Entwicklung von bioabbaubaren Implantaten zur ausdauernd Proteinwirkstofffreisetzung

Different drug delivery systems have been developed for peptides and proteins, with special interest on those that allow sustained parenteral release; they can prolong protein circulation, resulting in better therapeutic results and higher patient compliance. However, many of the manufacturing methods used involve conditions that can be detrimental for these molecules, such as the use of organic solvents, pH gradients, reduced pressure or heat. Besides, obtaining the desired formulation performance might be difficult since the dissolution profile might be far from optimal, or because the integrity of these bio-macromolecules cannot be preserved within the matrix. Thus, the development of these systems has followed a complex and challenging route, which have resulted in long development periods and few commercialized products. Therefore, gathering additional understanding of these systems and defining key parameters that impact the performance of them, is important to accelerate and assist the development of parenteral sustained release systems. This work focused on the development of biodegradable implants for protein delivery, using ovalbumin (OVA) as acid-labile model protein. Hot melt extrusion (HME) was selected as the manufacturing method, since it does not involve the use of solvents and the protein can be incorporated on its more stable dry state, avoiding the creation of interfaces that compromise protein stability. Nonetheless, the use of high temperatures and presence of shear forces might impact protein stability. Therefore, the feasibility of HME to prepare OVA-loaded implants was investigated. With this purpose, a mini-ram extruder (syringe-die device) was used as a rapid screening tool. Moreover, two types of biodegradable matrices (PLGA- and lipid-based) were used to assess differences on formulation performances, with special emphasis on dissolution and protein stability. OVA was stable during extrusion as indicated by SEC-HPLC, FTIR, CD and fluorescence spectroscopy. It showed comparable behaviors when analyzed as received and after extraction from the freshly prepared implants. This was observed for the two types of matrices used, indicating that they might have a protective effect on the protein. As they melt or soften during the process, they may act as a “protective cushion” against heat and shear forces. In the case of PLGA-based implants, OVA release was characterized by a low burst and a slow release up to day 21 which plateaued thereafter, resulting in incomplete release for all evaluated protein loadings. The percolation threshold of this matrix was reached at loadings ≥25%, but cumulative releases did not reach 100% either. Release incompleteness was accompanied by the formation of an insoluble residual mass. Characterization of this mass indicated that it consisted of non-covalent protein aggregates and polymer, where ovalbumin was ionically bound as the pH inside the degrading matrix felt below the pI of the protein. Exposure to the acidic microclimate created by the degradation process of the matrix induced structural changes in OVA by increasing its hydrophobicity and its -sheet content. Furthermore, the autocatalytic rate of hydrolysis of PLGA was decreased in the presence of OVA since the later has certain buffering capacity. These changes increased the likeliness of protein-protein ad protein-polymer/oligomer interactions. To create a viscous environment within the implant, PVP and Poloxamer were used as protein co-excipients. For this, these compounds were dissolved together with the protein and freeze-dried prior blending with the polymer and extrusion. The release of OVA during the first days was increased, indicating that these excipients effectively decreased the adsorption of the protein on the PLGA. Nonetheless, they were not able to control the acidic inner pH and thus, OVA aggregation was not prevented and release incompleteness was still observed. Furthermore, there were changes in OVA secondary structure caused by the freeze-drying process and the interaction of the co-excipients and the protein when they were in solution. The optimization of the freeze-drying conditions and co- excipient:protein ratio could improve the stability of the protein. However, strategies to control