I N VITRO RELEASE STUDIES

The addition of PEG to the lipidic matrix was an efficient tool to adjust the desired release kinetics of disc shaped lipid implants prepared by compression (see Chapter IV.1). Therefore, the in-vitro release behaviour of tristearin extrudates comprising various amounts of PEG was initially investigated.

It is obvious from Figure 62 that the addition of PEG to the lipidic formulation significantly affects the in-vitro release kinetics of IFN-α. Extrudates containing no

PEG liberated just 32.71 % (n=3, SD= 1.17 %) of the incorporated protein within 16 days. The protein was delivered mainly in the first seven days.

Admixing of 5 % PEG prolonged the liberation period to 16 days. Furthermore, the amount of totally delivered protein was raised to 66.43 % (n=3, SD=1.54 %). Complete protein recovery was achieved after 16 days when using a formulation with 10 % PEG or more. 0 25 50 75 100 0 5 10 15 20 time, d c u m u la ti ve I F N α -2 a r e leas e, % 15% PEG 6000 10% PEG 6000 5% PEG 6000 0% PEG 6000

Figure 62: Effects of the addition of various amounts of PEG on the release of IFN-α from extrudates (average, +/- SD; n = 3).

The extrudates were prepared by ram extrusion. All devices were loaded with 10 % IFN-α co- lyophilised with HP-β-CD and the indicated PEG amount (average +/- SD; n = 3).

In contrast to the results obtained with lipid implants prepared by compression, the addition of PEG enhanced the burst effects. For instance, PEG-free extrudates liberate 21.62 % (n=3, SD=1.97 %) within the first 24 hours, whereas admixing of 10 % PEG enhanced the burst effect to 32.36 % (n=3, SD=0.77 %).

It has to be noted that the released protein almost exclusively existed in its monomeric form. The amount of dimer fraction detected with size exclusion chromatography was less than 1.5 % during the entire in-vitro release study and irrespective of the PEG amount. To confirm this excellent protein integrity, liberated IFN-α was analysed by means of gel electrophoreses followed by silver staining. As visualised in Figure 63, gel electrophoresis featured that the protein was delivered in its monomeric form.

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8

Figure 63: Protein stability during in-vitro release.

SDS-PAGE of IFN-α liberated from tristearin implants comprising 10 % PEG. Lane 1: molecular weight marker, lane 2: IFN-α standard material, lane 3: IFN-α released after 24 hours, lane 4: IFN-α released after 4 days, lane 5: IFN-α released after 7 days, lane 6: IFN-α released after 10 days, lane 7: IFN-α

released after 13 days, lane 8: IFN-α released after 16 days.

The elevated amounts of liberated drug as well as the accelerated burst release observed with increasing PEG loadings are often ascribed to the ability of PEG to act as a porogen. When water soluble hydrophilic excipients, such as PEG, are added to an inert sustained release matrix these substances facilitate the creation of an interconnected pore network (see Chapter I.5). As a result, a larger portion of the incorporated protein has access to water-filled pores and can diffuse out of the matrix, which accounts for increased recovery rates. On the other hand, the amplified pore formation reduces the geometric hindrance of the pore network, consequently the release is accelerated and the burst effect is often elevated [40, 40, 138, 174]. To investigate whether such effects occur due to the addition of PEG, the external morphology of the lipidic extrudates was studied by scanning electron microscopy before and after exposure to the release medium.

Before in-vitro release studies no significant differences in the morphology were visible between extrudates containing different amounts of PEG (data not shown). During incubation water-soluble substances (IFN-α, together with HP-β-CD and PEG) were leaching out of the matrix into the bulk fluid. Accordingly, the SEM pictures of incubated extrudates revealed the formation of pores. A few small pores with a size between 2 to 5 µm were formed at the surface of extrudates comprising no PEG (Figure 64 A). By increasing the PEG content the pore number per area and the pore size increased (Figure 64 B, C, D).

