HU-444, a Novel, Potent Anti-Inflammatory, Nonpsychotropic Cannabinoid.

Cannabidiol (CBD) is a component of cannabis, which does not cause the typical marijuana-type effects, but has a high potential for use in several therapeutic areas. In contrast to Δ(9)-tetrahydrocannabinol (Δ(9)-THC), it binds very weakly to the CB1 and CB2 cannabinoid receptors. It has potent activity in both in vitro and in vivo anti-inflammatory assays. Thus, it lowers the formation of tumor necrosis factor (TNF)-α, a proinflammatory cytokine, and was found to be an oral antiarthritic therapeutic in murine collagen-induced arthritis in vivo. However, in acidic media, it can cyclize to the psychoactive Δ(9)-THC. We report the synthesis of a novel CBD derivative, HU-444, which cannot be converted by acid cyclization into a Δ(9)-THC-like compound. In vitro HU-444 had anti-inflammatory activity (decrease of reactive oxygen intermediates and inhibition of TNF-α production by macrophages); in vivo it led to suppression of production of TNF-α and amelioration of liver damage as well as lowering of mouse collagen-induced arthritis. HU-444 did not cause Δ(9)-THC-like effects in mice. We believe that HU-444 represents a potential novel drug for rheumatoid arthritis and other inflammatory diseases.

Blockade of NKG2D ameliorates disease in mice with collagen-induced arthritis: a potential pathogenic role in chronic inflammatory arthritis.

OBJECTIVE: To assess the role of the activating receptor NKG2D in arthritis. METHODS: Levels of NKG2D and its ligands were determined by fluorescence-activated cell sorting, real-time polymerase chain reaction, and immunohistochemistry in rheumatoid arthritis (RA) synovial membrane tissue and in paw tissue from arthritic mice. Arthritis was induced in DBA/1 mice by immunization with type II collagen, and mice were treated intraperitoneally with a blocking anti-NKG2D antibody (CX5) on days 1, 5, and 8 after clinical onset and were monitored for 10 days. RESULTS: We demonstrated expression of NKG2D and its ligands on human RA synovial cells and extended this finding to the paws of arthritic mice. Expression of messenger RNA for the NKG2D ligand Rae-1 was up-regulated, and NKG2D was present predominantly on natural killer (NK) and CD4+ T cells, in arthritic paw cell isolates. NKG2D was down-modulated during the progression of collagen-induced arthritis (CIA). NKG2D expression in arthritic paws was demonstrated by immunohistochemistry. Blockade of NKG2D ameliorated established CIA, with significant reductions in clinical scores and paw swelling. Histologic analysis of arthritic joints from anti-NKG2D-treated mice demonstrated significant joint protection, compared with control mice. Moreover, anti-NKG2D treatment significantly reduced both interleukin-17 production from CD4+ T cells in arthritic paws and splenic NK cell cytotoxic effector functions in vivo and in vitro. CONCLUSION: Our findings indicate that blockade of NKG2D in a murine model and in human explants has beneficial therapeutic potential that merits further investigation in RA.

Regulation of the angiopoietin-Tie ligand-receptor system with a novel splice variant of Tie1 reduces the severity of murine arthritis.

