Effect of Ointment Base on the Skin Wound-Healing Deficits in Streptozotocin-Induced Diabetic Rat.

Wound-healing deficits of the skin, one of the most common complications in patients with diabetes, delay wound healing, significantly reducing the patient's QOL. Therefore, the topical treatment of wound areas with drug-containing ointments and dressings is important. In this study, we investigated the effect of various ointment bases on skin wound healing in normal and streptozotocin-induced diabetic rats (STZ rats). Three ointment bases were used: white ointment (oil-based), absorbent cream (emulsion-based, w/o), and macrogol ointment (water-based). Skin wound healing in STZ rats was delayed compared with that in normal rats. Each of the three ointment bases was applied to the skin wound area in normal rats, and there was no difference in the therapeutic effect. The therapeutic effect of both white ointment and absorbent cream was higher in the STZ rats group than that in the non-treated group, and delayed wound healing was observed in STZ rats treated with macrogol ointment. In conclusion, skin wound healing in STZ rats is affected by the properties of the ointment base, and it is important to use an ointment base that controls the drying of the wound area in STZ rats. These findings provide information for the selection of ointment bases useful for application to skin wounds in patients with diabetes.

An Ophthalmic Formulation of Disulfiram Nanoparticles Prolongs Drug Residence Time in Lens.

Disulfiram (DSF) is a dimer of diethyldithiocarbamate (DDC) that we previously added to a solution of 2-hydroxypropyl-ß-cyclodextrin (DSF solution). We found that the instillation of this DSF solution delayed lens opacification in a hereditary cataractous ICR/f rat. In this study, we attempted to design an ophthalmic formulation containing DSF nanoparticles for use as a lens targeted drug delivery system (nano-DSF suspension), and investigated the changes in drug content in the lens after the instillation of DSF solution or nano-DSF suspension. The nano-DSF suspension was prepared by a bead mill method to yield a mean particle size of nano-DSF of 181 nm. Following the instillation of 1.4% DSF solution or the nano-DSF suspension, DDC was detected only in the aqueous humor and lens; in both, the area under the curve (AUC) and mean residence time (MRT) for the nano-DSF suspension were higher than for the DSF solution. In addition, we found that the DDC residence time in the cortex and nucleus of the lens was higher than in the capsule-epithelium. Although DDC was not detected in the cortex and nucleus of lenses following the instillation of the 1.4% DSF solution, the instillation of a 1.4% nano-DSF suspension led to the accumulation of DDC in both areas. In conclusion, it is possible that the instillation of a nano-DSF suspension can supply more DDC into the aqueous humor and lens than a conventional formulation, and these findings provide information significant for the prevention of cataracts and the design of a lens targeted drug delivery system.

Sequence and structural determinants of human APOBEC3H deaminase and anti-HIV-1 activities.

BACKGROUND: Human APOBEC3H (A3H) belongs to the A3 family of host restriction factors, which are cytidine deaminases that catalyze conversion of deoxycytidine to deoxyuridine in single-stranded DNA. A3 proteins contain either one (A3A, A3C, A3H) or two (A3B, A3D, A3F, A3G) Zn-binding domains. A3H has seven haplotypes (I-VII) that exhibit diverse biological phenotypes and geographical distribution in the human population. Its single Zn-coordinating deaminase domain belongs to a phylogenetic cluster (Z3) that is different from the Z1- and Z2-type domains in other human A3 proteins. A3H HapII, unlike A3A or A3C, has potent activity against HIV-1. Here, we sought to identify the determinants of A3H HapII deaminase and antiviral activities, using site-directed sequence- and structure-guided mutagenesis together with cell-based, biochemical, and HIV-1 infectivity assays. RESULTS: We have constructed a homology model of A3H HapII, which is similar to the known structures of other A3 proteins. The model revealed a large cluster of basic residues (not present in A3A or A3C) that are likely to be involved in nucleic acid binding. Indeed, RNase A pretreatment of 293T cell lysates expressing A3H was shown to be required for detection of deaminase activity, indicating that interaction with cellular RNAs inhibits A3H catalytic function. Similar observations have been made with A3G. Analysis of A3H deaminase substrate specificity demonstrated that a 5' T adjacent to the catalytic C is preferred. Changing the putative nucleic acid binding residues identified by the model resulted in reduction or abrogation of enzymatic activity, while substituting Z3-specific residues in A3H to the corresponding residues in other A3 proteins did not affect enzyme function. As shown for A3G and A3F, some A3H mutants were defective in catalysis, but retained antiviral activity against HIV-1vif (-) virions. Furthermore, endogenous reverse transcription assays demonstrated that the E56A catalytic mutant inhibits HIV-1 DNA synthesis, although not as efficiently as wild type. CONCLUSIONS: The molecular and biological activities of A3H are more similar to those of the double-domain A3 proteins than to those of A3A or A3C. Importantly, A3H appears to use both deaminase-dependent and -independent mechanisms to target reverse transcription and restrict HIV-1 replication.

