Takatoshi Sakamoto

Improvement of dermatitis by iontophoretically delivered antisense oligonucleotides for interleukin-10 in NC/Nga mice

IL-10 is overexpressed in skin lesions of atopic dermatitis (AD) patients and believed to be an important factor in the pathogenesis of the disease. Thus the regulation of IL-10 production is a potential solution for immunotherapeutic intervention in AD. We examined the topical delivery of an antisense oligonucleotide for mouse IL-10 (AS6) and the therapeutic effect on the skin lesions of NC/Nga mice, a human AD model. Using an iontophoresis system, about 30% of the applied dose of AS6 penetrated the skin and was distributed in the epidermis and upper dermis. Topically delivered AS6 decreased the levels of mRNA and protein of IL-10 in the lesions of NC/Nga mice, with no effect on IL-4 levels. The dorsal lesions of NC/Nga mice disappeared with repeated topical application of AS6. Topically delivered AS6 showed an inhibitory effect on the production of IL-10 in the skin lesions of NC/Nga mice and had a therapeutic effect on the established dermatitis.

Changes in Immune Responses to Antigen Applied to Tape-Stripped Skin with CpG-Oligodeoxynucleotide in NC/Nga Mice

PurposeCpG-oligodeoxynucleotide (CpG-ODN) plays a critical role in immunity via the augmentation of Th1 and the suppression of Th2 responses. We examined here the effect of CpG-ODN on the immune response to an antigen applied to a tape-stripped skin of NC/Nga mouse, a human atopic dermatitis (AD) model, by evaluating the production of cytokines and immunoglobulin isotypes.MethodsModel antigen, ovalbumin (OVA), and CpG-ODN were applied on to the shaved skin. The penetration of OVA and CpG-ODN was evaluated using confocal laser scanning microscopy (CLSM). Secretion of cytokine from splenocytes and changes in immunoglobulin isotype levels were determined by enzyme-linked immunosorbent assay (ELISA).ResultsThrough CLSM it was revealed that the model antigen, OVA, and CpG-ODN easily penetrated the tape-stripped skin. Coadministration of CpG-ODN and OVA to the skin elicited an antigen-specific, Th1-predominant immune response and enhanced the production of IFN-γ. On the other hand, the production of a Th2-type cytokine, IL-4, was drastically suppressed. In terms of antigen-specific antibody production, the level of IgG2a regulated by IFN-γ was increased by CpG-ODN, but IgE production regulated by IL-4 was suppressed.ConclusionsAdministration of CpG-ODN with antigen through the skin may shift the immune response from a Th2- to Th1-like response. These results suggested that administration of CpG-ODN via skin is a simple strategy for patients with diseases such as AD, which is characterized by Th2-dominated inflammation.

Design of potent phosphorothioate antisense oligonucleotides directed to human interleukin 10 gene product and their evaluation of antisense activity in U937 cells.

AbstractPurpose. The two objectives of this study were to design potent phosphorothioate antisense oligonucleotides (AS-S-oligos) directed against the human interleukin-10 (IL-10) gene product and to reveal the DNA sequence which best activates antisense effects. Methods. The design of potent AS-S-oligo was performed by using melting temperature (Tm) value of a DNA/RNA hybrid calculated by the nearest neighbor method and a secondary structure of human IL-10 mRNA suggested by RNA folding algorithms. U937 cells were used to estimate the antisense effect of the AS-S-oligos. Results. Of the eight candidates selected as potent AS-S-oligos on the basis of having higher Tm values and favorable secondary structures of the IL-10 mRNA, AS-S-oligos directed against the translated (AS367-S-oligo) and 3′-untranslated (AS637-S-oligo) region of IL-10 mRNA showed the strongest inhibitory effects on IL-10 production and this inhibition was dose- and time-dependent. Reverse transcription-polymerase chain reaction (RT-PCR) revealed that the antisense effects of AS-S-oligos originated from a specific reduction of target IL-10 mRNA by hybridization with AS367- and AS637-S-oligos. In addition, these AS-S-oligos did not affect human tumor necrosis factor-∝ (TNF-∝) production in the cells stimulated by lipopolysaccharide (LPS). Strong positive correlations between the inhibitory effect of AS-S-oligos on the IL-10 production and not only Tm values calculated by nearest neighbor method but also Tm values determined by absorbance versus temperature profiles were demonstrated except for AS25-S-oligo and AS1249-S-oligo. Conclusions. These findings suggest AS367- and AS637-S-oligos powerfully inhibit IL-10 production in U937 cells via an antisense mechanism. In addition, it is suggested efficiency of AS-S-oligo directed against the sequence of the target gene product can be explained by these Tm values and the proposed secondary structures of the target gene product.

