Characterizations of a neutralizing antibody broadly reactive to multiple gluten peptide:HLA-DQ2.5 complexes in the context of celiac disease.

In human celiac disease (CeD) HLA-DQ2.5 presents gluten peptides to antigen-specific CD4+ T cells, thereby instigating immune activation and enteropathy. Targeting HLA-DQ2.5 with neutralizing antibody for treating CeD may be plausible, yet using pan-HLA-DQ antibody risks affecting systemic immunity, while targeting selected gluten peptide:HLA-DQ2.5 complex (pHLA-DQ2.5) may be insufficient. Here we generate a TCR-like, neutralizing antibody (DONQ52) that broadly recognizes more than twenty-five distinct gluten pHLA-DQ2.5 through rabbit immunization with multi-epitope gluten pHLA-DQ2.5 and multidimensional optimization. Structural analyses show that the proline-rich and glutamine-rich motif of gluten epitopes critical for pathogenesis is flexibly recognized by multiple tyrosine residues present in the antibody paratope, implicating the mechanisms for the broad reactivity. In HLA-DQ2.5 transgenic mice, DONQ52 demonstrates favorable pharmacokinetics with high subcutaneous bioavailability, and blocks immunity to gluten while not affecting systemic immunity. Our results thus provide a rationale for clinical testing of DONQ52 in CeD.

Development of a Method for Converting a TAK1 Type I Inhibitor into a Type II or c-Helix-Out Inhibitor by Structure-Based Drug Design (SBDD).

We have developed a method for converting a transforming growth factor-ß-activated kinase 1 (TAK1) type I inhibitor into a type II or c-helix-out inhibitor by structure-based drug design (SBDD) to achieve an effective strategy for developing these different types of kinase inhibitor in parallel. TAK1 plays a key role in inflammatory and immune signaling, and is therefore considered to be an attractive molecular target for the treatment of human diseases (inflammatory disease, cancer, etc.). We have already reported novel type I TAK1 inhibitor, so we utilized its X-ray information to design a new chemical class type II and c-helix-out inhibitors. To develop the type II inhibitor, we superimposed the X-ray structure of our reported type I inhibitor onto a type II compound that inhibits multiple kinases, and used SBDD to design a new type II inhibitor. For the TAK1 c-helix-out inhibitor, we utilized the X-ray structure of a b-Raf c-helix-out inhibitor to design compounds, because TAK1 is located close to b-Raf in the Sugen kinase tree, so we considered that TAK1 would, similarly to b-Raf, form a c-helix-out conformation. The X-ray crystal structure of the inhibitors in complex with TAK1 confirmed the binding modes of the compounds we designed. This report is notable for being the first discovery of a c-helix-out inhibitor against TAK1.

Discovery of a potent and highly selective transforming growth factor ß receptor-associated kinase 1 (TAK1) inhibitor by structure based drug design (SBDD).

A novel thienopyrimidinone analog was discovered as a potent and highly selective TAK1 inhibitor using the SBDD approach. TAK1 plays a key role in inflammatory and immune signaling, so TAK1 is considered to be an attractive molecular target for the treatment of human diseases (inflammatory disease, cancer, etc.). After the hit compound had been obtained, our modifications successfully increased TAK1 inhibitory activity and solubility, but metabolic stability was still unsatisfactory. To improve metabolic stability, we conducted metabolic identification. Although the obtained metabolite was fortunately a potent TAK1 inhibitor, its kinase selectivity was low. Subsequently, to achieve high kinase selectivity, we used SBDD to follow two strategies: one targeting unique amino acid residues in TAK1, especially the combination of Ser111 and Asn114; the other decreasing the interaction with Tyr106 at the hinge position in TAK1. As expected, our designed compound showed an excellent kinase selectivity profile in both an in-house and a commercially available panel assay of over 420 kinases and also retained its potent TAK1 inhibitory activity (TAK1 IC50=11nM).

A facile preparation method of a PFC-containing nano-sized emulsion for theranostics of solid tumors.

Theranostics means a therapy conducted in a diagnosis-guided manner. For theranostics of solid tumors by means of ultrasound, we designed a nano-sized emulsion containing perfluoropentane (PFC5). This emulsion can be delivered into tumor tissues through the tumor vasculatures owing to its nano-size, and the emulsion is transformed into a micron-sized bubble upon sonication through phase transition of PFC5. The micron-sized bubbles can more efficiently absorb ultrasonic energy for better diagnostic images and can exhibit more efficient ultrasound-driven therapeutic effects than nano-sized bubbles. For more efficient tumor delivery, smaller size is preferable, yet the preparation of a smaller emulsion is technically more difficult. In this paper, we used a bath-type sonicator to successfully obtain small PFC5-containing emulsions in a diameter of ca. 200nm. Additionally, we prepared these small emulsions at 40°C, which is above the boiling temperature of PFC5. Accordingly, we succeeded in obtaining very small nano-emulsions for theranostics through a very facile method.

