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From a biopharmaceutical point of view, poor oral bioavailability of a drug is one of the greatest challenges for formulation scientists. The majority of new chemical entities (NCEs) are weakly basic drugs. Consequently, these drugs exhibit pH-dependent solubility, being higher under acidic conditions in the fasted stomach and lower under neutral conditions in the small intestine, the main site of drug absorption. For theses compounds, pH-dependent precipitation testing represents a key parameter during early development stages. In this development phase, the amount of drug available is limited, and fast and detailed investigations of simulated drug solubility are desired. Therefore, an automated small-scale in vitro transfer model, simulating drug transfer from a donor (stomach; simulated gastric fluid, SGF pH 2.0) to an acceptor (small intestine; fasted state simulated intestinal fluid, FaSSIF-phosphate pH 6.5) compartment, has been developed. In contrast to the originally published transfer model, this model allowed a detailed investigation of drug supersaturation and precipitation in a small-scale, feasible for pre-formulation purposes, through miniaturization and automation in an in-line analytical set-up. In-line drug concentration analysis in turbid samples, due to pH-dependent drug precipitation, was achieved by a pre-filtration step, the use of flow-through cuvettes and the application of UV derivative spectroscopy. Compared to the common procedure of manual sampling followed by HPLC-UV analysis for concentration determination, the supersaturation and precipitation of the model drug ketoconazole was more accurately captured by the newly developed in-line analytical set-up. In addition, the newly developed small-scale model was compared to a USP II-based transfer model, representing an established scale of the transfer model. Using a physiologically relevant simulated gastric emptying rate of 5 min half-time, supersaturation and precipitation of the model drugs ketoconazole and a new chemical entity from the research laboratories of Merck Healthcare KGaA, MSC-A, were observed to be highly comparable. Following miniaturization and automation, the developed small-scale model was used to establish eight physiologically relevant test-sets. These test-sets were used to assess the impact of gastrointestinal (GI) variability, i.e. gastric pH, gastric emptying, and GI fluid volumes, on supersaturation and precipitation of two weakly basic model compounds, ketoconazole and MSC-A. The experiments revealed that variations in all GI parameters investigated affected the in vitro supersaturation and precipitation of ketoconazole. For example, faster gastric emptying yielded higher supersaturation and faster precipitation of ketoconazole. In contrast, MSC-A supersaturation and precipitation was only affected by variability in gastric pH. Consequently, the effect of varying GI parameters was found to be drug-specific. Elevated gastric pH, as it can result from co-medication with acid-reducing drugs, resulted in lower degrees of supersaturation for both substances. For ketoconazole, this result is in agreement with the observation that the oral bioavailability of ketoconazole is lowered when proton pump inhibitors are co-administered. In addition to the physiological considerations, the small-scale model developed herein was used to establish an in vitro screening assay for precipitation inhibitors (PIs). The use of PIs represents one option of reducing the process of pH-dependent drug precipitation during simulated GI transfer. For this purpose, ketoconazole and five orally administered kinase inhibitors (i.e. pazopanib, gefitinib, lapatinib, vemurafenib, and MSC-A) were analyzed with and without the polymeric PIs HPMC, HPMCAS, PVPK17 and K30, PEG6000, and Soluplus® in the small-scale transfer model. This screening revealed that at least one effective PI could be identified for each model drug. Moreover, HPMCAS and Soluplus® were the most effective PIs. Another outcome of these studies was that gefitinib expressed highly variable amorphous precipitation which was confirmed by powder X-ray diffraction (PXRD). During the transfer model experiments, the intermediate amorphous and supersaturated state of gefitinib was stabilized using HPMCAS and Soluplus®. After the polymer investigations, the impact of the buffer species in the simulated intestinal medium on drug supersaturation and precipitation was assessed. Since luminal fluids are mainly buffered by hydrogen carbonate ions, a USP II-based transfer model equipped with the pHysio-grad® device was proposed. This allowed the use of a complex bicarbonate buffer for the preparation of FaSSIF-bicarbonate in an in vitro transfer model. Results of transfer model experiments using standard phosphate-based FaSSIF and a more physiologically relevant bicarbonate-based FaSSIF were compared. Therefore, ketoconazole, pazopanib, and lapatinib were analyzed with and without the precipitation inhibitor HPMCAS. While HPMCAS was found to be an effective precipitation inhibitor for all drugs in FaSSIF-phosphate, the effect in FaSSIF-bicarbonate was much less pronounced. Additionally, performed rat PK studies revealed that HPMCAS did not increase the exposure of any of the model compounds significantly, indicating that the transfer model employing bicarbonate-buffered FaSSIF was more predictive compared to the model using phosphate-buffered FaSSIF. The in vitro and in vivo results of these studies demonstrated that the supersaturation precipitation of poorly soluble weakly basic drugs can be significantly affected by GI variability. Furthermore, the use of the automated small-scale transfer model enabled the identification of effective precipitation inhibitors for the model drugs involved in these studies. At the same time the buffer species has been observed to be especially important to reliably predict the in vivo solubility/dissolution behavior of HPMCAS and the weakly basic model drugs.
