Intraocular distribution of intravitreally administered amphotericin B in normal and vitrectomized eyes.

A study was performed to determine how amphotericin B is distributed in the eye after direct intravitreal injection. Radiolabeled amphotericin B was administered by direct injection into the vitreous space of unmodified phakic control or vitrectomized aphakic eyes in the rabbit model. The eyes were removed at different post-injection times, frozen and dissected into anatomical subparts of cornea, aqueous, iris, lens, vitreous and sclera-choroid-retina. The parts were assayed for total radioactivity (expressed as remaining amphotericin B). No accumulation of drug was observed in the cornea, lens, iris or aqueous region. The majority of drug was found in the vitreous cavity. The rate of disappearance of radiolabeled drug or radiolabeled drug degradation products from the vitreous space was similar to the rate of disappearance from the eye. However, progressive accumulation of radioactivity was observed in the sclera-choroid-retina tissue in the unmodified phakic eyes. This was not observed in vitrectomized aphakic eyes. The accumulated radioactivity could have represented drug degradation products or active drug. These results shed light on the distribution of amphotericin B in the eye after direct intravitreal injection, a procedure often employed clinically for fungal endophthalmitis.

Immobilization of a primary amine-containing drug, adriamycin. Coupling to crosslinked polyvinyl alcohol and mechanistic comparison of hydrolytic stability.

Literature reports have described the covalent coupling of the primary amine-containing anticancer drug, adriamycin, to polymeric supports through the amine group on the drug. These reports also have described drug mechanism studies with the immobilized adriamycin, where the release of the drug would undermine the validity of the conclusions. In the present paper, detailed experimental conditions are given for preparation of nonwater-soluble particles of polyvinyl alcohol by crosslinking water-soluble polyvinyl alcohol with 1,4-benzenedicarboxaldehyde, and for activation with cyanuric chloride and covalent attachment of adriamycin. The expected stability of this drug-support linkage against hydrolytic cleavage is compared mechanistically to that expected for less stable coupling through a carbamate linkage or for less stable coupling via an azomethine link.

Formation of propylene oxide by Nocardia corallina immobilized in liquid paraffin.

Nocardia corallina B276 cells were immobilized by emulsification with liquid paraffin and an antifoam agent at room temperature. The immobilized cells were studied for their ability to carry out the formation of propylene oxide from propylene and oxygen. The evaluations were done with the cells in a bubble-type reactor with a continuous gas feed of 5% propylene and 11.6-95% oxygen, with the balance nitrogen. By using liquid paraffin and antifoam, both the epoxidation activity and the stability were improved, especially for the P-1-200 strain, over that for nonimmobilized cells. The N. corallina cells showed an apparent preference for a hydrophobic, as compared to a hydrophilic, environment. The propylene-oxide-forming activity of the immobilized cells was higher at 40 than at 30 degrees C reactor temperature and with 20% (versus 95%) oxygen in the feed. The stability was markedly better at 30 degrees C and with 20% oxygen. High gas flowrates gave increased apparent activity probably because of less resistance to substrate mass transfer. The effects of pH were minor. The role of glucose as the energy source for regeneration of cofactors for the monooxygenase system also is discussed.

Amphotericin clearance in vitrectomized versus nonvitrectomized eyes.

Ocular clearance of amphotericin B after direct intravitreal injection was studied in a rabbit model. Unmodified phakic eyes, Candida-infected eyes, aphakic eyes, and aphakic vitrectomized eyes were employed. Using high pressure liquid chromatography to assess drug level, the half-lives of drug disappearance after single 10-microgram (microgram) intravitreal injections were 9.1, 8.6, 4.7, and 1.4 days, respectively. The disappearance slope for vitrectomized eyes was significantly different from all nonvitrectomized eyes with P less than 0.001. The rapid disappearance of amphotericin from vitrectomized eyes must be considered in clinical management of patients with fungal endophthalmitis.

Cell surface effects of adriamycin and carminomycin immobilized on cross-linked polyvinyl alcohol.

