Release mechanisms of the incorporated excipients – HP-β-CD and PEG

Table 5: Diffusion coefficients of IFN-α within phosphate-buffer filled capillaries comprising various amounts of PEG.

The diffusion coefficients were determined by fitting Equation {13} to the release data shown in Figure 21.

IFN-α/HP-β-CD:PEG ratio D, x 10^-6 cm²/s

1:0 1.8 1:1 1.8 1:2 1.7

However, the drug diffusivity within a controlled release device is not only a function of the composition of the liquid within the pores. In addition, the characteristics of the generated pores might be even more important for the diffusivity of the drug (see Chapter I.5).

It has to be taken into account that the continuously leaching out of the protein itself and of the incorporated hydrophilic excipients (HP-β-CD and PEG) progressively increases the porosity of the implants. Such a time-dependent increase in porosity might enhance the diffusivities during release. However, the mathematical solution of Fick´s second law of diffusion used to analyse the release data assumed constant diffusion coefficients over the entire release period. This means, a possible explanation for the observed deviations between the calculated release curves and the experimental obtained protein liberation might be a time-dependent increase in the protein mobility due to an increase in matrix porosity.

In order to evaluate if such a scenario is of relevance, the release of PEG and HP-β-CD was monitored simultaneously to the protein delivery. If a time-dependent increase in diffusivity by changes of the pore structure is of importance, systematic deviations between the mathematical predicted release data and the experimentally obtained data should be observed also for HP-β-CD and PEG.

allow also the quantification of PEG in the concentration range from 0.025 mg/mL to 2 mg/mL (see Chapter III).

Concentrations of HP-β-CD were determined by following the fading of an alkaline phenolphthalein solution spectrophotometrically [11]. The original assay needed to be adapted to allow the determination of small quantities of released cyclodextrin (see Chapter III).

Since the hydrophobic side chains of proteins [105] as well as of PEG [87] are able to interact with the cyclodextrin cavity the released protein or the liberated PEG might interfere with the developed assay. However, it could be shown that the calibration curve obtained with protein- and PEG-free HP-β-CD solutions overlapped with that obtained in the presence of IFN-α and PEG (data not shown). Thus, any interference of released IFN-α or PEG with the formation of the phenolphthalein/HP-β-CD inclusion complex was negligible in the present study.

0 25 50 75 100

0 2 5 7 10 12 15

time, d

cumulative release, %

PEG HP-ß-CD IFN-α

Figure 22: Release of HP-β-CD, PEG, and IFN-α from the tristearin-based implants.

The implants were loaded with 10 % IFN-α / HP-β-CD co-lyophilisate and 10 % (average +/- SD; n = 3).

Figure 22 compares the in-vitro release kinetics of IFN-α, PEG, and HP-β-CD from tristearin implants containing 10 % PEG. Clearly, PEG leaching was fastest, followed by HP-β-CD and IFN-α liberation. Importantly, the shape of the release curves of PEG and HP-β-CD was different from that of IFN-α: the slope monotonically decreased with time for the two excipients, whereas it remained almost constant during at least 7 d for the protein. This already indicated differences in the underlying mass transport mechanisms. In order to judge the importance of diffusional mass

transport of the release of the excipients, the above described mathematical model (Equation 9, Chapter I.5.3) was fitted to the experimentally determined release rates considering PEG and HP-β-CD as the diffusing species.

In Figure 23 the obtained releases profiles of PEG from implants with initial PEG contents of 5 - 20 % as well as the respective fittings are illustrated. Clearly, good agreement between the applied theory and experiments was obtained in all cases (R² > 0.99), indicating that pure diffusion (in axial and radial direction) with constant diffusion coefficients governed the transport of this excipient. As it can be seen, the release rate significantly increased with higher initial PEG loading.

0 25 50 75 100

0 5 10 15 20

time, d

cumulaive PEG release, %

20 % PEG 15 % PEG 10 % PEG 5 % PEG theory

Figure 23: Effects of the initial PEG loading on the release of PEG.