A nanoparticle formulation of disulfiram prolongs corneal residence time of the drug and reduces intraocular pressure.

The goal in the search for successful therapies for glaucoma is the reduction of intraocular pressure (IOP), and the search for effective eye drops that reduce IOP is a high priority. We previously reported the potential of a 2-hydroxypropyl-ß-cyclodextrin (HPßCD) solution containing 0.5% DSF (DSF solution) to provide effective anti-glaucoma treatment in eye drop form. In this study, we designed new ophthalmic formulations containing 0.5% DSF nanoparticles prepared by a bead mill method (DSFnano dispersion; particle size 183 ± 92 nm, mean ± S.D.), and compared the IOP-reducing effects of a DSFnano dispersion with those of a DSF solution. The high stability of the DSFnano dispersion was observed until 7 days after preparation, and the DSFnano dispersion showed high antimicrobial activity against Escherichia coli (ATCC 8739). In transcorneal penetration experiments using rabbit corneas, only diethyldithiocarbamate (DDC) was detected in the aqueous humor, while no DSF was detected. The DDC penetration level (area under the curve, AUC) and corneal residence time (mean residence time, MRT) of the DSFnano dispersion were approximately 1.45- and 1.44-fold higher than those of the DSF, respectively. Moreover, the IOP-reducing effects of the DSFnano dispersion were significantly greater than those of the DSF solution in rabbits (the IOP was enhanced by placing the rabbits in a dark room for 5 h). In addition, DSFnano dispersion are tolerated better by a corneal epithelial cell than DSF solution and commercially available timolol maleate eye drops. It is possible that dispersions containing DSF nanoparticles will provide new possibilities for the effective treatment of glaucoma, and that an ocular drug delivery system using drug nanoparticles may expand their usage as therapy in the ophthalmologic field. These findings provide significant information that can be used to design further studies aimed at developing anti-glaucoma drugs.

Recovery from indomethacin-induced gastrointestinal bleeding by treatment with teprenone.

BACKGROUND: Gastrointestinal injuries caused by nonsteroidal anti-inflammatory drugs (NSAIDs) is a serious side effect in patients with rheumatoid arthritis (RA). However, effective therapeutic strategies have yet to be established. In this study, we investigated the therapeutic effects of teprenone (TEP), a gastric mucosal protective drug, on NSAID-induced gastrointestinal injuries in rats with RA (AA rats). METHODS: Gastrointestinal injury was induced by oral administration of indomethacin (IMC), a typical NSAID. TEP was orally administered after IMC-induced gastrointestinal bleeding, and the stomach, jejunum, and ileum were excised. RESULTS: On day 14 of IMC administration, lesion areas in the stomach, jejunum, and ileum were significantly larger in AA rats than in normal rats. When TEP was orally administered to AA rats, the lesion areas in the stomach, jejunum, and ileum significantly decreased compared with those in control rats (IMC-induced AA rats). Therefore, we measured NOS2 mRNA and NO levels, which were significantly decreased in rats with IMC-induced AA after treatment with TEP. CONCLUSIONS: These results suggest that the oral administration of TEP may be useful for the treatment of NSAID-induced gastrointestinal injuries in patients with RA.