the acidic microclimate may still be required in order to obtain an optimal formulation. PEG of various molecular weights were used as “pore formers” (10% loading). At lower PEG’s molecular weight, higher OVA cumulative release was obtained. This was attributed to the differences on PEG hydrophilicity, which is higher at lower molecular weights. However, the release plateaued after 21 days in all the cases, most probably caused by pore closure of the matrix after PEG had diffused out rapidly during the first weeks, as well as by the uncontrolled acidic microclimate. Sugars and inorganic salts were also evaluated as pore forming agents (at 10% loading). Unfortunately, incomplete release was still observed; they were not able to control the acidic pH and induced OVA aggregation by increasing the ionic strength within the matrix. Hence, they were unsuitable to improve the release completeness of OVA from PLGA-based implants Since the acidic microclimate was found to be the primary trigger for the incomplete release of OVA, pH modifiers were incorporated within the matrix. The use of MgCO3 and Mg(OH)2 (3.5% loading) increased the released fraction of OVA in a 20% and 10% respectively. They also delayed by one week the degradation onset of PLGA thanks to their neutralization effect during the first weeks of dissolution. Nevertheless, release incompleteness was still observed since this effect did not last long and the pH was not kept above OVA’s pI. Higher amounts of these co-excipients should be used to achieve a long-lasting effect. However, this increase may result in an increase in the inner ionic strength and the induction of OVA aggregation, which is unfavorable. Changing the polymer matrix to a slower-degrading one, where the pH drops slower, could work as well, but care should be taken to avoid greater increase in hydrophobicity that might hinder the protein. With the use of shellac, a well-known enteric and biocompatible polymer, as protective co-excipient (1:5 and 1:10 protein:shellac ratio), a distinct late release phase occurred and release completeness was increased to more than 75% (cumulative release). Shellac seemed to protect the protein against the acidic microclimate due to its low solubility at low pH. The so protected OVA was then released once the pH increased, due to the declining PLGA-oligomer formation. The result was a triphasic release profile, consisting of an initial burst, a slow diffusion phase over about 7 weeks, and an erosion-controlled dissolution phase over the next three weeks. An acid-labile protein like OVA was thus effectively protected from an interaction with PLGA and its degradation products, resulting in a controlled delivery of more than 90% of the original payload. One of these formulations (1:5 OVA:shellac ratio) was up-scaled from the mini- ram extruder to a twin-screw extruder using a scale-up size factor of 15. No substantial differences on OVA integrity between both extrusion methods were detected. The dissolution for the up-scaled formulation showed also a triphasic release profile, but with a lower burst release (10% vs. 30%), probably caused by a densification of the matrix after extrusion. The dissolution of the protein during the erosion-controlled phase of release was slightly slower for the up-scaled formulation and lasted longer (8 vs. 3 weeks) reaching also more than 75% of cumulative release. These differences were attributed to the lower burst release and less pore formation, which slowed down the protein’s diffusion. In both cases an insoluble mass remained after the release. However, it was identified as undissolved shellac together with the small portion of unreleased protein. A further characterization of the degradation properties of these implants will be interesting to assess their suitability as a depo formulation intended for multiple applications. Storage stability of the up-scaled formulation indicated a change in thermal properties (decrease in Tg) and dissolution profile over the time when the implants were stored at 25°C, being more drastic for storage temperatures above the formulation’s Tg (37°C) since mobility of the polymer chains and re- arrangements into a less rigid structure occurred. No substantial changes in OVA’s hydrophobicity or formation of additional aggregates were observed when the implants were stored at 25°C. Protein