Preparation of Polymeric Submicron Particle-Containing Microparticles Using a 4-Fluid Nozzle Spray Drier

Purpose We studied a novel method for preparing polymeric submicron particle-containing microparticles using a 4-fluid nozzle spray drier. Method Ethylcellulose (EC) and poly(lactic-co-glycolic acid) (PLGA), either alone or in combination with polyethylenimine (PEI), were used as polymers to produce submicron particles, and mannitol (MAN) was used as a water-soluble carrier for the microparticles. The polymer and MAN solutions were supplied through different liquid passages of a 4-fluid nozzle and then dried to obtain MAN microparticles containing EC or PLGA submicron particles. The polymer/MAN ratio was controlled by changing the concentration of the polymer and MAN solutions. EC or PLGA microparticles were observed via scanning electron microscopy, and the size of microparticles was determined by image analysis. The particle size distribution of EC or PLGA submicron particles was measured with a super dynamic light scattering spectrophotometer. Results The method generated submicron-sized (<1 μm) particles of EC and PLGA. The mean diameters of EC and PLGA particles at a polymer/MAN ratio of 1:10 were 631 and 490 nm, respectively. The mean diameter of PLGA particles decreased as the PLGA/MAN ratio was reduced, reaching ∼200 nm at a PLGA/MAN ratio of 1:100. The mean diameter of PLGA/PEI particles at PLGA/PEI/MAN ratios of 1:0.5:10 and 1:0.5:100 were 525 and 223 nm, respectively, and their zeta potentials were +50.8 and +58.2 mV, respectively. The size of EC submicron particles could be controlled by varying the spray conditions. Conclusions This study demonstrated that it is possible to prepare polymeric submicron particles dispersed in MAN microparticles in a single process using the 4-fluid nozzle spray drying method. Cationic PLGA particles with a diameter of ∼200 nm could be prepared by adding PEI, suggesting the possibility of its use as a carrier for delivering DNA into cells. The precipitation of EC may occur by the mutual dispersion and mixing of solvents after collision of EC and MAN mists by antisolvent effect, thereby producing MAN microparticles containing EC submicron particles.     

Molecular mechanisms and therapeutic strategies of chronic renal injury: renoprotective effect of rho-kinase inhibitor in hypertensive glomerulosclerosis

Among the GTP-binding proteins, Rho is known to function as a molecular switch in various cellular functions. Among the Rho effectors, the cellular function and signal transduction of Rho-kinase have been extensively studied. However, information about its in vivo functions is still limited. With the recent development of a specific Rho-kinase inhibitor such as Y-27632 and fasudil, the understanding of the role of the Rho/Rho-kinase pathway in vitro and in vivo has advanced. However, to date, there have been few studies investigating the role of Rho-kinase in renal disease. Recent studies have shown that Rho-kinase inhibitor significantly attenuated the tubulointerstitial fibrosis in kidney induced by unilateral ureteral obstruction. However, there have been few studies investigating the role of the Rho/Rho-kinase pathway in hypertensive glomerular sclerosis. In this review, we described the role of the Rho/Rho-kinase pathway in the progression of renal glomerulosclerosis in several forms of hypertensive rats. Our results suggest that chronic inhibition of the Rho-kinase pathway may be a new therapeutic approach for hypertensive glomerulosclerosis. Our results also suggest that the mechanism of the renoprotective effect of Rho-kinase inhibitor is partly mediated via inhibition of extracellular matrix gene expression, monocytes/macrophages infiltration, oxidative stress, and upregulation of eNOS gene expression.     

Evaluation of antipsychotic and relative drugs using disruption of prepulse inhibition as an animal model for schizophrenia

The prepulse inhibition (PPI) is a phenomenon in which a weak prepulse attenuates the response to a subsequent startling stimulus. The PPI, a model of sensorimotor gating, is deficient in patients with schizophrenia and some other psychiatric disorders. In rodents, PPI can be disrupted by methamphetamine or phencyclidine, which causes psychotomimetic symptoms, and the dopaminergic agonist-induced PPI is reversed by dopamine D2 receptor antagonists and a dopaminergic partial agonist aripiprazole. However, in general, the glutamate receptor antagonist-induced PPI is reversed by atypical antipsychotics such as clozapine, but not by typical antipsychotics such as haloperidol. Therefore, PPI is believed to have face, construct, and predictive validity for the PPI disruption in schizophrenia, and it is widely used as a model to study the neurobiology of this disorder and for screening antipsychotics. Recently, various inbred mouse strains and genetically modified mouse lines have been examined and the studies using PPI indicated the involvement of various neurotransmitters such as dopamine, glutamate, serotonin, GABA and neuropeptide in the biological basis of sensorimotor gating. In addtition, mood stabilizers such as valproate and lamotrigine or alpha7 nicotinic receptor agonists have reported to reverse the PPI disruption.     