Enhancement of Amyloid ß1-43 Production in the Lens Epithelium of Japanese Type 2 Diabetic Patients.

We investigated whether the accumulation of amyloid ß-protein (Aß) is enhanced in the lenses of diabetic patients. Lens epithelium samples were collected from Japanese patients during cataract surgery, and the Aß levels and gene expression of Aß-producing and -degrading enzymes in the samples were measured by ELISA and real-time RT-PCR, respectively. The Aß 1-43 levels in lenses of non-diabetic patients were low (0.11 pmol/g protein), while the levels in lenses of diabetic patients were significantly (6-fold) higher. Moreover, the Aß1-43/total-Aß ratio in the lenses of diabetic patients was also significantly higher than non-diabetic patients (p < 0.05). In addition, the mRNA levels for Aß-producing enzymes were also enhanced in the lenses of diabetic patients. In contrast to the results for Aß-producing enzymes, the mRNAs for the Aß-degrading enzymes in the lenses of diabetic patients were significantly lower than in non-diabetic patients (p < 0.05). Furthermore, Aß 1-43/total-Aß ratio in lenses was found to increase with plasma glucose level. In conclusion, these results suggest that high glucose levels cause both an increase in Aß production and a decrease in Aß degradation, and these changes lead to the enhancement in Aß1-43 accumulation in the lenses of diabetic patients. These findings are useful for developing therapies for diabetic cataracts and for developing anti-cataract drugs.

Changes in mitochondrial cytochrome c oxidase mRNA levels with cataract severity in lens epithelia of Japanese patients.

We previously reported that the collapse of ATP production via mitochondrial damage causes ATPase dysfunction, resulting in the onset or progression of lens opacification in cataracts in model rats. In the present study, it was investigated whether the mRNA expression levels of the three subtypes of mitochondrial cytochrome c oxidase (MTCO)1, 2 and 3 and ATP content change with the type and severity of cataracts in human lens. Samples of lens epithelium were collected from Japanese patients during cataract surgery, and the type and severity of the cataracts (grade) were determined according to the WHO classification [cortical (COR), nuclear (NUC), posterior subcapsular (PSC) opacification]. The MTCO1­3 mRNA expression levels in patients with grade­1 COR, NUC and PSC opacification were significantly enhanced compared with those of normal patients. The enhanced MTCO1­3 mRNA levels subsequently decreased in patients with COR, and the MTCO1­3 mRNA levels and ATP levels in patients with grade­3 COR were similar to those in normal patients. However, the mRNA expression levels of MTCO3 in patients with grade 3­NUC opacification and MTCO1­3 in patients with grade­3 PSC opacification, along with the ATP content, were significantly lower than in patients without cataracts. In conclusion, it was revealed that ATP production in lens epithelium is enhanced in early­stage cataracts (grade­1) in Japanese patients with COR, NUC and PSC opacification. In addition, in severe cataracts (grade­3), ATP production and content are strongly decreased in Japanese patients with PSC opacification. ATP depletion in human lens epithelium with PSC opacification may promote lens opacification by ATPase dysfunction.

Amyloid ß1-43 Accumulates in the Lens Epithelium of Cortical Opacification in Japanese Patients.