conformational changes and increase of interactions between the matrix and the protein were induced by higher storage temperatures. The dissolution profile was comparable for all the evaluated implants prior and after storage during the burst release and lag phase. Differences were observed during the erosion phase, which could be attributed to the decrease on Tg after storage, with a faster release upon storage for longer time. In conclusion, the implants should be stored at refrigerated conditions to guarantee their proper performance and avoid undesirable changes. Compared with the case of PLGA, OVA was generally more stable both within the lipid-based matrix and during its release from it, since no acidic environment was created (pH was always 6.8). The extrusion process did not alter the polymorphism of D114 or D118, but it induced a reordering of the lipidic chains in the case of DP60. However, this reordering was always observed, indicating that this lipid adopted a more stable conformation after extrusion. The release of OVA from the lipid-based implants was mainly driven by diffusional processes: influx of water that dissolved the protein allowing its efflux. It was also dependent on the crystallinity degree of the lipidic matrix used: the release was slower and longer when a more amorphous and heterogeneous lipid, like DP60, was used. In contrast, the presence of crystallites within the structure of more crystalline materials, like D114 and D118, led to a higher anisotropy of the protein distribution yielding to a faster release. Incomplete release from the DP60 matrix was observed when OVA was loaded at 5%, but without the formation of insoluble aggregates. This behavior was attributed to possible stronger physical interactions between the protein and the matrix. By increasing the protein loading, faster and more complete release was observed, which can be ascribed to the formation of more and/or bigger pores at higher protein loadings. Loading lysozyme, a smaller protein, instead of OVA resulted in a more prolonged release with similar time-frame for D118 and DP60. This was probably caused by the creation of smaller and less interconnected pores, and a stronger physical interaction between the matrix and the protein. A deeper investigation on these interactions and the distribution of the protein within the matrix might be helpful to determine their possible impact on microstructural rearrangement upon storage. The use of PLA as a co-excipient on the D118-lipid matrix was a suitable approach to prolong the release of OVA from 1 week to 1 month without the detrimental effects that the polymeric matrix could have on the protein. In contrast, incorporation of PEG in the DP60-based matrix accelerated the diffusion rate of the protein during the first weeks of release. This effect was correlated with PEG’s molecular weight: the lower it was, the faster the release obtained. The release from DP60-based implants was also faster when binary lipid blends were used. The rate was related to the fatty acid chain length of the secondary lipid: by being shorter the release was faster. This could be ascribed to the decrease in lipid wettability with the increase on its chain length, since diffusivity of the protein depends on the influx of water. Thermal characterization of these blends indicated that the lipids did not mixed at a molecular level, presenting some phase separation. In this case, it would be recommended to use an extruder that compounds the materials while they are in their molten state. This could avoid changes on the lipidic microstructure after storage. In general, the dissolution of OVA was successfully tuned by the inclusion of more hydrophobic or more hydrophilic excipients, leading to different dissolution speeds with times ranging from 2 to 15 weeks. The versatility of these systems without the inclusion of many components makes them an attractive option for protein delivery with various applications. Crystallinity changes in the lipid matrices were observed after curing under different conditions; these changes also had a significant effect on the dissolution profile of OVA. All the cured DP60-based implants as well as the D118-ones cured at 50°C presented a faster dissolution, ascribed to the creation of larger channels that increased the diffusivity of OVA. In contrast, slower