Properties of poly(lactic-co-glycolic acid) nanospheres containing protease inhibitors: camostat mesilate and nafamostat mesilate.

Poly(lactic-co-glycolic acid) (PLGA) nanospheres containing protease inhibitors, camostat mesilate (CM) and nafamostat mesilate (NM), were prepared by the emulsion solvent diffusion methods in water or in oil, and the w/o/w emulsion solvent evaporation method. The average diameter of PLGA nanospheres prepared in the water system were about 150-300 nm, whereas those prepared in the oil system were 500-600 nm. Among the three methods, these drugs were the most efficiently encapsulated up to 60-70% in PLGA nanospheres in the oil system. Other factors that may influence drug encapsulation efficiency and in vitro release such as drug load, molecular weight of polymer were also investigated. Both the CM- and NM-loaded nanospheres prepared in the water system immediately released about 85% of the drug upon dispersed in the release medium while the drug initial burst of nanospheres prepared by the emulsion solvent diffusion in oil method reduced to 30% and 60% for CM and NM, respectively. Poly(aspartic acid) (PAA), a complexing agent for cationic water soluble drugs, showed little effect on the encapsulation efficiency and release behavior for CM and NM. The DSC study and AFM pictures of nanospheres demonstrated that temperature-dependent drug release behavior was ascribable to the glass transition temperature of the polymer, which also affected the morphology of nanospheres upon dispersed in the release medium and influenced the drug release consequently.

Role of cholecystokinin in cholestyramine-induced changes of the exocrine pancreas.

This study was an investigation of the role of cholecystokinin (CCK) in the stimulatory action of cholestyramine on rat exocrine pancreas. Postprandial CCK release was significantly enhanced by acute administration of cholestyramine (12.7 +/- 1.8 vs 3.7 +/- 0.5 pmol/L in controls). Over four weeks, rats were fed either regular diet or diet containing 6% cholestyramine, and were treated with the specific CCK receptor antagonist L-364,718 (2 x 0.5 mg/kg body weight/day s.c.) or DMSO (vehicle for the antagonist). Cholestyramine significantly increased pancreatic weight and trypsin and chymotrypsin contents. L-364,718 abolished these effects. Concomitant administration of antagonist and cholestyramine elevated amylase content, compared to controls. CCK levels in fasted animals did not differ between the four groups. The effect of the same dose of L-364,718 on pancreatic enzyme depletion, induced by the protease inhibitor camostate, was studied in a control experiment. A single dose of camostate (200 mg/kg) caused a 44-68% decrease in enzyme content. L-364,718 reversed this effect for all enzymes. We conclude that CCK is the mediator of cholestyramine-induced pancreatic hypertrophy and increase in content of proteases. After long-term administration, the CCK receptor antagonist, in combination with cholestyramine revealed an agonistic effect on individual, pancreatic enzyme content.

Changes of the Met-enkephalin-like peptide content induced by noxious stimuli in the rat incisor pulp.

It was examined what mechanism involved in an increase of met-enkephalin (met-EK)-like peptide content in the pulp induced by noxious stimuli. The increased content of the peptides by cavity formation as noxious stimulation was not influenced by cycloheximide, but attenuated by FOY-305 [N,N-dimethyl carbamoyl-methyl 4-(4-guanidinobenzoyloxy) phenyl acetate methanesulfonate], a trypsin-like enzyme inhibitor, and enhanced by captopril, and attenuated by infusion of saline in the pulp cavity. From these results, it was suggested that noxious stimuli on the pulp led to activation of trypsin-like enzymes followed by an increased content of met-EK-like peptides, and thereafter, the peptides, such as met-EK, might be degraded by angiotensin converting enzyme (ACE). Furthermore, an immunohistochemical study demonstrated that met-EK-like immunoreactivity (met-EK-IR) of cells in the rat incisor pulp was clearly increased following tryptic digestion of the pulp section, supporting a suggestion mentioned above.

Preparation and characterization of biodegradable or enteric-coated microspheres containing the protease inhibitor camostat.

We have produced biodegradable or enteric-coated microspheres containing camostat mesylate, a protease inhibitor, using a water-oil-water emulsion solvent evaporation method. The characteristics of the microspheres were determined. When polylactic acid, a biodegradable polymer, was used as a wall material, the optimized microsphere obtained showed a loading efficiency of almost 95% and had a mean diameter of 30 microm. This microsphere showed a sustained-release profile, with nearly 25% of drug being released at seven days in a dissolution test. When hypromellose acetate succinate (AS-HG type, with a high content of succinyl group) was used as an enteric wall material, optimized microspheres showed a loading efficiency of almost 80%. In this case, pH 3.0 citrate buffer was used as an internal aqueous phase, and citrate buffer containing 0.5% polyvinylalcohol was used as the external aqueous phase. These microspheres showed a rapid release profile in pH 6.8 buffer, whereas the release was extremely slow in pH 1.2 buffer. Hypromellose acetate succinate microspheres were also prepared containing 10% (w/w) N-benzoyl-dl-arg-4-nitroanilide as a model substrate for trypsin, with or without 5% (w/w) camostat. These microspheres were incubated in pH 6 or 7 buffer containing trypsin at 37 degrees C. When camostat was included in the microspheres, the substrate was protected from attack by trypsin, while in the absence of camostat, the released substrate was immediately attacked by trypsin to produce the degradation product N-benzoyl-dl-arginine.

