Discovery of GLPG3667, a Selective ATP Competitive Tyrosine Kinase 2 Inhibitor for the Treatment of Autoimmune Diseases.

Tyrosine kinase 2 (TYK2) mediates cytokine signaling through type 1 interferon, interleukin (IL)-12/IL-23, and the IL-10 family. There appears to be an association between TYK2 genetic variants and inflammatory conditions, and clinical evidence suggests that selective inhibition of TYK2 could produce a unique therapeutic profile. Here, we describe the discovery of compound 9 (GLPG3667), a reversible and selective TYK2 adenosine triphosphate competitive inhibitor in development for the treatment of inflammatory and autoimmune diseases. The preclinical pharmacokinetic profile was favorable, and TYK2 selectivity was confirmed in peripheral blood mononuclear cells and whole blood assays. Dermal ear inflammation was reduced in an IL-23-induced in vivo mouse model of psoriasis. GLPG3667 also completed a phase 1b study (NCT04594928) in patients with moderate-to-severe psoriasis where clinical effect was shown within the 4 weeks of treatment and it is now in phase 2 trials for the treatment of dermatomyositis (NCT05695950) and systemic lupus erythematosus (NCT05856448).

IRAK4 inhibition dampens pathogenic processes driving inflammatory skin diseases.

Innate immunity not only shapes the way epithelial barriers interpret environmental cues but also drives adaptive responses. Therefore, modulators of innate immune responses are expected to have high therapeutic potential across immune-mediated inflammatory diseases. IRAK4 is a kinase that integrates signaling downstream of receptors acting at the interface between innate and adaptive immune responses, such as Toll-like receptors (TLRs), interleukin-1R (IL-1R), and IL-18R. Because effects of IRAK4 inhibition are stimulus, cell type, and species dependent, the evaluation of the therapeutic potential of IRAK4 inhibitors requires a highly translational approach. Here, we profiled a selective IRAK4 inhibitor, GLPG2534, in an extensive panel of models of inflammatory skin diseases, translationally expanding evidence from in vitro to in vivo and from mouse to human. In vitro, IRAK4 inhibition resulted in substantial inhibition of TLR and IL-1 responses in dendritic cells, keratinocytes, granulocytes, and T cells but only weakly affected dermal fibroblast responses. Furthermore, disease activity in murine models of skin inflammation (IL-23-, IL-33-, imiquimod-, and MC903-induced) was markedly dampened by IRAK4 inhibition. Last, inhibiting IRAK4 reversed pathogenic molecular signatures in human lesional psoriasis and atopic dermatitis biopsies. Over the variety of models used, IRAK4 inhibition consistently affected central mediators of psoriasis (IL-17A) and atopic dermatitis (IL-4 and IL-13). Overall, our data highlight IRAK4 as a central player in skin inflammatory processes and demonstrate the potential of IRAK4 inhibition as a therapeutic strategy in chronic inflammatory skin diseases.

Discovery and Optimization of Orally Bioavailable Phthalazone and Cinnolone Carboxylic Acid Derivatives as S1P2 Antagonists against Fibrotic Diseases.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease. Current treatments only slow down disease progression, making new therapeutic strategies compelling. Increasing evidence suggests that S1P2 antagonists could be effective agents against fibrotic diseases. Our compound collection was mined for molecules possessing substructure features associated with S1P2 activity. The weakly potent indole hit 6 evolved into a potent phthalazone series, bearing a carboxylic acid, with the aid of a homology model. Suboptimal pharmacokinetics of a benzimidazole subseries were improved by modifications targeting potential interactions with transporters, based on concepts deriving from the extended clearance classification system (ECCS). Scaffold hopping, as a part of a chemical enablement strategy, permitted the rapid exploration of the position adjacent to the carboxylic acid. Compound 38, with good pharmacokinetics and in vitro potency, was efficacious at 10 mg/kg BID in three different in vivo mouse models of fibrotic diseases in a therapeutic setting.

Discovery of the S1P2 Antagonist GLPG2938 (1-[2-Ethoxy-6-(trifluoromethyl)-4-pyridyl]-3-[[5-methyl-6-[1-methyl-3-(trifluoromethyl)pyrazol-4-yl]pyridazin-3-yl]methyl]urea), a Preclinical Candidate for the Treatment of Idiopathic Pulmonary Fibrosis.