OBJECTIVES: To determine the function of the angiopoietin (Ang)-Tie ligand-receptor system, and to assess the effect of Tie1-751, a naturally occurring extracellular domain of the Tie1 receptor derived by alternative splicing, in an in vivo model of RA. METHODS: In the murine CIA model, expression of endogenous Ang1, Ang2, Tie1 and Tie2 in whole paws was analysed by quantitative RT-PCR. To assess the effect of inhibition of the Ang-Tie axis, Tie1-751 was expressed and fused to the Fc fragment of human IgG1. The effect of Tie1-751-Fc on human umbilical vein endothelial cell (HUVEC) cytoprotection and migration in response to Ang1, either alone or in combination with VEGF, was investigated. Furthermore, an in vitro angiogenesis assay was used to determine the effect of Tie1-751-Fc on Ang1-mediated angiogenesis. Finally, Tie1-751-Fc was administered in CIA, and the effects on clinical disease and joint architecture of hind foot specimens were determined. RESULTS: Gene expression levels of Ang1, Ang2, and receptors Tie1 and Tie2 in whole paws were significantly increased during the progression of arthritis. Tie1-751-Fc significantly inhibited HUVEC cytoprotection and migration in response to Ang1 alone, or Ang1 in combination with VEGF. Tie1-751-Fc also significantly inhibited angiogenesis induced by a combination of Ang1 plus VEGF. Finally, Tie1-751-Fc, when administered intra-peritoneally to arthritic mice, reduced clinical signs of arthritis, damage to joint architecture and infiltration of blood vessels into the synovium. CONCLUSIONS: Our data demonstrate that the Ang-Tie ligand-receptor system is dysregulated in CIA. Tie1-751, a novel splice variant of the Tie1 receptor, inhibits Ang1/VEGF signalling, suggesting that Ang inhibition may be of therapeutic benefit in inflammatory arthritis.

Oral activated charcoal prevents experimental cerebral malaria in mice and in a randomized controlled clinical trial in man did not interfere with the pharmacokinetics of parenteral artesunate.

BACKGROUND: Safe, cheap and effective adjunct therapies preventing the development of, or reducing the mortality from, severe malaria could have considerable and rapid public health impact. Oral activated charcoal (oAC) is a safe and well tolerated treatment for acute poisoning, more recently shown to have significant immunomodulatory effects in man. In preparation for possible efficacy trials in human malaria, we sought to determine whether oAC would i) reduce mortality due to experimental cerebral malaria (ECM) in mice, ii) modulate immune and inflammatory responses associated with ECM, and iii) affect the pharmacokinetics of parenteral artesunate in human volunteers. METHODS/PRINCIPAL FINDINGS: We found that oAC provided significant protection against P. berghei ANKA-induced ECM, increasing overall survival time compared to untreated mice (p<0.0001; hazard ratio 16.4; 95% CI 6.73 to 40.1). Protection from ECM by oAC was associated with reduced numbers of splenic TNF(+) CD4(+) T cells and multifunctional IFNgamma(+)TNF(+) CD4(+) and CD8(+) T cells. Furthermore, we identified a whole blood gene expression signature (68 genes) associated with protection from ECM. To evaluate whether oAC might affect current best available anti-malarial treatment, we conducted a randomized controlled open label trial in 52 human volunteers (ISRCTN NR. 64793756), administering artesunate (AS) in the presence or absence of oAC. We demonstrated that co-administration of oAC was safe and well-tolerated. In the 26 subjects further analyzed, we found no interference with the pharmacokinetics of parenteral AS or its pharmacologically active metabolite dihydroartemisinin. CONCLUSIONS/SIGNIFICANCE: oAC protects against ECM in mice, and does not interfere with the pharmacokinetics of parenteral artesunate. If future studies succeed in establishing the efficacy of oAC in human malaria, then the characteristics of being inexpensive, well-tolerated at high doses and requiring no sophisticated storage would make oAC a relevant candidate for adjunct therapy to reduce mortality from severe malaria, or for immediate treatment of suspected severe malaria in a rural setting. TRIAL REGISTRATION: Controlled-Trials.com ISRCTN64793756.

Antagonism of the human epidermal growth factor receptor family controls disease severity in murine collagen-induced arthritis.

