Enhanced percutaneous absorption of cilostazol nanocrystals using aqueous gel patch systems and clarification of the absorption mechanism.

Cilostazol (CLZ), an anti-platelet agent, is primarily used following the onset of cerebral infarction. However, as CLZ is only marginally soluble in water, a strategy for patients with serious secondary conditions, such as impaired consciousness or aphagia, is required. In the present study, topical formulations containing CLZ nanocrystals (CLZnano) were designed to enhance percutaneous absorption. In addition, the mechanism of penetration of CLZnano through rat skin was investigated. A topical formulation containing CLZ nanoparticles (CLZnano gel patch) was prepared using a combination of recrystallization and ball milling of an aqueous gel. The particle size of CLZnano was 74.5±6.2 nm (mean ± standard deviation). The concentration of permeated CLZnano and penetration mechanism of the nanocrystals were measured in a percutaneous absorption experiment. The amount of penetrated CLZ, the penetration rate (Jc), the penetration coefficient through the skin (Kp) and the skin/preparation partition coefficient (Km) for the CLZnano gel patch were all significantly higher than those of the CLZ powder (CLZmicro) gel patch, the CLZnano ointment and the CLZmicro ointment. In in vitro percutaneous penetration experiments on the CLZnano gel patches, there was a positive correlation between the number of CLZnano. Following the application of the CLZnano gel patch on rat skin, 98% of penetrated CLZ was observed in nanoparticle form; for the CLZmicro gel patch, this figure was 9%. In addition, the CLZ concentrations in the plasma of rats administered the CLZnano gel patches were significantly higher than those of rats administered the CLZnano CP gel and PEG ointments. It was suggested that CLZnano (diameter <100 nm) were transferred through the intracellular spaces in the skin and then into peripheral blood vessels. To the best of our knowledge, this is the first report to elucidate the mechanism of the percutaneous penetration of nanocrystal medicines.

An oral formulation of cilostazol nanoparticles enhances intestinal drug absorption in rats.

Cilostazol (CLZ) is an anti-platelet agent that is generally used after the onset of cerebral infarction. However, CLZ is a poorly water-soluble drug and a strategy for increasing its bioavailability is required. In the present study, novel oral formulations were designed containing CLZ solid nanoparticles to improve bioavailability. The present study investigated the therapeutic effect of the oral formulations containing CLZ nanoparticles on ischemic stroke using a cerebral ischemia/reperfusion-induced injury model (MCAO/reperfusion mice). The oral formulation containing CLZ nanoparticles (CLZ/Rnano tablet) was prepared using a combination of recrystallization and ball milling with the following ingredients: CLZ, docusate sodium, methylcellulose, 2-hydoxypropyl-ß-cyclodextrin, gum arabic, polyvinylpyrrolidone, and mannitol. The particle size after re-dispersion of the CLZ/Rnano tablet was 64±47 nm (mean ± standard deviation). The CLZ areas under the concentration-time curve (AUC) and mean residence time (MRT) in rats that were administered CLZ/Rnano tablets were significantly greater compared with those in rats that were administered CLZ/Rmicro tablets. Results indicated, the AUC after administration of CLZ/Rnano tablets was 3.1-fold higher compared with that after administration of the commercially available CLZ OD tablet. In addition, oral administration with CLZ/Rnano tablets ameliorated neurological deficits caused by ischemic stroke in MCAO/reperfusion mice. It is possible that the oral formulation containing CLZ nanoparticles will be useful for the treatment of patients with ischemic stroke and that these findings will provide significant information that can be used to improve the drug with low bioavailability.

Evaluation of Retinal Function in Streptozotocin-induced Diabetic Rats by Using the Electroretinography and Immunohistochemistry Methods.

Streptozotocin-induced diabetic rat (STZ rat) was used in many studies for the diabetic mellitus. In this study, we demonstrated whether the electroretinograms (ERG) was changed in the retina of STZ rats. In addition, we investigated the histopathological alteration in the retina of STZ rats by using the immunological method. The 100 mg/kg of STZ was injected continuously for 2 d (100 mg/kg×2). The insulin level was decreased, and the glucose level was enhanced 14 d after the injection of STZ. Moreover, the levels of a-wave, b-wave and OP amplitude were decreased in the rat at 14 d after the injection of STZ. Although, the damage and apoptosis was not observed in the retinal ganglion cell of STZ rats by the immunological experiment using the phospho-H2A.X and cleaved caspase-3, the distance between cell and cell was increased in both of outer- and inner- nuclear (granule) layer in retina of STZ rats. In conclusion, we showed that the enhanced thickening in retina was caused by the injection of excessive STZ. The thickening in retina of STZ rats may lead to the dysfunction of retina, resulting in the decrease in ERG. These findings provide significant information that can be used in the design of a model of diabetic retinopathy.