Mounting evidence from the literature suggests that blocking S1P2 receptor (S1PR2) signaling could be effective for the treatment of idiopathic pulmonary fibrosis (IPF). However, only a few antagonists have been so far disclosed. A chemical enablement strategy led to the discovery of a pyridine series with good antagonist activity. A pyridazine series with improved lipophilic efficiency and with no CYP inhibition liability was identified by scaffold hopping. Further optimization led to the discovery of 40 (GLPG2938), a compound with exquisite potency on a phenotypic IL8 release assay, good pharmacokinetics, and good activity in a bleomycin-induced model of pulmonary fibrosis.

Discovery of 9-Cyclopropylethynyl-2-((S)-1-[1,4]dioxan-2-ylmethoxy)-6,7-dihydropyrimido[6,1-a]isoquinolin-4-one (GLPG1205), a Unique GPR84 Negative Allosteric Modulator Undergoing Evaluation in a Phase II Clinical Trial.

GPR84 is a medium chain free fatty acid-binding G-protein-coupled receptor associated with inflammatory and fibrotic diseases. As the only reported antagonist of GPR84 (PBI-4050) that displays relatively low potency and selectivity, a clear need exists for an improved modulator. Structural optimization of GPR84 antagonist hit 1, identified through high-throughput screening, led to the identification of potent and selective GPR84 inhibitor GLPG1205 (36). Compared with the initial hit, 36 showed improved potency in a guanosine 5'-O-[γ-thio]triphosphate assay, exhibited metabolic stability, and lacked activity against phosphodiesterase-4. This novel pharmacological tool allowed investigation of the therapeutic potential of GPR84 inhibition. At once-daily doses of 3 and 10 mg/kg, GLPG1205 reduced disease activity index score and neutrophil infiltration in a mouse dextran sodium sulfate-induced chronic inflammatory bowel disease model, with efficacy similar to positive-control compound sulfasalazine. The drug discovery steps leading to GLPG1205 identification, currently under phase II clinical investigation, are described herein.

Discovery of 2-[[2-Ethyl-6-[4-[2-(3-hydroxyazetidin-1-yl)-2-oxoethyl]piperazin-1-yl]-8-methylimidazo[1,2-a]pyridin-3-yl]methylamino]-4-(4-fluorophenyl)thiazole-5-carbonitrile (GLPG1690), a First-in-Class Autotaxin Inhibitor Undergoing Clinical Evaluation for the Treatment of Idiopathic Pulmonary Fibrosis.

Autotaxin is a circulating enzyme with a major role in the production of lysophosphatic acid (LPA) species in blood. A role for the autotaxin/LPA axis has been suggested in many disease areas including pulmonary fibrosis. Structural modifications of the known autotaxin inhibitor lead compound 1, to attenuate hERG inhibition, remove CYP3A4 time-dependent inhibition, and improve pharmacokinetic properties, led to the identification of clinical candidate GLPG1690 (11). Compound 11 was able to cause a sustained reduction of LPA levels in plasma in vivo and was shown to be efficacious in a bleomycin-induced pulmonary fibrosis model in mice and in reducing extracellular matrix deposition in the lung while also reducing LPA 18:2 content in bronchoalveolar lavage fluid. Compound 11 is currently being evaluated in an exploratory phase 2a study in idiopathic pulmonary fibrosis patients.

Discovery and optimization of an azetidine chemical series as a free fatty acid receptor 2 (FFA2) antagonist: from hit to clinic.

FFA2, also called GPR43, is a G-protein coupled receptor for short chain fatty acids which is involved in the mediation of inflammatory responses. A class of azetidines was developed as potent FFA2 antagonists. Multiparametric optimization of early hits with moderate potency and suboptimal ADME properties led to the identification of several compounds with nanomolar potency on the receptor combined with excellent pharmacokinetic (PK) parameters. The most advanced compound, 4-[[(R)-1-(benzo[b]thiophene-3-carbonyl)-2-methyl-azetidine-2-carbonyl]-(3-chloro-benzyl)-amino]-butyric acid 99 (GLPG0974), is able to inhibit acetate-induced neutrophil migration strongly in vitro and demonstrated ability to inhibit a neutrophil-based pharmacodynamic (PD) marker, CD11b activation-specific epitope [AE], in a human whole blood assay. All together, these data supported the progression of 99 toward next phases, becoming the first FFA2 antagonist to reach the clinic.

