DOTAM derivatives as active cartilage-targeting drug carriers for the treatment of osteoarthritis.

Targeted drug-delivery methods are crucial for effective treatment of degenerative joint diseases such as osteoarthritis (OA). Toward this goal, we developed a small multivalent structure as a model drug for the attenuation of cartilage degradation. The DOTAM (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid amide)-based model structure is equipped with the cathepsin D protease inhibitor pepstatin A, a fluorophore, and peptide moieties targeting collagen II. In vivo injection of these soluble probes into the knee joints of mice resulted in 7-day-long local retention, while the drug carrier equipped with a scrambled peptide sequence was washed away within 6-8 h. The model drug conjugate successfully reduced the cathepsin D protease activity as measured by release of GAG peptide. Therefore, these conjugates represent a promising first drug conjugate for the targeted treatment of degenerative joint diseases.

The role of spinal nuclear factor-kappa B in spinal hyperexcitability.

In behavioral experiments, inhibition of nuclear factor-kappaB activation by systemic administration of the IkappaB kinase inhibitor S1627 has been shown to attenuate inflammatory and neuropathic pain. Here, we specifically investigated with electrophysiological recordings in anesthetized rats whether spinal application of S1627 influences hyperexcitability of dorsal horn neurons during an acute knee joint inflammation. Spinal application of S1627 before and early during development of inflammation totally prevented spinal hyperexcitability suggesting an important role of spinal nuclear factor-kappaB in this process. During established inflammation, however, S1627 did not reduce the responses of neurons to mechanical stimulation of the inflamed knee within 2.5 h after spinal administration, thus suggesting that spinal hyperexcitability is not maintained by continuous nuclear factor-kappaB activation.

Protein kinase C pathway is involved in transcriptional regulation of C-reactive protein synthesis in human hepatocytes.

OBJECTIVE: C-reactive protein (CRP) is the prototype acute phase protein and a cardiovascular risk factor. Interleukin-1beta (IL-1beta) and IL-6 stimulate CRP synthesis in hepatocytes. We searched for additional pathways regulating CRP expression. METHODS AND RESULTS: Primary human hepatocytes (PHHs) were treated with IL-1beta, IL-6, and protein kinase C (PKC) activator phorbol 12,13-dibutyrate (PDBu). CRP was analyzed by quantitative RT-PCR and ELISA. PDBu significantly induced CRP transcription by 21.0+/-9.24-fold and protein release by 2.9+/-0.5-fold. Transcriptional regulation was studied in detail in hepatoma G2 (HepG2) cells stably transfected with the 1-kb CRP promoter (HepG2-ABEK14 cells). In these cells, PDBu significantly induced CRP transcription by 5.39+/-0.66-fold. Competitive inhibition with bisindolylmaleimide derivative LY333531 abolished PDBu-mediated promoter activation. Competitive inhibition with IkappaB kinase inhibitor I229 also inhibited PDBu effects. Importantly, IL-8 significantly induced CRP release in PHHs by 58.675+/-19.1-fold, which was blockable by LY333531. CONCLUSIONS: This study describes a novel PKC-dependent transcriptional regulation of CRP gene expression, which, in analogy to the classical IL-1beta and IL-6 pathways, is operational in hepatocytes only. It also identifies IL-8 as a potential physiological PKC activator. HepG2-ABEK14 cells may be useful for high throughput screening to identify inhibitors of CRP synthesis for the prevention of cardiovascular disease.

Specific Inhibition of IkappaB kinase reduces hyperalgesia in inflammatory and neuropathic pain models in rats.

Phosphorylation of IkappaB through IkappaB kinase (IKK) is the first step in nuclear factor kappaB (NF-kappaB) activation and upregulation of NF-kappaB-responsive genes. Hence, inhibition of IKK activity may be expected to prevent injury-, infection-, or stress-induced upregulation of various proinflammatory genes and may thereby reduce hyperalgesia and inflammation. In the present study, we tested this hypothesis using a specific and potent IKK inhibitor (S1627). In an IKK assay, S1627 inhibited IKK activity with an IC50 value of 10.0 +/- 1.2 nm. In cell culture experiments, S1627 inhibited interleukin (IL)-1beta-stimulated nuclear translocation and DNA-binding of NF-kappaB. Plasma concentration time courses after intraperitoneal injection revealed a short half-life of 2.8 hr in rats. Repeated intraperitoneal injections were, therefore, chosen as the dosing regimen. S1627 reversed thermal and mechanical hyperalgesia at 3x 30 mg/kg in the zymosan-induced paw inflammation model and reduced the inflammatory paw edema at 3x 40 mg/kg. S1627 also significantly reduced tactile and cold allodynia in the chronic constriction injury model of neuropathic pain at 30 mg/kg once daily. The drug had no effect on acute inflammatory nociception in the formalin test and did not affect responses to heat and tactile stimuli in naive animals. As hypothesized, S1627 prevented the zymosan-induced nuclear translocation of NF-kappaB in the spinal cord and the upregulation of NF-kappaB-responsive genes including cyclooxygenase-2, tumor necrosis factor-alpha, and IL-1beta. Our data indicate that IKK may prove an interesting novel drug target in the treatment of pathological pain and inflammation.

Suppression of NF-kappaB increases bone formation and ameliorates osteopenia in ovariectomized mice.

Bone degenerative diseases, including osteoporosis, impair the fine balance between osteoclast bone resorption and osteoblast bone formation. Single-agent therapy for anabolic and anticatabolic effects is attractive as a drug target to ameliorate such conditions. Inhibition of nuclear factor (NF)-κB reduces the osteoclast bone resorption. The role of NF-κB inhibitors on osteoblasts and bone formation, however, is minimal and not well investigated. Using an established NF-κB inhibitor named S1627, we demonstrated that inhibition of NF-κB increases osteoblast differentiation and bone formation in vitro by up-regulating the mRNAs of osteoblast-specific genes like type I collagen, alkaline phosphatase, and osteopontin. In addition, S1627 was able to increase bone formation and repair bone defect in a murine calvarial defect model. To determine the effect of NF-κB on a model of osteoporosis, we injected two doses of inhibitor (25 and 50 mg/kg·d) twice a day in sham-operated or ovariectomized 12-wk-old mice and killed them after 4 wk. The anabolic effect of S1627 on trabecular bone was determined by micro focal computed tomography and histomorphometry. Bone mineral density of inhibitor-treated ovariectomized animals was significantly increased compared with sham-operated mice. Osteoblast-related indices like osteoblast surface, mineral apposition rate, and bone formation rate were increased in S1627-treated animals in a dose-dependent manner. NF-κB inhibition by S1627 increased the trabecular bone volume in ovariectomized mice. Furthermore, S1627 could inhibit the osteoclast number, and osteoclast surface to bone surface. In vitro osteoclastogenesis and bone resorbing activity were dose-dependently reduced by NF-κB inhibitor S1627. Taken collectively, our results suggest that NF-κB inhibitors are effective in treating bone-related diseases due to their dual anabolic and antiresorptive activities.