Discovery of NXT-10796, an orally active, intestinally restricted EP4 agonist prodrug for the treatment of inflammatory bowel disease.

A property-focused optimization strategy was employed to modify the carboxylic acid head group of a class of EP4 agonists in order to minimize its absorption upon oral administration. The resulting oxalic acid monohydrazide-derived carboxylate isostere demonstrated utility as a class of prodrug showing colon-targeted delivery of parent agonist 2, with minimal exposure observed in the plasma. Oral administration of NXT-10796 demonstrated tissue specific activation of the EP4 receptor through modulation of immune genes in the colon, without modulation of EP4 driven biomarkers in the plasma compartment. Although further in depth understanding of the conversion of NXT-10796 is required for further assessment of the developability of this series of prodrugs, using NXT-10796 as a tool molecule has allowed us to confirm that tissue-specific modulation of an EP4-modulated gene signature is possible, which allows for further evaluation of this therapeutic modality in rodent models of human disease.

A fluorogenic near-infrared imaging agent for quantifying plasma and local tissue renin activity in vivo and ex vivo.

The renin-angiotensin system (RAS) is well studied for its regulation of blood pressure and fluid homeostasis, as well as for increased activity associated with a variety of diseases and conditions, including cardiovascular disease, diabetes, and kidney disease. The enzyme renin cleaves angiotensinogen to form angiotensin I (ANG I), which is further cleaved by angiotensin-converting enzyme to produce ANG II. Although ANG II is the main effector molecule of the RAS, renin is the rate-limiting enzyme, thus playing a pivotal role in regulating RAS activity in hypertension and organ injury processes. Our objective was to develop a near-infrared fluorescent (NIRF) renin-imaging agent for noninvasive in vivo detection of renin activity as a measure of tissue RAS and in vitro plasma renin activity. We synthesized a renin-activatable agent, ReninSense 680 FAST (ReninSense), using a NIRF-quenched substrate derived from angiotensinogen that is cleaved specifically by purified mouse and rat renin enzymes to generate a fluorescent signal. This agent was assessed in vitro, in vivo, and ex vivo to detect and quantify increases in plasma and kidney renin activity in sodium-sensitive inbred C57BL/6 mice maintained on a low dietary sodium and diuretic regimen. Noninvasive in vivo fluorescence molecular tomographic imaging of the ReninSense signal in the kidney detected increased renin activity in the kidneys of hyperreninemic C57BL/6 mice. The agent also effectively detected renin activity in ex vivo kidneys, kidney tissue sections, and plasma samples. This approach could provide a new tool for assessing disorders linked to altered tissue and plasma renin activity and to monitor the efficacy of therapeutic treatments.

Design and synthesis of potent, isoxazole-containing renin inhibitors.

The design and optimization of a novel isoxazole S(1) linker for renin inhibitor is described herein. This effort culminated in the identification of compound 18, an orally bioavailable, sub-nanomolar renin inhibitor even in the presence of human plasma. When compound 18 was found to inhibit CYP3A4 in a time dependent manner, two strategies were pursued that successfully delivered equipotent compounds with minimal TDI potential.

Renin inhibitors for the treatment of hypertension: design and optimization of a novel series of tertiary alcohol-bearing piperidines.

The design and optimization of a novel series of renin inhibitor is described herein. Strategically, by committing the necessary resources to the development of synthetic sequences and scaffolds that were most amenable for late stage structural diversification, even as the focus of the SAR campaign moved from one end of the molecule to another, highly potent renin inhibitors could be rapidly identified and profiled.

Renin inhibitors for the treatment of hypertension: design and optimization of a novel series of pyridone-substituted piperidines.

An SAR campaign aimed at decreasing the overall lipophilicity of renin inhibitors such as 1 is described herein. It was found that replacement of the northern appendage in 1 with an N-methyl pyridone and subsequent re-optimization of the benzyl amide handle afforded compounds with in vitro and in vivo profiles suitable for further profiling. An unexpected CV toxicity in dogs observed with compound 20 led to the employment of a time and resource sparing rodent model for in vivo screening of key compounds. This culminated in the identification of compound 31 as an optimized renin inhibitor.

Characterization of a stable, hypertensive rat model suitable for the consecutive evaluation of human renin inhibitors.