OBJECTIVE: To evaluate the therapeutic potential of the human epidermal growth factor receptor (HER) family inhibitor, herstatin, in an animal model of arthritis. METHODS: Constructs of herstatin and modified tissue plasminogen activator (tPA)-herstatin were expressed in HEK 293T cells, and secreted protein was analyzed by Western blotting. Tissue PA-herstatin adenovirus (Ad-tPA-Her) was prepared, and titers established. Gene expression of Ad-tPA-Her was determined by polymerase chain reaction using HeLa cells. Pharmacokinetics of gene and protein expression in vivo in liver tissue and serum samples were confirmed via intravenous administration of Ad-tPA-Her. Clinical signs of disease were monitored in arthritic DBA/1 mice after therapeutic administration of Ad-tPA-Her, and histologic analysis of hind foot specimens was performed. RESULTS: Native herstatin was not secreted in supernatants, while modified tPA-herstatin was detected in abundance. HeLa cells stably expressed the tPA-herstatin gene when infected with virus. Additionally, tPA-herstatin gene and protein expression was observed over time in mice treated with virus. Importantly, Ad-tPA-Her, when administered therapeutically to arthritic mice, controlled clinical and histologic signs of disease and reduced the number of joints with severe damage. CONCLUSION: Our results support the notion that the human epidermal growth factor receptor family has a role in the progression of collagen-induced arthritis. The novel tPA-herstatin fusion protein could be used as an effective therapeutic tool for control of inflammatory disorders involving an angiogenic component.

Novel splice variants derived from the receptor tyrosine kinase superfamily are potential therapeutics for rheumatoid arthritis.

INTRODUCTION: Despite the advent of biological therapies for the treatment of rheumatoid arthritis, there is a compelling need to develop alternative therapeutic targets for nonresponders to existing treatments. Soluble receptors occur naturally in vivo, such as the splice variant of the cell surface receptor for vascular endothelial growth factor (VEGF)--a key regulator of angiogenesis in rheumatoid arthritis. Bioinformatics analyses predict that the majority of human genes undergo alternative splicing, generating proteins--many of which may have regulatory functions. The objective of the present study was to identify alternative splice variants (ASV) from cell surface receptor genes, and to determine whether the novel proteins encoded exert therapeutic activity in an in vivo model of arthritis. METHODS: To identify novel splice variants, we performed RT-PCR using an mRNA pool representing major human tissue types and tumors. Novel ASV were identified by alignment of each cloned sequence to its respective genomic sequence in comparison with full-length transcripts. To test whether these ASV have biologic activity, we characterized a subset of them for ligand binding, and for efficacy in an animal model of arthritis. The in vivo study was accomplished using adenoviruses expressing secreted ASV. RESULTS: We cloned 60 novel human ASV from 21 genes, encoding cell surface receptors--many of which are known to be important in the regulation of angiogenesis. The ASV were characterized by exon extension, intron retention and alternative exon utilization. Efficient expression and secretion of selected ASV--corresponding to VEGF receptor type 1, VEGF receptor type 2, VEGF receptor type 3, angiopoietin receptor Tie1, Met (receptor for hepatocyte growth factor), colony-stimulating factor 1 receptor, platelet-derived growth factor receptor beta, fibroblast growth factor receptor 1, Kit, and RAGE--was demonstrated, together with binding to their cognate ligands. Importantly, ASV derived from VEGF receptor type 1 and Tie1, and to a lesser extent from VEGF receptor type 2 and fibroblast growth factor receptor 1, reduced clinical signs of arthritis in vivo. The reduction was paralleled by decreased joint inflammation and destruction. CONCLUSION: The present study shows that unique ASV derived from receptors that play key roles in angiogenesis--namely, VEGF receptor type 1 and, for the first time, Tie1--can markedly reduce arthritis severity. More broadly, our results demonstrate that ASV are a source of novel proteins with therapeutic potential in diseases in which angiogenesis and cellular hyperplasia play a central role, such as rheumatoid arthritis.

Analysing the effect of novel therapies on cytokine expression in experimental arthritis.

Type II collagen-induced arthritis (CIA) is an animal model of rheumatoid arthritis that has been used extensively to address questions of disease pathogenesis and to validate novel therapeutic targets. Susceptibility to CIA is strongly associated with major histocompatibility complex class II genes, and the development of arthritis is accompanied by a robust T- and B-cell response to type II collagen. The main pathological features of CIA include proliferative synovitis with infiltration of inflammatory cells, pannus formation, cartilage degradation, erosion of bone and fibrosis. Pro-inflammatory cytokines, such as tumour necrosis factor alpha and interleukin-1beta, are expressed in the arthritic joints in both murine CIA and human rheumatoid arthritis, and blockade of these molecules results in amelioration of disease. Hence, there is a great deal of interest in the development of small-molecular-weight inhibitors of pro-inflammatory cytokines. There is also interest in the development and testing of drugs with the capacity to modulate the immune pathways involved in driving the inflammatory response in arthritis. For these reasons, there is a need to monitor the effect of novel treatments on cytokine expression in vivo. In this review, we outline the various techniques used to detect cytokines in experimental arthritis and describe how these techniques have been used to quantify changes in cytokine expression following therapeutic intervention.