Effect of molecular weight on the dermatopharmacokinetics and systemic disposition of drugs after intracutaneous injection

The aim of this study was to investigate the effect of molecular weight on the topical migration and systemic absorption of drugs, sodium salicylate (SA), calcein sodium (CAL) and FITC-dextrans (FD-4, FD-10, FD-20 and FD-40), after intracutaneous (i.c.) injection. The apparent diffusion coefficients in the skin (D(s)) of SA, CAL and FD-4 after i.c. injection were almost the same and slightly less than 1x10(-4)cm(2)/min. The D(s) value drastically decreased with molecular weight more than 10kDa. The plasma concentration-time curve after i.c. injection was predicted by the convolution method. The ratio of AUC (observed/predicted) was approximately unity for SA, CAL and FD-4 although the systemic absorption of only FD-4 was delayed. In contrast, the AUC ratios of FD-10, FD-20 and FD-40 were about 0.5, 0.2 and 0.06, respectively, and obviously decreased with the molecular weight. The elimination of FD-10, FD-20 and FD-40 from the skin was slow and the drugs tended to remain in the muscle. These results indicated the importance of diffusion in the skin and permeability through the blood vessels for local migration and systemic absorption of drugs after penetration into viable skin.     

Long-term inhibition of Rho-kinase ameliorates diastolic heart failure in hypertensive rats

Diastolic heart failure (DHF) is a major cardiovascular disorder with poor prognosis; however, its molecular mechanism still remains to be fully elucidated. We have previously demonstrated the important roles of Rho-kinase pathway in the molecular mechanisms of cardiovascular fibrosis/hypertrophy and oxidative stress, but not examined in the development of heart failure. Therefore, we examined in this study whether Rho-kinase pathway is also involved in the pathogenesis of DHF in Dahl salt-sensitive rats, an established animal model of DHF. They were maintained with or without fasudil, a Rho-kinase inhibitor (30 or 100 mg/kg/day, PO) for 10 weeks. Untreated DHF group exhibited overt heart failure associated with diastolic dysfunction but with preserved systolic function, characterized by increased myocardial stiffness, cardiomyocyte hypertrophy, and enhanced cardiac fibrosis and superoxide production. Fasudil treatment significantly ameliorated those DHF-related myocardial changes. Western blot analysis showed that cardiac Rho-kinase activity was significantly increased in the untreated DHF group and was dose-dependently inhibited by fasudil. Importantly, there was a significant correlation between the extent of myocardial stiffness and that of cardiac Rho-kinase activity. These results indicate that Rho-kinase pathway plays an important role in the pathogenesis of DHF and thus could be an important therapeutic target for the disorder.     

The effects of atropine on changes in the sleep patterns induced by psychological stress in rats

The present study was designed to investigate the acute effects of psychological stress on the sleep patterns of rats and the potential mechanisms underlying these effects. Psychological stress was induced using a communication box. In a preliminary study, sleep recording was performed for 6 h immediately after 1-h stress in rats. The results showed that psychological stress significantly enhanced the total amount of rapid eye movement (REM) sleep by prolonging the average duration of REM sleep episodes during first 3 h of sleep, whereas the total amount of non-rapid eye movement sleep was not influenced. In a second study, three doses of atropine (3, 10, or 30 nmol) were intracerebroventricularly microinjected into the rats. Total REM sleep was inhibited by the highest dose of atropine during the first 3 h of sleep in the stressed group but not in the control group; the inhibition of total REM sleep was due to a reduction in the average duration of REM sleep episodes. These results suggest that the increase in total REM sleep induced by stress was related to the activation of cholinergic neurons. In addition, plasma corticosterone--an index of hypothalamic-pituitary-adrenal (HPA) axis activity in response to stress--was examined after psychological stress. The concentration of corticosterone did not change significantly. Therefore, the abnormal sleep patterns could not be simply attributed to the HPA axis. These results indicate that the changes in sleep patterns induced by stress were related to cholinergic neurons, but not the HPA axis.     

