IL-23 regulation of myeloid cell biology during inflammation.

Interleukin (IL)-23 is implicated in the pathogenesis of several inflammatory diseases and is usually linked with helper T cell (Th17) biology. However, there is some data linking IL-23 with innate immune biology in such diseases. We therefore examined the effects of IL-23p19 genetic deletion and/or neutralization on in vitro macrophage activation and in an innate immune-driven peritonitis model. We report that endogenous IL-23 was required for maximal macrophage activation by zymosan as determined by pro-inflammatory cytokine production, including a dramatic upregulation of granulocyte-colony stimulating factor (G-CSF). Furthermore, both IL-23p19 genetic deletion and neutralization in zymosan-induced peritonitis (ZIP) led to a specific reduction in the neutrophil numbers, as well as a reduction in the G-CSF levels in exudate fluids. We conclude that endogenous IL-23 can contribute significantly to macrophage activation during an inflammatory response, mostly likely via an autocrine/paracrine mechanism; of note, endogenous IL-23 can directly up-regulate macrophage G-CSF expression, which in turn is likely to contribute to the regulation of IL-23-dependent neutrophil number and function during an inflammatory response, with potential significance for IL-23 targeting particularly in neutrophil-associated inflammatory diseases.

The CSF-1-receptor inhibitor, JNJ-40346527 (PRV-6527), reduced inflammatory macrophage recruitment to the intestinal mucosa and suppressed murine T cell mediated colitis.

BACKGROUND & AIMS: Originally believed to be primarily a disorder of T-cell signaling, evidence shows that macrophage-lineage cells also contribute to the pathogenesis of Crohn's disease (CD). Colony stimulating factor-1 (CSF-1) is a key regulator of the macrophage lineage, but its role in CD has not been well established. We examined transcriptional data from CD mucosa for evidence of CSF-1 pathway activation and tested JNJ-40346527 (PRV-6527), a small molecule inhibitor of CSF-1 receptor kinase (CSF-1R), for its ability to inhibit disease indices in murine colitis. METHODS: A CSF-1 pathway gene set was created from microarray data of human whole blood cultured ex vivo with CSF-1 and compared to a TNFα-induced gene set generated from epithelial-lineage cells. Gene set variation analysis was performed using existing Crohn's mucosa microarray data comparing patients who either responded or failed to respond to anti-TNFα therapy. Commencing day 14 or day 21, mice with T-cell transfer colitis were treated with vehicle or JNJ-40346527 until study termination (day 42). Endpoints included colon weight/length ratios and histopathology scores, and macrophage and T cells were assessed by immunohistochemistry. Mucosal gene expression was investigated using RNAseq. RESULTS: Both the CSF-1 and the TNFα gene sets were enriched in the colonic mucosal transcriptomes of Crohn's disease and in mouse colitis, and expression of both gene sets was highest in patients who did not respond to anti-TNFα therapy. In these patients neither set was reduced by therapy. In the mouse model, JNJ-40346527 inhibited the increase in colon weight/length ratio by ∼50%, reduced histological disease scores by ∼60%, and reduced F4/80+ mononuclear cell and CD3+ lymphocyte numbers. RNAseq analysis confirmed the CSF-1 gene set was sharply reduced in treated mice, as were gene sets enriched in "M1" inflammatory and "M0" resident macrophages and in activated T cells. CONCLUSIONS: CSF-1 biology is activated in Crohn's disease and in murine T cell transfer colitis. Inhibition of CSF-1R by JNJ-40346527 was associated with attenuated clinical disease scores and reduced inflammatory gene expression in mice. These data provide rationale for testing JNJ-40346527 (PRV-6527) in human inflammatory bowel disease.

Enhancement of kinase selectivity in a potent class of arylamide FMS inhibitors.

