Weakly acidic pH-responsive liposomal content release induced by histidine-modified agents.

Internal stimuli-responsive controlled release from liposomal vesicles is an innovative approach for site-specific delivery of therapeutic drugs. In this study, to enhance the endosomal pH control of drug release from liposomes, a series of histidine-modified pH-sensitive Cn-His (n = 8, 12, 18) agents were designed and utilized as triggers for liposomal content release. The pH-dependent properties of Cn-His-incorporated liposomes were characterized using dynamic light scattering, ζ-potential, and fluorescence spectroscopy. The liposomes maintained a relatively uniform size across all pH conditions. However, the ζ-potential exhibited positive values at endosomal acidic pH levels and neutral or negative values at physiological pH levels. Furthermore, acidic pH-dependent release of both polar content (carboxyfluorescein) and nonpolar content (Nile red) was observed from the Cn-His-incorporated liposomes. Notably, with C12-His as the pH sensitizer, the pH dependence of liposomal content release was significantly evident. This work establishes endosomal pH-controllable liposome platforms, laying the groundwork for developing clinically applicable triggered release formulations.

Anti-Inflammatory Effects of Miyako Bidens pilosa in a Mouse Model of Amyotrophic Lateral Sclerosis and Lipopolysaccharide-Stimulated BV-2 Microglia.

Neuroinflammation is a fundamental feature in the pathogenesis of amyotrophic lateral sclerosis (ALS) and arises from the activation of astrocytes and microglial cells. Previously, we reported that Miyako Bidens pilosa extract (MBP) inhibited microglial activation and prolonged the life span in a human ALS-linked mutant superoxide dismutase-1 (SOD1G93A) transgenic mouse model of ALS (G93A mice). Herein, we evaluated the effect of MBP on microglial activation in the spinal cord of G93A mice and lipopolysaccharide-stimulated BV-2 microglial cells. The administration of MBP inhibited the upregulation of the M1-microglia/macrophage marker (interferon-γ receptor (IFN-γR)) and pro-inflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6) in G93A mice. However, MBP did not affect the increase in the M2-microglia/macrophage marker (IL-13R) and anti-inflammatory cytokines (transforming growth factor (TGF)-ß and IL-10) in G93A mice. BV-2 cell exposure to MBP resulted in a decrease in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) reduction activity and bromodeoxyuridine incorporation, without an increase in the number of ethidium homodimer-1-stained dead cells. Moreover, MBP suppressed the production of lipopolysaccharide-induced pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-6) in BV-2 cells. These results suggest that the selective suppression of M1-related pro-inflammatory cytokines is involved in the therapeutic potential of MBP in ALS model mice.

Efficacy of Zinc Acetate in the Treatment of Zinc Deficiency in Elderly Inpatients and Effect of Total Dose on Its Replacement Therapy.

We aimed to determine the efficacy of zinc acetate hydrate (ZAH) treatment for hypozincemia in elderly inpatients and to identify the factors affecting its therapeutic effect. We enrolled 79 patients with a mean age of 82 years. The mean serum zinc level before ZAH administration was 53.4 ± 11.5 µg/dL. More than half of the patients (67%) had zinc deficiency (<60 µg/dL), whereas 33% had subclinical zinc deficiency (60-80 µg/dL). The median increase in serum zinc level per ZAH tablet (25 mg) was 1.00 µg/dL. Based on the cutoff value, two groups were identified: slight increase (<1.00 µg/dL) and marked increase (≥1.00 µg/dL) groups; the difference between the two groups was significant (0.57 ± 0.22 µg/dL, n = 39 vs. 1.68 ± 0.70 µg /dL, n = 40; p < 0.0001, Wilcoxon rank sum test). Logistic regression analysis using total zinc dose, serum albumin level, impaired renal function, and diuretics as multivariate variables revealed a significant difference in total zinc dose (total number of tablets per 25 mg tablet: odds ratio 1.056, 95% confidence interval 1.019-1.095, p = 0.003). A significant increase in serum zinc levels was observed in the group with a total zinc dose of less than 1000 mg. The results suggest that an increasing trend in total zinc dose is associated with a low increase in serum zinc levels. Therefore, for the treatment of zinc deficiency in elderly inpatients, serum zinc levels need to be measured once, at a total dose of 1000 mg after initiation of ZAH.

