Novel 2,7-Diazaspiro[4,4]nonane Derivatives to Inhibit Mouse and Human Osteoclast Activities and Prevent Bone Loss in Ovariectomized Mice without Affecting Bone Formation.

Osteoporosis is currently treated with drugs targeting the differentiation or viability osteoclasts, the cells responsible for physiological and pathological bone resorption. Nevertheless, osteoporosis drugs that target only osteoclast activity are expected to preserve bone formation by osteoblasts in contrast to current treatments. We report here the design, synthesis, and biological characterization of a series of novel N-arylsufonamides featuring a diazaspiro[4,4]nonane nucleus to target the guanine nucleotide exchange activity of DOCK5, which is essential for bone resorption by osteoclasts. These compounds can inhibit both mouse and human osteoclast activity. In particular, 4-chlorobenzyl-4-hydroxy-2-phenyl-1-thia-2,7-diazaspiro[4,4]nonane 1,1-dioxide (compound E197) prevented pathological bone loss in mice. Most interestingly, treatment with E197 did not affect osteoclast and osteoblast numbers and hence did not impair bone formation. E197 could represent a lead molecule to develop new antiosteoporotic drugs targeting the mechanism of osteoclast adhesion onto the bone.

Radium-223 Inhibits Osseous Prostate Cancer Growth by Dual Targeting of Cancer Cells and Bone Microenvironment in Mouse Models.

Purpose: Radium-223 dichloride (radium-223, Xofigo), a targeted alpha therapy, is currently used for the treatment of patients with castration-resistant prostate cancer (CRPC) with bone metastases. This study examines the mode-of-action and antitumor efficacy of radium-223 in two prostate cancer xenograft models.Experimental Design: Mice bearing intratibial LNCaP or LuCaP 58 tumors were randomized into groups (n = 12-17) based on lesion grade and/or serum PSA level and administered radium-223 (300 kBq/kg) or vehicle, twice at 4-week intervals. X-rays and serum samples were obtained biweekly. Soft tissue tumors were observed macroscopically at sacrifice. Tibiae were analyzed by gamma counter, micro-CT, autoradiography and histology.Results: Radium-223 inhibited tumor-induced osteoblastic bone growth and protected normal bone architecture, leading to reduced bone volume in LNCaP and abiraterone-resistant LuCaP 58 models. Furthermore, radium-223 resulted in lower PSA values and reduced total tissue and tumor areas, indicating that treatment constrains prostate cancer growth in bone. In addition, radium-223 suppressed abnormal bone metabolic activity as evidenced by decreased number of osteoblasts and osteoclasts and reduced level of the bone formation marker PINP. Mode-of-action studies revealed that radium-223 was deposited in the intratumoral bone matrix. DNA double-strand breaks were induced in cancer cells within 24 hours after radium-223 treatment, and PSA levels were significantly lower 72 hours after treatment, providing further evidence of the antitumor effects.Conclusions: Taken together, radium-223 therapy exhibits a dual targeting mode-of-action that induces tumor cell death and suppresses tumor-induced pathologic bone formation in tumor microenvironment of osseous CRPC growth in mice. Clin Cancer Res; 23(15); 4335-46. ©2017 AACR.

ZP2307, a novel, cyclic PTH(1-17) analog that augments bone mass in ovariectomized rats.

Daily injections of human parathyroid hormone (1-34), hPTH(1-34), provide a highly effective treatment option for severe osteoporosis. However, PTH analogs shorter than 28 amino acids do not retain any bone augmenting potential. Here, we present ZP2307 ([Ac5c¹, Aib³, Leu8, Gln¹°, Har¹¹, Ala¹², Trp¹4, Asp¹7]PTH(1-17)-NH2), a novel, chemically modified and cyclized hPTH(1-17) analog, that augments bone mass in ovariectomized, osteopenic rats. Subcutaneous administration of this structurally constrained, K¹³-D¹7 side-chain-to-side-chain cyclized peptide reversed bone loss and increased bone mineral density (BMD) up to or above baseline levels in rat long bones and vertebrae. Highly significant effects of ZP2307 were achieved at doses of 40-320 nmol/kg. Micro-CT and histomorphometric analyses showed that ZP2307 improved quantitative and qualitative parameters of bone structure. Biomechanical testing of rat femora confirmed that ZP2307 dramatically increased bone strength. Over a broad maximally effective dose range (40-160 nmol/kg) ZP2307 did not increase serum concentrations of ionized free calcium above normal levels. Only at the highest dose (320 nmol/kg) ZP2307 induced hypercalcemic calcium levels in the ovariectomized rats. To our knowledge ZP2307 is the smallest PTH peptide analog known to exert augmentation of bone. Our findings suggest that ZP2307 has the potential to effectively augment bone mass over a broad dose range without a concomitant increase in the serum concentration of ionized free calcium above the normal range.