The biological decontamination and sterilization is a crucial processing step in producing and reprocessing of medical devices. Since polymer-based materials are increasingly used for the production of medical devices, the application of conventional sterilization processes are restricted to a certain extent. Conventional sterilization techniques on the basis of high temperatures, toxic gases, or ionizing radiation can be detrimental to the functionality and performance of polymeric materials. For this reason, alternative, gentle, and efficient decontamination processes are required. One possible approach is the use of non-thermal physical plasmas. Especially atmospheric pressure plasma is receiving great interest due to the absence of vacuum systems which is highly attractive for the practical applicability. Its mechanisms of action enable the efficient killing and inactivation of micro-organisms which are attributed to the interaction of plasma-generated reactive oxygen and nitrogen species (ROS, RNS) as well as plasma-emitted (V)UV radiation. Owing to the moderate gas temperatures (near or at room temperature) so-called cold plasmas are well-suitable for the treatment of heat-sensitive materials, such as polymers, without affecting their bulk properties. The present work focuses on the investigation of atmospheric pressure plasma processes for the biological decontamination of polymers. The objective is to help elucidate on the one hand the impact of varied plasma process parameters on the inactivation of micro-organisms and on the other hand the influence of plasma on the surface properties of the substrate. The investigations were performed by means of a high-frequency driven plasma jet (from the product line kINPen) operated with argon and argon-oxygen mixtures. Three main aspects were analyzed: 1. The effect of plasma on the viability of micro-organisms dependent on working gas, treatment time, and the sample distance (distance between the jet nozzle and the substrate). 2. The plasma-based removal of microbial biofilms. 3. The effects of the plasma treatment on the surface properties of selected polymers. Additionally to the capability of the applied plasma jet in killing microbes the efficacy of this plasma jet for the removal of complex biological systems (e.g. biofilms) is shown. To model cell constituents of bacteria different synthetic polymers were chosen to gain insight into the decomposition process responsible for biofilm degradation. By investigating the impact of atmospheric pressure plasma on physico-chemical surface properties of various synthetic aliphatic and aromatic polymers the interaction mechanisms between plasma and plasma-exposed material are discussed. These studies are accompanied by applying different optical plasma diagnostic techniques (optical emission spectroscopy and two-photon absorption laser induced fluorescence spectroscopy) to obtain information on the plasma gas phase which contributes to the elucidation of the reaction mechanisms occurring during plasma exposure. Moreover, it is presented to which extent the plasma treatment influences the surface properties of polymers during the plasma-based bio-decontamination process and further, the benefits of surface-functionalized polymers for biomedical application is discussed.