Monitoring of Cocrystal Dissociation during the Wet Granulation Process in the Presence of Disintegrants by Using Low-Frequency Raman Spectroscopy.

The aim of this study was to evaluate the effect of three coformers and five disintegrants in the granulation formulation on the dissociation of cocrystal during the granulation process by monitoring wet granulation with probe-type low-frequency Raman (LF-Raman) spectroscopy. As model cocrystals, paracetamol (APAP)-oxalic acid (OXA), APAP-maleic acid (MLA), and APAP-trimethylglycine (TMG) were used. The monitoring of the granulation recipe containing cocrystals during wet granulation was performed over time with high-performance LF-Raman spectrometry and the dissociation rate was calculated from the results of multivariate analysis of LF-Raman spectra. The dissociation rate decreased in the order of APAP-TMG, APAP-OXA, and APAP-MLA, showing the same order as observed in Powder X-ray diffraction measurements. Furthermore, to compare the effect of disintegrants on the dissociation rate of APAP-OXA, LF-Raman monitoring was performed for the granulation recipes containing five typical disintegrants (two low-substitution hydroxypropyl cellulose (HPC), cornstarch (CSW), carmellose sodium (CMC), and crospovidone (CRP)). The dissociation rate of APAP-OXA decreased in the order of CSW, HPCs, CMC, and CRP. This difference in the dissociation rate of APAP-OXA was thought to be due to the disintegration mechanism of the disintegrants and the water absorption ratio, which was expected to affect the water behavior on the disintegrant surface during wet granulation. These results suggested that probe-type LF-Raman spectroscopy is useful to monitor the dissociation behavior of cocrystals during wet granulation and can compare the relative stability of cocrystal during wet granulation between different formulations.

Molecular State of Active Pharmaceutical Ingredients in Ketoprofen Dermal Patches Characterized by Pharmaceutical Evaluation.

The molecular states of ketoprofen and the interaction between ketoprofen and other pharmaceutical excipients in the matrix layer were examined to determine their effect on the pharmaceutical properties of original and generic ketoprofen dermal patches (generic patches A and B). Molecular states of ketoprofen were evaluated using polarized light microscopy, Raman spectroscopy and powder X-ray diffraction. For the original ketoprofen patch, crystalline components were not observed in the matrix layer. However, crystalline ketoprofen was observed in the two generic ketoprofen patches. Moreover, the ketoprofen exhibited hydrogen bonding with the pharmaceutical excipients or patch materials in the generic products. Skin permeation of ketoprofen from the patches was evaluated using hairless mouse skin. Twelve hours after application, the original patch demonstrated the highest level of cumulative skin permeation of ketoprofen. This was followed by generic patch B while generic patch A showed the lowest level of permeation. Fluxes were calculated from the skin permeation profiles. The original patch was approx. 2.4-times faster compared with generic patch A and approximately 1.9-times faster compared with generic patch B. This investigation suggested that pharmaceutical properties such as skin permeability for these types of products are affected by the precipitation of crystalline ketoprofen in the matrix layer and the interaction of ketoprofen with the pharmaceutical excipients or patch materials.

Comparison of the relative stability of pharmaceutical cocrystals consisting of paracetamol and dicarboxylic acids.

OBJECTIVE: The aim of this study is to evaluate the relative stability of pharmaceutical cocrystals consisting of paracetamol (APAP) and oxalic acid (OXA) or maleic acid (MLA). SIGNIFICANCE: These observations of cocrystal stability under various conditions are useful coformer criteria when cocrystals are selected as the active pharmaceutical ingredient in drug development. METHOD: The relative stability was determined from the preferentially formed cocrystals under various conditions. RESULT: Cocrystal of APAP-OXA was more stable than that of APAP-MLA in a ternary cogrinding system and possessed thermodynamical stability. On the other hand, when grinding with moisture or maintaining at high temperatures and relative humidity conditions, APAP-MLA was more stable, and OXA converted to OXA dihydrate. In the slurry method, APAP-OXA was more stable in aprotic solvents because the APAP-OXA with low-solubility product precipitated. CONCLUSIONS: The relative stability order was affected by preparing conditions of presence of moisture. This order might attribute to the small difference of crystal structure in the extension of the hydrogen bond network.