Predicting the Occurrence of Sticking during Tablet Production by Shear Testing of a Pharmaceutical Powder

Sticking is a failure of pharmaceutical production that occurs when a powder containing a large amount of adhesive is being tableted. This is most frequently observed when long-term tableting is carried out, making it extremely difficult to predict its occurrence during the tablet formula design stage. The efficiency of the pharmaceutical production process could be improved if it were possible to predict whether a particular formulation was likely to stick during tableting. To address this issue, in the present study we prepared tablets composed of blended ibuprofen (Ibu), a highly adhesive drug, and measured the degree of adherence of powder particles to the surface of the tablet punch. We also measured the shear stress of the powder to determine the practical angle of internal friction (Φp) of the powder bed as well as the angle of wall friction (Φw) relative to the punch surface. These values were used to define a sticking index (SI), which showed a high correlation with the amount of Ibu that adhered to the punch during tableting; sticking occurred at SI >0.3. When the amount of lubricant added to the formulation was changed to yield tablets exhibiting different SI values without changing the compounding ratio, sticking did not occur at SI ≤0.3. These results suggest that determining the SI of a pharmaceutical powder before tableting allows prediction of the likelihood of sticking during tableting.

Evaluation of Sucrose Fatty Acid Esters as Lubricants in Tablet Manufacturing

Lubricants are essential additives in tablet formulations. Magnesium stearate (Mg-St) is the most commonly used lubricant in tableting. Here, we used sucrose fatty acid ester (SE) as an additive to manufacture tablets by direct compression. We evaluated the effects of hydrophile-lipophile balance (HLB) and the amount of SE on the flowability of a pharmaceutical powder using angle of repose and practical angle of internal friction measurements. In addition, we investigated the effects of SE on tablet properties. When SEs with an HLB ≥3 were added, the angle of repose was approximately the same as that of a pharmaceutical powder containing Mg-St, with no major differences in flowability. However, the practical angle of internal friction became closer to pharmaceutical powder containing Mg-St as HLB decreased. As HLB increased, the practical angle of internal friction approached the value of additive-free pharmaceutical powder. Tablets containing 2.0% Mg-St had a mean hardness of 40 N and disintegrated in approximately 6 min, whereas tablets containing 2.0% SE (low HLB) had a mean hardness of approximately ≥80 N and disintegrated within 3 min. The results indicate that SEs can be used as lubricants in tablet production by direct compression and to reduce problems associated with the use of Mg-St. In particular, we suggest that SEs with low HLB values can be used as excipients to achieve high tablet hardness and short disintegration time.

Mechanism by Which Magnesium Oxide Suppresses Tablet Hardness Reduction during Storage

This study investigated how the inclusion of magnesium oxide (MgO) maintained tablet hardness during storage in an unpackaged state. Tablets were prepared with a range of MgO levels and stored at 40°C with 75% relative humidity for up to 14 d. The hardness of tablets prepared without MgO decreased over time. The amount of added MgO was positively associated with tablet hardness and mass from an early stage during storage. Investigation of the water sorption properties of the tablet components showed that carmellose water sorption correlated positively with the relative humidity, while MgO absorbed and retained moisture, even when the relative humidity was reduced. In tablets prepared using only MgO, a petal- or plate-like material was observed during storage. Fourier transform infrared spectrophotometry showed that this material was hydromagnesite, produced when MgO reacts with water and CO2. The estimated level of hydromagnesite at each time-point showed a significant negative correlation with tablet porosity. These results suggested that MgO suppressed storage-associated softening by absorbing moisture from the environment. The conversion of MgO to hydromagnesite results in solid bridge formation between the powder particles comprising the tablets, suppressing the storage-related increase in volume and increasing tablet hardness.

Selective and enhanced transgene expression in hepatocellular carcinoma cells by asialofetuin-labelled liposomes and AFP promoter

Abstract In the present study, it was showed that combination with the plasmid DNA of genes under the control of α-fetoprotein (AFP) promoter and asialofetuin-labelled cationic liposomes (AF-liposomes) achieved specific and enhanced gene expression in AFP high-producing HCC cells. The plasmids, containing luciferase gene (pAFP-luc) or herpes simplex virus thymidine kinase gene (pAFP-tk) under the human AFP enhancer/silencer/promoter sequence, were complexed with AF-liposomes and transfected to established cancer cell lines. The transfection of pAFP-luc with AF-liposomes led to higher luciferase expression than that with control cationic liposomes in AFP high-producing hepatocellular carcinomas (HCC). Conversely, almost no expression was observed in AFP non-producing cell lines. Exposure of HepG2 cells to acyclovir following transfection of pAFP-tk with AF-liposomes decreased viability of the cells significantly. These results suggest AF-liposomes significantly enhance specific transgene expression driven by AFP promoter and have the potentiality of a non-viral vector in gene therapy for AFP-producing HCC.