diffusion was observed for the D118-based implants cured at 40°C. In this case the partial melting of the lipid might have created a denser matrix. In general, the DP60 matrix showed less polymorphic changes, probably due to its more heterogeneous and amorphous character. This could represent a more stable formulation without unpredictable variations upon storage. A sustained and prolonged OVA release formulation with optimized dissolution was obtained after the application of a Design of Experiments (DoE). The used Taguchi method allowed to study four factors at three different levels, with a reduction of experimental runs from 81 to 27, thanks to the use of an orthogonal array. It was found that the release of OVA was significantly influenced by the matrix composition and protein loading, and to a lesser extent by the curing conditions (temperature and time). Based on the results, a formulation with 5% protein loading, DP60/D118 (50:50) as matrix and curing at 40°C during 60 minutes was found to be the optimal one with a constant release over 28 weeks at a rate of ~2.8%/week. DoE is a valuable tool that allows the evaluation of various formulation and process parameters without incurring on unnecessary experiments. Gamma-irradiation affected the performance of both prepared formulations, PLGA:shellac- and lipid-based, and it proved to be unsuitable for the OVA-loaded implants. Increase in protein hydrophobicity, changes in OVA dissolution and induction of rearrangements within the biodegradable matrix were caused by the sterilization process. The use of a cooling agent during the irradiation decreased the magnitude of these changes, because molecule mobility was reduced, but it did not avoid them. Further evaluation of the polymers’ molecular weight distribution, protein and matrix distribution and free radical formation would be ideal to fully understand the effect of irradiation on the implants and to assess whether the incorporation of other co-excipients (e.g. antioxidants) could maintain the physicochemical characteristics of the formulation after irradiation. A suitable alternative may be the use of a low-bioburden sterilization process, which could produce sterile implants that maintain their physicochemical properties after irradiation at a lower dose. In conclusion, HME was a suitable process for the preparation of biodegradable implants with sustained and prolonged release of OVA. Different types of matrices were evaluated, obtaining different release patterns and various releasing times. Protein stability and release completeness were improved after understanding its degradation mechanism and the corresponding key formulation parameters. The ability to obtain tailored properties out of the same dosage form, makes biodegradable implants an interesting and promising candidate for protein delivery.Verschiedene Systeme für die kontrollierte Freisetzung von Peptid- und Proteinwirkstoffen aus Arzneimittelpräparaten sind entwickelt worden. Der Fokus wurde auf diejenigen gesetzt, die eine anhaltende parenterale Freisetzung ermöglichen, sie können die Proteinfreisetzung verlängern. Dies führt zu einer Verbesserung der therapeutischen Ergebnisse, sowie der Compliance der Patienten. Viele von den aktuellen Herstellungsmethoden werden unter Bedingungen durchgeführt, die schädlich für solche Moleküle sein können, z. B. die Anwendung von organischen Lösemitteln, pH-Gradienten, Vakuum und/oder Hitze. Auch die Herstellung der Formulierung mit den gewünschten Freisetzungsparametern kann schwierig sein, weil das gewünschte Freisetzungsprofil nicht optimal ist, oder weil die Integrität dieser Biomakromoleküle in der Matrix nicht haltbar ist. Die Entwicklung dieser Systeme ist eine komplexe und herausfordernde Aufgabe mit langen Entwicklungszeiten und wenigen kommerzialisierten Produkten als Schlussfolge. Sowohl das Sammeln von neuen Erkenntnissen über diese Systeme, als auch die Feststellung der wichtigsten Parametern, die das Verhalten der Präparate beeinflussen, ist daher von Bedeutung um die Entwicklung von Präparaten zur Parenteral-Wirkstofffreisetzung zu unterstützen und zu beschleunigen. Diese Arbeit beschäftigte sich mit der Entwicklung von bioabbaubaren Implantaten zur Proteinwirkstofffreisetzung. Es wurde Ovalbumin (OVA) als Säure-labiles Modellprotein verwendet. Die Herstellungsmethode der Wahl war