Structure-activity relationship (SAR) studies on a highly potent series of arylamide FMS inhibitors were carried out with the aim of improving FMS kinase selectivity, particularly over KIT. Potent compound 17r (FMS IC50 0.7 nM, FMS cell IC50 6.1 nM) was discovered that had good PK properties and a greater than fivefold improvement in selectivity for FMS over KIT kinase in a cellular assay relative to the previously reported clinical candidate 4. This improved selectivity was manifested in vivo by no observed decrease in circulating reticulocytes, a measure of bone safety, at the highest studied dose. Compound 17r was highly active in a mouse pharmacodynamic model and demonstrated disease-modifying effects in a dose-dependent manner in a strep cell wall-induced arthritis model of rheumatoid arthritis in rats.

Optimization of a potent class of arylamide colony-stimulating factor-1 receptor inhibitors leading to anti-inflammatory clinical candidate 4-cyano-N-[2-(1-cyclohexen-1-yl)-4-[1-[(dimethylamino)acetyl]-4-piperidinyl]phenyl]-1H-imidazole-2-carboxamide (JNJ-28312141).

A class of potent inhibitors of colony-stimulating factor-1 receptor (CSF-1R or FMS), as exemplified by 8 and 21, was optimized to improve pharmacokinetic and pharmacodynamic properties and potential toxicological liabilities. Early stage absorption, distribution, metabolism, and excretion assays were employed to ensure the incorporation of druglike properties resulting in the selection of several compounds with good activity in a pharmacodynamic screening assay in mice. Further investigation, utilizing the type II collagen-induced arthritis model in mice, culminated in the selection of anti-inflammatory development candidate JNJ-28312141 (23, FMS IC(50) = 0.69 nM, cell assay IC(50) = 2.6 nM). Compound 23 also demonstrated efficacy in rat adjuvant and streptococcal cell wall-induced models of arthritis and has entered phase I clinical trials.

Matrix metalloproteinase-9 inhibition reduces inflammation and improves motility in murine models of postoperative ileus.

BACKGROUND & AIMS: Matrix metalloproteinase (MMP)-9, a member of the gelatinase family of MMPs, mediates leukocyte migration during inflammation. Inflammation contributes to development of postoperative ileus (POI), which is caused by physical disturbances to the bowel during abdominal surgery. We evaluated the role of MMP-9 in POI and investigated whether disruption of MMP-9 or administration of an inhibitor of MMP-9 activity reduced cellular inflammation and bowel dysmotility in rat and mouse models of POI. METHODS: Mice and rats underwent laparotomy and bowel manipulation; bowel tissues were collected 3 to 24 hours later and analyzed by real-time reverse-transcriptase polymerase chain reaction, immunoblot, in situ zymography, and functional analyses. RESULTS: Bowel manipulation resulted in a time-dependent increase in MMP-9 expression within the intestinal muscularis; increases in MMP-9 messenger RNA were inducible nitric oxide synthase dependent. Immunoblot analyses confirmed the presence of the proenzyme and the catalytically active form of MMP-9. Administration of MMP-2/MMP-9 II, a dual active-site inhibitor, reduced the number of myeloperoxidase-positive immune cells that infiltrated the muscularis and prevented the surgically induced reduction in bowel smooth muscle contractility. Zymography analysis, performed in muscularis whole mounts in situ, indicated that MMP-9 and not MMP-2 mediated the gelatinase activity observed in infiltrating cells. MMP-9 knockout mice were protected from the inflammation and dysmotility associated with POI. CONCLUSIONS: MMP-9 mediates cellular inflammatory responses within the intestinal muscularis in mouse and rat models of POI. Inhibition of MMP-9 activity reduced recruitment of immune cells to the intestinal muscularis, preventing loss of smooth muscle contractility. Induction of MMP-9 expression requires inducible nitric oxide synthase.

c-fms blockade reverses glomerular macrophage infiltration and halts development of crescentic anti-GBM glomerulonephritis in the rat.