INTRODUCTION: The hypertensive double-transgenic (dTG) rat strain, expressing human renin and angiotensinogen, develops severe hypertension and organ damage and 50% of individuals die by 7 weeks of age. Here, we characterise a variation of this model in which animals present stable hypertension. MATERIALS AND METHODS: The effect of renin-angiotensin system blockers on blood pressure was determined with adult dTG rats treated with enalapril from 3 to 12 weeks of age. Tissue expression levels of renin and angiotensinogen were determined in dTG rats and rhesus monkeys by quantitative PCR. RESULTS: Upon withdrawal from enalapril, mean arterial pressure (MAP) rose to 160-180 mmHg, with 95% of the female dTG rats surviving for 6 to 12 months, In Sprague-Dawley (SD) rats and rhesus monkeys, renin mRNA was absent or weakly expressed in most tissues, except for the kidneys and adrenals. In dTG rats, human renin expression was high in many additional tissues. The expression of human angiotensinogen in dTG rats followed a similar tissue pattern to SD and rhesus monkey angiotensinogen. Oral dosing of aliskiren, enalapril or losartan provided a similar maximal reduction in MAP and duration of efficacy in telemetrised dTG rats. CONCLUSIONS: Enalapril-pretreated dTG rats are suitable for long-term MAP monitoring and sequential evaluation of human renin inhibitors.

The discovery and synthesis of potent zwitterionic inhibitors of renin.

The incorporation of a carboxylic acid within in a series of 3-amido-4-aryl substituted piperidines (represented by general structure 32) led to the discovery of potent, zwitterionic, renin inhibitors with improved off-target profiles (CYP3A4 time-dependent inhibition and hERG affinity) relative to analogous non-zwitterionic inhibitors of the past (i.e., 3). Strategies to address the oral absorption of these zwitterions are also discussed within.

Therapeutic dosing of an orally active, selective cathepsin S inhibitor suppresses disease in models of autoimmunity.

The purpose of the study was to examine the potential of inhibition of cathepsin S as a treatment for autoimmune diseases. A highly selective cathepsin S inhibitor, CSI-75, was shown to upregulate levels of the cathepsin S substrate, invariant chain Lip10, in vitro as well as in vivo in C57Bl/6 mice after oral administration. Functional activity of the compound was shown by a reduction in the OVA-specific response of OVA-sensitized splenocytes from C57Bl/6 mice as well as from OVA-TCR transgenic mice (DO11.10). Since these studies revealed a selective suppression of the Th1 and Th17 cytokines causing a shift to Th2, CSI-75 was tested in the murine HC-gp39-immunization model. Indeed, CSI-75 specifically reduced the circulating HC-gp39-specific IgG2a in these mice indicating selective inhibition of the Th1 type of response in vivo. The importance of especially the Th1 and Th17 cell subsets in the pathology of autoimmune diseases, renders CatS inhibition a highly interesting potential therapeutic treatment of autoimmune diseases. Therefore, CSI-75 was tested in a murine model of multiple sclerosis (i.e. experimental autoimmune encephalomyelitis (EAE)) in a semi-therapeutic setting (ie. oral treatment after initial sensitization to antigen). Finally, in a murine model with features resembling rheumatoid arthritis (the collagen-induced arthritis (CIA) model), CSI-75 was tested in a therapeutic manner (after disease development). CSI-75 caused a significant reduction in disease score in both disease models, indicating a promising role for CatS inhibitors in the area of therapeutic treatments for autoimmune diseases.

Lack of cathepsin activities alter or prevent the development of lung granulomas in a mouse model of sarcoidosis.

BACKGROUND: Remodeling of lung tissues during the process of granuloma formation requires significant restructuring of the extra-cellular matrix and cathepsins K, L and S are among the strongest extra-cellular matrix degrading enzymes. Cathepsin K is highly expressed in various pathological granulomatous infiltrates and all three enzymes in their active form are detected in bronchoalveolar lavage fluids from patients with sarcoidosis. Granulomatous inflammation is driven by T-cell response and cathepsins S and L are actively involved in the regulation of antigen presentation and T-cell selection. Here, we show that the disruption of the activities of cathepsins K, L, or S affects the development of lung granulomas in a mouse model of sarcoidosis. METHODS: Apolipoprotein E-deficient mice lacking cathepsin K or L were fed Paigen diet for 16 weeks and lungs were analyzed and compared with their cathepsin-expressing littermates. The role of cathepsin S in the development of granulomas was evaluated using mice treated for 8 weeks with a potent and selective cathepsin S inhibitor. RESULTS: When compared to wild-type litters, more cathepsin K-deficient mice had lung granulomas, but individually affected mice developed smaller granulomas that were present in lower numbers. The absence of cathepsin K increased the number of multinucleated giant cells and the collagen content in granulomas. Cathepsin L deficiency resulted in decreased size and number of lung granulomas. Apoe-/- mice treated with a selective cathepsin S inhibitor did not develop lung granulomas and only individual epithelioid cells were observed. CONCLUSIONS: Cathepsin K deficiency affected mostly the occurrence and composition of lung granulomas, whereas cathepsin L deficiency significantly reduced their number and cathepsin S inhibition prevented the formation of granulomas.

