Pharmacological characterization of GLPG1972/S201086, a potent and selective small-molecule inhibitor of ADAMTS5.

OBJECTIVE: A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) is a key enzyme in degradation of cartilage in osteoarthritis (OA). We report the pharmacological characterization of GLPG1972/S201086, a new, potent and selective small-molecule ADAMTS5 inhibitor. METHODS: Potency and selectivity of GLPG1972/S201086 for ADAMTS5 were determined using fluorescently labeled peptide substrates. Inhibitory effects of GLPG1972/S201086 on interleukin-1α-stimulated glycosaminoglycan release in mouse femoral head cartilage explants and on interleukin-1ß-stimulated release of an ADAMTS5-derived aggrecan neoepitope (quantified with ELISA) in human articular cartilage explants were determined. In the destabilization of the medial meniscus (DMM) mouse and menisectomized (MNX) rat models, effects of oral GLPG1972/S201086 on relevant OA histological and histomorphometric parameters were evaluated. RESULTS: GLPG1972/S201086 inhibited human and rat ADAMTS5 (IC50 ± SD: 19 ± 2 nM and <23 ± 1 nM, respectively), with 8-fold selectivity over ADAMTS4, and 60->5,000-fold selectivity over other related proteases in humans. GLPG1972/S201086 dose-dependently inhibited cytokine-stimulated aggrenolysis in mouse and human cartilage explants (100% at 20 µM and 10 µM, respectively). In DMM mice, GLPG1972/S201086 (30-120 mg/kg b.i.d) vs vehicle reduced femorotibial cartilage proteoglycan loss (23-37%), cartilage structural damage (23-39%) and subchondral bone sclerosis (21-36%). In MNX rats, GLPG1972/S201086 (10-50 mg/kg b.i.d) vs vehicle reduced cartilage damage (OARSI score reduction, 6-23%), and decreased proteoglycan loss (∼27%) and subchondral bone sclerosis (77-110%). CONCLUSIONS: GLPG1972/S201086 is a potent, selective and orally available ADAMTS5 inhibitor, demonstrating significant protective efficacy on both cartilage and subchondral bone in two relevant in vivo preclinical OA models.

IL-17C amplifies epithelial inflammation in human psoriasis and atopic eczema.

BACKGROUND: Key pathogenic events of psoriasis and atopic eczema (AE) are misguided immune reactions of the skin. IL-17C is an epithelial-derived cytokine, whose impact on skin inflammation is unclear. OBJECTIVE: We sought to characterize the role of IL-17C in human ISD. METHODS: IL-17C gene and protein expression was assessed by immunohistochemistry and transcriptome analysis. Primary human keratinocytes were stimulated and expression of cytokines chemokines was determined by qRT-PCR and luminex assay. Neutrophil migration towards supernatant of stimulated keratinocytes was assessed. IL-17C was depleted using a new IL-17C-specific antibody (MOR106) in murine models of psoriasis (IL-23 injection model) and AE (MC903 model) as well as in human skin biopsies of psoriasis and AE. Effects on cell influx (mouse models) and gene expression (human explant cultures) were determined. RESULTS: Expression of IL-17C mRNA and protein was elevated in various ISD. We demonstrate that IL-17C potentiates the expression of innate cytokines, antimicrobial peptides (IL-36G, S100A7 and HBD2) and chemokines (CXCL8, CXCL10, CCL5 and VEGF) and the autocrine induction of IL-17C in keratinocytes. Cell-free supernatant of keratinocytes stimulated with IL-17C was strongly chemotactic for neutrophils, thus demonstrating a critical role for IL-17C in immune cell recruitment. IL-17C depletion significantly reduced cell numbers of T cells, neutrophils and eosinophils in murine models of psoriasis and AE and led to a significant downregulation of inflammatory mediators in human skin biopsies of psoriasis and AE ex vivo. CONCLUSION: IL-17C amplifies epithelial inflammation in Th2 and Th17 dominated skin inflammation and represents a promising target for the treatment of ISD.

Anti-rheumatic activities of histone deacetylase (HDAC) inhibitors in vivo in collagen-induced arthritis in rodents.

BACKGROUND AND PURPOSE: Rheumatoid arthritis (RA) is a chronic inflammatory disease. Histone deacetylase inhibitors (HDACi), a new class of anti-cancer agents, have recently been reported to exhibit potent anti-inflammatory activities. A proof of concept study was carried out with suberoylanilide hydroxamic acid (SAHA) and MS-275, two HDACi currently undergoing clinical investigations for various oncological indications. EXPERIMENTAL APPROACH: The anti-rheumatic effects of SAHA and MS-275 were assessed in both mouse and rat collagen induced arthritis (CIA) models. KEY RESULTS: SAHA exhibited moderate prophylactic efficacy. It attenuated paw swelling due to inflammation, decreased bone erosion in both mice and rats and reduced slightly the RA-induced bone resorption in rats. However, SAHA could not inhibit the onset of arthritis. In contrast, MS-275 displayed dramatic anti-rheumatic activities. In prophylactic intervention, high doses of MS-275 prevented bone erosion and markedly delayed the onset of arthritis; at low doses, MS-275 strongly attenuated paw swelling, bone erosion, and bone resorption associated with RA. Furthermore, the therapeutic efficacy of MS-275 was also documented. After the onset of arthritis, it could stop the disease progression and joint destruction. An anti inflammatory effect of MS-275 was also confirmed through its capacity to decrease serum IL-6 and IL-1beta levels in the CIA induced mouse model. The anti-rheumatic activity of MS-275 was also confirmed through histological observation. No synovial hyperplasia, pannus formation, cartilage or bone destruction were observed in the high dose prophylactic intervention in mice. CONCLUSION AND IMPLICATION: This study strongly supported HDACi as an innovative therapeutic strategy for RA.