Involvement of a proton-coupled organic cation antiporter in the blood-brain barrier transport of amantadine.

The blood-to-brain transport of amantadine, a weak N-methyl-d-aspartate (NMDA) antagonist, has been shown previously to participate in the cationic drug-sensitive transport system across the mouse blood-brain barrier (BBB). The purpose of the present study was to characterize the influx transport system by means of both an in situ mouse brain perfusion technique and in vitro studies using rat immortalized brain capillary endothelial cells (GPNT). The observed concentration-dependent initial uptake rate of [(3) H]amantadine suggested the involvement of a carrier-mediated transport mechanism. The normal uptake at physiological pH 7.4 was decreased by 72.9% in acidic perfusate, while it was increased by 35.3% in alkaline perfusate. These results suggest that pH-dependent transport is regulated by utilizing an oppositely directed proton gradient as a driving force. In addition, the [(3) H]amantadine uptake was moderately inhibited by the adamantane structural analogs (rimantadine and memantine) and other cationic drugs (pyrilamine, clonidine, nicotine, etc.), but not by substrates or inhibitors of the well-characterized organic cation transporters (tetraethylammonium, l-carnitine and choline). A similar inhibition pattern was observed between the in vivo studies and the in vitro experiments. These results indicate that the influx transport for amantadine across the BBB involves a proton-coupled organic cation antiporter. Copyright © 2016 John Wiley & Sons, Ltd.

Analysis of the Stability of External-Application Dermatologic Preparations: Consideration from Rheological Measurements.

The present study examined the stability of mixtures of various combinations of moisturizers, water in oil (w/o)-type or oil in water (o/w)-type cream preparations containing heparinoids, and steroidal ointments or creams (o/w-type) frequently used in children. Centrifugation at room temperature led to separation of mixtures of w/o-type moisturizers and steroidal ointments into three layers. Polarized microscopic observations, near-infrared (NIR) spectroscopy, and dye-based analyses revealed the presence of oily components in the upper and middle layers and water-soluble components in the lower layer. Separation into three layers upon centrifugation was also observed for mixtures of o/w-type moisturizers and steroidal ointments. In contrast, neither the o/w-type moisturizer and steroidal cream nor the w/o-type moisturizer and steroidal cream mixtures separated into layers upon centrifugation. Consideration of the characteristics of each preparation is necessary when mixing external-application dermatologic preparations. Centrifugation at 4°C did not result in layer separation of the w/o-type moisturizer and steroidal ointment mixture, suggesting that cold storage of such mixtures provides superior stability compared with room temperature storage. However, despite no obvious layer separation, the NIR spectra indicated that water movement was induced within the mixture. These results clearly indicate that methods such as NIR spectroscopy are useful for early determinations of the stability of mixed external-application dermatologic preparations.

Possible involvement of cationic-drug sensitive transport systems in the blood-to-brain influx and brain-to-blood efflux of amantadine across the blood-brain barrier.

The purpose of this study was to characterize the brain-to-blood efflux transport of amantadine across the blood-brain barrier (BBB). The apparent in vivo efflux rate constant for [(3) H]amantadine from the rat brain (keff ) was found to be 1.53 × 10(-2) min(-1) after intracerebral microinjection using the brain efflux index method. The efflux of [(3) H]amantadine was inhibited by 1-methyl-4-phenylpyridinium (MPP(+) ), a cationic neurotoxin, suggesting that amantadine transport from the brain to the blood across the BBB potentially involves the rat plasma membrane monoamine transporter (rPMAT). On the other hand, other selected substrates for organic cation transporters (OCTs) and organic anion transporters (OATs), as well as inhibitors of P-glycoprotein (P-gp), did not affect the efflux transport of [(3) H]amantadine. In addition, in vitro studies using an immortalized rat brain endothelial cell line (GPNT) showed that the uptake and retention of [(3) H]amantadine by the cells was not changed by the addition of cyclosporin, which is an inhibitor of P-gp. However, cyclosporin affected the uptake and retention of rhodamine123. Finally, the initial brain uptake of [(3) H]amantadine was determined using an in situ mouse brain perfusion technique. Notably, the brain uptake clearance for [(3) H]amantadine was significantly decreased with the co-perfusion of quinidine or verapamil, which are cationic P-gp inhibitors, while MPP(+) did not have a significant effect. It is thus concluded that while P-gp is not involved, it is possible that rPMAT and the cationic drug-sensitive transport system participate in the brain-to-blood efflux and the blood-to-brain influx of amantadine across the BBB, respectively.