Effects of diet-induced obesity and voluntary wheel running on bone properties in young male C57BL/6J mice.

Both physical activity and body mass affect bone properties. In this study we examined how diet-induced obesity combined with voluntary physical activity affects bone properties. Forty 7-week-old male C57BL/6J mice were assigned to four groups evenly: control diet (C), control diet + running (CR), high-fat diet (HF, 60% energy from fat), and high-fat diet + running (HFR). After 21-week intervention, all mice were killed and the left femur was dissected for pQCT and mechanical measurements. Body mass increased 80% in HF and 62% in HFR, with increased epididymal fat pad weight and impaired insulin sensitivity. Except for total and trabecular volumetric bone mineral density (BMD), bone traits correlated positively with body mass, fat pad, leptin, and osteoprotegerin. Obesity induced by a high-fat diet resulted in increased femoral bone cross-sectional area, mineral content (BMC), polar moment of inertia, and mechanical parameters. Of the mice accessing the running wheel, those fed the control diet had thinner cortex and less total metaphyseal BMC and BMD, with enlarged metaphyseal marrow cavity, whereas mice fed the high-fat diet had significantly higher trabecular BMD and smaller marrow cavity. However, the runners had a weaker femoral neck as indicated by decreased maximum flexure load. These results suggest that voluntary running exercise affects bone properties in a site-specific manner and that there is a complex interaction between physical activity and obesity. Thus, both diet and exercise should be considered when optimizing the effects on body composition and bone, even though the underlying mechanisms remain partly unknown.

Overexpression of cathepsin K accelerates the resorption cycle and osteoblast differentiation in vitro.

Bone resorption is a multistep process including osteoclast attachment, cytoskeletal reorganization, formation of four distinct plasma membrane domains, and matrix demineralization and degradation followed by cell detachment. The present study describes the intracellular mechanisms by which overexpression of cathepsin K in osteoclasts results in enhanced bone resorption. Osteoclasts and bone marrow-derived osteoclast and osteoblast precursors were isolated from mice homozygous (UTU17(+/+)) and negative for the transgene locus. Cells cultured on bovine cortical bone slices were analyzed by fluorescence and confocal laser scanning microscopy, and bone resorption was studied by measurements of biochemical resorption markers, morphometry, and FESEM. Excessive cathepsin K protein and enzyme activity were microscopically observed in various intracellular vesicles and in the resorption lacunae of cathepsin K-overexpressing osteoclasts. The number of cathepsin K-containing vesicles in UTU17(+/+) osteoclasts was highly increased, and co-localization with markers for the biosynthetic and transcytotic pathways was observed throughout the cytoplasm. As a functional consequence of cathepsin K overexpression, biochemical resorption markers were increased in culture media of UTU17(+/+) osteoclasts. Detailed morphometrical analysis of the erosion in bone slices indicated that the increased biosynthesis of cathepsin K was sufficient to accelerate the osteoclastic bone resorption cycle. Cathepsin K overexpression also enhanced osteogenesis and induced the formation of exceptionally small, actively resorbing osteoclasts from their bone marrow precursors in vitro. The present study describes for the first time how enhancement in one phase of the osteoclastic resorption cycle also stimulates its other phases and further demonstrate that tight control and temporal coupling of mesenchymal and hematopoietic bone cells in this multistep process.

Overexpression of cathepsin K in mice decreases collagen deposition and lung resistance in response to bleomycin-induced pulmonary fibrosis.