Difluoroethylamines as an amide isostere in inhibitors of cathepsin K.

The trifluoroethylamine group found in cathepsin K inhibitors like odanacatib can be replaced by a difluoroethylamine group. This change increased the basicity of the nitrogen which positively impacted the log D. This translated into an improved oral bioavailability in pre-clinical species. Difluoroethylamine compounds exhibit a similar potency against cathepsin K and selectivity profile against other cathepsins when compared to trifluoroethylamine analogs.

Genetic and pharmacological evaluation of cathepsin s in a mouse model of asthma.

Cathepsin S (Cat S) is predominantly expressed in antigen-presenting cells and is up-regulated in several preclinical models of antigen-induced inflammation, suggesting a role in the allergic response. Prophylactic dosing of an irreversible Cat S inhibitor has been shown to attenuate pulmonary eosinophilia in mice, supporting the hypothesis that Cat S inhibition before the initiation of airway inflammation is beneficial in airway disease. In addition, Cat S has been shown to play a role in more distal events in the allergic response. To determine where Cat S inhibition may affect the allergic response, we used complementary genetic and pharmacological approaches to investigate the role of Cat S in the early and downstream allergic events in a murine model of antigen-induced lung inflammation. Cat S knockout mice did not develop ovalbumin-induced pulmonary inflammation, consistent with a role for Cat S in the development of the allergic response. Alternatively, wild-type mice were treated with a reversible, highly selective Cat S inhibitor in prophylactic and therapeutic dosing paradigms and assessed for changes in airway inflammation. Although both treatment paradigms resulted in potent Cat S inhibition, only prophylactic Cat S inhibitor dosing blocked lung inflammation, consistent with our findings in Cat S knockout mice. The findings indicate that although Cat S is up-regulated in allergic models, it does not appear to play a significant role in the downstream effector inflammatory phase in this model; however, our results demonstrate that Cat S inhibition in a prophylactic paradigm would ameliorate airway inflammation.

Probing cathepsin S activity in whole blood by the activity-based probe BIL-DMK: cellular distribution in human leukocyte populations and evidence of diurnal modulation.

Using the cell-permeable, radioiodinated, irreversible inhibitor BIL-DMK, we probed active cysteine cathepsins in blood. Incubation of the probe in human whole blood followed by separation of white blood cells by dextran sedimentation led to the labeling of one major band at 24kDa. Two-dimensional gel electrophoresis showed that the band resolved in a single protein spot and corresponded to cathepsin S based on its molecular mass, isoelectric point, and Western blot analysis using anti-human cathepsin S antibodies. Cathepsin S activity in human whole blood was dependent on the time of blood collection, suggesting that cathepsin S activity is subject to circadian variations. Separation of white blood cell populations using a magnetic cell sorter and further characterization by FACS (fluorescent-activated cell sorting) analysis demonstrated that the majority of active cathepsin S resided in the monocyte and neutrophil populations, whereas on a cell basis cathepsin S activity in granulocytes is 10-fold lower than that in monocytes. A whole blood cathepsin S assay was developed and used to measure cathepsin S inhibition in both in vitro and ex vivo conditions. To determine the correlation between the in vitro and ex vivo assays, a reversible cathepsin S inhibitor was dosed intravenously to a rhesus monkey. The inhibitor concentration required to inhibit 50% of the cathepsin S activity ex vivo correlated well with the concentration required to inhibit the enzyme in rhesus monkey whole blood in vitro. The results reported here demonstrate the utility of the activity-based probe BIL-DMK for the ex vivo assessment of cathepsin S inhibition.

Pharmacological and genetic evidence that cathepsin B is not the physiological activator of rodent prorenin.

Renin is the first enzyme in the renin-angiotensin-aldosterone system which is the principal regulator of blood pressure and hydroelectrolyte balance. Previous studies suggest that cathepsin B is the activator of the prorenin zymogen. Here, we show no difference in plasma renin activity, or mean arterial blood pressure between wild-type and cathepsin B knockout mice. To account for potential gene compensation, a potent, selective, reversible cathepsin B inhibitor was developed to determine the role of cathepsin B on prorenin processing in rats. Pharmacological inhibition of cathepsin B in spontaneously hypertensive and double transgenic rats did not result in a reduction in renal mature renin protein levels or plasma renin activity. We conclude that cathepsin B does not play a significant role in this process in rodents.