Depletion and adoptive transfer studies have demonstrated that macrophages induce glomerular lesions in experimental anti-glomerular basement membrane (anti-GBM) glomerulonephritis. However, there is no current therapeutic strategy that can rapidly and selectively remove these cells from the glomerulus in order to halt disease development. This study examined whether inhibition of the receptor for macrophage colony-stimulating factor (known as c-fms), which is selectively expressed by monocyte/macrophages, can eliminate the macrophage infiltrate in a rat model of crescentic anti-GBM glomerulonephritis. Wistar-Kyoto rats were treated with 10 or 30 mg/kg bid of fms-I (a selective c-fms kinase inhibitor) from the time of anti-GBM serum injection until being killed 1, 5 or 14 days later. fms-I treatment had only a minor effect upon the glomerular macrophage infiltrate on day 1 and did not prevent the subsequent induction of proteinuria. However, fms-I treatment reduced the glomerular macrophage infiltrate by 60% at day 5 and completely reversed the macrophage infiltrate by day 14. In addition, fms-I treatment downregulated the glomerular expression of pro-inflammatory molecules (TNF-α, NOS2, MMP-12, CCL2 and IL-12) on days 1 and 5, suggesting a suppression of the macrophage M1-type response. Despite a significant early loss of glomerular podocytes, ongoing proteinuria and glomerular tuft adhesions to Bowman's capsule, the reversal of the macrophage infiltrate prevented the development of glomerulosclerosis, crescent formation, tubulointerstitial damage and renal dysfunction. In conclusion, this study has identified c-fms kinase inhibition as a selective approach to target infiltrating macrophages in acute glomerular injury, which may have therapeutic potential in rapidly progressive crescentic glomerulonephritis.

Reducing ion channel activity in a series of 4-heterocyclic arylamide FMS inhibitors.

During efforts to improve the bioavailability of FMS kinase inhibitors 1 and 2, a series of saturated and aromatic 4-heterocycles of reduced basicity were prepared and evaluated in an attempt to also improve the cardiovascular safety profile over lead arylamide 1, which possessed ion channel activity. The resultant compounds retained excellent potency and exhibited diminished ion channel activity.

JNJ-28312141, a novel orally active colony-stimulating factor-1 receptor/FMS-related receptor tyrosine kinase-3 receptor tyrosine kinase inhibitor with potential utility in solid tumors, bone metastases, and acute myeloid leukemia.

There is increasing evidence that tumor-associated macrophages promote the malignancy of some cancers. Colony-stimulating factor-1 (CSF-1) is expressed by many tumors and is a growth factor for macrophages and mediates osteoclast differentiation. Herein, we report the efficacy of a novel orally active CSF-1 receptor (CSF-1R) kinase inhibitor, JNJ-28312141, in proof of concept studies of solid tumor growth and tumor-induced bone erosion. H460 lung adenocarcinoma cells did not express CSF-1R and were not growth inhibited by JNJ-28312141 in vitro. Nevertheless, daily p.o. administration of JNJ-28312141 caused dose-dependent suppression of H460 tumor growth in nude mice that correlated with marked reductions in F4/80(+) tumor-associated macrophages and with increased plasma CSF-1, a possible biomarker of CSF-1R inhibition. Furthermore, the tumor microvasculature was reduced in JNJ-28312141-treated mice, consistent with a role for macrophages in tumor angiogenesis. In separate studies, JNJ-28312141 was compared with zoledronate in a model in which MRMT-1 mammary carcinoma cells inoculated into the tibias of rats led to severe cortical and trabecular bone lesions. Both agents reduced tumor growth and preserved bone. However, JNJ-28312141 reduced the number of tumor-associated osteoclasts superior to zoledronate. JNJ-28312141 exhibited additional activity against FMS-related receptor tyrosine kinase-3 (FLT3). To more fully define the therapeutic potential of this new agent, JNJ-28312141 was evaluated in a FLT3-dependent acute myeloid leukemia tumor xenograft model and caused tumor regression. In summary, this novel CSF-1R/FLT3 inhibitor represents a new agent with potential therapeutic activity in acute myeloid leukemia and in settings where CSF-1-dependent macrophages and osteoclasts contribute to tumor growth and skeletal events.

Pyrido[2,3-d]pyrimidin-5-ones: a novel class of antiinflammatory macrophage colony-stimulating factor-1 receptor inhibitors.

