The antioxidant system beta-D(+) glucose-glucose oxidase-catalase: tests for pyrogenicity and antigenicity.

PURPOSE: beta-D(+) glucose-glucose oxidase (E.C.# 1.1.3.4)-catalase (E.C.# 1.11.1.6) (GO-CAT) is being investigated as a new antioxidant system for use in pharmaceutical solutions. This study reports the results of tests for pyrogenicity and antigenicity of GO-CAT derived from Aspergillus niger when used parenterally in autoclaved preparations. METHODS: The Limulus amebocyte lysate (LAL) method was used to test the pyrogenicity of native GO-CAT. Pyrogenicity/antigenicity was evaluated in vivo by injecting autoclaved GO-CAT into New Zealand white rabbits. Antigenicity was also evaluated by Ouchterlony and Western blotting. RESULTS: None of the native GO-CAT concentrations tested (up to 30.83 u/ml) produced a positive gel clot in the LAL test, thereby suggesting its non-pyrogenicity. The rabbits, which received seven injections of autoclaved GO-CAT over a period of eleven weeks, remained healthy during and after the GO-CAT injections. All Ouchterlony and Western blot assays using sera from rabbits injected with autoclaved GO-CAT were negative. Furthermore, autoclaved GO-CAT could not be detected in Ouchterlony assays using a mouse monoclonal antibody (GO40 mAb) to native A. niger glucose oxidase. Control samples containing native GO-CAT produced an antigen-antibody complex reaction in Ouchterlony assays against the GO40 mAb. Antigen-antibody complexes could be detected by non-denaturing PAGE in samples containing native GO-CAT/GO40 and boiled GO-CAT/GO40, but not in samples containing autoclaved GO-CAT/GO40. These results indicate autoclaved GO-CAT is neither pyrogenic nor antigenic. CONCLUSIONS: Based on these results, there is potential for the use of beta-D(+) glucose-glucose oxidase-catalase as an antioxidant system in pharmaceutical solutions, particularly in terminally autoclaved aqueous formulations for parenteral use.

Release kinetics of a controlled-release multiparticulate dosage form prepared using a hot-melt fluid bed coating method.

Dissolution profiles of various multiparticulate controlled-release dosage forms prepared by applying lipophilic materials using a hot-melt coating technique were analyzed to determine release kinetics. The dosage forms were prepared using a novel solid dispersion hot-melt fluid bed coating method. Various lipophilic materials were investigated and those showing promise as potential controlled-release coating agents were analyzed further. Dissolution data were used as the primary basis of comparison. A dual equation combining first-order and square-root-of-time release kinetics was used to fit data. The statistical output from nonlinear regression software suggests that this equation describes the data better than the individual equations alone. Particular parameters used to compare the various models include R2, the model selection criteria, and the coefficient of determination. The high degree of fit offered by the dual equation implies that two distinct kinetic processes may be involved in the release of drug from the dosage forms. A possible release mechanism based on this information is offered. The dual equation was shown to be a superior model for the controlled-release system chosen for study. The equation also appears to be applicable to a variety of other controlled-release systems reported in the literature.

Evaluation of extended-release applications for solid dispersion hot-melt fluid bed coatings utilizing hydrophobic coating agents.

A new hot-melt fluid bed coating method was evaluated for potential extended-release applications. Chlorpheniramine maleate (CPM) USP was chosen as a model drug. The assays for drug release and content uniformity were dictated by the USP Official Monograph for a Chlorpheniramine Maleate Extended-Release Capsule. The fluid bed chamber was charged with CPM-loaded nonpareils and hydrophobic coating agents in the solid state. The method consists of four processing stages: (a) warming, (b) preheating, (c) melting-spreading, and (d) cooling-congealing. Various hydrophobic coating agent candidates were evaluated for extended-release potential by a preliminary screen at a coating agent level of 1.5% (w/w). A beeswax coating agent was identified as the most promising candidate of the preliminary screen. After the level of beeswax was increased to 2.0%, the dissolution profile met all of the specifications of the USP Drug Release Test 1 for a CPM Extended-Release Capsule. The potency and content uniformity remained unchanged by the process. Dual coatings demonstrated a cumulative extension of release superior to the capability of a single coat. The new method is a viable alternative to hot-melt spray-coating methodologies. Organic solvents, spraying equipment, steam jackets, and/or heating tape are eliminated from the process. A reduction of equipment costs, setup time, and cleanup time may be realized. The method has demonstrated extended-release capabilities. No excessive attrition of potency or content uniformity has been noted. Additive, multiple coatings that have a cumulative effect on release retardation are feasible.