BACKGROUND: Lung fibrosis is a devastating pulmonary disorder characterized by alveolar epithelial injury, extracellular matrix deposition and scar tissue formation. Due to its potent collagenolytic activity, cathepsin K, a lysosomal cysteine protease is an interesting target molecule with therapeutic potential to attenuate bleomycin-induced pulmonary fibrosis in mice. We here tested the hypothesis that over-expression of cathepsin K in the lungs of mice is protective in bleomycin-induced pulmonary fibrosis. METHODS: Wild-type and cathepsin K overexpressing (cathepsin K transgenic; cath K tg) mice were challenged intratracheally with bleomycin and sacrificed at 1, 2, 3 and 4 weeks post-treatment followed by determination of lung fibrosis by estimating lung collagen content, lung histopathology, leukocytic infiltrates and lung function. In addition, changes in cathepsin K protein levels in the lung were determined by immunohistochemistry, real time RT-PCR and western blotting. RESULTS: Cathepsin K protein levels were strongly increased in alveolar macrophages and lung parenchymal tissue of mock-treated cathepsin K transgenic (cath K tg) mice relative to wild-type mice and further increased particularly in cath K tg but also wild-type mice in response to bleomycin. Moreover, cath K tg mice responded with a lower collagen deposition in their lungs, which was accompanied by a significantly lower lung resistance (RL) compared to bleomycin-treated wild-type mice. In addition, cath K tg mice responded with a lower degree of lung fibrosis than wild-type mice, a process that was found to be independent of inflammatory leukocyte mobilization in response to bleomycin challenge. CONCLUSION: Over-expression of cathepsin K reduced lung collagen deposition and improved lung function parameters in the lungs of transgenic mice, thereby providing at least partial protection against bleomycin-induced lung fibrosis.

Short-term changes in serum PINP predict long-term changes in trabecular bone in the rat ovariectomy model.

Serum procollagen I N-terminal propeptide (PINP) is a sensitive bone formation marker in humans. We have developed a nonradioactive immunoassay for rat PINP and studied PINP as a bone formation marker in the rat ovariectomy (OVX) model. Two OVX studies were performed with 3-month-old rats, both including measurement of PINP, C-terminal cross-linked telopeptide of type I collagen (CTX), and N-terminal mid-fragment of osteocalcin. A pilot 14-day study contained a sham-operated control group and an OVX group, and an extensive 8-week study contained a sham-operated control group and OVX groups receiving vehicle and 17 beta-estradiol (E2, 10 microg/kg/day s.c.). The bone markers were measured before the operation and at days 2, 4, 7, 10, and 14 in the pilot study and before the operations and at 2 and 8 weeks in the extensive study. Trabecular bone parameters were determined by peripheral quantitative computed tomography and histomorphometry from tibial metaphysis in the extensive study. The rat PINP immunoassay had the following characteristics: intra-assay coefficient of variation (CV) 2.8%, interassay CV 7.5%, dilution linearity 95%, and recovery 107%. PINP increased significantly during the first 2 weeks after OVX and returned to sham level at 8 weeks. E2 prevented the increase caused by OVX. Changes in PINP at 2 weeks correlated strongly with changes in CTX and osteocalcin at 2 weeks and with trabecular bone parameters at 8 weeks. As a conclusion, short-term changes in PINP predict long-term changes in trabecular bone parameters, suggesting that PINP is a reliable marker of bone formation in the rat OVX model.

Spontaneous development of synovitis and cartilage degeneration in transgenic mice overexpressing cathepsin K.

OBJECTIVE: Several recent studies have demonstrated that cathepsin K, a proteolytic enzyme capable of degrading native fibrillar collagen, is overexpressed in osteoarthritic cartilage and inflamed synovial tissue. However, it is not known whether increased cathepsin K production is a primary or a secondary event in these diseases. The availability of transgenic UTU17 mice, which exhibit constitutive overexpression of the cathepsin K gene, prompted us to study possible arthritic changes in their knee joints. METHODS: Progression of synovitis and articular cartilage degeneration in the knee joints of UTU17 mice and their nontransgenic littermates was monitored by histologic analyses at 7 and 12 months of age. Distribution of cathepsin K in the knee joints was studied by immunohistochemistry. RESULTS: At the age of 7 months, UTU17 mice exhibited clear signs of synovitis, with strong immunostaining for cathepsin K in the synovial lining and the stroma, while control knee joints appeared normal. At 12 months, marked synovial thickening and fibrosis and severe degradation of cartilage and subchondral bone were observed in UTU17 mouse knee joints. In areas of cartilage degeneration, both chondrocytes and cells of hypertrophic synovia were positive for cathepsin K. At 12 months, synovia of control mice revealed only a few isolated cathepsin K-positive cells and mild changes in articular cartilage. CONCLUSION: Our findings demonstrate that overexpression of the cathepsin K gene under its own promoter in transgenic mice makes them susceptible to progressive synovitis, which, upon aging, results in synovial hyperplasia and fibrosis and subsequent destruction of articular cartilage and bone.