A series of pyrido[2,3-d]pyrimidin-5-ones has been synthesized and evaluated as inhibitors of the kinase domain of macrophage colony-stimulating factor-1 receptor (FMS). FMS inhibitors may be useful in treating rheumatoid arthritis and other chronic inflammatory diseases. Structure-based optimization of the lead amide analogue 10 led to hydroxamate analogue 37, which possessed excellent potency and an improved pharmacokinetic profile. During the chronic phase of streptococcal cell wall-induced arthritis in rats, compound 37 (10, 3, and 1 mg/kg) was highly effective at reversing established joint swelling. In an adjuvant-induced arthritis model in rats, 37 prevented joint swelling partially at 10 mg/kg. In this model, osteoclastogenesis and bone erosion were prevented by low doses (1 or 0.33 mg/kg) that had minimal impact on inflammation. These data underscore the potential of FMS inhibitors to prevent erosions and reduce symptoms in rheumatoid arthritis.

Targeting renal macrophage accumulation via c-fms kinase reduces tubular apoptosis but fails to modify progressive fibrosis in the obstructed rat kidney.

The role of macrophages in promoting interstitial fibrosis in the obstructed kidney is controversial. Macrophage depletion studies in the unilateral ureter obstruction (UUO) model have produced opposing results, presumably reflecting the subtleties of the individual depletion methods used. To address this question, we targeted the macrophage colony-stimulating factor receptor, c-fms, which is uniquely expressed by cells of the monocyte/macrophage lineage. Administration of 5, 12.5, or 30 mg/kg (bid) of a selective inhibitor of c-fms kinase activity (fms-I) resulted in a dose-dependent inhibition of renal macrophage accumulation in the rat UUO model. This was due to inhibition of local macrophage proliferation in the obstructed kidney and, at higher doses, to depletion of circulating blood monocytes. To determine the contribution of macrophages to renal pathology in the obstructed kidney, groups of animals were treated with 30 mg/kg fms-I and killed 3, 7, or 14 days later. Complete inhibition of renal macrophage accumulation prevented upregulation of the macrophage-associated proinflammatory mediators, tumor necrosis factor (TNF)-alpha and matrix metalloproteinase-12, and significantly reduced tubular apoptosis. Macrophage depletion caused a minor reduction of interstitial myofibroblast accumulation and deposition of interstitial collagen IV at day 3, but no difference was seen in renal fibrosis on day 7 or 14. Similarly, the upregulation of collagen IV, fibronectin, transforming growth factor-beta1 and connective tissue growth factor mRNA levels on day 7 and 14 in the obstructed kidney was unaffected by macrophage depletion. In conclusion, c-fms blockade was shown to selectively prevent interstitial macrophage accumulation and to reduce tubular apoptosis in the obstructed kidney, but it had no significant impact on the development of interstitial fibrosis.

Structure-based optimization of a potent class of arylamide FMS inhibitors.

An anti-inflammatory 1,2,4-phenylenetriamine-containing series of FMS inhibitors with a potential to form reactive metabolites was transformed into a series with equivalent potency by incorporation of carbon-based replacement groups. Structure-based modeling provided the framework to efficiently effect this transformation and restore potencies to previous levels. This optimization removed a risk factor for potential idiosyncratic drug reactions.

Design and synthesis of a pyrido[2,3-d]pyrimidin-5-one class of anti-inflammatory FMS inhibitors.

A series of pyrimidinopyridones has been designed, synthesized and shown to be potent and selective inhibitors of the FMS tyrosine kinase. Introduction of an amide substituent at the 6-position of the pyridone core resulted in a significant potency increase. Compound 24 effectively inhibited in vivo LPS-induced TNF in mice greater than 80%.

Carboxylic acid bioisosteres acylsulfonamides, acylsulfamides, and sulfonylureas as novel antagonists of the CXCR2 receptor.

A series of novel acylsulfonamide, acylsulfamide, and sulfonylurea bioisosteres of carboxylic acids were prepared as CXCR2 antagonists. Structure-activity relationships are reported for these series. One potent orally bioavailable inhibitor had excellent PK properties and was active in a lung injury model in hyperoxia-exposed newborn rats.