A multiparameter approach to monitor disease activity in collagen-induced arthritis.

INTRODUCTION: Disease severity in collagen-induced arthritis (CIA) is commonly assessed by clinical scoring of paw swelling and histological examination of joints. Although this is an accurate approach, it is also labour-intensive and the application of less invasive and less time-consuming methods is of great interest. However, it is still unclear which of these methods represents the most discriminating measure of disease activity. METHODS: We undertook a comparative analysis in which different measurements of inflammation and tissue damage in CIA were studied on an individual mouse level. We compared the current gold standard methods - clinical scoring and histological examination - with alternative methods based on scoring of X-ray or micro-computed tomography (CT) images and investigated the significance of systemically expressed proteins, involved in CIA pathogenesis, that have potential as biomarkers. RESULTS: Linear regression analysis revealed a marked association of serum matrix metalloproteinase (MMP)-3 levels with all features of CIA including inflammation, cartilage destruction and bone erosions. This association was improved by combined detection of MMP-3 and anti-collagen IgG2a antibody concentrations. In addition, combined analysis of both X-ray and micro-CT images was found to be predictive for cartilage and bone damage. Most remarkably, validation analysis using an independent data set proved that variations in disease severity, induced by different therapies, could be accurately represented by predicted values based on the proposed parameters. CONCLUSIONS: Our analyses revealed that clinical scoring, combined with serum MMP-3, anti-collagen IgG2a measurement and scoring of X-ray and micro-CT images, yields a comprehensive insight into the different aspects of disease activity in CIA.

Role of dietary phosphorus and degree of uremia in the development of renal bone disease in rats.

The remnant kidney rat model has been extensively used for the evaluation of bone changes due to uremia. The present study aimed to assess the effect of the dietary phosphorus availability and of the severity of renal failure on bone histomorphometric changes and various biochemical markers over time in this model. Chronic renal failure (CRF) was induced in male Wistar rats by 5/6th nephrectomy. Half of the number of animals received a standard rat diet (STD) (0.67% P, containing low bioavailable phosphorus of plant origin); the other animals were fed a high phosphorus diet (HPD) (0.93% P, containing inorganic phosphorus with high bioavailability). Every two weeks, blood and urine samples were collected. At sacrifice after 6 or 12 weeks, bone samples were taken for the measurement of histological and histodynamic parameters. Serum creatinine measurements indicated the development of mild to moderate renal failure in both diet groups. Phosphaturia was unexpectedly low in all animals that received the STD, indicating relative phosphorus depletion despite the normal dietary phosphorus content. In the HPD CRF group, a decrease in calcemia and a rise in phosphatemia were seen after 12 weeks of CRF, which were more pronounced in animals with higher serum creatinine. Serum iPTH levels were distinctly increased in CRF rats fed a HPD, especially those with more pronounced renal failure. Serum osteocalcin and to a lesser extend tartrate-resistant acid phosphatase and urinary pyridinoline and deoxypyridinoline crosslinks were higher in the CRF animals compared to the shams, particularly in the animals of the HPD group with more pronounced CRF. In both diet groups, the CRF animals had significantly higher amounts of osteoid compared to shams. Only the animals that received a HPD developed distinct histological signs of secondary hyperparathyroidism (sHPTH), that is, an increased bone formation rate, mineral apposition rate, osteoblast perimeter, and eroded perimeter. Again, this effect was most prominent in rats with more severe CRF. In conclusion, data of the present study indicate that in experimental studies using the remnant kidney rat model, both the dietary phosphorus bioavailability and the degree of renal failure in the development of hyperparathyroidism should be considered.

Development and reversibility of impaired mineralization associated with lanthanum carbonate treatment in chronic renal failure rats.

