Evaluation of the model

Before the actual release tests in the static model, control experiments are performed to evaluate the loss of activity and total content during the actual release tests.

Therefore, a solution of 60µg/ml ACT formulated in a 50mM potassium phosphate buffer pH 7.2 with 0.1% Poloxamer® _{188 is exposed to the tubing}

material, the chamber material, and the membrane, respectively. A control is kept in a glass vial. For analysis by activity assay and ELISA, samples are drawn after 18 hours and 75 hours.

The loss of activity is minimal in the glass vials. But, in the samples exposed to tubing, chamber, and membrane loss of activity is observed between 10% to 25% after 75 hours (Fig. 3-72).

60 70 80 90 100 110

tubing chamber membrane glass

re la tiv e a ctiv ity [ % ] _

Fig. 3-72 Residual activity of a 60µg/ml ACT solution formulated in a 50mM potassium phosphate buffer pH 7.2 with 0.1% Poloxamer® 188 during exposure to tubing material, chamber material, membrane material, and glass, respectively, measured by activity assay; left: control before exposure; centre: exposure for 18 hours, right: exposure for 75 hours;

Chapter 3 – Results and Discussion

The total content measured by ELISA shows a decrease over time as well, but to much lower extent. In detail, glass again shows almost no loss, but in the other samples only 90% to 95% of total content are recovered (Fig. 3-73).

60 70 80 90 100 110

tubing chamber membrane glass

re la ti ve am ount [ % ] _

Fig. 3-73 Residual total content of a 60µg/ml ACT solution formulated in a 50mM potassium phosphate buffer pH 7.2 with 0.1% Poloxamer® 188 during exposure to tubing material, chamber material, membrane material, and glass, respectively, measured by ELISA; left: control before exposure; centre: exposure for 18 hours, right: exposure for 75 hours;

Therefore, it can be concluded that during the release experiments in the static model ACT activity and total content is lost as well but to much lower extent compared to the dynamic model. Moreover, the loss of ACT seems to follow two mechanisms. The loss of total content is supposed to be rooted in absorption phenomena which result in ACT molecules remaining in the system. Besides, this mechanism also leads to reduced recovered activity values. However, the 2-fold higher values of lost activity compared to total content loss can solely be explained by an additional deactivation of non-absorbed ACT molecules.

Indeed, based on the results it is to be expected that during release experiments with ideally releasing formulations 75% to 90% of relative activity can be recovered at best. As well, a maximum of 90% of total content is likely to be recovered at most.

Chapter 3 – Results and Discussion

methods being amplified in such an operation. Thus, a result gained after correction would imply an unsatisfactory inaccuracy.

Moreover, the control experiments most notably on the tubing material have to be understood as worst case conditions because the surface of tubing presented to the protein containing medium is very low in the release model (Fig 3-65) compared to control experiments. For the latter, of course, a certain amount of solution (0.5ml) had to be filled in the tubing for technical and analytical reasons which also requires a certain tubing volume that naturally is correlated with a higher surface area compared to the small pieces of tubing used for the release chamber. This difference in the relation of solution amount and exposed tubing surface in control and actual release situation leads to an exaggerated impression about the influence of the tubing material. To conclude, a simple multiplication of the particular recovered activities and amounts, respectively, of Fig. 3-72 and Fig. 3-73 will not result in a correct overall value for the maximum recovered amount of ACT to be expected in release tests.

For those reasons, the uncorrected values are used for the comparison of the release results of the different formulations.

0 20 40 60 80 0 20 40 60 80 100 120 time [h] re le as ed a m ount of A C T [ % ] _

Fig. 3-74 Release diagram of ACT from a xerogel formulation in the static model; release profiles are given as active ACT gained by activity assay () and total ACT content measured by ELISA (0); underlying hydrogel contains 60µg/ml ACT, 10mM potassium phosphate buffer pH 7.2, 10mM arginine, 0.1% Poloxamer® 188, 0.05% PVP 17, and 2.5% hydroxyethyl cellulose 100.000 in water – xerogel formulation (1) of Tab. 3-18; release medium is a 10mM potassium phosphate buffer pH 7.2 with 0.1% Poloxamer® 188;

Fortunately, for the estimation of future in vivo experiments the situation is different. The constant recovered amounts in the glass samples for both analytical methods (Fig. 3-72, 3-73) indicate that the release system with its plastic

Chapter 3 – Results and Discussion

components is responsible for the losses. In an in vivo situation, however, these materials and surfaces are not present in the application site why it can be stated that the here detected losses are unlikely to occur.

After the control experiments actual release tests on ACT xerogel formulations are performed. In fact, in release tests of ACT in the static model the complete loss of activity detected in the dynamic model is not observed. However, the curves measured by activity assay flattens after about 50 hours of testing time in comparison to the total content curve obtained from ELISA measurements (Fig. 3- 74).

This corresponds to the control experiments predicting a higher loss of active ACT compared to total ACT content. As well, the magnitude of recovered contents in the release test concur with the results gained from control experiments.

Nevertheless, when drawn in the square root of time plot both curves are linear up to 50 hours releasing time. In that period, a linear fitting can be conducted delivering slopes for measuring release rates (Fig. 3-75).

0 20 40 60 80 2 4 6 8 10 12

square root of time [h]

re le as ed a m ount of A C T [ % ] _

Fig. 3-75 Linearised release diagram of ACT from a xerogel formulation in the static model; release profiles are given as active ACT gained by activity assay (, slope=6.6, R2=0.98) and total ACT content measured by ELISA (0, slope 7.8, R2=0.99); underlying hydrogel contains 60µg/ml ACT, 10mM potassium phosphate buffer pH 7.2, 10mM arginine, 0.1% Poloxamer® 188, 0.05% PVP 17, and 2.5% hydroxyethyl cellulose 100.000 in water – xerogel formulation (1) of Tab. 3-18; release medium is a 10mM potassium phosphate buffer pH 7.2 with 0.1% Poloxamer® _188;

Chapter 3 – Results and Discussion

values are read after 120 hours of releasing time because this represents the common change of dressing interval in clinical practice.

One may notice that slopes of release curves are lower in the static method – 6.6 vs. 10.8 for activity assay readout and 7.8 vs. 12.8 in the ELISA readout - and therefore the release is delayed in comparison with the studies on the dynamic model.