Enhanced Production of Nitric Oxide Leads to ATP Collapse in the Retinas of Otsuka Long-Evans Tokushima Fatty Rats, a Model of Human Diabetes.

PURPOSE: We determined nitric oxide (NO) production via inducible NO synthase (iNOS) by hyperglycemia using the retina of Otsuka Long-Evans Tokushima Fatty rats (OLETF rats), and investigated the relationship between ATP contents and NO production in the retinas of OLETF rats. METHODS: Long-Evans Tokushima Otsuka rats (LETO rats, normal rats) and OLETF rats (model rat for diabetes mellitus) aged 60 weeks of age were used. Plasma glucose (Glu) levels were determined using an Accutrend GCT System, and NO levels were measured by the microdialysis method as nitrite ([Formula: see text]). Cytochrome c oxidase (CCO) activity was measured using a Mitochondrial Isolation Kit and Cytochrome c Oxidase Assay Kit, and ATP levels were determined using a Sigma ATP Bioluminescent Assay Kit and a luminometer AB-2200. RESULTS: [Formula: see text] levels in the retinas of OLETF rats were significantly higher than in LETO rats, and the [Formula: see text] levels in the retinas of 60-week-old OLETF rats increased with increasing Glu. CCO activity in the retinas of OLETF rats showed no significant difference from that in LETO rats; however, ATP levels in the retinas of OLETF rats were significantly lower than those in LETO rats. The oral administration of aminoguanidine or disulfiram, an iNOS inhibitor, attenuated the decrease in ATP levels in the retinas of 60-week-old OELTF rats. CONCLUSION: The present study demonstrates that NO production via iNOS in the retinas of 60-week-old OLETF rats is caused by hyperglycemia, and that NO causes a decrease in ATP contents in the retinas of 60-week-old OELTF rats. It is possible that the low ATP contents caused by NO may affect the normal functioning of the retina in OLETF rats.

Pharmacokinetics and Antiinflammatory Effect of a Novel Gel System Containing Ketoprofen Solid Nanoparticles.

We previously reported that dermal application using nanoparticles improves skin penetration. In this study, we prepared novel topical formulations containing ketoprofen (KET) solid nanoparticles (KETnano gel ointment) and investigated the antiinflammatory effect of the KET nanoparticle formulations on rheumatoid arthritis using adjuvant-induced arthritis (AA) rats. The KETnano gel ointment was prepared using a bead mill method and additives including methylcellulose and Carbopol 934; the mean particle size of the KET nanoparticles was 83 nm. In the in vitro skin penetration experiment, the penetration rate (Jc) and penetration coefficient through the skin (Kp) values of the KETnano gel ointment were significantly higher than those of gel ointment containing KET microparticles (KETmicro gel ointment; mean particle size 7.7 µm). On the other hand, in the in vivo percutaneous absorption experiment, the apparent absorption rate constant (ka) and the areas under the KET concentration-time curve values in the skin of rats receiving the KETnano gel ointment were significantly higher than those of rats receiving the KETmicro gel ointment, and the amounts of KET in the skin tissues of rats receiving the KETnano gel ointment were also significantly higher than those of rats receiving the KETmicro gel ointment. In addition, the application of the KETnano gel ointment attenuated the enhancement of paw edema of the hind feet of AA rats more than the application of the KETmicro gel ointment. Our findings suggest that a topical drug delivery system using nanoparticles could lead to expansion in the therapeutic use of KET.

Intravenous Administration of Cilostazol Nanoparticles Ameliorates Acute Ischemic Stroke in a Cerebral Ischemia/Reperfusion-Induced Injury Model.

It was reported that cilostazol (CLZ) suppressed disruption of the microvasculature in ischemic areas. In this study, we have designed novel injection formulations containing CLZ nanoparticles using 0.5% methylcellulose, 0.2% docusate sodium salt, and mill methods (CLZnano dispersion; particle size 81 ± 59 nm, mean ± S.D.), and investigated their toxicity and usefulness in a cerebral ischemia/reperfusion-induced injury model (MCAO/reperfusion mice). The pharmacokinetics of injections of CLZnano dispersions is similar to that of CLZ solutions prepared with 2-hydroxypropyl-ß-cyclodextrin, and no changes in the rate of hemolysis of rabbit red blood cells, a model of cell injury, were observed with CLZnano dispersions. In addition, the intravenous injection of 0.6 mg/kg CLZnano dispersions does not affect the blood pressure and blood flow, and the 0.6 mg/kg CLZnano dispersions ameliorate neurological deficits and ischemic stroke in MCAO/reperfusion mice. It is possible that the CLZnano dispersions will provide effective therapy for ischemic stroke patients, and that injection preparations of lipophilic drugs containing drug nanoparticles expand their therapeutic usage.