A novel synthetic, nonpsychoactive cannabinoid acid (HU-320) with antiinflammatory properties in murine collagen-induced arthritis.

OBJECTIVE: To explore the antiarthritic potential of a novel synthetic cannabinoid acid, Hebrew University-320 (HU-320), in the DBA/1 mouse model of arthritis, and to investigate in vitro antiinflammatory and immunosuppressive effects of HU-320 on macrophages and lymphocytes. METHODS: DBA/1 mice were immunized with bovine type II collagen (CII) to induce arthritis and then injected intraperitoneally daily with HU-320. The effects of treatment on arthritic changes in hind feet were assessed clinically and histologically, and draining lymph node responses to CII were assayed. Murine splenic and human blood lymphocytes were cultured to study the effect of HU-320 on polyclonal mitogenic stimulation. Macrophage cultures were set up to evaluate in vitro effects of HU-320 on production of tumor necrosis factor alpha (TNF alpha) and reactive oxygen intermediates (ROIs). The effect of HU-320 administration on lipopolysaccharide-induced serum TNF levels was assayed using C57BL/6 mice. Bioactive TNF production was measured using BALB/c clone 7 target cells. Evaluation of HU-320 psychoactivity was performed using established laboratory tests on Sabra mice. RESULTS: Systemic daily administration of 1 and 2 mg/kg HU-320 ameliorated established CII-induced arthritis. Hind foot joints of treated mice were protected from pathologic damage. CII-specific and polyclonal responses of murine and human lymphocytes were down-modulated. HU-320 inhibited production of TNF from mouse macrophages and of ROIs from RAW 264.7 cells and suppressed the rise in serum TNF level following endotoxin challenge. HU-320 administration yielded no adverse psychotropic effects in mice. CONCLUSION: Our studies show that the novel synthetic cannabinoid acid HU-320 has strong antiinflammatory and immunosuppressive properties while demonstrating no psychoactive effects. The profound suppressive effects on cellular immune responses and on the production of proinflammatory mediators all indicate its usefulness as a novel nonpsychoactive, synthetic antiinflammatory product.

The angiogenesis inhibitor protease-activated kringles 1-5 reduces the severity of murine collagen-induced arthritis.

During rheumatoid arthritis there is enlargement and increased cellularity of the synovial lining of joints, before invasion by the synovium of the underlying cartilage and bone. This increased tissue mass requires a network of blood vessels to supply nutrients and oxygen. Disruption of synovial angiogenesis is thus a desirable aim of antiarthritic therapies. Protease-activated kringles 1-5 (K1-5) is an angiogenesis inhibitor related to angiostatin. In common with angiostatin, K1-5 contains the first four kringle domains of plasminogen, but also encompasses the kringle 5 domain, which confers enhanced antiangiogenic activity when compared with angiostatin. The purpose of the present study was to assess the effect on murine arthritis of K1-5. Arthritis was induced in DBA/1 mice by a single injection of bovine collagen. Treatment with K1-5 was commenced on the day of arthritis onset and continued for 10 days, until the end of the experiment. Daily intraperitoneal administration of K1-5 (2 mg/kg body weight) significantly reduced both paw swelling and clinical score (a composite index of the number of arthritic limbs and the severity of disease). The clinical efficacy of this treatment was reflected by a reduction in joint inflammation and destruction, as assessed histologically. These data suggest that antiangiogenic therapies, which block formation of new blood vessels and hence reduce synovial expansion, might be effective in treating rheumatoid arthritis.