Purpose: We investigated the accumulation of amyloid ß (Aß1-40, Aß1-42, Aß1-43) in the lens epithelium of patients with opacification of five different types (cortical cataract [COR]; nuclear cataract [NUC]; posterior subcapsular cataract [PSC]; retrodots [RD]; and water clefts [WC]). Methods: Samples were collected from Japanese patients taken during cataract surgery; Aß levels and mRNA expression were determined by ELISA and a real-time RT-PCR method, respectively. Results: Levels of Aß1-40 and Aß1-42 in the lens epithelium of patients with COR, NUC, PSC, RD, and WC showed no significant differences in comparison with transparent lens epithelium. Levels of Aß1-43 in the lens epithelium of patients with PSC and WC were not detected, and NUC and RD were slightly elevated. In contrast to the results in these cataract types, high Aß1-43 levels were observed in the lens epithelium of patients with COR, and a close relationship was observed between Aß1-43 levels and the degree of lens opacification (R = 0.8229, n = 6). The levels of Aß1-43 were also higher in the lens epithelium of patients with mixed-cataract showing cortical opacification, and the Aß1-43 levels in the lens epithelium of mixed-cataract patients with cortical opacification was significantly higher than in that of mixed-cataract patients without cortical opacification. In addition, the level of an amyloid precursor protein mRNA in the lens epithelium of mixed-cataract patients with cortical opacification was significantly higher than in transparent lens and mixed-cataract patients without cortical opacification. Conclusions: We found high levels of Aß1-43 accumulation in the lens epithelium of Japanese patients with cortical opacification.

Ferulic Acid Suppresses Amyloid ß Production in the Human Lens Epithelial Cell Stimulated with Hydrogen Peroxide.

It is well known that oxidative stresses induce the production of amyloid ß (Aß) in the brain, lens, and retina, leading to age-related diseases. In the present study, we investigated the effects of ferulic acid on the Aß levels in H2O2-stimulated human lens epithelial (HLE) SRA 01/04 cells. Three types of Aß peptides (Aß1-40, Aß1-42, and Aß1-43) were measured by ELISA, and the levels of mRNA for the expressed proteins related to Aß production (APP, BACE1, and PS proteins) and degradation (ADAM10, NEP, and ECE1 proteins) were determined by quantitative real-time RT-PCR. H2O2 stimulation augmented gene expression of the proteins related to Aß production, resulting in the production of three types of Aß peptides. Treatment with 0.1 µM ferulic acid attenuated the augmentations of gene expression and production of the proteins related to the secretion of three types of Aß peptides in the H2O2-stimulated HLE cells. These results provided evidence of antioxidative functions of ferulic acid for lens epithelial cells.

Effect of Eye Drops Containing Disulfiram and Low-Substituted Methylcellulose in Reducing Intraocular Pressure in Rabbit Models.

PURPOSE: We attempted to develop anti-glaucoma eye drops using 0.5% disulfiram (DSF), 5% 2-hydroxypropyl-ß-cyclodextrin, 0.1% hydroxypropylmethylcellulose, and 2% methylcellulose (MC) (DSF eye drops with MC), and tested the ability of a DSF eye drops with MC to reduce intraocular pressure (IOP) in rabbit models. METHODS: Elevated IOP was induced by the rapid infusion of 5% glucose solution (15 ml/kg of body weight) through the marginal ear vein or by keeping rabbits in the dark for 5 h. IOP and the nitric oxide (NO) level in the aqueous humor were measured with an electronic tonometer and by a microdialysis method, respectively. ΔIOP and ΔNO values were analyzed as the differences in IOP and NO in rabbits instilled with saline or eye drops, respectively. RESULTS: Increased IOP in rabbit models was reduced by the instillation of DSF eye drops with or without MC, and a close relationship was observed between IOP and NO levels in rabbit receiving a rapid infusion of isotonic glucose. We present kinetic parameters [secondary AUC (prolonged drug effect) and secondary MRT (prolonged effective time)] analyzed as the area under the curve (AUC) of ΔIOP or ΔNO versus time using rabbits instilled with eye drops 10, 50, or 90 min prior to the infusion of the isotonic glucose solution. The elevations in IOP and NO level were reduced by the instillation of DSF eye drops with or without MC; the addition of MC increased the secondary AUC and MRT of DSF eye drops. CONCLUSIONS: The present study demonstrates that 0.5% DSF eye drops suppress increased IOP in rabbit models, probably by inhibiting the elevation in NO levels. In addition, we propose a kinetic analysis method to predict drug effects and effective time. These findings suggest that a low-substituted MC-based drug delivery system promotes drug effectiveness and effective time.