Yasuo Seta

Delivery of siRNA to the brain using a combination of nose-to-brain delivery and cell-penetrating peptide-modified nano-micelles.

The potential for RNA-based agents to serve as effective therapeutics for central nerve systems (CNS) disorders has been successfully demonstrated in vitro. However, the blood-brain barrier limits the distribution of systemically administered therapeutics to the CNS, posing a major challenge for drug development aimed at combatting CNS disorders. Therefore, the development of effective strategies to enhance siRNA delivery to the brain is of great interest in clinical and pharmaceutical fields. To improve the efficiency of small interfering RNA (siRNA) delivery to the brain, we developed a nose-to-brain delivery system combined with cell-penetrating peptide (CPP) modified nano-micelles comprising polyethylene glycol-polycaprolactone (PEG-PCL) copolymers conjugated with the CPP, Tat (MPEG-PCL-Tat). In this study, we describe intranasal brain delivery of siRNA or dextran (Mw: 10,000 Da) as a model siRNA, by using MPEG-PCL-Tat. Intranasal delivery of dextran with MPEG-PCL-Tat improved brain delivery compared to intravenous delivery of dextran either with or without MPEG-PCL-Tat. We also studied the intranasal transfer of MPEG-PCL-Tat to the brain via the olfactory and trigeminal nerves, the putative pathways to the brain from the nasal cavity. We found that MPEG-PCL-Tat accelerated transport along the olfactory and trigeminal nerve pathway because of its high permeation across the nasal mucosa.

Design and preparation of captopril sustained-release dosage forms and their biopharmaceutical properties

Abstract To obtain a prolonged-action dosage form of captopril, 7 types of formulations were prepared and administered to beagle dogs. They were coated slow-release (CSR) granules, 4 kinds of modified release tablets, enteric coated granules and oily semisolid matrix (OSSM). Each formulation was evaluated by the area under the curve ( AUC ) and time course of plasma captopril concentration after oral administration. The CSR granules and the modified release tablets showed them to be markedly inefficient under fasting conditions. The AUC s of these products were 0.64–1.69 (μg · h/ml), which is about 20–40% of the AUC obtained by conventional tablets under fasting conditions. Under non-fasting conditions, the CSR granules and enteric coated granules suffered additional decreases in AUC . The AUC of the enteric coated granules under non-fasting conditions was only 20% of the AUC with the uncoated granules under fasting conditions. The OSSM maintained higher plasma captopril level for a long time as compared with the CSR granules under the same non-fasting conditions. Although the AUC (0.83 μg · h/ml) of OSSM was only half of the conventional tablets (non-fasting condition), this formulation is expected to offer an effective prolonged-action dosage form of captopril.

Measurement of agitation force in dissolution test and mechanical destructive force in disintegration test

The purpose of this study was to investigate the effect of the agitation force and mechanical destructive force on the drug dissolution of a tablet in the paddle rotation dissolution test and in the disintegration test. The agitation in the paddle method and the mechanical destructive force in the disintegration test were considered to be conclusive factors for drug dissolution. The dissolution rate of planar-constant-release tablets increased with increasing paddle rotation speed and increased with increasing distance from the center of the vessel bottom. Separately, the fluid resistance (agitation force) in the vessel was measured using a modified paddle method apparatus equipped with a fluid resistance sensor. The fluid resistance was 0.03 x 10(-3) N/(64 mm(2)) when the paddle rotation speed was 50 rpm at a position 4 mm away from the center. A considerable position-dependent change in agitation force intensity was seen with the fluid resistance sensor. The impulsive force (mechanical destructive force) in the disintegration test apparatus was measured using a modified basket-rack assembly with a strain gauge transducer. The fluid resistance was measured using the basket-rack assembly with a different sensor probe and amplifier. The impulsive force applied by the auxiliary disk was 0.31 N and the fluid resistance at the bottom of the basket-rack assembly was 1.66 x 10(-3) N/(64 mm(2)).

Topical drug delivery to retinal pigment epithelium with microfluidizer produced small liposomes.

