An artifactual component of drug-protein binding generated in vitro

In the course of investigating the binding of imipramine to soluble cellular fractions from brain and leukocytes using equillibrium dialysis, an artifactual binding component was produced. On Scatchard and double-reciprocal plots of the data, the component appeared as a homogeneous population of sites displaying a binding affinity of 170 nM. The obtained pattern of biphasic interaction bore a marked resemblance to reported Scatchard plots representing the interaction of drugs with bovine serum albumin,and depicting two components of widely differing binding affinity and capacity. The artifact occurred when solutions were transferred after dialysis and before quantitation to intermediate containers, and resulted from binding of H-imirpramine to the walls of these containers. The latter interaction decreased the concentration of radiolabeled drug in the dialusate but not in the dialyzed solution, and thus mimicked increased imipramine binding to the biological material under study. The effect was particularly pronounced at low drug concentrations, and was prevented by the presence of either proteinaceous material, or of an excess of another basic compound such as methadone. The concentration dependence of the phenomenon led to its appearance as a discrete binding component. The artifact was eliminated either by applying an appropriate correction factor, or by transferring the dialyzed solutions directly into scintillation vials for counting.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25164/1/0000600.pd

In vitro retinoid binding and release from a collagen sponge material in a simulated intravaginal environment

Four in vitro preparations were constructed to simulate the intravaginal release of two retinoids, all-trans-retinoic acid (t-RA) and 13-cis-retinoic acid (c-RA), from a 0.7% collagen sponge diaphragm insert. Four t-RA concentrations, 0.019, 0.05, 0.1, and 0.15% in methanol were added to the sponge. The release into an artificial vaginal fluid was monitored serially over 72 h by serial analysis for t-RA and c-RA using high-pressure liquid chromatography. In each preparation, retinoid release was immediate and noncontinuous. At 37 degrees C, the retinoids were stable for at least 48 h. Trans-retinoic acid was the predominant retinoid recovered. Only trace amounts of the cis-isomer were released. Peak t-RA levels were 20 microM after 0.01%, 60-80 microM after 0.05%, 100-200 microM after 0.1%, and 320 microM after 0.15%. When the vaginal fluid bath was changed after 5 h, no further significant retinoid release occurred. There was significant loss of up to 70% of the applied t-RA into the collagen sponge. The retinoid binding was concentration dependent (higher binding with higher concentrations) and was maximal only after 24 h of co-incubation. The discontinuous release of t-RA and the high degree of binding to collagen would seem to preclude use of the diaphragm insert as a vaginal drug delivery system, at least for retinoids