Further studies on lipid drug depot degradation

In the first part of this chapter it was shown, that the rapid degradation of D112 in the presence of lipases is a consequence of the generation of fatty acid/triglyceride mixtures exhibiting a melting point beneath the human body temperature. Since the employed RP-HPLC described in the previous chapter is capable of separating and quantifying the FFA-components of all triglycerides used in the described experiments, characterization of the degradation behaviour of depot systems consisting of different triglycerides is possible.

Therefore, in this section, the degradation of an extrudated lipid implant is investigated with respect to the amount and the ratio of different FFA released over the time of lipase incubation. Scanning electron microscopy was used to characterize the changes in extrudate morphology occurring during the incubation. In addition to that, an experiment dealing with the comparison of the degradation behaviour of extrudated implants and compressed implants with the same surface area is presented. As shown in the previous sections the surface area has a great influence on the degradation of lipid based depot systems, since the employed enzymes have to adsorb on the lipid surface in order to start lipid cleavage. As a consequence of their manufacturing process extrudated implants exhibit a rod-shaped, cylindrical form, whereas compressed implants show a more tablet-like shape. It is understood that both systems are different concerning their surface area and thus these differences should be taken in account when comparing their degradation behaviour. Therefore, compressed implants with a similar surface area were prepared in order to compare the degradation of these two systems.

Materials and methods Implant manufacturing

Implants were prepared by using a 5 ton hydraulic press (Maassen, Eningen, Germany).

The implant components - tristearin powder 80% (D118) and H12 powder- were ground in an agate mortar after thoroughly cooling with liquid nitrogen. The resulting mixture was compressed with a pressure of 2 tons for 30 seconds. The obtained implants had a diameter of 5 mm and a height of 7 mm. The dimensions of the prepared implants were measured with a digital caliper. According to the dimensions of the implants the surface area was calculated to be 1.49 cm².

CHAPTER THREE Studies on the mechanisms of lipid matrix disintegration

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Extrudate preparation

The extrudated implants were prepared as described in chapter 2 using a Thermo Haake MiniLab microcompounder (Thermo Fisher Scientific, Inc. Waltham, U.S.A.). The prepared extrudates used for the study consisted of 80% D118 and 20% H12. The produced lipid extrudates had a diameter of 1.45 mm and were cut to a length of 32 mm in order to obtain the same surface area as the implants described above.

Scanning Electron Microscopy (SEM)

Samples were analyzed using a Field Emission Scanning Electron Microscope Joel JSM-6500F (Joel Inc., Peabody, USA). The samples were put on adhesive carbon tape (BAL-TEC AG, Balzers, Principality of Liechtenstein) and attached to a custom made brass stub, carbon-sputtered and analyzed.

HPLC analysis

The samples withdrawn from the various degradation experiments were treated and analyzed as explicitly described in chapter 2 and 3. Release of the different FFA was analyzed and the obtained FFA release was compared to the theoretical values.

Experimental setup

Implants and extrudates with the same surface area were incubated in 2ml PBS pH 7.4 containing 100U lipoprotein lipase from pseudomonas sp. After 3 days of incubation the buffer media was replenished. FFA were isolated from the drawn samples and, after derivatization, analyzed. Another set of extrudates was treated in the same manner after 1, 15 and 30 days samples were drawn and analyzed with SEM. All experiments were run in triplicate.

Results and discussion Surface are

Analysis of the tested implants and extrudates exhibiting the same surface area revealed that both systems exhibit the same degree of degradation at least for the first days of incubation as seen in Fig. 1.

Extrudates and compressed implants with the same surface area

0 1000 2000 3000 4000 5000 6000

0 5 10 15 t [d] 20 25 30 35

cumulated amount of FFA

Extrudates

Compressed implants

Figure 1: Implants and extrudates with a surface area of 1.49 cm² incubated in lipase containing buffer (Average + S.D., n = 3).

