Targeting murine alveolar macrophages by the intratracheal administration of locked nucleic acid containing antisense oligonucleotides.

The pulmonary delivery of locked nucleic acid containing antisense oligonucleotides (LNA-ASOs) is expected to be a novel therapeutic approach for lung diseases. However, there are two concerns to be considered: immune responses, as the lung has a distinct immune mechanism to protect it from inhaled pathogens; and leakage into blood, since the lung is permeable to small molecules. As phagocytic alveolar macrophages reside in the alveolar space, it is hypothesized that inhaled LNA-ASOs effectively accumulate and exert a knockdown (KD) effect on these cells at low doses. Thus, targeting alveolar macrophages by inhaled LNA-ASOs may reduce these risks. To test this hypothesis, LNA-ASOs targeting Scarb1 or Hprt1 were intratracheally administered to mice. We confirmed the remarkable accumulation of intratracheally administered LNA-ASOs in murine alveolar macrophages and found that they exerted a significant and sequence-dependent KD effect. The dose required for KD in alveolar macrophages was lower than that required to induce KD in the whole lung. Furthermore, when a KD effect was observed in alveolar macrophages, no KD effect was observed in the liver or kidney; however, several inflammatory cytokines were increased in the lung. These results suggest the potential application of LNA-ASOs as an inhaled drug specific to alveolar macrophages. However, further studies on the immuno-stimulatory effects of LNA-ASOs will be necessary for the development of novel inhaled therapeutic agents.

The efficiency of lipid nanoparticles with an original cationic lipid as a siRNA delivery system for macrophages and dendritic cells.

Small interfering of RNA (siRNA) technology has the potential to be a next-generation therapy. However, naked siRNA does not have high transfection efficiency and is rapidly degraded after systemic injection, so an appropriate drug delivery system (DDS) is required for clinical use. Several potential systems have been assessed, clinically focusing on hepatocyte or cancer tissue using siRNA. However, targeting immune cells using siRNA is still challenging, and a new DDS is required. In this study, we prepared lipid nanoparticles (LNP) composed of original cationic lipid, neutral lipid of DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) and PEG2000-DMPE (N-(carbonyl-methoxypolyethyleneglycol 2000)-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, sodium salt). Our LNP encapsulating siRNA (LNP/siRNA) exerted a knock-down (KD) effect on mouse inflammatory peritoneal macrophages in vitro. In addition, an in vivo KD effect by systemic administration of LNP/siRNA was observed in macrophages and dendritic cells (DCs) in mice. Furthermore, our LNP/siRNA showed in vitro KD effects not only on murine cells but also on human cells like monocyte-derived macrophages (MDMs) and monocyte-derived DCs (MDDCs). These results indicate the potential utility of our LNP for siRNA-based therapy targeting macrophages and DCs. Because these cells are known to have a significant role in several kinds of diseases, and siRNA can specifically suppress target genes that are closely associated with disease states and are untreatable by small molecules or antibodies. Therefore, delivering siRNA by our LNP to macrophages and DCs could provide novel therapies.

The intratracheal administration of locked nucleic acid containing antisense oligonucleotides induced gene silencing and an immune-stimulatory effect in the murine lung.

Locked nucleic acid containing antisense oligonucleotides (LNA-ASOs) have the potential to modulate the disease-related gene expression by the RNaseH-dependent degradation of mRNAs. Pulmonary drug delivery has been widely used for the treatment of lung disease. Thus, the inhalation of LNA-ASOs is expected to be an efficient therapy that can be applied to several types of lung disease. Because the lung has a distinct immune system against pathogens, the immune-stimulatory effect of LNA-ASOs should be considered for the development of novel inhaled LNA-ASOs therapies. However, there have been no reports on the relationship between knock-down (KD) and the immune-stimulatory effects of inhaled LNA-ASOs in the lung. In this report, LNA-ASOs targeting Scarb1 (Scarb1-ASOs) or negative control LNA-ASOs targeting ApoB (ApoB-ASOs) were intratracheally administered to mice to investigate the KD of the gene expression and the immune-stimulatory effects in the lung. We confirmed that the intratracheal administration of Scarb1-ASOs exerted a KD effect in the lung without a drug delivery system. On the other hand, both Scarb1-ASOs and ApoB-ASOs induced neutrophilic infiltration in the alveoli and increased the expression levels of G-CSF and CXCL1 in the lung. The dose required for KD was the same as the dose that induced the neutrophilic immune response. In addition, in our in vitro experiments, Scarb1-ASOs did not increase the G-CSF or CXCL1 expression in primary lung cells, even though Scarb1-ASOs exerted a strong KD effect. Hence, we hypothesize that inhaled LNA-ASOs have the potential to exert a KD effect in the lung, but that they may be associated with a risk of immune stimulation. Further studies about the mechanism underlying the immune-stimulatory effect of LNA-ASOs is necessary for the development of novel inhaled LNA-ASO therapies.

The selective M-CSF receptor tyrosine kinase inhibitor Ki20227 suppresses experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), can be induced by the immunization of mice with myelin antigens in the form of myelin oligodendrocyte glycoprotein (MOG). Macrophage colony-stimulating factor (M-CSF) is required for the development of individual mononuclear phagocyte populations and is involved in the immune response. We previously reported that Ki20227 (N-{4-[(6,7-dimethoxy-4-quinolyl)oxy]-2-methoxyphenyl}-N'-[1-(1,3-thiazole-2-yl)ethyl]urea) is a highly selective M-CSF receptor (c-fms) tyrosine kinase inhibitor. In our current study, we investigated whether Ki20227 has suppressive effects upon EAE and indeed found that this drug significantly reduced the severity of this disease both preventively and therapeutically. Notably also, Ki20227 treatments inhibited the turn-over/expansion of myeloid cells provoked by the immunization and subsequent MOG-specific T cell responses in our EAE animal model. These findings suggest that M-CSF plays a pivotal role in the development of EAE and that Ki20227 and its derivatives may be candidate drugs for the treatment of human MS.