Discovery of novel FMS kinase inhibitors as anti-inflammatory agents.

The optimization of the arylamide lead 2 resulted in identification of a highly potent series of 2,4-disubstituted arylamides. Compound 8 (FMS kinase IC(50)=0.0008 microM) served as a proof-of-concept candidate in a collagen-induced model of arthritis in mice.

Synthesis and evaluation of novel 3,4,6-substituted 2-quinolones as FMS kinase inhibitors.

A series of 3,4,6-substituted 2-quinolones has been synthesized and evaluated as inhibitors of the kinase domain of macrophage colony-stimulating factor-1 receptor (FMS). The fully optimized compound, 4-(4-ethyl-phenyl)-3-(2-methyl-3H-imidazol-4-yl)-2-quinolone-6-carbonitrile 21b, has an IC(50) of 2.5 nM in an in vitro assay and 5.0 nM in a bone marrow-derived macrophage cellular assay. Inhibition of FMS signaling in vivo was also demonstrated in a mouse pharmacodynamic model.

Identification of novel short chain 4-substituted indoles as potent alphavbeta3 antagonist using structure-based drug design.

The vitronectin receptor alpha(v)beta(3) has been identified as a promising potential target for the treatment of osteoporosis, diabetic retinopathy and cancer. We have recently reported 5-substituted indoles 3-[5-[2-(5,6,7,8-tetrahydro[1,8]naphthyridin-2-yl)ethoxy]indol-1-yl]-3-(3-pyridyl)propionic acid 3 and 3-[5-[2-(5,6,7,8-tetrahydro[1,8]naphthyridin-2-yl)ethoxy]indol-1-yl]-3-(3,4-methylenedioxyphenyl)propionic acid 4, as an original series of potent alpha(v)beta(3) antagonists with subnanomolar activity. Ligand-protein docking analyses have been performed to generate binding models of three different chemical classes of known alpha(v)beta(3) antagonists with alpha(v)beta(3). Results of this docking study suggested that indoles bearing the basic tetrahydronaphthyridine group at position 4 can easily adopt the correct binding conformation and should be as potent as our current 5-substituted indole leads 3 and 4. This hypothesis was nicely demonstrated by the synthesis of a series of 1,4-disubstituted indoles through a tandem of reactions involving: (i) the N-alkylation of indoles 15 and 22 with propargyl esters and cesium fluoride, and (ii) a Heck coupling reaction between 4-bromoindole and 7-vinyl-3,4-dihydro-2H-[1,8]naphthyridine-1-carboxylic acid tert-butyl ester 12, or (iii) a reductive amination involving the N-substituted-4-aminoindole 23 and the BOC-protected tetrahydro[1,8]naphthyridine aldehyde 13. Among the compounds assayed, 3-(3-pyridyl)-3-[4-[2-(5,6,7,8-tetrahydro[1,8]naphthyridin-2-yl)ethyl]indol-1-yl]propionic acid 21 showed the most promising activity on alpha(v)beta(3) (IC(50)=0.5 nM), and was found to have the same potency as our current leads 3 and 4, while maintaining selectivity over alpha(IIb)beta(IIIa). Moreover, based on the reasonable apparent permeability coefficient in an in vitro CACO-2 cell monolayer assay (P(app) apical/basolateral=2.2 x 10(-6)cm/s, P(app) basolateral/apical=2.5 x 10(-6)cm/s), compound 21 is expected to be absorbed through the intestine in human. Thus, 1,4-disubstituted indole 21 represents a new lead for this novel class of conformationally restricted alpha(v)beta(3) antagonists. Additionally, this study validates the pharmacophore model previously postulated and provides an improved basis for further structure-based drug design in the field of alpha(v)beta(3).

Novel potent and selective alphavbeta3/alphavbeta5 integrin dual antagonists with reduced binding affinity for human serum albumin.