After day 15 the lipid structure of the extrudate collapsed resulting in different degradation behaviour (Fig.2). Presumably due to the preparation process and its dimensions the compressed implant maintained its structure and showed no signs of structural damage

Figure 2:SEM pictures of an extrudated lipid implant incubated for different periods of time in lipase solution.

a) extrudated lipid implant incubated for 1 day (50X magnification) b) lipid extrudate after 15 days of lipase incubation: loss of structural integrity (50X magnification) c) fragments derived from the lipid matrix collapse of the extrudated implants, drawn after 30 days of incubation (50X magnification)

CHAPTER THREE Studies on the mechanisms of lipid matrix disintegration

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However after 30 days of incubation the surface of the compressed implant showed distinct signs of degradation in addition to that the diameter and length of the implant was significantly reduced. As seen in Fig.3 porous regions were visible when examining the implant with scanning electron microscopy. Presumably these porous regions are a consequence of the rapid degradation of the contained H12 component.

Figure 3: SEM photographs of the compressed implant incubated in lipase solution. a) Overview (50X magnification) b) porous area on the implant surface (150X magnification)

As a result it can be concluded that, concerning the first 15 days, manufacturing technique has no major influence on the degradation of lipid based drug delivery systems. Although a pressure of 2 tons was applied during the preparation of the compressed implants these systems showed no difference in the degradation rate compared to depot systems prepared by twin screw extrusion. Nevertheless the preparation technique especially the application of high pressure seems to stabilize the lipid matrix. As the detected lipid matrix collapse is highly desirable with regard to bioerosion processes the so far observed stability of the compressed lipid matrix would interfere with the requirement for bioerodible drug depot systems.

HPLC-Analysis of extrudate degradation

Free fatty acids (FFA) generated during the degradation of lipid extrudate formulation E1 (80:20 D118/H12) were separated and quantified with RP-HPLC as seen in Fig. 4.

Figure 4:HPLC chromatogram of an extrudate degradation study. 1) lauric acid 2)myristic acid 3) palmitic acid 4) stearic acid.

After the first 3 days of incubation it was found that the release of lauric acid and myristic acid was almost 2 times higher than theoretically expected whereas the release of the stearic acid, i.e. the corresponding fatty acid of the main component, was significantly below the expected level (Fig. 5).

Figure 5:Theoretical and experimentally obtained FFA values after 3 days of lipase incubation

CHAPTER THREE Studies on the mechanisms of lipid matrix disintegration

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Figure 6:Theoretical and experimentally obtained FFA values after 12 days of lipase incubation

After 12 days of lipase incubation the amount of low melting fatty acids released from the extrudate was decreased, however it was still on a slightly higher level than theoretically expected (Fig. 6). According to this finding the release of stearic acid was increased and almost reaching the theoretical level. This decrease in low melting fatty acids indicates a depletion of H12 in the outer areas of the extrudate since the H12 erodes quickly as seen in the experiments described in the previous sections of this chapter. This decreasing trend for low melting fatty acids was continued regarding the FFA release data of day 30 (Fig. 7).

Lauric acid release was decreased by two thirds of the theoretical level, whereas myristic acid release was still slightly increased. Concerning the release of stearic acid from the incubated extrudate the obtained values were almost identical to the theoretically expected values. Once again, these findings are in accordance to the results of 3.2 indicating a quick and preferential cleavage of the low melting component H12 -consisting mainly of triglycerides esterified with lauric acid – and a subsequent constant release of the main component stearic acid.

Figure 7:Theoretical and experimentally obtained FFA values after 30 days of lipase incubation

Conclusion

It was shown that the degradation of lipid based drug delivery systems isn’t affected significantly by the preparation method of the lipid depot systems. In short, depot systems with a similar surface area exhibit similar degradation rates. However, as a consequence of the applied high pressure when using the hydraulic press as production method, compressed implants maintain their structure and show only minor changes in morphology upon degradation. Therefore since lipid matrix collapse is desirable extrusion methods should be chosen in order to obtain erodible drug depot systems. In addition to that the presented results of RP-HPLC analysis clearly show that the phenomenon of lipid matrix breakdown of certain extrudates – proposed in chapter 3- is a direct consequence of the heterogeneous FFA release during lipase incubation. Firstly, lauric acid release is high, leading to the formation of lauric acid/triglyceride mixtures which promote the erosion of the lipid matrix, then, after this initial phase, the lipid matrix is depleted on lauric acid and D118 degradation is mainly occurring.

CHAPTER FOUR Investigations on spider silk proteins

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