Studies on uniformity of the active ingredients in acetaminophen suppositories re-solidified after melting under high temperature conditions.

The target of the present pharmaceutical study was the antipyretic analgesic, acetaminophen; its suppository form is usually split when used in pediatric patients. We focused on the active ingredient uniformity in these products, which were re-solidified after melting under high temperature condition. When sections of the cut surfaces of the seven acetaminophen suppository products (SUP-A-G) commercially available in Japan were visualized by polarized microscopy, acetaminophen crystals that were dispersed in the base were identified. The results of the quantitative determination of agent concentration for each cut portion (mg/g) suggested uniform dispersion of these crystals in the base of each product. The agent concentration in each portion of the suppositories that was re-solidified after melting at high temperatures was measured. Segregation of the active ingredient was observed in four products at a temperature of 40°C for 1 h, while active ingredient uniformity was maintained in the other three products (SUP-C, SUP-F and SUP-G). The latter three products also showed high viscosity at 40°C. At 50°C for 4 h, only the uniformity of the active ingredient in SUP-C was maintained. These results suggest that the uniformity of the active ingredient is lost in some acetaminophen suppositories that were re-solidified after melting under high temperature conditions. The degree of loss varies depending on the product.

Effect of terpenes on the skin permeation of lomerizine dihydrochloride.

PURPOSE: Lomerizine dihydrochloride (LOM) is a Ca2+ channel blocker used as an antimigraine drug, which is currently administered orally in Japan. We therefore investigated the effect of terpenes in propylene glycol (PG) solvent on the percutaneous absorption of LOM by hairless mouse skin. METHODS: Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), confocal laser scanning microscopy (CLSM), and small angle X-ray scattering (SAXS) were carried out to analyze the effects of terpene enhancers on the biophysical properties of the stratum corneum (SC) of the skin. RESULTS: Of the terpenes tested, the highest permeation rate of LOM (28.8 mg/cm2/h) was observed with 1,8-cineole, while nerolidol conferred the lowest enhancement of LOM flux (14.2 mg/cm2/h). ATR-FTIR studies revealed that terpenes/PG induced higher CH2 stretching frequencies of SC lipids than PG alone. The extent of penetration of the lipophilic fluorescence probes Nile Red and DiI was measured by CLSM in in vitro skin permeation studies, using either PG or terpenes/PG as skin permeation enhancers. With PG alone, both fluorescence dyes were undetectable in the skin. In contrast, when co-administered with terpenes/PG, both probes were distributed into the intercellular space between corneocytes and detected in the deeper layers of the skin. SAXS measurements showed that in SC treated with a combination of 1,8-cineole and PG, the scattering peak of the SC was broad and very weak in intensity compared to untreated SC, whereas pretreatment with PG alone did not alter the peak profile. CONCLUSION: A combination of terpenes and PG enhance the skin permeation of LOM. Our findings suggest that the mechanism for this effect involves the ability of terpenes to increase the fluidity of SC lipids, thus enhancing the distribution of LOM into the intercellular region of the SC. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Effects of slow-releasing colistin microspheres on endotoxin-induced sepsis.