The orally-active and selective c-Fms tyrosine kinase inhibitor Ki20227 inhibits disease progression in a collagen-induced arthritis mouse model.

Macrophage colony-stimulating factor (M-CSF) is important in the development of macrophages and osteoclasts. Previous studies have also shown that CD11b(+) myeloblasts and osteoclasts play key roles during inflammation and bone destruction in arthritic lesions. In this study, we investigated whether N-{4-[(6,7-dimethoxy-4-quinolyl)oxy]-2-methoxyphenyl}-N'-[1-(1,3-thiazole-2-yl)ethyl] urea (Ki20227), an inhibitor of the M-CSF receptor (c-Fms), suppressed disease progression in a type II collagen (CII)-induced arthritis (CIA) mouse model. We found that Ki20227 inhibited M-CSF-dependent reactions, such as lipopolysaccharide-induced tumor necrosis factor-alpha production, which were enhanced by M-CSF in vitro. Oral administration of Ki20227 in vivo prevented inflammatory cell infiltration and bone destruction, and consequently suppressed disease progression. In addition, the number of CD11b(+), Gr-1(+), and Ly-6G(+) cells in the spleen decreased in the Ki20227-treated mice, and the CII-induced cytokine production in splenocytes isolated from the Ki20227-treated arthritic mice was also reduced. These observations indicate that Ki20227 might exert its therapeutic effects in the CIA mouse model by suppressing the M-CSF-dependent accumulation of both inflammatory and osteoclast cells, as well as by inhibiting inflammatory cytokine production. Hence, inhibitors of the c-Fms tyrosine kinase might act as anti-inflammatory or anti-osteolytic agents against arthritis.

Treatment with anti-gamma-glutamyl transpeptidase antibody attenuates osteolysis in collagen-induced arthritis mice.

UNLABELLED: The effectiveness of a new antibody treatment on arthritis-associated osteolysis was studied by using CIA mice. GGT, a newly identified bone-resorbing factor, was upregulated in arthritic joints. We generated monoclonal antibodies against GGT and injected them into CIA mice. Mice treated with antibodies showed a reduction in osteoclast number and bone erosion. INTRODUCTION: Gamma-glutamyl transpeptidase (GGT) acts as a bone-resorbing factor that stimulates osteoclast formation. GGT expression has been detected in active lymphocytes that accumulate at inflammation sites, such as rheumatoid arthritis (RA). We hypothesize that GGT is an effective target for suppression of arthritis-related osteoclastogenesis and joint destruction. Here, we describe the therapeutic effect of neutralizing antibodies against GGT on joint destruction using a collagen-induced arthritis (CIA) mouse model. MATERIALS AND METHODS: GGT expression in the synovium of RA patients and CIA mice was determined by immunohistochemistry and RT-PCR. Monoclonal antibodies were generated against recombinant human GGT (GGT-mAbs) using BALB/c mice. Antibody treatment was performed by intraperitoneal injections of GGT-mAbs into CIA mice. Effects of antibody treatment on arthritis and bone erosion were evaluated by incidence score, arthritis score, and histopathological observations. The role of GGT in osteoclast development was examined by using the established osteoclastogenic culture system. RESULTS: GGT expression was significantly upregulated in inflamed synovium. Immunohistochemistry revealed that GGT was present in lymphocytes, plasma cells, and macrophages, as well as capillaries. Injection of GGT-mAbs significantly decreased the number of osteoclasts and attenuated the severity of joint destruction in CIA mice. In vitro examination showed that GGT enhanced RANKL-dependent osteoclast formation. GGT stimulated the expression of RANKL in osteoblasts and its receptor RANK in osteoclast precursors, respectively. CONCLUSIONS: This study indicates that inflamed synovial tissue-derived GGT acts as a risk factor for joint destruction and that the antibody-mediated inhibition of GGT significantly decreases osteoclast number and bone erosion in CIA mice. GGT antagonists might be novel therapeutic agents for attenuating joint destruction in RA patients.

CD52 is a novel costimulatory molecule for induction of CD4+ regulatory T cells.

We previously reported that 4C8 monoclonal antibody (mAb) provides a costimulatory signal to human CD4+ T cells and consequently induces regulatory T (Treg) cells, which are hypo-responsive and suppress the polyclonal response of bystander CD4+ cells in a contact-dependent manner. In this study, we identified the antigen of 4C8 mAb as CD52. Costimulation with Campath-1H, a humanized anti-CD52 mAb, also induced Treg cells. Anti-CD52-induced Treg cells suppressed the proliferation of both CD4+ and CD8+ T cells provided with polyclonal or allogeneic stimulation. When Treg cells were induced from Staphylococcal enterotoxin B (SEB) treated cells, they suppressed the response to SEB more efficiently than that to another superantigen, SEA. Furthermore, anti-CD52-induced Treg cells could be expanded by culture with IL-2 followed by CD52-costimulation, and co-injection of expanded Treg cells suppressed lethal xenogeneic graft versus host disease (GvHD) reactions in SCID mice caused by human peripheral blood mononuclear cells (PBMCs).