Pretreatment effects of moxibustion on the skin permeation of FITC-dextran

This study was conducted to evaluate the pretreatment effects of different in vivo moxibustion on the permeation of a model high molecular compound, FITC-dextran, with a mean molecular weight of 4 kDa (FD-4), through excised hairless rat skin. Direct or indirect moxibustion (0.10 g moxa) was pretreated consecutively 4 times every 5 min on the abdomen of hairless rats, and the permeation of FD-4 was determined through the excised skin over 8h from 30 min after starting the first moxibustion. This consecutive moxibustion pretreatment showed a significant increase in the skin temperature as well as skin permeation of FD-4 compared with the control group (no moxibustion pretreatment). Quantitative parameters showed an increase in skin temperature and skin permeation: the area under the skin temperature over control temperature-time curve during one burning cycle (5.0 min) (AUCtemp) or the maximum skin temperature during moxibustion (Tmax) and the cumulative amount of FD-4 permeated through skin over 8h (Q8) or steady-state flux were increased by moxibustion pretreatment. Then, the effect of pedestal thickness (distance from the moxa cylinder and skin surface), shape of the moxa cylinder (5mm diameter, 13 mm height or 9 mm diameter, 7 mm height), burning materials (moxa or aromatic incense), pedestal component (paper, potato or ginger) and moxibustion pretreatment method (direct or indirect moxibustion) was evaluated on the AUCtemp or Tmax and Q8 or flux. The amount of protein leached from the skin surface was also determined as an inflammatory index by this moxibustion pretreatment. When the skin temperature was increased to 60 degrees C, the Q8 or flux as well as the amount of protein leached were markedly increased. When the skin temperature was controlled to 42 to 45 degrees C by an adequate selection of pedestal thickness, shape of the moxa cylinder, burning materials, pedestal component and moxibustion pretreatment method, on the other hand, protein leaching remained unaltered, but the Q8 or flux significantly increased with the Tmax. This study thus provides credible evidence that moxibustion pretreatment increases the skin permeation of high molecular compounds.     

Analysis of skin disposition and metabolism of ethyl nicotinate after topical application using dual agar gel disc-inserted rats

The skin disposition and metabolism of topically applied ethyl nicotinate (EN) were evaluated in dual agar gel disc-inserted hairless rats, which have two agar gel discs subcutaneously inserted into the abdominal region as drug receptors, and a topical formulation containing EN placed on either side of the gel disc through the skin. Plasma and agar levels of EN and its metabolite, nicotinic acid (NA), were followed every 2 h over 6 h. EN permeated through the skin barrier and partly metabolized to NA with 89.4% of the metabolite ratio [NA/(EN+NA)] at 6 h. Some EN and NA in the skin moved to the systemic circulation, and the remainder migrated into the agar gel below the formulation. The total amount (EN+NA) in the skin that distributed from the formulation directly to the systemic circulation and the application side of the gel corresponded to 95.2% and 4.8% of the total skin permeation at 6 h, respectively. Only NA was distributed from the systemic circulation to both agar gel discs. The NA fraction in the application side of the gel from the circulation was only 1% of the total amount in the agar gel. The metabolite ratio on the application side of the agar gel was higher than that in the receiver for the in vitro skin permeation using excised hairless rat skin. This difference was probably related to a lower EN ratio in viable skin in situ than in vitro. These results suggest that the present in situ method is useful to evaluate the skin disposition and metabolism of topically applied drugs.     

Effect of preparation method on properties of orally disintegrating tablets made by phase transition

In order to evaluate the effect of preparation method on the properties of orally disintegrating (OD) tablets, OD tablets were prepared by compressing a mixture of high melting point sugar alcohol (HMP-SA) and low melting point sugar alcohol (LMP-SA) and subsequent heating. In the direct compression method (DCM) where the LMP-SA was added as a powder, both hardness and disintegration time were increased by decreasing the particle size of the LMP-SA. In the wet granule compression method (WGCM), where the LMP-SA was added as an aqueous binder solution, the tablets became harder with less heating compared to tablets prepared by DCM. Using 1% xylitol as the LMP-SA provided tablets with sufficient hardness when prepared by WGCM, as opposed to DCM where 5% xylitol was necessary to prepare tablets with similar hardness. These results suggest that uniformly distributed LMP-SA on the surface of HMP-SA particles in WGCM might diffuse more easily during the heating process compared to mechanically mixed LMP-SA in DCM, resulting in an increase in tablet hardness even with a short heating time and low content of LMP-SA. In addition, disintegration and hardness stability of the tablets were affected by the LMP-SA content when prepared by WGCM, suggesting that the LMP-SA content should be regulated to assure the stability of OD tablet characteristics.     

Inhibitory effect of juniperonic acid (Delta-5c,11c,14c,17c-20:4, omega-3) on bombesin-induced proliferation of Swiss 3T3 cells

Juniperonic acid (Delta-5c,11c,14c,17c-20:4, JA) is a polymethylene-interrupted (PMI) fatty acid that occurs in Biota orientalis. In this study, we found that JA has an antiproliferative activity. Swiss 3T3 cells were preloaded with fatty acids before stimulation with bombesin, a mitogenic neuropeptide, and proliferation of the cells was assessed by [(3)H]thymidine incorporation. Preloading of linoleic acid (Delta-9c,12c-18:2) significantly enhanced bombesin-induced proliferation. In contrast, preloading of eicosapentaenoic acid (Delta-5c,8c,11c,14c,17c-20:5, EPA) suppressed proliferation. Likewise, cells preloaded with JA showed a significantly curtailed response to bombesin. The antiproliferative potency of JA was equivalent to that of EPA. Sciadonic acid (Delta-5c,11c,14c-20:3), an omega-6 analogue of JA did not show antiproliferative activity, suggesting the importance of the omega-3 double bond rather than the PMI structure. The EPA-like activity of JA may be involved in the pharmaceutical activity of biota seeds, a psychoactive Chinese traditional medicine.