Decrease in Corneal Damage due to Benzalkonium Chloride by the Addition of Mannitol into Timolol Maleate Eye Drops.

We investigated the protective effects of mannitol on corneal damage caused by benzalkonium chloride (BAC), which is used as a preservative in commercially available timolol maleate eye drops, using rat debrided corneal epithelium and a human cornea epithelial cell line (HCE-T). Corneal wounds were monitored using a fundus camera TRC-50X equipped with a digital camera; eye drops were instilled into rat eyes five times a day after corneal epithelial abrasion. The viability of HCE-T cells was calculated by TetraColor One; and Escherichia coli (ATCC 8739) were used to measure antimicrobial activity. The reducing effects on transcorneal penetration and intraocular pressure (IOP) of the eye drops were determined using rabbits. The corneal wound healing rate and rate constant (kH), as well as cell viability, were higher following treatment with 0.005% BAC solution containing 0.5% mannitol than in the case BAC solution alone; the antimicrobial activity was approximately the same for BAC solutions with and without mannitol. In addition, the kH for rat eyes instilled with commercially available timolol maleate eye drops containing 0.5% mannitol was significantly higher than that for eyes instilled with timolol maleate eye drops without mannitol, and the addition of mannitol did not affect the corneal penetration or IOP reducing effect of the timolol maleate eye drops. A preservative system comprising BAC and mannitol may provide effective therapy for glaucoma patients requiring long-term treatment with anti-glaucoma agents.

Topical therapies for rheumatoid arthritis by gel ointments containing indomethacin nanoparticles in adjuvant-induced arthritis rat.

Indomethacin (IMC), a nonsteroidal anti-inflammatory drug, has been used in the treatment of rheumatoid arthritis (RA), although its clinical use has been limited by its systemic side effects that include gastrointestinal lesions. Therefore, the development of IMC formulations that do not cause gastrointestinal lesions is highly anticipated. In this study, we designed new topical formulations containing IMC solid nanoparticles (IMCnano gel ointment), and investigated their pharmacokinetics. In addition, we demonstrate the preventive effects of this topical application of IMC nanoparticles on inflammation in adjuvant-induced arthritis rat (AA rat). The IMCnano gel ointment was prepared using Bead Smash 12 (a bead mill) and additives including 2-hydroxypropyl-ß-cyclodextrin, methylcellulose and Carbopol 934; the mean particle size of the IMC nanoparticles was 173 ± 91 nm (means ± S.D.). The application of the IMCnano gel ointment attenuated the increase in paw edema of the hind feet of AA rats in comparison with AA rats treated with gel ointment containing IMC microparticles (IMCmicro gel ointment, particle diameter 17.1 ± 11.6 mm, means ± S.D). In addition, the accumulation of IMC from the IMCnano gel ointment in skin tissue was significantly large than for the IMCmicro gel ointment; however, the plasma IMC concentrations were similar for the IMCmicro and IMCnano gel ointments. Our findings suggest that the dermal application of nanoparticles may enable a medication to be applied without high-systemic drug levels, which could provide efficient and effective therapy that spares patients from unwanted side effects. A formulation of a topical drug delivery system using IMC nanoparticles may provide a delivery option for the clinical treatment of RA.

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.

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.

Pathological fracture due to vertebral osteonecrosis associated with pancreatitis.

A 50-year-old man with a history of alcohol-induced pancreatitis was admitted to a hospital with swelling and pain of the right ankle, and fever, and was suspected to have osteomyelitis. Radiographs of the fingers, ankles, and feet, in which pain and swelling were present, revealed multiple pathological fractures. The histological examination of the tissue sample in the right radius showed sequestrated fat necrosis. Bacterial culture test remained negative. Based on the findings mentioned above, a diagnosis of intraosseous fat necrosis associated with pancreatitis was made. Treatments proven to be effective in the literature for pancreatitis were started. Symptoms of the bones and joints gradually improved. However, pancreatitis relapsed, triggered by drinking, 4 months after discharge. He had complaints of back pain without any history of trauma. Radiographs showed fractures of the entire vertebral body from the 12th thoracic to 5th lumbar vertebrae. What was interesting about the present case was that, after the pathological fractures of the extremities were completely resolved, osteonecrosis relapsed as pancreatitis deteriorated, resulting in pancreatic cyst rupture into the intrathoracic cavity and vertebrae, differing from the previous lesions that were affected. There are very few cases of pathological fracture induced by pancreatitis that affect the vertebrae.

[Case of primary hepatic neuroendocrine carcinoma diagnosed by needle biopsy].