Activation of calcium ion-dependent proteinases by bradykinin in dental pulp of the rat.

The present study was aimed to examine whether BANA-degrading enzyme activities could be enhanced by bradykinin(BK) in dental pulp of the rat in vitro. The results showed that BK(0.1-10 microM) dose-dependently enhanced BANA-degrading enzyme activity at pH 7.4. The effects of BK(1 microM) were found to be most effective at both pH 7 and 8, with enhancement of the enzyme activities at a wide range of pH. The BK effects at both the pH were not inhibited by FOY-305(0.1 microM), an inhibitor of trypsin-like enzymes, differing from that at pH 6 in adrenal medulla of the rat. On the other hand, the effects of BK at both the pH were remarkably inhibited by EGTA (2 mM), followed by reversal with calcium ion (2.42 mM). These results suggested as follows: 1) there might be two kinds of BANA-degrading enzymes activated by BK in the pulp. 2) it was conceivable that BANA-degrading enzymes activated by BK were quite different from serine proteinases and were interfered with them in the pulp. 3) calcium ion might play a role in BK-induced enhancement of BANA-degrading enzyme activities which were regarded as met-enkephalin (ME) processing enzyme activities in the pulp.

Oral delivery system for two-pulse colonic release of protein drugs and protease inhibitor/absorption enhancer compounds.

It is well known that the intestinal stability and absorption of protein drugs are improved when enzyme inhibitors/permeation enhancers are coadministered. Recently, it was hypothesized that an increased effectiveness of these adjuvants might be achieved by timing their release prior to that of the protein, so that a more favorable environment would be established in advance. Therefore, an oral system was proposed for two-pulse colonic release of insulin and the protease inhibitor camostat mesilate/absorption enhancer sodium glycocholate. The device consisted of a drug-containing core, an inner swellable/erodible low-viscosity hydroxypropyl methylcellulose (HPMC) coating, an intermediate adjuvant layer, and an additional outer HPMC coating. HPMC coats and camostat mesilate/sodium glycocholate films with differing thicknesses were applied to immediate-release tablet cores by aqueous spray coating. The obtained units were characterized for weight, thickness, breaking force, and release performance. All systems showed satisfactory technological properties and the pursued pulsatile delivery behavior, with programmable delay phases preceding inhibitor/enhancer release and elapsing between inhibitor/enhancer and protein release, respectively. Indeed, both lag times linearly correlated with the relevant HPMC coating level. The system was thus proven suitable for yielding two-pulse release profiles, in which lag phases could be modulated to provide convenient concentration patterns for proteins and adjuvants.

Clinical experience with a protease inhibitor [N,N-dimethylcarbamoylmethyl 4-(4-guanidinobenzoyloxy)-phenylacetate] methanesulfate for prevention of recurrence of carcinoma of the mouth and in treatment of terminal carcinoma.

A protease inhibitor, (N,N-dimethylcarbamoylmethyl 4-(4-guanidinobenzoyloxy)-phenylacetate) methanesulfate was administered orally 3 times daily to 9 patients with squamous cell carcinoma of the oral cavity, at doses ranging from 0.6 g. to 7.2 g. Seven of 9 patients who had undergone all kinds of therapies were observed to see whether recurrence was prevented and in no case did the tumour recur. Also, in 2 terminal secondary cases tumour remission resulted. Histologically, after this therapy, carcinomatous lesions showed marked hyperkeratosis in 2 cases and the survival time in 1 case was markedly prolonged. Haematological and gastrointestinal toxicity was almost never observed at these doses.

Effect of bile salt binding or protease inactivation on plasma cholecystokinin and gallbladder responses to bombesin.

BACKGROUND/AIMS: Bombesin-stimulated plasma cholecystokinin levels decrease after an initial increase despite continuous infusion of bombesin. The aim of this study was to determine if a feedback mechanism, mediated by bile salts or proteolytic enzymes, is responsible for this decline. METHODS: Bombesin (1.0 ng.kg-1.min-1) was infused into volunteers for 180 minutes on separate occasions. Cholestyramine, colestipol, camostate, or saline were perfused intraduodenally during the second hour of the tests. Cholestyramine was also administered without infusion of bombesin. RESULTS: Colestipol and cholestyramine, dependent on their bile salt-binding capacity, markedly enhanced (P < 0.05) bombesin-stimulated plasma cholecystokinin from 2.1 +/- 0.5 pmol/L to 6.4 +/- 2.2 pmol/L and 12.1 +/- 3.3 pmol/L (P < 0.05 vs. colestipol), respectively, and further decreased gallbladder volume (P < 0.05) from 9.4 +/- 1.6 mL to 2.0 +/- 0.4 mL and 2.2 +/- 0.5 mL, respectively. The protease inhibitor camostate had no effect. Bile salt precipitation also enhanced plasma pancreatic polypeptide responses (P < 0.01) but did not alter gastrin responses. Plasma cholecystokinin responses to cholestyramine without bombesin infusion varied considerably, but increments were highly correlated to decreases in gallbladder volume (r = 0.91; P < 0.005). CONCLUSIONS: Bile salt sequestration but not protease inactivation enhances plasma cholecystokinin and gallbladder responses to bombesin infusion in humans.