Pharmaceutical Technology: Controlled Drug Release, Volume 2

CH. 14] COMPARISON OF TIMOLOL MALEATE RELEASE 157
Buffered matrices containing disodium phosphate (2.11 mmol) were prepared as above with the
following exception. The salt was first dissolved in a small amount of distilled water, then
methanol-polymer mixture and the drug were added. The amount of salt in the matrices was 12.5%
or 12.9% (w/w).
Studies of in vitro release
The release of timolol from isopropyl PVM-MA matrices used the rotating disk method [5]. The
dissolution medium was 100 ml of pH 7.42 mM phosphate buffer at 32°C. Ionic strength was
adjusted to 0.5 with sodium chloride. Samples of volume 3.0 ml were withdrawn and replaced by
dissolution medium. Timolol concentrations were analysed with a UV spectrophotometer at 294
nm and the pH was measured.
Release rates (%/min) were calculated from the best fits of released drug vs. time plots. The
slopes (k) of the log(released drug) vs. log(time) plots were calculated from the linear leastsquares
regression lines. A slope of 0.5 in the log-log plot indicates diffusional, square root of
time, dependence and a slope of 1.0 indicates zero-order release kinetics [6]. Times of 50% drug
release (t50%) were calculated from the best fits of drug released vs. time plots.
In vivo studies
Matrices of monoisopropyl ester of PVM-MA were carefully applied in the lower conjunctival sac
of rabbits. Inserts did not cause any irritation in rabbit eyes. Plasma and tear fluid samples were
collected at different times during a period of 8 h. Blood samples were taken from the cannulated
ear artery. Plasma was separated by centrifuging (2000 g, 4min) and kept at −20°C until analysed.
Tear fluid samples (1 µl) were collected with microcapillaries at 30, 120, 240 and 480 min after
application of the matrices. Tear fluid samples were diluted in 5 ml of phosphate buffer.
Timolol concentration in plasma and tear fluid samples was measured using a modified
radioreceptor assay [7]. In the assay, displacement of a β-antagonist, (–)- 3 H-CGP-12177, from β-
receptors of rat reticulocyte membranes by timolol was measured. Rat reticulocyte membranes were
obtained as described by Wellstein et al. [7].
In a total volume of 300 µl, 50µl reticulocyte membranes in phosphate buffer (500 µg protein)
were incubated for 60 min at 25°C with 50 µl (30 nCi) of (−)-3H-CGP-12177, 180 µl of plasma
and 20µ1 of 310 mOsm sodium phosphate buffer (pH 7.4). Standard curves were run for each
rabbit separately in order to avoid errors due to possible differences in protein binding. To
generate the standard curves, blank plasma was used and incubated in the presence of 1–20 nM
timolol. For the determination of non-specific binding, incubation in a 10 −5 M propranolol
solution was used.
Timolol concentration in tear fluid samples was measured in the same way, but 200 µl of the
diluted tear fluid in phosphate buffer were used instead of 180 µl plasma. After incubation, bound
and free radioligand were separated by vacuum filtration through Whatman GF/F glass fibre
filters. Filters were washed three times with 10 ml ice-cold 310 mOsm phosphate buffer, dried and
counted for retained radioactivity in 5 ml of Lipoluma-Lumasolve-water (10:1:0.2) mixture using