Convection-enhanced delivery of a synthetic retinoid Am80, loaded into polymeric micelles, prolongs the survival of rats bearing intracranial glioblastoma xenografts.

Prognosis for the patients with glioblastoma, the most common malignant brain tumor, remains dismal. A major barrier to progress in treatment of glioblastoma is the relative inaccessibility of tumors to chemotherapeutic agents. Convection-enhanced delivery (CED) is a direct intracranial drug infusion technique to deliver chemotherapeutic agents to the central nervous system, circumventing the blood-brain barrier and reducing systemic side effects. CED can provide wider distribution of infused agents compared to simple diffusion. We have reported that CED of a polymeric micelle carrier system could yield a clinically relevant distribution of encapsulated agents in the rat brain. Our aim was to evaluate the efficacy of CED of polymeric micellar Am80, a synthetic agonist with high affinity to nuclear retinoic acid receptor, in a rat model of glioblastoma xenografts. We also used systemic administration of temozolomide, a DNA-alkylating agent, which has been established as the standard of care for newly diagnosed malignant glioma. U87MG human glioma cells were injected into the cerebral hemisphere of nude rats. Rats bearing U87MG xenografts were treated with CED of micellar Am80 (2.4 mg/m(2)) on day 7 after tumor implantation. Temozolomide (200 mg/m(2)/day) was intraperitoneally administered daily for 5 days, starting on day 7 after tumor implantation. CED of micellar Am80 provided significantly longer survival than the control. The combination of CED of micellar Am80 and systemic administration of temozolomide provided significantly longer survival than single treatment. In conclusion, temozolomide combined with CED of micellar Am80 may be a promising method for the treatment of malignant gliomas.

Vital microscopic analysis of polymeric micelle extravasation from tumor vessels: macromolecular delivery according to tumor vascular growth stage.

Particles larger than a specific size have been thought to extravasate from tumor vessels but not from normal vessels. Therefore, various nanoparticles incorporating anticancer drugs have been developed to realize selective drug delivery to solid tumors. However, it is not yet clear whether nanoparticles extravasate readily from all tumor vessels including vessels of microtumors. To answer this question, we synthesized new polymeric micelles labeled with fluorescein isothiocyanate (FITC) and injected them into the tail vein of rats with implanted skinfold transparent chambers. We also analyzed, by means of time-lapse vital microscopy with image analysis, extravasation of FITC micelles from tumor vessels at different stages of growth of Yoshida ascites sarcoma LY80. Polymeric micelles readily leaked from vessels at the interface between normal and tumor tissues and those at the interface between tumor tissues and necrotic areas. The micelles showed negligible extravasation, however, from the vascular network of microtumors less than 1 mm in diameter and did not accumulate in the microtumor. Our results suggest that we must develop a novel therapeutic strategy that can deliver sufficient nanomedicine to microtumors.

The combretastatin derivative (Cderiv), a vascular disrupting agent, enables polymeric nanomicelles to accumulate in microtumors.

A previous study found almost no leakage of polymeric nanomicelles from vessels in microtumors. If such vessels become leaky, sufficient nanomedicines may be delivered to microtumors and large tumors. To create leaky vessels, a combretastatin derivative (Cderiv), a vascular disrupting agent, was used. Via vital microscopy with fluorescein isothiocyanate (FITC)-labeled nanomicelles, the effect of Cderiv pretreatment on changes in micelle extravasation was investigated. Whether such treatment would prolong microtumor retention of micelles was also examined. FITC-albumin was used for comparison. The degree of extravasation from intact vessels in microtumors (rat sarcoma LY80) was extremely low and comparable to that from normal vessels. Cderiv pretreatment (1 or 3 days before administration of FITC-labeled compounds) markedly enhanced extravasation of such nanomicelles and albumin from vessels that survived treatment and had restored blood flow. A high concentration of extravasated macromolecules remained even 24 h later in tissue areas whose microcirculatory function had collapsed. Tumors receiving 10 Gy irradiation 3 days before the macromolecules evidenced gradual removal of extravasated macromolecules, which did not accumulate in those areas, despite extravasation from tumor vessels. Our results strongly suggest that pretreatment with Cderiv is quite effective for maintaining microtumor concentrations of nanomicelles and albumin associated with anticancer or diagnostic drugs.

Histological study on side effects and tumor targeting of a block copolymer micelle on rats.

