Effective nose-to-brain drug delivery using a combination system targeting the olfactory region in monkeys.

The nose-to-brain (N2B) pathway has garnered attention because it transports drugs directly into the brain. Although recent studies have suggested the necessity of selective drug administration to the olfactory region for effective N2B drug delivery, the importance of delivering the formulation to the olfactory region and the detailed pathway involved in drug uptake in primates brain remain unclear. Here, we developed a combination system for N2B drug delivery comprising a proprietary mucoadhesive powder formulation and a dedicated nasal device (N2B-system) and evaluated it for nasal drug delivery to the brain in cynomolgus monkeys. This N2B-system demonstrated a much greater formulation distribution ratio in the olfactory region in an in vitro experiment using a 3D-printed nasal cast and in vivo experiment using cynomolgus monkeys, as compared to that in other nasal drug delivery systems that comprise of a proprietary nasal powder device developed for nasal absorption and vaccination and a commercially available liquid spray. Additionally, Texas Red-labeled dextran (TR-DEX, 3 kDa) was administered using the N2B-system to estimate the drug transition pathway from the nasal cavity to the brain. TR-DEX preferentially localized to the olfactory epithelium and reached the olfactory bulb through the cribriform foramina. Moreover, domperidone, a model drug with poor blood-brain barrier permeability, was administered to assess the brain uptake of medicine after olfactory region-selective administration by using the N2B-system. Domperidone accumulation in the brain was evaluated using positron emission tomography with intravenously administered [18F]fallypride based on competitive inhibition of the dopamine D2 receptor (D2R). Compared to other systems, the N2B-system significantly increased D2R occupancy and domperidone uptake in the D2R-expressing brain regions. The current study reveals that the olfactory region of the nasal cavity is a suitable target for efficient nasal drug delivery to the brain in cynomolgus monkeys. Thus, the N2B-system, which targets the olfactory region, provides an efficient approach for developing effective technology for nasal drug delivery to the brain in humans.

Experience with Photodynamic Therapy Using Indocyanine Green Liposomes for Refractory Cancer.

We reported the development of an effective cancer treatment using a multidisciplinary treatment, including photodynamic therapy (PDT) with indocyanine green (ICG) liposomes and a combination of Lentinula edodes mycelia (LEM) and hydrogen gas inhalation therapy. ICG liposomes were prepared by adding 5 mg of ICG to 50 mL liposomes. Later, 25 mL of ICG liposomes were diluted with 250 mL of 5% glucose solution and administered intravenously to the patient. We selected the multi-laser delivery system (MLDS), a laser irradiator for performing PDT. Further, the patients received a combination of LEM and hydrogen gas inhalation therapy throughout the treatment. We reported two cases of PDT therapy, one with middle intrathoracic esophagus carcinoma and the other with hypopharyngeal cancer. In the first case, the MLDS laser was directly attached to the endoscope and directed to the cancer area with wavelengths of 810 nm. After the treatment, a biopsy demonstrated no tumor recurrence. In the second case, the patient was treated with endovascular PDT using ICG liposomes and MLDS fiber optics. Later, tumor shrinkage was demonstrated after the first round and disappeared after six months. In conclusion, the present findings suggest that the effect of PDT using ICG liposomes with LEM and hydrogen gas may eradicate cancer without burdening patients by enhancing tumor immunity.

Indirect SPECT Imaging Evaluation for Possible Nose-to-Brain Drug Delivery Using a Compound with Poor Blood-Brain Barrier Permeability in Mice.

Single-photon emission computed tomography (SPECT) imaging using intravenous radioactive ligand administration to indirectly evaluate the time-dependent effect of intranasal drugs with poor blood-brain barrier permeability on brain drug distributions in mice was evaluated. The biodistribution was examined using domperidone, a dopamine D2 receptor ligand, as the model drug, with intranasal administration at 0, 15, or 30 min before intravenous [123I]IBZM administration. In the striatum, [123I]IBZM accumulation was significantly lower after intranasal (IN) domperidone administration than in controls 15 min after intravenous [125I]IBZM administration. [123I]IBZM SPECT was acquired with intravenous (IV) or IN domperidone administration 15 min before [123I]IBZM, and time-activity curves were obtained. In the striatum, [123I]IBZM accumulation was clearly lower in the IN group than in the control and IV groups. Time-activity curves showed no significant difference between the control and IV groups in the striatum, and values were significantly lowest during the first 10 min in the IN group. In the IN group, binding potential and % of receptor occupancy were significantly lower and higher, respectively, compared to the control and IV groups. Thus, brain-migrated domperidone inhibited D2R binding of [123I]IBZM. SPECT imaging is suitable for research to indirectly explore nose-to-brain drug delivery and locus-specific biological distribution.