Schmelzextrusion (Hot melt extrusion, HME), da organische Lösemittel dabei nicht benötigt werden und das Protein in seinem stabilsten, trockenen Zustand hinzugefügt werden kann. Dabei wird auch die Erzeugung von weiteren Grenzflächen vermieden, die die Proteinstabilität gefährden können. Nichtsdestotrotz, die Verwendung von hohen Temperaturen und die dazugehörigen Scherkräfte können eine negative Wirkung auf die Proteinstruktur haben. Daher wurde untersucht, ob OVA enthaltende Implantate mit Hilfe der HME hergestellt werden können. Um dies zu untersuchen wurde ein Mini-Ram Extruder (Syringe-die-Gerät) als Screening-Werkzeug genutzt. Zum Vergleich wurden zwei verschiedene bioabbaubaren Matrix-Bildner (PLGA- und Lipide-basierte) ausgewählt, um die Freisetzung und Proteinstabilität zu untersuchen. Laut SEC-HPLC-, FTIR-, CD-, und Fluoreszenzspektroskopie war das OVA während der Extrusion stabil. Vergleichbare Ergebnissen wurden beim neu vom Lieferant erhaltenen OVA und dem OVA ermittelt, das aus den hergestellten Implantaten extrahiert wurde. Dieses Ergebnis ist bei beiden Matrizen beobachtet worden, was ein Hinweis auf den Schutzeffekt auf das Protein sein kann. Wenn sie während das Prozess schmelzen oder weich werden, können sie als „Schutzkissen“ gegen Hitze oder Schubkräfte fungieren. Bei PLGA-basierten Implantaten zeigte die OVA-Freisetzung eine anfängliche kurze schnelle Freisetzung (burst release) auf die eine langsame Freisetzung folgte, die nach 21 Tagen in einem Plateau endete. Keine der untersuchten Formulierungen zeigte eine 100%ige Freisetzung. Die Perkolationsschwelle dieser Matrizen wurde bei Ladungen von 25% erreicht, selbst die kumulative Freisetzung erzielte keine 100%. Die unvollständige Freisetzung ging mit der Absetzung von einem unlöslichen Niederschlag einher. Die Charakterisierung von diesem Feststoff zeigte, dass es aus nicht- kovalenten Proteinaggregaten und Polymer bestand, in dem Ovalbumin ionisch gebunden wurde, als der pH in der abbaubauenden Matrix unter den isoelektrischen Punkt des Proteins fiel. Der Kontakt zu dem sauren Mikroklima, das der Abbauprozess der Matrix erzeugt hat, kann strukturelle Änderungen in der OVA-Struktur verursachen, z.B. die Erhöhung der hydrophoben Eigenschaften und der -Faltblatt-Anteile. Außerdem ist die autokatalysierte Hydrolysegeschwindigkeit des PLGAs bei der Anwesenheit von OVA gesunken, da das Protein gewisse Puffer-Eigenschaften besitzt. Diese Änderungen erhöhen die Wahrscheinlichkeit von Protein-Protein und Protein-Polymer/Oligomer Wechselwirkungen. Um eine viskose Umgebung innerhalb die Implantate zu generieren, wurden PVP und Poloxamer als Hilfsstoff angewendet. Diese Zusatzstoffe wurden zusammen mit dem Protein in Lösung gebracht und dann gefriergetrocknet, bevor sie dann mit dem Polymer gemischt wurden um die Extrusion durchzuführen. Die Freisetzung von OVA war während der ersten Tage erhöht. Dies weist darauf hin, dass diese Träger gewissermaßen die Adsorption des Proteins auf dem PLGA herabsetzen. Trotzdem waren sie nicht in der Lage, das Ansäuern des inneren pHs zu kontrollieren um die Aggregation von OVA zu vermeiden, sodass auch in diesem Fall keine vollständige Freisetzung beobachtet wurde. Außerdem gab es Änderungen in der Sekundärstruktur des OVAs, die sowohl von dem Gefriertrocknungsprozess als auch von der Wechselwirkung mit dem gelösten Träger verursacht worden sind. Die Optimierung der Prozessparameter bei der Gefriertrocknung und des Protein/Träger-Verhältnisses können die Proteinstabilität verbessern. Strategien um das saure Mikroklima zu kontrollieren sind allerdings immer noch notwendig, damit eine optimale Formulierung hergestellt werden kann. PEG mit verschiedenen Molmassen wurde als „Poren-Bilder“ (10%-Ladung) genutzt. Je kleiner die Molmasse des PEGs war, desto höher war die erhaltene Kumulative-Freisetzung des OVAs. Dies ist eine Konsequenz der unterschiedlichen hydrophoben Eigenschaften des PEGs, die größer werden, wenn das Molgewichte steigt. Nichtsdestotrotz hat die Freisetzung nach 21 Tagen in jedem Fall ihr Maximum erreicht. Die Gründe dafür liegen höchstwahrscheinlich an dem Poren-Verschluss der Grundmasse, nach dem PEG während der ersten Wochen rausdiffundierte, sowie an dem unkontrollierten sauren Mikroklima. Zucker und anorganische Salze wurden auch als „Poren- Bilder“ getestet (10%-Ladung). Leider ist die unvollst