Histological examinations were performed with polymeric micelle-injected rats for evaluations of possible toxicities of polymeric micelle carriers. Weight of major organs as well as body weight of rats was measured after multiple intravenous injections of polymeric micelles forming from poly(ethylene glycol)-b-poly(aspartate) block copolymer. No pathological toxic side effects were observed at two different doses, followed only by activation of the mononuclear phagocyte system (MPS) in the spleen, liver, lung, bone marrow, and lymph node. This finding confirms the absence of--or the very low level of--in vivo toxicity of the polymeric micelle carriers that were reported in previous animal experiments and clinical results. Then, immunohistochemical analyses with a biotinylated polymeric micelle confirmed specific accumulation of the micelle in the MPS. The immunohistochemical analyses also revealed, first, very rapid and specific accumulation of the micelle in the vasculatures of tumor capsule of rat ascites hepatoma AH109A, and second, the micelle's scanty infiltration into tumor parenchyma. This finding suggests a unique tumor-accumulation mechanism that is very different from simple EPR effect-based tumor targeting.

Therapeutic efficacy of a polymeric micellar doxorubicin infused by convection-enhanced delivery against intracranial 9L brain tumor models.

Convection-enhanced delivery (CED) with various drug carrier systems has recently emerged as a novel chemotherapeutic method to overcome the problems of current chemotherapies against brain tumors. Polymeric micelle systems have exhibited dramatically higher in vivo antitumor activity in systemic administration. This study investigated the effectiveness of CED with polymeric micellar doxorubicin (DOX) in a 9L syngeneic rat model. Distribution, toxicity, and efficacy of free, liposomal, and micellar DOX infused by CED were evaluated. Micellar DOX achieved much wider distribution in brain tumor tissue and surrounding normal brain tissue than free DOX. Tissue toxicity increased at higher doses, but rats treated with micellar DOX showed no abnormal neurological symptoms at any dose tested (0.1-1.0 mg/ml). Micellar DOX infused by CED resulted in prolonged median survival (36 days) compared with free DOX (19.6 days; p = 0.0173) and liposomal DOX (16.6 days; p = 0.0007) at the same dose (0.2 mg/ml). This study indicates the potential of CED with the polymeric micelle drug carrier system for the treatment of brain tumors.

A cost-effective method for the optical transduction of chemical reactions. Application to hyaluronidase inhibitor screening with polyarginine-counteranion complexes in lipid bilayers.

We report the systematic elaboration of a cost-effective, interference-minimized assay for the label-free optical transduction of chemical reactions. Recently, we have found that certain complexes formed by arginine-rich cell-penetrating peptides (CPPs) and amphiphilic counteranions can act as synergistic anion carriers in lipid bilayer membranes. Application of this discovery to rapid and reversible cytosolic CPP delivery has been described (Futaki, S.; et al. ACS Chem. Biol. 2006, 1, 299). Here, we report the complementary use of polyarginine (pR)-counteranion complexes as general optical transducers of chemical reactions. Counterion screening revealed dodecyl phosphate (DP) as an ideal pR activator. Carboxyfluorescein (CF)-loaded vesicles with a shelf life of 3.5 years served best for the detection of fluorogenic CF release by pR-DP complexes with the naked eye. Inactivation of pR-DP complexes by counterion exchange with hyaluronan (HA) caused no CF emission, while HA removal by hyaluronidase (HAase) did. pR-DP complexes were further compatible with the optical detection of HA immobilization on solid support as well as inhibitor screening for HAase (cromolyn, heparin) with and without substrate immobilization. Controls concerning binary ATP/ADP discrimination for naked-eye kinase detection are mentioned to delineate scope but also limitations of this simple and quite universal method.

Anionic fullerenes, calixarenes, coronenes, and pyrenes as activators of oligo/polyarginines in model membranes and live cells.

We report that the efflux of 5(6)-carboxyfluorescein anions from neutral egg yolk phosphatidylcholine vesicles is mediated by oligo/polyarginines only in the presence of activating amphiphilic anions. Screening of anion activators reveals best synergism for amphiphilic carboxylates (fullerene > calix[4]arene approximately coronene > pyrene > calix[6]arene > alkyl), whereas amphiphilic sulfates show less satisfactory activation despite often lower effective concentrations. The analogous alcohols and one calix[4]arene diphosphate were inactive. These results are discussed in the context of a tentative anion carrier mechanism, where interactions with bilayer (interface-directed translocation) and carrier (arene-templated carboxylate-guanidinium pairing) contribute to activator efficiencies. Applied to HeLa cells, pyrenebutyrate is shown to significantly increase the uptake of a fluorescently labeled octaarginine in a concentration-dependent manner.