Drug delivery from topically instilled eye drops to the posterior segment of the eye has long been one of the greatest challenges of ocular drug development. We developed methods of liposome preparation utilizing a microfluidizer to achieve adjustable nanoparticle size (even less than 80 nm) and high loading capacity of plasmid DNA. The microfluidizing process parameters were shown to affect the size of the liposomes. Higher operating pressures and passage for at least 10 times through the microfluidizer produced small liposomes with narrow size distribution. The liposomes were physically stable for several months at +4°C. In vivo distribution of the optimized liposome formulations in the rat eyes was investigated with confocal microscopy of the histological specimens. Transferrin was used as a targeting ligand directed to retinal pigment epithelium. Size dependent distribution of liposomes to different posterior segment tissues was seen. Liposomes with the diameter less than 80 nm permeated to the retinal pigment epithelium whereas liposomes with the diameter of 100 nm or more were distributed to the choroidal endothelium. Active targeting was shown to be necessary for liposome retention to the target tissue. In conclusion, these microfluidizer produced small liposomes in eye drops are an attractive option for drug delivery to the posterior segment tissues of the eye.

Cytoplasm-responsive nanocarriers conjugated with a functional cell-penetrating peptide for systemic siRNA delivery

To develop a gene carrier for cancer therapy by systemic injection, we synthesized methoxypolyethylene glycol-polycaprolactone (MPEG-PCL) diblock copolymers conjugated with a cytoplasm-responsive cell-penetrating peptide (CPP), CH2R4H2C (C, Cys; H, His; R, Arg). The carrier/small interfering RNA (siRNA) complexes (N/P ratio of 20) had a particle size of approximately 50 nm and stabilized the siRNA against RNase. The cellular uptake ability of the carrier/FAM-siRNA complexes with fetal bovine serum was significantly higher than that of naked FAM-siRNA. In addition, the carrier/anti-vascular endothelial growth factor siRNA (siVEGF) complexes attained a significantly greater silencing effect than naked siVEGF with low cytotoxicity, resulting from higher uptake, early endosomal escape, and efficient release from the complexes in the cytoplasm. Furthermore, intravenous injection of MPEG-PCL-CH2R4H2C/siVEGF complexes had a significantly higher anti-tumor effect in S-180 tumor-bearing mice, which could be attributed to the rigid compaction of siRNA by ionic interactions and disulfide linkages in the CPP polymer micelles in the blood, as well as higher release following cleavage of the disulfide bonds in the reductive cytosol. Taken together, our data demonstrated that these cytoplasm-responsive polymer micelles conjugated with multi-functional CPP, could facilitate siVEGF delivery to tumor tissues after systemic injection and could exert an extremely strong anti-tumor effect.

Preparation and pharmacological evaluation of captopril sustained-release dosage forms using oily semisolid matrix

Abstract The stability of captopril in the gastrointestinal (GI) tract was estimated by means of in vitro experiments to clarify the reasons why bioavailability of captopril was reduced when administered after meals. It was found that captopril became dramatically unstable in the presence of food, and that the addition of ascorbic acid to the system improved its stability. Oily semisolid matrix (OSSM) and enteric coated granules, ascorbic acid was formulated in both dosage forms, were prepared and administered to beagle dogs to ascertain the stabilization effect in vivo. Both prolonged-action dosage forms showed bioavailability improved, but ascorbic acid worked much more effectively with OSSM. The time courses of captopril plasma concentration, inhibition of angiotensin-converting enzyme (ACE) activity and the inhibition of presser response to angiotension I (AI) were measured following single oral administration of captopril OSSM and conventional tablets (25 mg/T × 2) to non-fasting beagle dogs. The OSSM contained 50 mg of captopril and 250 mg of ascorbic acid. The beagle dogs were pretreated with i.v. administration of AI to obtain a simulated hypertensional state. The OSSM showed longer pharmacological action of captopril than the conventional tablets. The OSSM maintained more than 80% inhibition of ACE activity for over 8.5 h, while conventional tablets could maintain the same effect for only 2.5 h.

Design of captopril sustained-release preparation with oily semisolid matrix intended for use in human subjects

Abstract A captopril sustained-release dosage form using oily semisolid matrix (OSSM) has been studied to develop a formulation useful in human treatment. Four OSSMs which had different in vitro dissolution rates were administered to human subjects. The resulting bioavailabilities revealed that the best OSSM suitable for humans had a faster in vitro dissolution rate than that for dogs. Another series of administrations of OSSM also showed that formulated ascorbic acid improved the bioavailability of OSSM, and that the bioavailability was sufficient for a sustained-release dosage form so long as the amount of ascorbic acid in the OSSM was more than 5 times that of the captopril by weight. Pharmacokinetic analysis was performed based on plasma concentrations of captopril in humans ( n = 8) after a single oral administration of conventional tablets or OSSM reformulated for humans. Calculated areas under the curve ( AUC s, 0-∞ h) of plasma captopril concentration were 250.5 for conventional tablets and 283.5 (ng·h/ml) for OSSM. The mean residence times (MRTs) obtained by both formulations were 1.75 h and 3.59 h, respectively. The duration time in which plasma captopril concentration stayed above 50% inhibitory concentration of angiotensin converting enzyme activity was calculated. Total duration time (TDT) per day of conventional tablets (12.5 mg) taken 3 times daily was calculated to be 10.95 h. The TDT of OSSM was 17.00 h when the OSSM (18.75 mg of captopril) was administered twice a day at 12-h intervals. Consequently, OSSM dosed twice a day is expected to show a greater efficiency than conventional tablets taken 3 times daily.