Pharmacological inhibition of cathepsin S decreases atherosclerotic lesions in Apoe-/- mice.

Recent studies provided evidence for a significant role of cathepsin S during extracellular remodeling in atherosclerosis. In this study, we investigated the effect of a specific cathepsin S inhibitor on atherosclerotic plaque progression in the brachiocephalic artery. Male and female Apoe-/- mice on a cholate-containing high-fat diet containing or lacking a specific cathepsin S inhibitor were evaluated for the remodeling of atherosclerotic lesions. The in vivo efficacy of the cathepsin S inhibitor was demonstrated by the inhibition of invariant chain processing in spleen. After 8 weeks of diet, brachiocephalic arteries were analyzed for plaque size, collagen, macrophage, and smooth muscle cell content, for elastic lamina breaks, and the number of buried fibrous caps. The size of atherosclerotic plaques in inhibitor-treated mice was reduced by 36% in male and 68% in female mice, and they showed significantly smaller numbers in elastin lamina breaks (60% less in males; 75% less in females), plaque macrophages (47% less in males; 40% less in females), and buried fibrous caps (50% less in males; 86% less in females). In conclusion, the inhibition of cathepsin S showed a strong atheroprotective activity, demonstrating the potential benefits of a small molecule anti-cathepsin therapy.

Therapeutic utility and medicinal chemistry of cathepsin C inhibitors.

The lysosomal cysteine protease cathepsin C (Cat C), also known as dipeptidyl peptidase I, activates a number of granule-associated serine proteases with pro-inflammatory and immune functions by removal of their inhibitory N-terminal dipeptides. Thus, Cat C is a therapeutic target for the treatment of a number of inflammatory and autoimmune diseases. Cathepsin C null mice and humans with Cat C loss of function mutations (Papillon-Lefèvre syndrome) show deficiencies in disease-relevant proteases including neutrophil elastase, cathepsin G, chymases and granzymes and the Cat C mice are protected in a number of disease models. Several methodologies have been recently reported for assessing the effects of Cat C inhibitors on serine protease activities in cellular assays and prolonged treatment of rats with a reversible, selective Cat C inhibitor reduced the activity of three leukocyte serine proteases. Nearly all potent and selective Cat C inhibitors described are based on the preferred dipeptide substrates bearing either irreversible (e.g. diazomethylketone, acyloxymethyl ketone, o-acyl hydroxamic acid and vinyl sulfone) or reversible (e.g. semicarbazide, nitrile and cyanamide) electrophilic warheads. While potent and highly selective, the best inhibitors described to date still have poor stability and/or rodent pharmacokinetics, likely resulting from their peptidic nature. The lack of selective compounds with appropriate rodent pharmacokinetic properties has hampered the assessment of the effects of Cat C inhibitors on the activation of disease-relevant proteases in vivo and the full evaluation of the therapeutic utility of Cat C inhibitors.

The discovery of MK-0674, an orally bioavailable cathepsin K inhibitor.

MK-0674 is a potent and selective cathepsin K inhibitor from the same structural class as odanacatib with a comparable inhibitory potency profile against Cat K. It is orally bioavailable and exhibits long half-life in pre-clinical species. In vivo studies using deuterated MK-0674 show stereoselective epimerization of the alcohol stereocenter via an oxidation/reduction cycle. From in vitro incubations, two metabolites could be identified: the hydroxyleucine and the glucuronide conjugate which were confirmed using authentic synthetic standards.

Pharmacological inhibitors to identify roles of cathepsin K in cell-based studies: a comparison of available tools.

Cathepsin K (Cat K) degrades bone type I collagen and is a target for the pharmacological treatment of osteoporosis. Further roles for Cat K have been recently described, some of which are supported by the use of purportedly selective Cat K inhibitors in human and rodent cell-based assays. Twelve commercial and non-commercial Cat K inhibitors were profiled against a panel of purified human, rat, and mouse cysteine cathepsins and in two cell-based enzyme occupancy assays for activity against Cat K, B, and L. Ten inhibitors, including the carbohydrazide Cat K inhibitor II (Boc-Phe-Leu-NHNH-CO-NHNH-Leu-Z), the non-covalent K4b, and the epoxide NC-2300, have either little Cat K selectivity, or appear poorly cell penetrant. The amino-acetonitrile-containing inhibitors L-873724 and odanacatib show greater than 100-fold human Cat K enzyme selectivity and have similar IC(50) values against each cathepsin in cell-based and enzyme assays. The basic inhibitor balicatib has greater cellular potencies than expected on the basis of purified enzyme assays. The accumulation of [(14)C]-balicatib in fibroblasts is blocked by prior treatment of the cells with NH(4)Cl, consistent with balicatib having lysosomotropic properties. These results support the use of L-873724 and odanacatib as tools to identify novel roles for Cat K using human cell-based systems, but suggest using caution in the interpretation of studies employing the other compounds.

