscitation, the patient died. Review of the Literature. From 1991 to 2017, 29 cases of A urinae-induced endocarditis have been described in PubMed and Medline. One or 2 new cases are published annually. We review all reported cases of A urinae endocarditis, with an emphasis on the predisposing factors, course, and outcomes of the disease. Conclusion. A urinae endocarditis is a rare disease primarily affecting elderly men with urinary tract pathologies and comorbidities. The course of the disease is severe, and the outcome is often fatal. A 16S rDNA polymerase chain reaction investigation of bacterial genome provides proof of the presence of A urinae. Because of the high risk of embolism, rapid treatment should focus on the diseased heart valve. Based on existing data and the experience gained from handling cases, treatment with ß-lactam and aminoglycosides is recommended. It is also recommended that operative therapy take place as soon as possible.

Development of orally bioavailable and CNS penetrant biphenylaminocyclopropane carboxamide bradykinin B1 receptor antagonists.

A series of biphenylaminocyclopropane carboxamide based bradykinin B1 receptor antagonists has been developed that possesses good pharmacokinetic properties and is CNS penetrant. Discovery that the replacement of the trifluoropropionamide in the lead structure with polyhaloacetamides, particularly a trifluoroacetamide, significantly reduced P-glycoprotein mediated efflux for the series proved essential. One of these novel bradykinin B1 antagonists (13b) also exhibited suitable pharmacokinetic properties and efficient ex vivo receptor occupancy for further development as a novel approach for the treatment of pain and inflammation.

Cyclopropylamino acid amide as a pharmacophoric replacement for 2,3-diaminopyridine. Application to the design of novel bradykinin B1 receptor antagonists.

Antagonism of the bradykinin B1 receptor represents a potential treatment for chronic pain and inflammation. Novel antagonists were designed that display low-nanomolar affinity for the human bradykinin B1 receptor and good bioavailability in the rat.

Tetrabutylammonium salt induced denitration of nitropyridines: synthesis of fluoro-, hydroxy-, and methoxypyridines.

An efficient method for the synthesis of fluoropyridines via the fluorodenitration reaction is reported. The reaction is mediated by tetrabutylammonium fluoride (TBAF) under mild conditions without undue regard to the presence of water. The fluorodenitration is general for 2- or 4-nitro-substituted pyridines, while 3-nitropyridines require attendant electron-withdrawing groups for the reaction to proceed efficiently. Nitropyridines also undergo hydroxy- and methoxydenitration under mild conditions in the presence of the corresponding tetrabutylammonium species. [reaction: see text]

Inhibition of acute nociceptive responses in rat spinal cord by a bradykinin B1 receptor antagonist.

This study used behavioural and in vivo electrophysiological paradigms to examine the effects of systemic and spinal administration of a bradykinin B1 receptor antagonist, compound X, on acute nociceptive responses in the rat. In behavioural experiments, compound X significantly increased the latency to withdraw the hindpaw from a radiant heat source after both intravenous and intrathecal administration, without affecting motor performance on the rotarod. In electrophysiological experiments, both intravenous and direct spinal administration of compound X attenuated the responses of single dorsal horn neurones to noxious thermal stimulation of the hindpaw. These data show that the antinociceptive effects of a bradykinin B1 receptor antagonist are mediated, at least in part, at the level of the spinal cord and suggest a role for spinal bradykinin B1 receptors in acute nociception.

From ergolines to indoles: improved inhibitors of the human H3 receptor for the treatment of narcolepsy.

Ergolines were recently identified as a novel class of H3 receptor (H3R) inverse agonists. Although their optimization led to drug candidates with encouraging properties for the treatment of narcolepsy, brain penetration remained low. To overcome this issue, ergoline 1 ((6aR,9R,10aR)-4-(2-(dimethylamino)ethyl)-N-phenyl-9-(pyrrolidine-1-carbonyl)-6,6a,8,9,10,10a-hexahydroindolo[4,3-fg]quinoline-7(4H)-carboxamide)) was transformed into a series of indole derivatives with high H3R affinity. These new molecules were profiled by simultaneous determination of their brain receptor occupancy (RO) levels and pharmacodynamic (PD) effects in mice. These efforts culminated in the discovery of 15 m ((R)-1-isopropyl-5-(1-(2-(2-methylpyrrolidin-1-yl)ethyl)-1H-indol-4-yl)pyridin-2(1H)-one), which has an ideal profile showing a strong correlation of PD effects with RO, and no measurable safety liabilities. Its desirably short duration of action was confirmed by electroencephalography (EEG) measurements in rats.