Evaluation of Systemic and Mucosal Immune Responses Induced by a Nasal Powder Delivery System in Conjunction with an OVA Antigen in Cynomolgus Monkeys.

An immune response for a nasal ovalbumin (OVA) powder formulation with an applied nasal delivery platform technology, consisting of a powdery nasal carrier and a device, was evaluated in monkeys with similar upper respiratory tracts and immune systems to those of humans, in order to assess the applicability to a vaccine antigen. Nasal distribution and retention studies using a 3D nasal cavity model and manganese-enhanced MRI were conducted by administering nasal dye and manganese powder formulations with the applied technology. Systemic and mucosal immune responses for the nasal OVA powder formulation were evaluated by determining serum IgG and nasal wash IgA antibody titers. The nasal dye and manganese powder formulations showed wider distribution and longer retention time than did a nasal liquid formulation. The nasal OVA powder formulation also showed comparable and higher antigen-specific IgG antibody titer to an injection and nasal liquid formulation, respectively. Furthermore, antigen-specific IgA antibody response was detected only for the nasal OVA powder formulation. The present study suggests that the technology, originally designed for drug absorption, is promising for nasal vaccines, enabling both a mucosal immunity response as the first line of defense and systemic immunity response as a second line of defense against infection.

Epitope determination of anti rat thy-1 monoclonal antibody that regulates neurite outgrowth.

Glycosylphosphatidylinositol-anchored protein Thy-1 is abundantly expressed on the cell surface of neurons and T lymphocytes in rodents. Although Thy-1 is known to bind integrins as a ligand and to mediate neurite outgrowth and immune responses, its precise function is not fully understood. Previously we produced several anti rat Thy-1 monoclonal antibodies and identified one, 2E11, which induces PC12 cell neurite outgrowth. Here we screened antibodies that inhibit 2E11-induced neurite outgrowth and stimulate or inhibit rat thymocyte aggregation. Since Thy-1 lacks an intracellular region, it requires other membrane-bound molecules for the signal transduction. Hence these antibodies are hypothesized to play key roles in the interaction between Thy-1 and signaling molecules. To elucidate the mechanisms of antibody-induced Thy-1 functions, antibody characterization and epitope determination were carried out. Thy-1 cleavage and mutation revealed that the antibodies recognize not only amino acid sequences, but also the three-dimensional structures consisting of immunoglobulin-like domains. Two antibodies were suggested to bind spatially close to the integrin binding site and crosslink Thy-1 molecules, while a third antibody is believed to inhibit Thy-1 crosslinking and subsequent Thy-1 signaling. The antibodies reported here may therefore function as crosslinkers, agonists, or antagonists that modify Thy-1 signaling.

Characteristics of troponins as myocardial damage biomarkers in cynomolgus monkeys.

Recently, troponin T (TnT) and troponin I (TnI) have been reported as suitable biomarkers of myocardial injury for pre-clinical toxicity studies. The purpose of the present study was to investigate the characteristics of troponins as myocardial damage biomarkers in cynomolgus monkeys. Initially, tissue distribution of biomarkers was investigated in nine organs (including the heart, liver, and kidneys) collected from naive cynomolgus monkeys. The results showed that TnT and TnI were distributed specifically in the heart, and were not detected in other tissues. Secondly, changes in blood biomarker levels and histopathological changes in cardiac tissue were investigated following myocardial injury induced by concomitant administration of isoproterenol (ISO) and vasopressin (VASO). Compared with pre-dosing, TnT and TnI were markedly increased in the ISO + VASO groups, in which severe histopathological changes including necrosis and vacuolation of muscle fibers were observed. In order to investigate the relationship of biomarker levels with the severity of myocardial injury, Spearman's correlation coefficient was calculated between C(max) and AUC and necrosis and vacuolation scores in the heart. A high correlation between necrosis and vacuolation in the heart and TnT and TnI levels was noted. These results suggest that TnT and TnI possess high sensitivity and specificity for myocardial injury in cynomolgus monkeys, and are useful biomarkers for detection of drug-induced myocardial injury in cynomolgus monkeys.