Renin and prorenin activate pathways implicated in organ damage in human mesangial cells independent of angiotensin II production.

BACKGROUND: The mechanism by which an activated renin-angiotensin system (RAS) leads to the development of renal diseases, such as fibrosis, is only partially explained by the downstream effects of angiotensin II. The discovery of a receptor that binds renin and prorenin, and the consequent production of profibrotic molecules, revealed a novel axis within the RAS pathway that may contribute to the pathogenesis of organ damage in patients with elevated renin and/or prorenin levels. METHODS: To better understand the genes and networks underlying the receptor-mediated effects of renin and prorenin, a gene expression profiling study was performed on human mesangial cells in the presence of angiotensin-II-blocking agents. RESULTS: Renin and prorenin induce highly overlapping gene expression signatures that are dependent, only in part, on the presence of the (pro)renin receptor. We found that 2 distinct pathways were activated by renin and prorenin: a TGFbeta-dependent pathway and a TGFbeta-independent pathway. Bioinformatic analysis was used to show that both pathways are highly enriched with genes implicated in fibrosis, hypertrophy and atherosclerosis. CONCLUSIONS: This study suggests that both renin and inactive prorenin are capable of inducing genetic programs that could contribute to end-organ damage and atherogenesis, through receptor-mediated angiotensin-independent mechanisms.

Development of a homogeneous immunoassay for the detection of angiotensin I in plasma using AlphaLISA acceptor beads technology.

Plasma renin activity (PRA) is a well-established biomarker for assessing the efficacy of various antihypertensive agents such as direct renin inhibitors, angiotensin receptor blockers, and angiotensin-converting enzyme inhibitors (ACEIs). PRA measurements are obtained through the detection and quantification of angiotensin I (Ang I) produced by the action of renin on its natural substrate angiotensinogen. The most accepted and reproducible method for PRA measurement uses an antibody capture Ang I methodology that employs specific antibodies that recognize and protect Ang I against angiotensinase activities contained in plasma. The amount of Ang I is then quantified by either radioimmunoassay (RIA) or enzyme immunoassay (EIA). In the current report, we describe the optimization of a novel homogeneous immunoassay based on the AlphaScreen technology for the detection and quantification of antibody-captured Ang I using AlphaLISA acceptor beads in buffer and in the plasma of various species (human, rat, and mouse). Ex vivo measurements of renin activity were performed using 10 microl or less of a reaction mixture, and concentrations as low as 1 nM Ang I were quantified. Titration curves obtained for the quantification of Ang I in buffer and plasma gave similar EC(50) values of 5.6 and 14.4 nM, respectively. Both matrices generated an equivalent dynamic range that varies from approximately 1 to 50 nM. Renin inhibitors have been successfully titrated and IC(50) values obtained correlated well with those obtained using EIA methodology (r(2)=0.80). This assay is sensitive, robust, fast, and less tedious than measurements performed using nonhomogeneous EIA. The AlphaLISA methodology is homogeneous, does not require wash steps prior to the addition of reagents, and does not generate radioactive waste.

Identification of a nonbasic, nitrile-containing cathepsin K inhibitor (MK-1256) that is efficacious in a monkey model of osteoporosis.

Herein, we report on the identification of nonbasic, potent, and highly selective, nitrile-containing cathepsin K (Cat K) inhibitors that are built on our previously identified cyclohexanecarboxamide core structure. Subsequent to our initial investigations, we have found that incorporation of five-membered heterocycles as P2-P3 linkers allowed for the introduction of a methyl sulfone P3-substitutent that was not tolerated in inhibitors containing a six-membered aromatic P2-P3 linker. The combination of a five-membered N-methylpyrazole linker and a methyl sulfone in P3 yielded subnanomolar Cat K inhibitors that were minimally shifted (<10-fold) in our functional bone resorption assay. Issues that arose because of metabolic demethylation of the N-methylpyrazole were addressed through introduction of a 2,2,2-trifluoroethyl substituent. This culminated in the identification of 31 (MK-1256), a potent (Cat K IC 50 = 0.62 nM) and selective (>1100-fold selectivity vs Cat B, L, S, C, H, Z, and V, 110-fold vs Cat F) inhibitor of cathepsin K that is efficacious in a monkey model of osteoporosis.