Differential turnover of cortical and trabecular bone in transgenic mice overexpressing cathepsin K.

Cathepsin K is a major osteoclastic protease. We have recently shown that overexpression of mouse cathepsin K gene in transgenic UTU17 mouse model results in high turnover osteopenia of metaphyseal trabecular bone at the age of 7 months. The present report extends these studies to a systematic analysis of cortical bone in growing and adult mice overexpressing cathepsin K. Mice homozygous for the transgene locus (UTU17+/+) and their control littermates were studied at the age of 1, 3, 7, and 12 months. Bone properties were analyzed using peripheral quantitative computed tomography (pQCT), histomorphometry, histochemistry, radiography, and biomechanical testing. In addition, the levels of biochemical markers of bone turnover were measured in the sera. Unexpectedly, cortical thickness and cortical bone mineral density were increased in the diaphyseal region of growing and adult UTU17+/+ mice. This was associated with an increased number of vascular canals leading to increased cortical porosity in UTU17+/+ mice without changes in the ultimate bending force or stiffness of the bone. In UTU17+/+ mice, osteopenia of metaphyseal trabecular bone was observed already at the age of 1 month. In sera of 1-month-old UTU17+/+ mice, the activity of tartrate-resistant acid phosphatase 5b was decreased and the levels of osteocalcin increased. Our results support the role of cathepsin K as a major proteinase in osteoclastic bone resorption. Excessive production of cathepsin K induced osteopenia of metaphyseal trabecular bone and increased the porosity of diaphyseal cortical bone. The increased cortical thickness and bone mineral density observed in diaphyses of UTU17+/+ mice demonstrate the different nature and reactivity of trabecular and cortical bone in mice. These results suggest that the biomechanical properties of cortical bone are preserved through adaptation as outlined in Wolff's law.

Impaired bone resorption in cathepsin K-deficient mice is partially compensated for by enhanced osteoclastogenesis and increased expression of other proteases via an increased RANKL/OPG ratio.

Previous reports indicate that mice deficient for cathepsin K (Ctsk), a key protease in osteoclastic bone resorption, develop osteopetrosis due to their inability to properly degrade organic bone matrix. Some features of the phenotype of Ctsk knockout mice, however, suggest the presence of mechanisms by which Ctsk-deficient mice compensate for the lack of cathepsin K. To study these mechanisms in detail, we generated Ctsk-deficient (Ctsk-/-) mice and analyzed them at the age of 2, 7, and 12 months using peripheral quantitative computed tomography, histomorphometry, resorption marker measurements, osteoclast and osteoblast differentiation cultures, and gene expression analyses. The present study verified the previously published osteopetrotic features of Ctsk-deficient mice. However, these changes did not exacerbate during aging indicating the absence of Ctsk to have its most severe effects during the rapid growth period. Resorption markers ICTP and CTX were decreased in the media of Ctsk-/- osteoclasts cultured on bone slices indicating impaired bone resorption. Ctsk-/- mice exhibited several mechanisms attempting to compensate for Ctsk deficiency. The number of osteoclasts in trabecular bone was significantly increased in Ctsk-/- mice compared to controls, as was the number of osteoclast precursors in bone marrow. The mRNA levels for receptor activator of nuclear factor (kappa)B ligand (RANKL) in Ctsk-/- bones were increased resulting in increased RANKL/OPG ratio favoring osteoclastogenesis. In addition, expression of mRNAs of osteoclastic enzymes (MMP-9, TRACP) and for osteoblastic proteases (MMP-13, MMP-14) were increased in Ctsk-/- mice compared to controls. Impaired osteoclastic bone resorption in Ctsk-/- mice results in activation of osteoblastic cells to produce increased amounts of other proteolytic enzymes and RANKL in vivo. We suggest that increased RANKL expression mediates enhanced osteoclastogenesis and increased protease expression by osteoclasts. These observations underline the important role of osteoblastic cells in regulation of osteoclast activity and bone turnover.