Lipopolysaccharide (LPS) is a major contributing factor to endotoxic shock. Colistin specifically binds to LPS. However, it has the disadvantages that adverse reactions are common and it has a short half-life. To overcome these disadvantages, we prepared slow-releasing colistin microspheres and examined the efficacy of these colistin microspheres in a mouse model of endotoxin-induced sepsis. We prepared the colistin microspheres using poly-lactic-co-glycolic acid. For acute toxicity investigations, mice were overdosed with colistin sulfate or colistin microspheres. The group administered with colistin microspheres was associated with less acute toxicity and fewer nephrotoxic changes on histopathological examination compared to the group administered with colistin sulfate alone. For pharmacokinetic analysis, mice were subcutaneously administered with colistin microspheres or colistin sulfate alone. The plasma concentration of colistin was higher in the colistin microspheres group than in the colistin sulfate group at 12 and 24 h after administration. Moreover, mice were intraperitoneally injected with LPS and then immediately subcutaneously administered with blank microspheres, colistin microspheres or colistin sulfate alone. The levels of endotoxin in the sera and cytokine in the spleens were then measured. A significant reduction in the serum endotoxin level in the colistin microspheres group was observed at 24 h. The reduced endotoxin levels in the sera were correlated with the lower cytokine levels in the spleens of mice treated with colistin microspheres. Our results suggest that the use of colistin microspheres may help to maintain a higher colistin concentration in blood, reduce the levels of endotoxin and cytokines in endotoxin-induced sepsis, and lead to decreased toxicity.

Blood-brain barrier transport of an essential amino acid after cerebral ischemia reperfusion injury.

Under pathophysiological conditions such as -cerebral ischemia-reperfusion (IR), damage to cerebrovascular endothelial cells causes alterations in the blood-brain barrier (BBB) function that can exacerbate neuronal cell injury and death. Clarifying changes in BBB transport in the early period of IR is important for understanding BBB function during therapy after cerebral ischemia. The present study was aimed at clarifying changes during IR in the BBB transport of L-phenylalanine (Phe) as a substrate of L-type amino acid transporter 1. An IR model was produced in mice by blood recirculation following occlusion of the middle cerebral artery. Permeability of the BBB to [(3)H]Phe was measured after IR injury using the brain perfusion method. Confocal microscopy of the IR injury showed no brain penetration of fluorescent tracer, thus confirming BBB integrity during 45 min of ischemia. Tight junction opening was not observed at 30 min after reperfusion following ischemia for 45 min. At the time of IR, [(3)H]Phe uptake into the brain appeared saturated. The Michaelis constant and maximum transport velocity in the IR group was reduced by 22 % compared with those in controls. These results suggest that the intrinsic transport clearance of Phe is slightly decreased in the early phase of IR.

Utility of a Novel Micro-Spraying Device for Intranasal Administration of Drug Solutions to Mice.

Intranasal administration has attracted attention as a means of delivering drugs because it bypasses the blood-brain barrier. However, conventional intranasal administration of drug solutions to mice using the micropipette method (MP method) is complicated and time-consuming because it requires small doses to be administered under inhalation anesthesia. This study evaluated the effectiveness of a novel intranasal administration method using Micro FPS™, a novel micro-spraying device (the MSD method). The MSD method allowed more reliable administration of the solution to the nasal mucosa than the MP method did. The transfer of inulin, a model water-soluble macromolecule compound, to the olfactory bulb and brain (cerebrum, cerebellum, brainstem, and striatum) was similar with the two methods. It also allowed the drug to be administered in a shorter time. These results suggest that the MSD method is simpler and more rapid than the MP method for intranasal administration of drugs to mice and achieves comparable delivery of inulin to the olfactory bulb and brain. Therefore, the Micro FPS™ device is a potentially useful tool for intranasal drug administration to rodents and could facilitate the development of intranasal formulations, contributing to drug development for central nervous system diseases.

Stabilization of a supersaturated solution of mefenamic acid from a solid dispersion with EUDRAGIT(®) EPO.

PURPOSE: The stabilization mechanism of a supersaturated solution of mefenamic acid (MFA) from a solid dispersion with EUDRAGIT(®) EPO (EPO) was investigated. METHODS: The solid dispersions were prepared by cryogenic grinding method. Powder X-ray diffractometry, in vitro dissolution test, in vivo oral absorption study, infrared spectroscopy, and solid- and solution-state NMR spectroscopies were used to characterize the solid dispersions. RESULTS: Dissolution tests in acetate buffer (pH 5.5) revealed that solid dispersion showed > 200-fold higher concentration of MFA. Supersaturated solution was stable over 1 month and exhibited improved oral bioavailability of MFA in rats, with a 7.8-fold higher area under the plasma concentration-versus-time curve. Solid-state (1)H spin-lattice relaxation time (T(1)) measurement showed that MFA was almost monomolecularly dispersed in the EPO polymer matrix. Intermolecular interaction between MFA and EPO was indicated by solid-state infrared and (13)C-T(1) measurements. Solution-state (1)H-NMR measurement demonstrated that MFA existed in monomolecular state in supersaturated solution. (1)H-T(1) and difference nuclear Overhauser effect measurements indicated that cross relaxation occurred between MFA and EPO due to the small distance between them. CONCLUSIONS: The formation and high stability of the supersaturated solution were attributable to the specifically formed intermolecular interactions between MFA and EPO.