The binding of lead compounds and drugs to human serum albumin (HSA) is a ubiquitous problem in drug discovery since it modulates the availability of the leads and drugs to their intended target, which is linked to biological efficacy. In our continuing efforts to identify small molecule alpha(V)beta(3) and alpha(V)beta(5) dual antagonists, we recently reported indoles 2-4 as potent and selective alpha(V)beta(3)/alpha(V)beta(5) antagonists with good oral bioavailability profile. In spite of subnanomolar binding affinity of these compounds to human alpha(V)beta(3) and alpha(V)beta(5) integrins, high HSA binding (96.5-97.3%) emerged as a limiting feature for these leads. Structure-activity HSA binding data of organic acids reported in the literature have demonstrated that the incorporation of polar groups into a given molecule can dramatically decrease the affinity toward HSA. We sought to apply this strategy by examining the effects of such modifications in both the central core constrain and the substituent beta to the carboxylate. Most of these derivatives were prepared in good yields through a cesium fluoride-catalyzed coupling reaction. This reaction was successful with a variety of nitrogen-containing scaffolds (20, 33, and 43) and selected acetylenic derivatives (16, 19, and 34). Among the compounds synthesized, the 3-[5-[2-(5,6,7,8-tetrahydro [1,8]naphthyridin-2-yl)ethoxy]indol-1-yl]-3-[5-(N,N-dimethylaminomethyl)-3-pyridyl]propionic acid (25) was found to be the most promising derivative within this novel series with a subnanomolar affinity for both alpha(v)beta(3) and alpha(v)beta(5) (IC(50) = 0.29 and 0.16 nM, respectively), similar to our initial lead receptor antagonists 2-4, and exhibiting a low HSA protein binding (40% bound, K(d) = 1.1+/-0.4 x 10(3) microM) and an improved in vitro stability profile toward human and mouse microsomes (99.9% and 98.7% remaining after 10 min). Moreover, the selectivity of 25 toward alpha(5)beta(1) and IIbIIIa integrins was perfectly maintained when compared to the parent leads 2-4. Thus, compound 25 was selected as a new lead with improved drug-like properties for further evaluations in the field of oncology and osteoporosis.

A functional radioreceptor assay of alpha-V-beta-3 (alphavbeta3) inhibitors in plasma: application as an ex vivo pharmacodynamic model.

Development of alphavbeta3-integrin inhibitors has been hampered by a lack of pharmacodynamic endpoints to identify doses that inhibit alphavbeta3 in vivo. To address this need, we developed an alphavbeta3 radioreceptor assay (RRA) that could be performed in 100% plasma. The RRA was based on 125I-echistatin binding to plate-immobilized alphavbeta3. Small molecule alphavbeta3 inhibitors efficiently competed echistatin binding to alphavbeta3 when the assay was carried out in buffer. However, when carried out in 100% plasma, the RRA revealed a 45 to >3000-fold loss in compound potencies. The losses in potency reflected, in part, the high plasma protein binding by the compounds examined. The RRA was adapted as an ex vivo pharmacodynamic model. Echistatin binding was measured in the presence of plasma harvested at timed intervals from rats dosed with select compounds. Using this pharmacodynamic model, compound and dose selection was optimized for further testing in models of corneal angiogenesis. Moderate anti-angiogenic activity was achieved when rats were dosed sufficient to achieve sustained (>50%) plasma inhibition through the trough interval. Thus, the RRA provided a simple technique to rank order compound potency in plasma, and could find general use as an ex vivo pharmacodynamic assay to select compounds and doses for preclinical and clinical proof-of-principle studies.

Discovery and cocrystal structure of benzodiazepinedione HDM2 antagonists that activate p53 in cells.

HDM2 binds to an alpha-helical transactivation domain of p53, inhibiting its tumor suppressive functions. A miniaturized thermal denaturation assay was used to screen chemical libraries, resulting in the discovery of a novel series of benzodiazepinedione antagonists of the HDM2-p53 interaction. The X-ray crystal structure of improved antagonists bound to HDM2 reveals their alpha-helix mimetic properties. These optimized molecules increase the transcription of p53 target genes and decrease proliferation of tumor cells expressing wild-type p53.