Previous reports have claimed Adriamycin to be cytotoxic to cultured tumor cells when the drug is covalently immobilized on a solid support, thus suggesting a cell surface mechanism of action for the drug. Although these previous reports attempted to rule out released drug or endocytosis of drug-support particles as alternative explanations for the observed cytotoxicity, a more thorough analysis is necessary to substantiate fully the cell surface idea. In the present work, the stability of the drug-support linkage was increased by use of cross-linked polyvinyl alcohol as the support and cyanuric chloride or a diazonium salt for attachment of the drug. Different anthracycline orientations were tested by coupling Adriamycin at the amino sugar and carminomycin at the D-ring. The Adriamycin cross-linked polyvinyl alcohol and carminomycin cross-linked polyvinyl alcohol preparations had much lower drug release rates than did the earlier used carbamate-linked Adriamycin cross-linked agarose materials. All three immobilized drug preparations inhibited the growth of L1210 or S180 clones following 2- or 20-h incubation with cells at 37 degrees C. The results strongly support the concept that immobilized anthracyclines can be cytotoxic to cultured cells, for at least two different orientations of the drug on the support.

Site-specific immobilization of flavin adenine dinucleotide on indium/tin oxide electrodes through flavin adenine amino group.

A Mannich-type reaction was used to attach flavin adenine dinucleotide (FAD) covalently to aminosilane derivatized indium/tin oxide-coated glass plates. The aminosilane was activated with formaldehyde to give an intermediate that attached specifically to the adenine amino group of FAD. The presence of the intermediate also was demonstrated by coupling hydroquinone to the formaldehyde activated support. The immobilized FAD and hydroquinone were characterized by cyclic or differential pulse voltammetry. The immobilized FAD was shown to reduce the overpotential for NADH oxidation by 180 mV. In keeping with results for FAD on glassy carbon, FAD attached to indium/tin oxide at the adenine amino group did not lead to reconstitution of activity with apoglucose oxidase.

Electrochemical and glucose oxidase coenzyme activity of flavin adenine dinucleotide covalently attached to glassy carbon at the adenine amino group.

Flavin adenine dinucleotide (FAD) was covalently attached to an electron-conducting support, i.e., glassy carbon. The support was activated by oxidation to create surface carboxylic acid groups, followed by reaction with a water-soluble carbodiimide. FAD was then attached to the activated support by three different methods: (1) directly; (2) through 6-aminocaproic acid as a spacer; and (3) through ethylenediamine glutaraldehyde as a spacer. Coupling occurred at the FAD adenine amino group, or possibly at a ribityl OH group. Cyclic voltammetry was used to determine Eo' values and FAD loadings. The immobilized FAD also acted as a catalyst for the oxidation of reduced nicotinamide adenine dinucleotide (NADH) in that it reduced overpotential by about 195 mV. When the apoenzyme of glucose oxidase was added to the glassy carbon-FAD or glassy carbon-spacer-FAD preparations, no reconstitution of enzyme activity could be observed. This suggests strongly that the adenine amino group of FAD cannot be modified by attachment of something as large as easily visible solid particles. However, it leaves unanswered the question of larger molecular weight material can be accommodated in the FAD-apoenzyme cleft and retain glucose oxidase activity.

Electrochemical and enzymatic activity of flavin adenine dinucleotide and glucose oxidase immobilized by adsorption on carbon.

Flavin adenine dinucleotide (FAD) and glucose oxidase were adsorbed on medium porosity spectroscopic graphite (SG) and on low porosity glassy carbon (GC) with retention of electrochemical activity, as measured by cyclic and differential pulse voltammetry. Adsorption on the SG was very strong, while that on GC was much weaker. Enzyme activity could be partially restored by the addition of the apoenzyme of glucose oxidase to the SG-adsorbed FAD preparation. The holoenzyme of glucose oxidase also was adsorbed on SG with retention of enzyme activity. The mechanism for the reconstitution of active enzyme from adsorbed FAD and soluble apoenzyme is not clear. The data suggest that the reconstituted enzyme stays adsorbed to the SG, but it is not clear whether the FAD or protein portions (or both) are adsorbed after reconstitution. The data also indicate that substrate mass transfer resistance may be important with the reconstituted-adsorbed enzyme.