Preliminary assessment of an image analysis method for the evaluation of pharmaceutical coatings.

This article describes the development and preliminary assessment of an innovative image analysis method for the evaluation of pharmaceutical coatings. Nonpareils previously hot-melt coated with a red water-soluble dye incorporated into polyethylene glycol were employed to develop and evaluate the new method. Digital images of batch samples were acquired and transferred to a PC for evaluation by image analysis software. Standard deviations generated for the optical densities of individual nonpareils in each batch sample were utilized to evaluate coating uniformity. Mean optical densities generated for each batch sample were used to evaluate the mean coating thickness. Method precision determinations, using an average sample size of 221 nonpareils, returned a relative standard deviation of 2.47% for coating uniformity and 1.71% for coating thickness. The new method is rapid, objective, and relatively inexpensive. It proved to be both qualitative and quantitative for coating uniformity, qualitative for coating thickness, and maintained a high degree of precision. The method can be used independently or in conjunction with other methods to eliminate their subjective aspects regarding sample selection.

beta-D(+) glucose-glucose oxidase-catalase for use as an antioxidant system.

The purpose of this study was to investigate the use of beta-D(+) glucose-glucose oxidase (EC 1.1.3.4)-catalase (EC 1.11.1.6) (BDG-GO-CAT) as a new antioxidant system in solutions. A novel method for estimation of activity of the system was developed using a dissolved oxygen (DO) meter and an oxygen probe. The method can be used to determine the enzymatic activity of the system at GO concentrations of 0.005 to 0.030 unit/ml, with an r2 of 0.995 for the linearity of the standard curve, and can be adapted for analysis in any solution. At room temperature of 23.0 degrees +/- 2.0 degrees C, the maximum activity of the BDG-GO-CAT system was found to occur at pH 5.40. The half-life values for the stability of GO-CAT in 0.10 M phosphate buffer solutions of pH 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 were 9.78, 49.43, 53.46, 36.51, 12.68, 1.84, and 0.80 weeks, respectively. Dextrose was used in place of beta-D(+) glucose for cost-saving purposes, and a standard curve for the activity of GO-CAT was obtained using 20 mg/ml dextrose. The BDG-GO-CAT was effective as a DO-scavenger in closed containers, when the containers were opened and exposed to atmosphere for 2 days between tests, and upon reclosing them. Pharmaceutical excipients such as ethanol, glycerin, propylene glycol, methylparaben, propylparaben, artificial strawberry flavor, and sodium benzoate did not show any adverse effect on the activity of BDG-GO-CAT. Sorbitol, high-fructose corn syrup, sucrose, and polyethylene glycol 3350 increased the rate of DO removal. Sodium carboxymethyl-cellulose (CMC) at 2.0% w/v decreased the rate of DO removal. These studies indicate that the BDG(dextrose)-GO-CAT system warrants serious consideration for use an antioxidant system in aqueous formulations.

Development and optimization of a solid dispersion hot-melt fluid bed coating method.

A new hot-melt fluid bed coating process has been developed, characterized, and optimized. Polyethylene glycol served as the model coating agent and was charged with substrate into the fluid bed chamber in the solid state. The processing stages included: (A) warm-up, (B) preheating, (C) melting-spreading, and (D) cooling-congealing. A central composite design was utilized to characterize and optimize the process. Substrate porosity and density evaluations were conducted by mercury intrusion. The method proved capable of coating nonpareils from 10 to 35 mesh (0.500 to 2.00 mm) and tablets up to 1 g. The nonpareils were coated as individual particles, while particle sizes significantly smaller than 40 mesh (0.420 mm) tended to agglomerate. The porosity and density values of dissimilar nonpareil batches showed a large degree of variation, affecting the method's reproducibility. Additive coatings were achieved by sequential runs using coating agents of diminishing melting points. The method is a viable alternative to hot-melt spray-coating processes. Organic solvents, spraying equipment, steam jackets, and/or heating tape are eliminated from the process.