Effects of 17 beta-estradiol and trimegestone alone, and in combination, on the bone and uterus of ovariectomized rats.

Trimegestone is a novel norpregnane progestin, which is being developed, in combination with 17 beta-estradiol, for the treatment of menopausal symptoms and prevention of postmenopausal osteoporosis. A model of osteoporosis in the ovariectomized rat has been used to evaluate the effects of 17 beta-estradiol and trimegestone, alone and in combination, on bone and uterus in these animals. Two treatment protocols were investigated, preventive with treatment starting immediately after ovariectomy and curative with treatment starting 1 or 6 months after ovariectomy. 17 beta-Estradiol was administered subcutaneously at a dose of 10 micrograms/kg/day with trimegestone or norethisterone being administered orally at a dose of 1 mg/kg/day; treatment was given 5 days per week. Treatment on both protocols was for 6 months. Given alone, 17 beta-estradiol maintained bone mass, either partially or completely, when given on the preventive protocol, or on the curative protocol with treatment starting 1 month after ovariectomy; it did not restore bone mass when given on the curative protocol with 6 months lapsing between ovariectomy and start of treatment. Trimegestone did not block the beneficial effects of 17 beta-estradiol on bone. 17 beta-Estradiol induced uterine hypertrophy on all these protocols and this was blocked completely by trimegestone. Trimegestone administered alone had no effect on bone or uterus but, when given in combination with 17 beta-estradiol, it did not inhibit the effect of 17 beta-estradiol in maintaining bone mass but completely blocked its uterotropic effect. Norethisterone at a similar dose did not inhibit the effects of 17 beta-estradiol on bone but also did not block its uterotropic effect.

Osteoblasts are a new target for prolactin: analysis of bone formation in prolactin receptor knockout mice.

Bone development is a multistep process that includes patterning of skeletal elements, commitment of hematopoietic and/or mesenchymental cells to chondrogenic and osteogenic lineages, and further differentiation into three specialized cell types: chondrocytes in cartilage and osteoblasts and osteoclasts in bone. Although PRL has a multitude of biological actions in addition to its role in the mammary gland, very little is known about its effect on bone. Mice carrying a germline null mutation for the PRL receptor gene have been produced in our laboratory and used to study the role of PRL in bone formation. In -/- embryos, we observed an alteration in bone development of calvaria. In adults, histomorphometric analysis showed that the absence of PRL receptors leads to a decrease in bone formation rate using double calcein labeling and a reduction of bone mineral density, measured by dual energy x-ray absorptiometry. In addition, serum estradiol, progesterone, testosterone, and PTH levels were analyzed. We also established that osteoblasts, but not osteoclasts, express PRL receptors. This suggests that an effect of PRL on osteoblasts could be required for normal bone formation and maintenance of bone mass. Thus, the PRL receptor knockout mouse model provides a new tool to investigate the involvement of PRL in bone metabolism.

A discrete helicoid of collagenous fibrils: the cuticle of deep-sea hydrothermal vent worms (Riftia pachyptila).

The fibrillar organization of the collagenous cuticle of the hydrothermal vent worm Riftia pachyptila is described. Fibrils in the posterior part of the cuticle are organized in a classical orthogonal plywood consisting of successive layers of fibrils: in this case, fibrils are oriented in only two directions which are orthogonal, as for pogonophoran. Our new data on the plume of Riftia pachyptila show a new type of fibrillar arrangement of the cuticle: 1) three sets of fibrils are arranged in an hexagonal pattern; 2) fibrils in successive plies are rotated by 60 degrees, and the organization of the fibrillar network is interpreted as a discrete helicoid when compared to continuously twisted plywoods; 3) a fourth set of fibrils crosses the hexagonally arranged plies and is oriented perpendicular to the surface of the body. X-ray diffraction studies of the cuticular fibrils reveal a triple helix which is characteristic of collagen molecules. Results obtained by differential scanning calorimetry (DSC) show that the denaturation temperature of the molecule is 54.7 degrees C for Riftia; whereas it is 58.9 degrees C for type I collagen measured under the same conditions. We discuss the origin of this plywood with respect to biomechanical constraints, self assembly processes, and compartmentation of the extracellular space. The involvement of the cell membrane in the fibrillogenesis of collagen is also discussed.

Spatial organization of collagen in annelid cuticle: order and defects.

The epidermis of Paralvinella grasslei (Polychaete, Annelida) is covered by an extracellular matrix, the cuticle, mainly composed as in other annelids of superimposed layers of non-striated collagen fibrils. The collagen fibrils of annelid cuticle are shown to be composed of parallel and sinuous microfibrils (thin sections and freeze-fracture replicas). The 3-dimensional organization of collagen is characterized by 2 different types of geometrical order: (a) Fibrils form a quasiorthogonal network, whose structure is comparable to that of a "plywood"; (b) Fibrils are helical, and goniometric studies show that microfibrils present a definite order within each fibril, which is termed "cylindrical twist". These 2 characteristics are those which have recently been evidenced in "blue phases", i.e., liquid crystals which are closely related to cholesteric liquid crystalline phases. Non-fluid analogues of cholesteric liquids are widespread among invertebrate cuticles and the presence of blue phase analogues suggests that a self-assembly mechanisms is involved in cuticle morpho-genesis, which is derived from that governing blue phase growth. The cuticular network presents local rearrangements of fibrils called "defects", despite the fact that they are elaborate structures which trigonal and pentagonal singularities. Branched fibrils are regularly observed. We discuss the involvement of these pattern disruptions in the cuticle growth process.