Masatoshi Hazama

Adjuvant-independent enhanced immune responses to recombinant herpes simplex virus type 1 glycoprotein D by fusion with biologically active interleukin-2.

A truncated herpes simplex virus (HSV) type 1 glycoprotein D (t-gD) gene was fused to the human interleukin-2 (IL-2) gene (t-gD-IL-2 gene) and introduced into mouse myeloma Sp2/0 cells. The gene product, t-gD-IL-2, secreted from the cells was immunoprecipitated with five monoclonal antibodies specific for native gD. Purified t-gD-IL-2 supported the growth of IL-2-dependent cells, with a specific activity almost comparable to that of recombinant human IL-2. Mice immunized with t-gD-IL-2 in an adjuvant-free form showed superior anti-HSV antibody responses, and were completely protected against HSV challenge, whereas immunization with t-gD adsorbed onto aluminum hydroxide (alum) partially failed to prevent the virus infection. The high immunogenicity of t-gD-IL-2 was due to the biological activity of the fused IL-2 rather than to a hapten-carrier effect of the IL-2 moiety, because mice primed with t-gD-IL-2 showed delayed-type hypersensitivity against stimulation with gD, but not against that with IL-2 antigen, and because a booster immunization with t-gD-IL-2 extensively augmented the response of anti-gD antibody, but not that of the anti-human IL-2 antibody. The serological half-life of IL-2 activity in mice injected with t-gD-IL-2 was prolonged to about four times that of rIL-2. However, when t-gD-IL-2 was co-administered with human albumin (HSA), the mouse anti-HSA antibody response was slightly enhanced.(ABSTRACT TRUNCATED AT 250 WORDS)

A novel strategy for converting recombinant viral protein into high immunogenic antigen

Interleukin 2 (IL‐2) is a lymphokine promoting immune response and therefore has been investigated as an immunological adjuvant. In order to enhance the immunogenicity of recombinant viral protein, herpes simplex virus type 1 (HSV‐1) glycoprotein D (gD), we gentically created a fusion protein consisting of gD and human IL‐2. The fusion protein, without any other adjuvants, induced high antibody responses and cell‐mediated immunity to HSV‐1 in mice. Mice immunized with the fusion protein were protected against HSV‐1 infection. The results indicate that IL‐2‐fusing can provide a means for converting a weak immunogenic protein into a high immunogenic antigen, and the strategy would be widely applicable to the other antigens for pathogens.

Immunogenicity of a new type of yeast-derived hepatitis B vaccine consisting of M (pre-S2 + S) protein particles

Genetically modified M (pre-S2+S; P31) protein (M-P31c) particles were formulated into a vaccine (TGP-943) through adsorption on to an alum adjuvant. The immunogenicity of this vaccine was investigated using guinea-pigs and various kinds of mice. In terms of anti-HBs(S) response, TGP-943 was found to be as immunogenic as the control plasma-derived vaccine (PDV) and yeast-derived S vaccine (YDSV) in Balb/c mice. TGP-943 induced anti-S antibodies even in S low-responder mice. In addition to the anti-S antibodies, TGP-943 was shown to elicit anti-pre-S2 antibodies dose-dependently, and the anti-pre-S2 antibodies were maintained at a high level after immunization with a high dose of TGP-943. The antibody response to pre-S2 had a tendency to appear earlier than that to S. M-P31c particles induced in vitro proliferation of TGP-943-primed lymph node cells, indicating that TGP-943 is more immunogenic than conventional vaccines in terms of T-cell levels. These results suggest that TGP-943 would be a promising candidate for a third generation hepatitis B vaccine.