A central point of this thesis is the investigation of surface structure and surface forces, which are created by single layers of linear polyelectrolytes (PE). In detail, the properties of cationic poly(allylamine)hydrochloride (PAH) and poly-l-lysine (PLL) and anionic sodium poly(styrene sulfonate) (PSS) are determined, which have been physisorbed onto oppositely charged silica surfaces in presence of a predefined salt concentration IAds. For these investigations, a new averaging method for colloidal probe (CP) force profiles is developed, which leads to an ultimate force resolution of 1 pN after the data processing, (signal to noise ratio of > 1000). Furthermore, a new kind of tapping mode imaging is presented (so called colloidal probe tapping mode, CPTM), which uses a CP instead of a sharp tip and hence which allows to resolve lateral inhomogeneously distributed surface forces. The basics to understand such-like obtained tapping mode images are developed. For adsorption from salt-free solution (IAds = 0) the dominance of an electrostatic double layer repulsion is observed, which is commonly attributed to the adsorption of the PE chains into a rather flat and compact layer and which is in full agreement with theoretical predictions and enormous experimental data available in literature. However, even a small addition of salt to the deposition solution (i.e. IAds > 1 mM NaCl) introduces a new contribution to the surface force, which is attributed to PE chains that are non-flatly physisorbed. Using scaling considerations, it is shown for all investigated PE that this non-flat conformation can be described by brush-like chain adsorption (cf. Section 3.3.5); other conformations like mushroom or pancake are excluded (cf. Section 5.3). Interestingly, these non-flatly physisorbed chains combine properties of neutral and PE brushes: (i) The force is very well described by the theory of Alexander and de Gennes (AdG, cf. Section 5.4). By fitting the AdG force law to the data, it is possible to determine the (brush) thickness L of the PE layer and the average distance s between brush-like physisorbed chains. Although the chains are charged the electrostatic contribution to the surface forces is too small to be noticeable (cf. Section 5.4.2). (ii) The thickness L of this PE layer is much larger compared to the compact layer (observed for salt-free adsorption) and is also subject to a pronounced swelling and shrinking if the bulk salt concentration I is decreased or increased, respectively. Surprisingly, all measurements indicate that L follows a scaling law known for salted end-grafted PE brushes, i.e. L ~ N (I s^2)^(-1/3) (with N denoting the degree of polymerization). Furthermore, the osmotic brush phase is never observed in the experiments, but chain stretching up to 1 / 3 of the contour length is regularly achieved. CPTM imaging applied to PSS shows that the brush-like physisorbed chains are not homogenously distributed over the surface, but form brush domains which coexist with flatly physisorbed chains (cf. sections 5.5 and 5.6). This clearly shows that PSS generally physisorbs in two distinct phases, which differ in conformation (flat vs. brush) and the surface force caused (electrostatic vs. steric repulsion). The force profile of the two phase system is in good approximation simply the superposition of a steric and an electrostatic repulsion, whereby their respective contribution to the composed force profile is given by their area fraction. The quantitative analysis reveals that L and s of the brush phase are independent on IAds. This is remarkable, as a change in IAds is known to induce a continuous transition between a stretched (low IAds) and coiled chain conformation (high IAds) in the deposition solution (cf. [Fleer1993, Yashiro2002]). Hence, one can conclude that the conformation in solution does not necessarily correspond to the conformation after adsorption. It is also shown that the area fraction A of the brush domains strongly depends on N and IAds. For example, for constant N the scaling relation A ~ sqrt(IAds) is determined, which is very similar to the common observation that the surface coverage %Gamma of adsorbed PE layers increases also with %Gamma ~ sqrt(IAds) [Schmitt1996, Cosgrove1986, Ahrens2001, Yim2000, Gopinadhan2007, Cornelson2010]. This suggest that brush-like physisorbed PE chains are responsible for the increase in %Gamma. In fact, Section 5.6 shows that the mass of the brush phase is approx. 0.5 mg/m² which is comparable to the increase in %Gamma reported in literature for IAds = 1 M NaCl [Cosgrove1986, Schmitt1996, Ahrens2001]. As a change in IAds does not affect L and s, but solely the brush area fraction A, it is argued in Section 5.6 that an increase in IAds can be understood as a phase transition from the (disordered) flat phase towards the (ordered and extended) brush phase. Here, further theoretical considerations would be desirable.