Influence of various anions on intestinal disappearance of hexamethonium chloride and pralidoxime chloride in rats.

The intestinal transfer of two poorly absorbed quaternary ammonium drugs, hexamethonium chloride (I) and pralidoxime chloride (II), in the presence of various organic and inorganic anions was investigated in the rat using a modified in situ gut technique. The results were in agreement with those of the conventional in situ loop and plasms drug level techniques. Of the anions investigated, cholate, desoxycholate, taurocholate, phoscholate, dehydrocholate, and hyodesoxycholate had the greatest effect on increasing the amount and rate of disappearance of I. Similarly, the amount and rate of disappearance of II were enhanced markedly in the presence of phoscholate and trichloroacetate. The effect of cholate and phoscholate was investigated in detail. The membrane permeability and histological studies indicated that these anions may compromise the structural integrity of the membrane tissue, thus enhancing drug transfer.

Separation of penicillin G potassium and its degradation products using high-pressure liquid chromatography.

A high-pressure liquid chromatographic technique was developed for the separation of penicillin G potassium and several of its decomposition products. The method utilized a buffered acetonitrile-phosphate mobile phase on a reversed-phase C18 column. Separation of penicillin G potassium and six degradation products was attained within 25 min.

Kinetics of absorption and elimination of pralidoxime chloride in dogs.

The kinetics of the absorption and elimination of pralidoxime chloride were investigated in the dog. Similar apparent elimination rate constants were obtained after intravenous, intramuscular, and oral administration. Although oral absorption occurred slowly, intramuscular absorption proceeded rapidly. With in situ techniques, it was found that no absorption occurred from the isolated stomach and duodenum but that absorption did take place from the jejunum and ileum.

Kinetics of intravenous theophylline.

The pharmacokinetics of theophylline was studied in 6 normal, nonsmoking, adult male volunteers. A constant-rate intravenous infusion of 3.84 to 4.98 mg/kg of theophylline (as the ethylenediamine salt, aminophylline) was administered over 40 min. Serum theophylline concentrations were measured for 24 hr by means of a gas chromatographic method specific for theophylline. Within 30 min of an average intravenous dose of 4.4 mg/kg of theophylline, serum levels reached 10 microgram/ml. The highest serum level at the end of the infusion was 17 microgram/ml. The serum concentration-time data were fitted to a two-compartment open model and yielded a mean serum half-life (t1/2) of 11.02 hr, a value longer than those previously reported. Our results indicated that after the original loading dose of 4.4 mg/kg was infused for 40min, an immediate infusion rate of 1.40 mg/kg/hr (1.65 mg/kg/hr aminophylline) would be necessary to maintain a serum level of 10 microgram/ml.

Interference by butyl rubber stoppers in GLC analysis for theophylline.

During a study of the pharmacokinetics of theophylline using GLC analysis, unexpectedly high values occurred in a random manner. The cause of there abnormal values was investigated, and significant interference was observed when blood samples were drawn using evacuated glass tubes sealed with butyl rubber stoppers. In vitro tests using distilled water showed no apparent theophylline levels due to the additives in three commonly used tubes. However, when water was allowed to remain in contact with the butyl rubber stoppers for 1 min, an apparent theophylline content of as high as 5.5 mug/ml was observed. A contact time of 60 min resulted in apparent theophylline levels of as high as 52.3 mug/ml. It was concluded that a substance leached from the butyl rubber stoppers accounted for the spurious results.

Rapid methods for bioavailability determination utilizing urinary excretion data.

Two equations were developed which enable urinary excretion data to be utilized for estimating drug bioavailability within 12 hr-starting between one and two half-lives of the drug, depending upon the relative rates of absorption, distribution, and elimination. Both equations were examined using simulated data for both the one- and two-compartment open models. One equation was tested using literature data with excellent results.