Tong-Rong Tsai

Gastroesophageal reflux disease (GERD) is a disorder that is related to an incompetent lower esophageal sphincter (LES). Previous studies showed that bombesin could increase LES pressure in humans and opossums. The aim of the present study was to characterize the effects of bombesin on porcine LES contraction. We used the selective agonists, neuromedin B (NMB), gastrin-releasing peptide (GRP), and [D-Tyr⁶,Apa-4Cl¹¹,Phe¹³,Nle¹⁴]bombesin-(6-14) (DTACPN-BN), as well as receptor antagonists of bombesin receptor subtype 2 (BB₂), and 3 (BB₃) for ex vivo contraction studies. Atropine, nifedipine, tetrodotoxin, and ω-conotoxin GVIA were used to explore the agonist-induced LES contraction mechanism. Reverse transcription polymerase chain reaction and immunohistochemistry were applied to detect bombesin receptor expression. Our results indicate that GRP and DTACPN-BN, but not NMB, induced tonic contractions of the porcine LES in a dose-dependent manner, and the contractions were inhibited with selective BB₂ and BB₃ antagonists. The GRP-induced contraction is mainly caused by L-type Ca²⁺ channel-mediated Ca²⁺ influx. However, DTACPN-BN-induced contractions are associated with neuronal conduction. RT-PCR and immunohistochemistry revealed that BB₂ and BB₃ were expressed in the porcine LES. Bombesin-induced tonic contraction of the LES is mediated through BB₂ and BB₃. Bombesin, BB_2, and BB₃ agonists might have the potential to treat GERD.

Up to 40% of patients with gastroesophageal reflux disease (GERD) suffer from proton pump inhibitor refractory GERD but clinically the medications to strengthen the lower esophageal sphincter (LES) to avoid irritating reflux are few in number. This study aimed to examine whether Salvia miltiorrhiza (SM) extracts induce tonic contraction of rat LES ex vivo and elucidate the underlying mechanisms. To investigate the mechanism underlying the SM extract-induced contractile effects, rats were pretreated with atropine (a muscarinic receptor antagonist), tetrodotoxin (a sodium channel blocker), nifedipine (a calcium channel blocker), and Ca²⁺-free Krebs-Henseleit solution with ethylene glycol tetraacetic acid (EGTA), followed by administration of cumulative dosages of SM extracts. SM extracts induced dose-related tonic contraction of the LES, which was unaffected by tetrodotoxin, atropine, or nifedipine. However, the SM extract-induced LES contraction was significantly inhibited by Ca²⁺-free Krebs-Henseleit solution with EGTA. Next, SM extracts significantly induce extracellular Ca²⁺ entry into primary LES cells in addition to intracellular Ca²⁺ release and in a dose-response manner. Confocal fluorescence microscopy showed that the SM extracts consistently induced significant extracellular Ca²⁺ influx into primary LES cells in a time-dependent manner. In conclusion, SM extracts could induce tonic contraction of LES mainly through the extracellular Ca²⁺ influx pathway.

This study aimed to develop optimal gelatin-based mucoadhesive nanocomposites as scaffolds for intravesical gene delivery to the urothelium. Hydrogels were prepared by chemically crosslinking gelatin A or B with glutaraldehyde. Physicochemical and delivery properties including hydration ratio, viscosity, size, yield, thermosensitivity, and enzymatic degradation were studied, and scanning electron microscopy (SEM) was carried out. The optimal hydrogels (H), composed of 15% gelatin A175, displayed an 81.5% yield rate, 87.1% hydration ratio, 42.9 Pa·s viscosity, and 125.8 nm particle size. The crosslinking density of the hydrogels was determined by performing pronase degradation and ninhydrin assays.In vitrolentivirus (LV) release studies involving p24 capsid protein analysis in 293T cells revealed that hydrogels containing lentivirus (H-LV) had a higher cumulative release than that observed for LV alone (3.7-, 2.3-, and 2.3-fold at days 1, 3, and 5, resp.). Lentivirus from lentivector constructed green fluorescent protein (GFP) was then entrapped in hydrogels (H-LV-GFP). H-LV-GFP showed enhanced gene delivery in AY-27 cellsin vitroand to rat urothelium by intravesical instillationin vivo. Cystometrogram showed mucoadhesive H-LV reduced peak micturition and threshold pressure and increased bladder compliance. In this study, we successfully developed first optimal gelatin-based mucoadhesive nanocomposites as intravesical gene delivery scaffolds.

Extrusion-spheronization and the fluid bed method are valuable commonly applied methods in microparticle production. However, the characteristics of resultants prepared by these two methods have seldom been compared. The aim of this study was to investigate differences in pharmaceutical properties and release kinetics of sustained-release ketoprofen microparticles prepared by different manufacturing processes. Microparticles prepared by extrusion-spheronization displayed slower release rate resulting in increased diffusion path of the drug, a behavior distinct for microparticles with less Surelease®. The effects of manufacturing method on release rate was also significant for microparticles with less Surelease®compared to microparticles with more Surelease®. The release profiles of coated microparticles fitted well to the Higuchi’s release model, but in cases of microparticles with larger Surelease® coating, the trend was towards a zero-order release model. These findings are valuable for comprehending the differences among different preparation processes and for choosing the optimal manufacturing method of sustained-release microparticles. Key words: Ketoprofen, microparticles, sustained drug delivery, extrusion-spheronization, fluid-bed, dissolution release kinetics.

Pyridostigmine bromide (PB) sustained-release (SR) pellets were developed by extrusion-spheronization and fluid-bed methods using Taguchi experimental and 2(3) full factorial design. In vitro studies, the 2(3) full factorial design was utilized to search for the optimal SR pellets with specific release rate at different time intervals (release percent of 2, 6, 12, and 24 hr were 6.24, 33.48, 75.18, and 95.26%, respectively) which followed a zero-order mechanism (n=0.93). The results of moisture absorption by Karl Fischer has shown the optimum SR pellets at 25 degrees C/60% RH, 30 degrees C/65% RH, and 40 degrees C/75% RH chambers from 1 hr-4 weeks, attributing that the moisture absorption was not significantly increased. In the in vivo study, the results of the bioavailability data showed the Tmax (from 0.65+/-0.082 hr-4.82+/-2.12 hr) and AUC0-30 hr (from 734.88+/-230.68 ng/mL.hr-1454.86+/-319.28 ng/mL.hr) were prolonged and increased, as well as Cmax (from 251.87+/-27.51 ng/mL-115.08+/-14.87 ng/mL) was decreased for optimum SR-PB pellets when compared with commercial immediate-release (IR) tablets. Furthermore, a good linear regression relationship (r=0.9943) was observed between the fraction dissolution and fraction absorption for the optimum SR pellets. In this study, the formulation design not only improved the hygroscopic character of PB but also achieved the SR effect.

The solid complex of gliclazide and beta-cyclodextrin was prepared by neutralization method and the precipitation solvent evaporation method was used to prepare gliclazide nanospheres. Fourier-transform infrared spectroscopy and differential scanning calorimetry were used to examine whether gliclazide solid complex and gliclazide nanospheres were successfully formed in this study. The dissolution rate of gliclazide from its nanospheres was faster than its solid complex and pure drug. The morphology of particles for nanospheres showed no crystal character of gliclazide. In summary, the results indicate that nanotechnology provides better effects in solubility and dissolution rate of gliclazide than neutralization method.