BACKGROUND: We have previously shown that administration of the new phosphate binder lanthanum (La) carbonate at high doses during 12 weeks induces a mineralization defect (MD) in chronic renal failure (CRF) rats most likely due to the powerful phosphate binding. In this study, we want to investigate the fate and possible biological activities of La once it is accumulated in bone. METHODS: CRF animals (5/6th nephrectomy) received La carbonate (2,000 mg/kg/day) via oral gavage for 2 or 6 weeks and were sacrificed immediately at the end of the treatment period and after a wash out period of 2 and 8 weeks. Bone histomorphometry and measurement of bone La content were performed. Control CRF animals received vehicle only. RESULTS: After 2 weeks of La treatment, 75% of the animals showed signs of MD compared to 14% in CRF controls despite similar bone La levels. Two weeks after arrest of La treatment, bone La levels remained unchanged, yet 87% showed normal bone histology. A similar evolution was noted in the animals treated for 6 weeks. Bone histology showed a reduction of number of animals with a MD from 62.5% at 6 weeks of La treatment to 20% and 28% 2 and 8 weeks after arrest of La treatment respectively. CONCLUSION: The phosphate-binder-induced MD may appear and disappear without any change in either the perimeter of active osteoblasts or in bone La levels. Bone histology in CRF animals normalized after arrest of the La administration, thereby presenting further arguments for the MD in La-treated animals to result from the high phosphate binding capacity of La rather than being the consequence of a direct effect of La on bone.

Localization of lanthanum in bone of chronic renal failure rats after oral dosing with lanthanum carbonate.

BACKGROUND: Lanthanum carbonate has been shown to be a safe, effective phosphate-binding agent. We have shown that an impaired mineralization in chronic renal failure rats treated with high doses of lanthanum carbonate develops secondary to phosphate depletion and is therefore pharmacologically mediated rather than a direct effect of lanthanum on bone. Although bulk bone lanthanum concentrations are low, it is important to consider the localization within a given tissue. METHODS: Using the scanning x-ray micro-fluorescence set-up at beamline ID21 of the European Synchrotron Radiation Facility, calcium and lanthanum distributions in bone samples were mapped. RESULTS: In chronic renal failure rats loaded orally with lanthanum carbonate (12 weeks) (2000 mg/kg/day), bulk bone lanthanum concentrations reached values up to 5 microg/g wet weight. Lanthanum could be demonstrated at the edge of the mineralized bone, at both actively mineralizing and quiescent sites, independent of the type of bone turnover. In the presence of hyperparathyroid bone disease, lanthanum was also distributed throughout the mineralized trabecular bone. No correlation with the presence of osteoid, or the underlying bone pathology could be demonstrated. After a 2- or 4-week washout period before sacrifice, lanthanum localization did not change significantly. CONCLUSION: The comparable localization of lanthanum in different types of bone turnover, and the unchanged localization after washout and consequent disappearance of the mineralization defect, indicates no relationship between the localization of lanthanum in bone and the presence of a mineralization defect.

Time-evolution and reversibility of strontium-induced osteomalacia in chronic renal failure rats.

BACKGROUND: Patients with impaired renal function can accumulate strontium in the bone, which has been associated with the development of osteomalacia. A causal role for strontium in the development of the disease was presented in chronic renal failure (CRF) rats. Strontium-ranelate has been put forward as a therapeutic agent in the treatment of osteoporosis. Since the target population for strontium treatment consists mainly in postmenopausal osteoporotic women, who may have a reduced renal function, the risk for osteomalacia should be considered. METHODS: To determine the time evolution and reversibility of the strontium-induced mineralization defect, CRF rats were loaded with strontium (2 g/L) (+/- 200 mg/kg/day) during 2, 6, and 12 weeks, followed by a washout period of 0, 2, 4, or 8 weeks. RESULTS: Histologic examination of the bone of the animals treated with strontium revealed signs of osteomalacia already after 2 weeks. Animals that received strontium during 6 and 12 weeks had a significantly higher osteoid perimeter, area and thickness as compared to CRF controls. After 12 weeks, the mineralization was significantly affected, as evidenced by a lower double-labeled surface, mineral apposition and bone formation rate in combination with an increased osteoid maturation time and mineralization lag time. The osteoblast perimeter was significantly lower in the strontium-treated animals. After the washout periods, these effects were reversed and the bone lesions evolved to the values of CRF controls. This went along with an 18% reduction of the bone strontium content. A significant rise in serum alkaline phosphatase (ALP) activity was apparent in the strontium-treated animals as compared to CRF controls. This was not only due to higher levels of the bone ALP but also to those of the liver and the intestinal isoenzymes. Serum parathyroid hormone (PTH) levels decreased during strontium treatment. After cessation of the treatment, the serum ALP activity and PTH concentration reversed to control levels. CONCLUSION: In this study evidence is provided for the rapid development of a mineralization defect in strontium-loaded CRF rats, accompanied by a reduced osteoblast number, reduced PTH synthesis or secretion, and increased serum ALP levels. These effects can be rapidly reversed after withdrawal of the compound.