Phosphodiesterase 4 inhibitor and phosphodiesterase 5 inhibitor combination therapy has antifibrotic and anti-inflammatory effects in mdx mice with Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is the most common inherited muscular dystrophy. Patients experience DMD in their 20s from cardiac or respiratory failure related to progressive muscle wasting. Currently, the only treatments for the symptoms of DMD are available. Muscle fibrosis, a DMD feature, leads to reduced muscle function and muscle mass, and hampers pharmaceutical therapeutic efficacy. Although antifibrotic agents may be useful, none is currently approved. Phosphodiesterase 4 (PDE4) inhibitors have exhibited antifibrotic effects in human and animal models. In this study, we showed beneficial effects of the PDE4 inhibitor piclamilast in the DMD mdx mouse. Piclamilast reduced the mRNA level of profibrotic genes, including collagen 1A1, in the gastrocnemius and diaphragm, in the mdx mouse, and significantly reduced the Sirius red staining area. The PDE5 inhibitors sildenafil and tadalafil ameliorated functional muscle ischemia in boys with DMD, and sildenafil reversed cardiac dysfunction in the mdx mouse. Single‐treatment piclamilast or sildenafil showed similar antifibrotic effects on the gastrocnemius; combination therapy showed a potent antifibrotic effect, and piclamilast and combination therapy increased peroxisome proliferator‐activated receptor γ coactivator‐1α mRNA in mouse gastrocnemius. In summary, we confirmed that piclamilast has significant antifibrotic effects in mdx mouse muscle and is a potential treatment for muscle fibrosis in DMD.—Nio, Y., Tanaka, M., Hirozane, Y., Muraki, Y., Okawara, M., Hazama, M., Matsuo, T. Phosphodiesterase 4 inhibitor and phosphodiesterase 5 inhibitor combination therapy has antifibrotic and anti‐inflammatory effects in mdx mice with Duchenne muscular dystrophy. FASEB J. 31, 5307–5320 (2017). www.fasebj.org

Design and synthesis of a novel series of orally active, selective somatostatin receptor 2 agonists for the treatment of type 2 diabetes

The discovery of a novel series of β-methyltryptophan (β MeTrp) derivatives as selective and orally active non-peptide somatostatin receptor 2 (SSTR2) agonists for the treatment of Type 2 diabetes is described. In our previous research, Compound A, β-MeTrp derivative with highly potent and selective SSTR2 agonistic activity IC50 (SSTR2/SSTR5)=0.3/>100 (nM), was identified asa drug candidate for treatment of Type 2 diabetes which lowers significantly plasma glucose level in Wistar fatty rats in its oral administrations. However, as serious increase in AUC and phospholipidosis (PLsis) were observed in its toxicological studies in rats, follow-up compounds were searched to avoid risk of PLsis with reference to their in vitro PLsis potentials evaluated on the basis of accumulation of phospholipids in HepG2 cells exposed to the compounds. It has been found that introduction of a carbonyl group onto the piperidine and piperazine or aniline moiety of compounds A and B reduced markedly the in vitro PLsis potentials. And further modification of the compounds and their evaluation led to a discovery of compounds 3k with lower in vitro PLsis potentials exhibiting lowering effect of hypoglycemia-induced glucagon secretion in SD rats (ED50=1.1mg/kg) and glucose excursion in meal tolerance test in Wistar fatty diabetic rats (MED=3.0mg/kg) in oral administrations. Compound 3k was selected asa new drug candidate of selective and orally active non-peptide SSTR2 agonists for treatment of Type 2 diabetes with low in vivo PLsis potential.

Phosphodiesterase 4 inhibition reduces lung fibrosis following targeted type II alveolar epithelial cell injury

Fibrosis of the lung constitutes a major clinical challenge and novel therapies are required to alleviate the associated morbidity and mortality. Investigating the antifibrotic efficacy of drugs that are already in clinical practice offers an efficient strategy to identify new therapies. The phosphodiesterase 4 (PDE4) inhibitors, approved for the treatment of chronic obstructive pulmonary disease, harbor therapeutic potential for pulmonary fibrosis by augmenting the activity of endogenous antifibrotic mediators that signal through cyclic AMP. In this study, we tested the efficacy of several PDE4 inhibitors including a novel compound (Compound 1) in a murine model of lung fibrosis that results from a targeted type II alveolar epithelial cell injury. We also compared the antifibrotic activity of PDE4 inhibition to the two therapies that are FDA‐approved for idiopathic pulmonary fibrosis (pirfenidone and nintedanib). We found that both preventative (day 0–21) and therapeutic (day 11–21) dosing regimens of the PDE4 inhibitors significantly ameliorated the weight loss and lung collagen accumulation that are the sequelae of targeted epithelial cell damage. In a therapeutic protocol, the reduction in lung fibrosis with PDE4 inhibitor administration was equivalent to pirfenidone and nintedanib. Treatment with this class of drugs also resulted in a decrease in plasma surfactant protein D concentration, a reduction in the plasma levels of several chemokines implicated in lung fibrosis, and an in vitro inhibition of fibroblast profibrotic gene expression. These results motivate further investigation of PDE4 inhibition as a treatment for patients with fibrotic lung disease.