Evaluation of the degree of mixing of combinations of dry syrup, powder, and fine granule products in consideration of particle size distribution using near infrared spectrometry.

We used near infrared (NIR) spectroscopy to evaluate the degree of mixing of blended dry syrup (DS) products whose particle sizes are not specified in the Revised 16th Edition of the Japanese Pharmacopoeia, and also evaluated the degree of mixing when powder products or fine granule products were added to DS products. The data obtained were used to investigate the relationship between the particle size distributions of the products studied and the degree of mixing. We found that the particle size distribution characteristics of the 15 DS products studied can be broadly classified into 5 types. Combinations of frequently prescribed products were selected to represent 4 of the 5 particle size distribution types and were blended with a mortar and pestle. The coefficient of variation (CV) decreased as the percent mass of Asverin® Dry Syrup 2% (Asverin-DS) increased in blends of Periactin® Powder 1% (Periactin) and Asverin-DS, indicating an improved degree of mixing (uniformity). In contrast, in blends of Periactin and Mucodyne® DS 33.3%, mixing a combination at a 1:1 mass ratio 40 times resulted in a CV of 20%. Other mixing frequencies and mass ratios resulted in a CV by 50% to 70%, indicating a very poor degree of mixing (poor uniformity). These results suggest that when combining different DSs, or a DS with a powder or fine granule product, the blending obtained with a mortar and pestle improves as the particle size distributions of the components approach each other and as the ranges of the distributions narrow.

Application of ionic liquid to enhance the nose-to-brain delivery of etodolac.

The purpose of this study was to enhance the delivery of Etodolac (ETD) to the brain through intranasal administration using an ionic liquid (IL) consisting of ETD and proline ethyl ester. The IL of ETD was prepared by mixing ETD with proline ethyl ester as a counterion in a molar ratio of 1:2.The formation of the IL was confirmed by differential scanning calorimetry (DSC), infrared spectroscopy (IR) and proton nuclear magnetic resonance (1H-NMR).The solubility of ETD in simulated nasal fluids was improved by approximately 200-fold due to the formation of IL. The intranasal administration of ETD-containing IL, which is viscous, increased the nose-to-brain delivery by approximately 7-fold 30 min after an administration of the ETD solution alone. The enhancement of ETD delivery to the brain from the nose was attributed to the enhanced retention of ETD in the nasal mucosal surface due to the viscosity of IL. The induction of prostaglandin E2 in the brain inflammation that was induced by lipopolysaccharides was significantly suppressed by up to 40% in the IL-treated group compared with the drug-untreated group. Therefore, ETD-containing IL were suggested to be useful in designing intranasal formulations for the nasal delivery of ETDs to the brain.

Intranasal Administration of N-acetyl-L-cysteine Combined with Cell-Penetrating Peptide-Modified Polymer Nanomicelles as a Potential Therapeutic Approach for Amyotrophic Lateral Sclerosis.

Intranasal administration is a promising route for direct drug delivery to the brain; its combination with nanocarriers enhances delivery. We have previously shown that intranasal administration combined with PEG-PCL-Tat (a nanocarrier) efficiently delivers drugs to the brain and exhibits excellent therapeutic efficacy against brain diseases. We aimed to clarify whether intranasal administration combined with PEG-PCL-Tat represents a useful drug delivery system (DDS) for amyotrophic lateral sclerosis (ALS) pharmacotherapy. We used N-acetyl-L-cysteine (NAC) as a model drug with low transferability to the spinal cord and determined the physicochemical properties of NAC/PEG-PCL-Tat. After intranasal administration of NAC/PEG-PCL-Tat, we measured the survival duration of superoxide dismutase-1 G93A mutant transgenic mice (G93A mice), widely used in ALS studies, and quantitatively analyzed the tissue distribution of NAC/PEG-PCL-Tat in ddY mice. The mean particle size and zeta potential of NAC/PEG-PCL-Tat were 294 nm and + 9.29 mV, respectively. Treatment with repeated intranasal administration of NAC/PEG-PCL-Tat considerably prolonged the median survival of G93A mice by 11.5 days compared with that of untreated G93A mice. Moreover, the highest distribution after a single administration of NAC/PEG-PCL-Tat was measured in the spinal cord. These results suggest that intranasal administration combined with PEG-PCL-Tat might represent a useful DDS for ALS therapeutics.