The patient was a 55-year-old man with a large hepatic tumor measuring 12 × 12 cm in the left lobe. To obtain the histological diagnosis, the target liver biopsy was performed. Histologically, the tumor revealed as a neuroendocrine carcinoma. After the diagnosis, he received the chemotherapy (CTX) with etoposide and cisplatin. Serum levels of NSE and the tumor size were decreased after the first course of CTX. We here report a case of primary hepatic neuroendocrine carcinoma treated with CTX following the diagnosis by the needle biopsy.

Poorly differentiated endocrine carcinoma of the pancreas responded to gemcitabine: Case report.

Poorly differentiated endocrine carcinoma (PDEC) of the pancreas is a rare and aggressive tumor. First-line treatment is commonly a combination of etoposide and cisplatin, but there is no consensus regarding further treatment recommendations. In this report, we describe a case of pancreatic PDEC treated with gemcitabine as third-line chemotherapy. A 62-year-old man with pancreatic PDEC was administered etoposide plus cisplatin as first-line treatment; he then received irinotecan for tumor relapse. However, because irinotecan induced ileus in this patient, we chose gemcitabine as third-line chemotherapy. After two cycles of gemcitabine (1000 mg/m(2) on days 1, 8 and 15 every 4 wk), a partial tumor response was noted by computed tomography (approximately 68% reduction in tumor size). Our patient survived for 15 mo after diagnosis. This is a rare case of unresectable pancreatic PDEC, which showed a partial response to gemcitabine after the failure of two other regimens. Gemcitabine could be an effective treatment option for pancreatic PDEC that is resistant to other treatments.

Tumor necrosis factor-alpha inhibits chondrogenic differentiation of synovial fibroblasts through p38 mitogen activating protein kinase pathways.

We previously reported that synovial fibroblast-like cells (SFs) can be differentiated into chondrocytes through activin receptor-like kinase (ALK) 3 activation. The aim of this study was to clarify the effect and signaling pathways of tumor necrosis factor (TNF)-alpha on the chondrogenic differentiation of SFs. Primary SFs from patients with rheumatoid arthritis (RA) were treated with recombinant human bone morphogenetic protein-2 or transduced with a constitutively active mutant of the ALK3 gene (ALK3CA) with or without TNF-alpha, and then cultured in pellets. Expression of chondrocyte-specific genes was analyzed by real-time polymerase chain reaction or by histological analysis. Inhibitors of mitogen-activating protein kinase (MAPK) pathways or adenovirus vectors carrying a dominant-negative mutant of the IkappaB kinase 2 gene (AxIKK2DN) were used to analyze the signaling pathways of TNF-alpha. Expression of chondrocyte-specific genes was induced in SFs either by rhBMP-2 treatment or by ALK3CA transduction, which was strongly suppressed by TNF-alpha treatment. TNF-alpha markedly increased the p38 MAPK pathways in SFs, and inhibition of p38 MAPK activation partially restored the inhibitory effect of TNF-alpha on the chondrogenic differentiation of SFs. Combination therapy BMP-2 and anti-TNF-alpha agents especially targeting p38 MAPK might be a good approach to stimulating neochondrogenesis in the damaged joints in RA.

Tumor necrosis factor-α inhibits chondrogenic differentiation of synovial fibroblasts through p38 mitogen activating protein kinase pathways.

We previously reported that synovial fibroblast-like cells (SFs) can be differentiated into chondrocytes through activin receptor-like kinase (ALK) 3 activation. The aim of this study was to clarify the effect and signaling pathways of tumor necrosis factor (TNF)-α on the chondrogenic differentiation of SFs. Primary SFs from patients with rheumatoid arthritis (RA) were treated with recombinant human bone morphogenetic protein-2 or transduced with a constitutively active mutant of the ALK3 gene (ALK3CA) with or without TNF-α, and then cultured in pellets. Expression of chondrocyte-specific genes was analyzed by real-time polymerase chain reaction or by histological analysis. Inhibitors of mitogen-activating protein kinase (MAPK) pathways or adenovirus vectors carrying a dominant-negative mutant of the IκB kinase 2 gene (AxIKK2DN) were used to analyze the signaling pathways of TNF-α. Expression of chondrocyte-specific genes was induced in SFs either by rhBMP-2 treatment or by ALK3CA transduction, which was strongly suppressed by TNF-α treatment. TNF-α markedly increased the p38 MAPK pathways in SFs, and inhibition of p38 MAPK activation partially restored the inhibitory effect of TNF-α on the chondrogenic differentiation of SFs. Combination therapy BMP-2 and anti-TNF-α agents especially targeting p38 MAPK might be a good approach to stimulating neochondrogenesis in the damaged joints in RA.