Experimental data are shown as symbols and theoretical data based on Equation 9, Chapter I.5.3 as solid lines. All tristearin-based implants were loaded with 10 % IFN-α/HP-β-CD co-lyophilisate and the indicated amount of PEG (for experimental results: average +/- SD; n = 3).

Since the used mathematical model described well the experimental release kinetics it was possible to determine the apparent diffusion coefficients of PEG within the lipidic matrices based on theoretical fittings shown in Figure 23. This approach allowed to quantify the impact of the initial PEG loading on the diffusivity of PEG. The dependence of the calculated apparent diffusion coefficients on the PEG content of the matrix is shown in Figure 24. Obviously, the diffusivity significantly increased with higher initial PEG loading and an exponential relationship between the diffusion coefficient D, and the initial PEG content could be established (R² = 0.99, equation indicated in Figure 24).

y = 0,7205e^0,1011x R² = 0,9913

0 2 4 6 8

0 5 10 15 20 25

PEG content, % DPEG, 10-8 cm²/s

Figure 24: Effects of the initial PEG loading on the apparent diffusion coefficient of PEG within the tristearin matrix.

The release patterns of HP-β-CD as well as the respective fittings based on Equation {9} (Chapter I.5.3) are illustrated in Figure 25 for various initial PEG contents. Like PEG release (and in contrast to IFN-α release), pure diffusion with constant diffusivities was the dominating mass transport mechanism of HP- β-CD.

This was indicated by the good agreement between theory and experiments (R² >0.98). In accordance with the liberation of PEG from matrices with various PEG loadings, the release rates increased with rising PEG contents.

0 25 50 75 100

0 5 10 15 20

time, d

cumulative HP-ß-CD release, %

20 % PEG 15 % PEG 10 % PEG 5 % PEG 0 % PEG theory

Figure 25: Effects of the initial PEG loading on the release of HP-β-CD.

Experimental data are shown as symbols and theoretical data based on Equation 9, Chapter I.5.3 as solid lines. All tristearin-based implants were loaded with 10 % IFN-α/HP-β-CD lyophilisate and the indicated amount of PEG (for experimental results: average +/- SD; n = 3).

Based on the theoretical fittings shown in Figure 25, the apparent diffusion coefficient of HP-β-CD in the lipidic matrices was calculated. The obtained D values are plotted versus the initial PEG loading of the matrix in Figure 26. Clearly, a higher PEG content of the matrix accounted for an increase in the diffusivity of HP-β-CD, which is in well accordance with the results obtained for PEG itself (Figure 24). Furthermore, similar to the diffusion coefficient of PEG an exponential relationship between the diffusivity of HP-β-CD and the initial PEG content could be established (R² = 0.98).

y = 0,7677e^0,0517x R² = 0,9843

0 1 2 3

0 5 10 15 20

PEG content, % DHP-ß-CD, 10-8 cm²/s

Figure 26: Effects of the initial PEG loading on the apparent diffusion coefficient of HP-β-CD within the tristearin matrix.

The observation that the diffusivities of both excipients increased with rising initial PEG loading is consistent with the SEM pictures of tristearin implants after in-vitro incubation (Figure 15). Enhanced levels of initial PEG amounts clearly resulted in a more pronounced pore formation. So, less restricted pore dimensions accounted for increased mobility of the excipients within the lipidic matrix. As a result the delivery rates of both excipients from matrices comprising higher initial PEG loadings were significantly accelerated.

Since release kinetics of PEG as well as of HP-β-CD were described well by a solution of Fick’s second law of diffusion assuming constant diffusivities during release, time-dependent variations of the diffusivity during the release period were not of importance for the tristearin implants studied here. This result clearly contradicts the assumption that the observed deviations of IFN-α release from purely diffusion-controlled mass transport from matrices containing PEG can be attributed to the time-dependent increase in the porosity of the matrices.