Nose-to-brain/spinal cord delivery kinetics of liposomes with different surface properties.

The administration of liposomes via nose-to-brain delivery is expected to become a strategy for efficient drug delivery to the central nervous system. Efficient nose-to-brain delivery and the kinetics of drugs administered in this manner depend on the properties of liposomes. However, there is a lack of basic knowledge of which liposomes are suitable for this purpose. Here, a qualitative study of intranasally administered liposomes (positively charged, neutral, and negatively charged, with or without polyethylene glycol [PEG] modification; particle size <100 nm) was performed to elucidate their dynamics in the brain and spinal cord. Additionally, a quantitative investigation was performed to ascertain their distribution in each part of the brain and spinal cord. The effects of liposome surface charge and PEG modification on the kinetics and distribution post intranasal administration were investigated via two experiments. Qualitative evaluation was performed via ex vivo observation after intranasal administration of fluorescently labeled liposomes. Neutral PEG-modified liposomes were distributed throughout the brain and spinal cord 60 min after administration, and the fluorescence intensity increased with time. By contrast, non-PEG-modified neutral liposomes showed particularly strong fluorescence in the olfactory bulb, and the fluorescence was localized in the anterior part of the brain. Positively charged liposomes showed low fluorescence around the lateral part of the brain and lumbar spinal cord 60 min after administration. Low fluorescence was observed in the whole brain and spinal cord, with strong fluorescence being observed in the olfactory bulb after 120 min of administration. Negatively charged liposomes showed no fluorescence at 60 min after administration, but low fluorescence was observed throughout the brain and spinal cord 120 min after administration. We quantified the radioactivity in the brain and spinal cord after intranasal administration of radioisotope-labeled liposomes. Neutral liposomes showed the highest distribution by area under the drug concentration-time curve (AUC60-120) in the brain and spinal cord compared to other liposomes. Compared with negatively charged liposomes, positively charged liposomes had a higher distribution in the olfactory bulb and forebrain, while negatively charged liposomes had a higher distribution in the hindbrain and bulbospinal tract cord. In addition, the distribution of PEG-modified neutral liposomes in the brain and spinal cord was significantly enhanced compared to that of non-PEG-modified neutral liposomes after 90 min of intranasal administration. These results indicate that surface charge and PEG modification strongly affect the efficiency of nose-to-brain delivery kinetics, and that PEG-modified neutral liposomes are excellent carriers for drug delivery to a wide area of the brain and spinal cord.

Quantitative analysis of inulin distribution in the brain focused on nose-to-brain route via olfactory epithelium by reverse esophageal cannulation.

This study aimed to determine the effect of intranasal dosing speed and administrating volume of nose-to-brain delivery on candidates for peptide drugs (molecular weight ca. 1-10 kDa). Using inulin as the model molecule of a peptide drug, intranasal administration by cannulation from the airway side through the esophagus was tested in mice. This was done to determine the quantitative distribution levels of the drug in the brain and cerebral spinal fluid (CSF). Distribution levels were increased with slower and constant speed (5 µL/min), with higher dosing volume equivalent to nasal volume per body weight in mice (25 µL), and were recorded 0.27% injected dose per gram of tissue (ID/g) in the brain, and 0.24% injected dose per milliliter (ID/mL) in the CSF at 60 min. Then, brain distribution resulting from reverse cannulation was two times more than that of the typical intranasal administration method using a micropipette. In addition, the percentage of inulin estimated to reach the brain via direct transport (%DTP) during reverse cannulation was estimated to be 93%, suggesting that ~95% of the total dose was transferred directly to the brain via the olfactory mucosa. These results show that distribution of the peptide drug in the brain was increased through constant administration at a slow and constant speed.