Mass transport and reaction kinetic parameters determined electrochemically for immobilized glucose oxidase.

Mass-transfer resistances often have pronounced effects on the overall reaction rates of enzymes immobilized at interfaces or in polymeric matrices. In the present work glucose oxidase was immobilized on the surface of a platinum disk electrode by one of three attachment techniques: silane-glutaraldehyde, allylamine-glutaraldehyde, and albumin-glutaraldehyde. In one group of studies the electrodes were rotated, and methods were employed to determine the diffusion and shielding coefficients for transport of a model electroactive compound, i.e., potassium ferrocyanide, through the enzyme matrix. A model electrochemically active compound was used because glucose exhibits a very slow rate of electron transfer at a platinum surface. The diffusion coefficient for ferrocyanide was reduced 7% by the silane-enzyme and 25% by the allylamine-enzyme matrices. In a second group of studies the electrodes were held stationary. Marked internal diffusional resistance was noted for the albumin-glutaraldehyde-enzyme matrix. The calculated flux of ferrocyanide was decreased by a factor of 2000-8500 for transport through albumin-enzyme matrices 0.21-0.063 cm thick, as compared to transport through free solution. In a third group of studies the rotating enzyme-matrix electrode was utilized in determining apparent values of the Michaelis constant for glucose. The velocity of the reaction was determined by amperometric measurement of the concentration of hydrogen peroxide reaching the ring electrode. The results, determined from Eadie-Hofstee type plots of reaction current and substrate concentration, gave values between 12 and 36 mM for the three methods of immobilization.(ABSTRACT TRUNCATED AT 250 WORDS)

Immobilized glucose oxidase in the potentiometric detection of glucose.

Previous work has shown that glucose oxidase can be immobilized on platinum to give an electrode that responds potentiometrically to glucose over the clinically useful range of about 10-250 mg glucose/100 mL. The present studies were carried out with electrochemically pretreated platinum and with gold or porous graphite substituted for the platinum support. The presence of the enzyme gave a significantly enhanced potentiometric response over that obtained with the bare support for both the pretreated platinum and the porous graphite, but not with gold. However, with platinum the potentiometric response became more negative with increasing glucose concentration. With porous graphite, the potential changed in the positive direction as the glucose concentration was increased. Hysteresis was demonstrated for the platinum-enzyme electrode. Mass transfer measurements with a rotating ring-disc electrode (RRDE) showed measurable diffusional resistances to the transport of a model electroactive compound (potassium ferrocyanide) through a matrix of immobilized enzyme attached to the disc of the RRDE. These results are part of a larger study to define the source of the potentiometric response by examining the roles of the support and the mass transfer resistances through the immobilized enzyme matrix.

Effect of enzyme-matrix composition on potentiometric response to glucose using glucose oxidase immobilized on platinum.

Glucose oxidase (beta-D-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) was immobilized in a crosslinked matrix of bovine serum albumin, catalase, glucose oxidase and glutaraldehyde on platinum foil. When placed in glucose solution, this enzyme-electrode elicited a potentiometric response that varied with the changes in glucose concentration. The immobilized glucose oxidase was present at 7.4-10.1 micrograms enzyme protein/ml of matrix, as determined with 125I-labelled enzyme. The coupled enzyme activity was stable over 120 h; however, the apparent activity of the immobilized glucose oxidase was markedly less than that for the same amount of enzyme free in solution. This indicated a significant level of diffusional resistance within the enzyme-matrix. The potentiometric response to glucose increased significantly as either the thickness of the enzyme-matrix or the glutaraldehyde content was reduced; this also was attributed to diffusional effects. Several enzyme-electrodes, constructed without exogenous catalase and with different amounts of glucose oxidase, showed greater sensitivity in potentiometric response at low glucose oxidase loadings. These results are consistent with the hypothesis that the potentiometric response arises from an interfacial reaction involving a hydrogen peroxide redox couple at a platinum surface. The data also suggest that an optimum range of hydrogen peroxide concentration exists for maximum electrode sensitivity.