Interestingly, the shape of the pores changed: on the surface of PEG-free matrices circular pores were found, whereas the surface of PEG-comprising extrudates revealed more slit-like pores. This geometry might be generated in the course of

extrusion. The mechanical properties of PEG range from soft and plastic material for low molecular weights to hard and brittle for high molecular weights. For PEGs with an intermediate molecular weight, such as the here used PEG 6000, it was shown that the particles were easy to deform at low compaction speed and pressures [135]. Thus, it can be assumed that the stretched pores were formed due to the deformation of PEG particles during extrusion. By forcing the lipid/PEG blend through the small extruder die the PEG particles are racked along the extrudate axis. This hypothesis is further backed by the comparison to the pore structure of PEG-loaded compressed implants. In contrast to the morphology observed for extrudates, compressed samples revealed large, irregular formed pores at the implant surface (see Chapter IV, Figure 15).

Figure 64: External morphology of extrudates containing no PEG (A), 5 %PEG (B), 15 % PEG (C) and 20 % PEG (D) after in-vitro release studies.

As the extrudates featured a low coherence after in-vitro incubation, it was not possible to obtain cross-sections of incubated extrudates to study the internal implant morphology. On the other hand, the samples were too small to determine reliable implant porosities with a helium pycnometer or with mercury porosimetry (data not shown). Nevertheless, to get an idea about the relative ability of the pores to percolate the inert lipid matrix the water uptake of PEG-free and PEG-loaded

extrudates was investigated (Figure 65). This attempt was based on the report of Rabelo and Coutinho, that showed that the water uptake was in good agreement with the pore volume determined by means of mercury porosimetry measurements [177]. The water uptake was obtained from the weight difference between dry and wetted samples [177]. Recently, Pongjanyakul et al. used this simple technique to estimate the water uptake of lysozyme loaded glyceryl palmitostearate implants after in-vitro release studies [174]. Accordingly, at predetermined point of time the implants were removed from the incubation buffer, blotted dry, and weighed. Afterwards the extrudates were dried to constant mass and weighed again (see Chapter III.2.6).

0 10 20 30 40 0.1 0.2 0.3 0.5 1.0 4.0 7.0 10.0 time, d w a te r up take, % 0% PEG 20% PEG

Figure 65: Effect of PEG loading on the water uptake of tristearin extrudates.

The extrudates were loaded with 10 % IFN-α/HP-β-CD lyophilisate and the indicated amount of PEG. (average +/- SD; n = 3),

PEG-free extrudates incorporated not more than 7 % water (relative to the implant weight). In generally, it was suggested that at least 30-35 % water-soluble drug (and excipient) are necessary to create percolating diffusion pathways within inert matrices (see Chapter I.5). As the extrudates were only loaded with 10 % IFN-α/HP-

β-CD co-lyophilisate, an incomplete pore network through the matrix seemed probable. This assumption was backed by the restricted entry of water which indicated that the observed pores of PEG-free extrudates (Figure 64 A) were only located at the implant surface and were not penetrating the matrix. As a result, not all of the incorporated IFN-α had access to water-filled pores connected to the implant surface. Since tristearin matrices themselves did not erode [85, 240] and the

permeability of IFN-α through crystalline lipid can be considered as negligible, IFN-α

was trapped within in the matrix. As a result, only 32.71 % (n=3, SD= 1.17 %) of the totally incorporated IFN-α were delivered from PEG-free extrudates (Figure 62). In contrast, extrudates loaded with 20 % PEG revealed a water uptake of up to 36 % (Figure 65). Therefore, inside the implant an increased space for the imbedding of water was provided which indicated the formation of pores penetrating the matrix. It can be concluded that the observed implant morphology as well as the water uptake agreed well with the protein release patterns shown in Figure 62. With a higher PEG content, the porosity of the lipidic matrices increased upon exposure to the release medium, resulting in enhanced IFN-α mobility and, hence, in increased protein release rates.