Howard Y. Ando

Immobilization of active urokinase on albumin microspheres : use of a chemical dehydrant and process monitoring

A method of immobilizing urokinase on albumin microspheres has been developed. Laser scattering, which was used to follow particle size from the initial emulsification stage to the final aqueous resuspension of the microsphere stage, showed that particle coalescence and crosslinking were critical parameters in manufacturing the microspheres. Chemical dehydration with 2,2-dimethoxypropane was used to convert an albumin emulsion into an albumin suspension and to reduce coalescence. An optimal amount of dehydrant produced 0.3-µm particles which resisted a 50°C temperature challenge. Since oil/glutaraldehyde emulsion resulted in large particles with no urokinase activity, the cross-linking concentration of glutaraldehyde was reduced by solubilizing 25% (w/v) glutaraldehyde in the oil phase with n-propanol. A concentration of 0.015% (v/v) glutaraldehyde effectively immobilized urokinase and stabilized albumin microspheres. Amidolytic activity using the specific chromogenic substrate for urokinase, S-2444, showed that enzyme activity could be retained during this glutaraldehyde cross-linking.

The influence of concentration of two salicylate derivatives on rectal insulin absorption enhancement

Abstract— 3,5‐Diiodosalicylate sodium (DIS), a highly lipophilic salicylate, was evaluated against 5‐methoxysalicylate sodium (MS) as a potential adjuvant absorption promoter for rectal insulin delivery. Comparative blood glucose measurements were made using the two adjuvants under identical conditions as promoters of rectal insulin absorption in rats. Concentrations of DIS greater than and including 0.1 M produced an unexpected, progressive decrease in adjuvant activity as determined by a decline in observed hypoglycaemic response. This was not due to formation of an insulin‐DIS complex. The adjuvant MS produced a classical, sigmoidal log‐dose response curve. Possible reasons for the occurrence of the DIS optimum phenomenon are discussed as well as are the observed differences in adjuvant potency of these agents in a propylene glycol‐containing vehicle.

Circulation time and body distribution of protein A-coated amino modified polystyrene nanoparticles in mice

Abstract Amino modified polystyrenc nanoparticles of size 240 nm were covalently coated with protein A (cell wall protein of Staphylococcus aureus, Cowan strain) using glutaraldehyde as a spacer. The protein A was then radiolabeled using [14C]formaldehyde and sodium cyanoborohydride by the reductive alkylation method. Blood circulation time and body distribution of 14C-labeled protein A-coated particles were studied in mice. After rapid intravenous injection through the tail vein, the coated particles showed higher blood concentrations for the first 3 min compared with that of the uncoated particles. The increased circulation time of protein A-coated particles appears to be the result of reduced uptake by the liver.

Predicting coordinated lipid biosynthesis: Application to the surfactant-accommodated epidermis

Factorialized correlation analysis is proposed as a method for predicting the coordination of multiple enzyme pathways. The approach can be used potentially to find new relationships and to predict relationships that have been established in other tissues. However, careful tracer studies are needed to verify the cause-and-effect relationships between precursor and products. In this study, guinea pigs that were chronically treated with an anionic, a nonionic and a cationic surfactant passed through an irritation stage to a clinical state that appeared normal. The method was used to examine binary coordination of lipid biosynthesis in the epidermis by using a factorialized table of regression coefficients. Coordinated lipid relationships that have been reported in other tissues were predicted between sphingomyelin and cholesterol, as well as between phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine. A new inverse relationship was found between triglycerides and both sphingomyelin and cholesterol, using this method. These data are discussed with respect to a membrane fluidization model for the accommodated state.

The Reversibility of Absorption Promoter Interaction with Red Blood Cell Membranes Studied with Differential Scanning Calorimetry

Absorption promoters, or adjuvants, are used to enhance the gastrointestinal absorption of poorly absorbed drugs such as macromolecules. In the present work, adjuvant–membrane interactions have been studied by differential scanning calorimetry (DSC) using red blood cell (RBC) membranes as model membrane. These interactions caused temperature shifts, amplitude changes, and broadening of the RBC transitions. Because more than one transition may be simultaneously affected by a given adjuvant, complex overlappings occur. Gaussian modeling and nonlinear regression analysis, therefore, were used to resolve these transitions. A correlation, which may serve as an indicator of adjuvant potency, was found between adjuvant concentration and induced transition temperature shifts. Further, these shifts recovered to baseline after successive washings with buffer (for most adjuvants). Sodium lauryl sulfate induced transition alterations, however, never recovered. Thus the DSC might be useful in monitoring reversible adjuvant–membrane interactions.

Resolving multiple overlapping calorimetric transitions by use of a microcomputer: studies on erythrocyte membranes

Buffer changes and certain drugs cause temperature shifts, amplitude changes, and transition broadening in the differential scanning calorimetric (DSC) analysis of erythrocyte membranes. However, it has been difficult to interpret and quantitate these shifts and changes because the scans are composed of multiple overlapping transitions and because more than one transition may be simultaneously affected. An empirical approach has been developed by using Gaussian modeling to resolve these calorimetric transitions. Data analysis was carried out on a microcomputer using a nonlinear regression program (PCNONLIN) to fit the data scans. These results show that changes in the calorimetric scans of erythrocyte membranes due to alterations in the buffer environment, such as pH and osmolarity, can be resolved by fitting the data scans with the proposed mathematical model and optimizing the resolution parameters with PCNONLIN. In addition, resolution uncovered hidden characteristics that may not have been readily evident. Under certain conditions, for example, apparent transition shifts were shown to actually be amplitude changes and transition broadening. Determination of the limitations and validity of this method was accomplished with simulation studies. This technique offers a simple means for fitting overlapping DSC transitions by use of a commercially available nonlinear regression program that can be run on a microcomputer.

The pharmacodynamic response of the basal skin potential to quinidine gluconate.

The basal skin potential (BSP) was explored as an indirect means of continuously monitoring the cardiac response of quinidine gluconate. A method was developed to follow the BSP using a high impedance, recording polygraph and nonpolarizing calomel electrodes. Intravenous administration of quinidine gluconate caused time dependent changes in the BSP. Further studies showed that blood levels during the elimination phase and the QT interval of the electrocardiogram (ECG) over their entire time period were highly correlated with the BSP. Optimal correlation with the QT interval occurred when the BSP curve was shifted to earlier times by approximately 10 minutes, reflecting possible differences in the accessibility or mechanism of the respective pharmacologic compartments. Further application of the BSP for pharmacodynamic monitoring will require electrode refinements and an increased understanding of its mechanism of action.