Topical Anti-Nuclear Factor-Kappa B Small Interfering RNA with Functional Peptides Containing Sericin-Based Hydrogel for Atopic Dermatitis

The small interfering RNA (siRNA) is suggested to offer a novel means of treating atopic dermatitis (AD) because it allows the specific silencing of genes related to AD pathogenesis. In our previous study, we found that siRNA targeted against RelA, an important nuclear factor-kappa B (NF-κB) subdomain, with functional peptides, showed therapeutic effects in a mouse model of AD. In the present study, to develop a topical skin application against AD, we prepared a hydrogel containing anti-RelA siRNA and functional peptides and determined the intradermal permeation and the anti-AD effects in an AD mouse model. We selected the silk protein, sericin (SC), which is a versatile biocompatible biomaterial to prepare hydrogel as an aqueous gel base. We found that the siRNA was more widely delivered to the site of application in AD-induced ear skin of mice after topical application via the hydrogel containing functional peptides than via the preparation without functional peptides. In addition, the ear thickness and clinical skin severity of the AD-induced mice treated with hydrogel containing anti-RelA siRNA with functional peptides improved more than that of mice treated with the preparation formulated with negative siRNA.

Functional peptide nanocarriers for delivery of novel anti-RelA RNA interference agents as a topical treatment of atopic dermatitis.

Small interfering RNAs (siRNAs) are a potential treatment of atopic dermatitis (AD) because they can specifically silence the gene expression of AD-related factors. However, siRNA alone cannot exert a sufficiently strong therapeutic effect due to low delivery efficiency to the target tissues and cells; simply increasing the amount used is not possible due to the possibility of off-target effects. We previously reported a novel class of therapeutic RNA interference (RNAi) agents called nkRNA(®) and PnkRNA(®), which have been shown to be effective in several disease models, have greater resistance to nuclease degradation than canonical siRNAs, and do not induce any immunotoxicity. In the present study, we describe a non-invasive and effective transdermal RNAi therapeutic system for atopic dermatitis that uses the functional cell-penetrating stearoyl-oligopeptide OK-102 as a cytoplasm-responsive nanocarrier for nkRNA(®) and PnkRNA(®). The two RNAi agents were targeted against RelA, a subclass of NF-κB (nuclear factor kappa B), and, as part of OK-102 complexes, they strongly silenced RelA mRNA in macrophage cells and demonstrated a significant therapeutic effect in a mouse model of AD. It was shown that OK-102-complexed RNAi agents were an efficient therapeutic system for AD and caused no adverse reactions.

Systemic delivery of small interfering RNA targeting nuclear factor κB in mice with collagen-induced arthritis using arginine-histidine-cysteine based oligopeptide-modified polymer nanomicelles

This study aimed to build an innovative system to deliver a systemic small interfering RNA (siRNA) treatment for rheumatoid arthritis. We combined arginine-histidine-cysteine based oligopeptide-modified polymer micelles with siRNA targeting the nuclear factor κB subunit, RelA (siRelA). This is a key molecule in the control of inflammation. We tested the cellular uptake of siRNA and its effects on inflammatory cytokine levels in vitro using synoviocytes, and siRNA distribution and therapeutic effects in vivo in mice with collagen-induced arthritis (CIA). These studies showed that arginine-histidine based oligopeptide modified micelles produced effective cellular siRNA uptake and suppressed inflammatory cytokine levels in synoviocytes. In vivo, these micelles produced marked accumulation of siRNAs in arthritic paws in CIA mice, with much less accumulation in healthy mice. The siRelA-polymer micelle complexes also produced more effective suppression of RelA mRNA expression and inflammatory cytokine levels in the arthritic paws of CIA mice and reduced their clinical symptom scores and paw thickness.