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Aggregatibacter actinomycetemcomitans is strongly associated with aggressive periodontitis and one of several virulence factors is a leukotoxin (LtxA). The toxin has a consequential impact on human leukocytes which leads to an interference with the host ́s defences due to a chain reaction involving activation and release of a pro-inflammatory cytokine; interleukin-1β (IL-1β). As an early phase in this reaction chain the toxin stimulates a massive release of adenosine triphosphate (ATP) from human leukocytes. We hypothesize that leukotoxin-induced pro- inflammatory cell death is initiated by ATP release through pannexin channels in the human monocyte cell membrane. The aim of this study was to investigate if blocking of pannexin channels with carbenoxolone (Cbx) results in a reduction of ATP release. A human monocyte cell line (THP-1 cells) was exposed to purified LtxA, Cbx and oxidized ATP. Colorimetric ATP kit was used to evaluate levels of ATP release and thereafter the samples were read in a spectrophotometer. This study confirms that LtxA induces an ATP release from THP-1 cells. Our conclusion is that blocking of pannexin channels does not result in a statistically significant reduction in ATP release, which indicates that ATP is released by one or several other undetected pathways.

Poly(L-lactide) (PLLA) is a well-recognised bioresorbable polymer known to degrade after 1.5 to 5 years by hydrolysis. For certain medical device or drug delivery applications, it would be desirable to reduce this degradation time as strategies for tailoring degradation and drug release rates remain limited. This work aimed to examine a consistent series of polymers based on a large block of PLLA and small quantities of hydrophilic poly(ethylene glycol) (PEG) initiator. The polymers had PLLA number average molecular weight (Mn) values ranging between about 60 kDa and 200 kDa and PEG Mns ranging between 550 Da and 5000 Da giving very low PEG wt% values ranging between 0.1 and 1.5 wt%. There are currently no studies which consider high molecular weight PLLA polymers with small quantities of PEG for potential use in structural implants. Furthermore, reports in the literature do not consider the individual effects of PEG addition and PEG and PLLA lengths. The focus of this project was on the impact of processing, hydrolytic degradation and drug release on the morphological aspects of the materials. The materials were thoroughly characterised in their as-synthesised and processed forms. The assynthesised polymers were semi-crystalline and retained the unit cell of PLLA. The glass transition temperature (Tg) was significantly reduced by PEG functionalisation. After injection moulding, nuclear magnetic resonance (NMR) indicated that the PEG component was still present. The Mn of the PEG functionalised samples decreased by approximately two-fold compared with the as-synthesised materials while the PLLA control polymers, processed beyond 200 °C, were more affected as the processing temperature was increased. The degradation properties of the materials were considered. The processed materials were submerged in phosphate buffered saline (PBS) (pH = 7.4) at 37 °C over an 8-month degradation study. During hydrolytic degradation, PEG functionalisation resulted in an increased water uptake. Mass loss began in all polymers when the Mn fell below a threshold of about 20 kDa. In the PEG functionalised samples, the degree of crystallinity increased with time, facilitated by plasticisation from PEG and the increased water content. The molecular degradation rate, k for the PEG-functionalised polymers was dependent on the presence of PEG functionalisation but was little affected by PEG length or PLLA length in the ranges studied. The time taken to reach the critical Mn, and hence the time for mass loss to begin, therefore depended on both the initial Mn and the presence or absence of PEG functionalisation. In the presence of PEG, k, was dramatically enhanced: k for PEG-functionalised polymers fell in the range of 6 x $10^{-4}$ $h^{-1}$ to 1 x $10^{-3}$ $h^{-1}$, as compared with that of the PLLA control of 2.9 x $10^{-5}$ $h^{-1}$. The mechanism of drug release from an analogous series of polymers was investigated. Propranolol. HCl was selected as a model drug for the drug release studies due to its thermal stability and solubility in PBS. Drug loading of propranolol.HCl was achieved by mixing the polymer and drug then injection moulding. A second method of drug incorporation using supercritical CO2 to load propranolol into as-synthesised polymer granules before injection moulding was examined for comparison. The materials processed through injection moulding showed that while drug crystals were present at the surface and in the polymer matrix, a level of drug solubility was also achieved in the PEG-functionalised polymers whereas the PLLA control showed no signs of polymer-drug interaction and only a distribution of drug crystals confined to the surface. The presence of drug crystals on the surface of the PLLA control resulted in the instant dissolution of propranolol.HCl and gave a burst release compared with an initial burst release in the PEG-functionalised polymers followed by a gradual release of the drug. This initial burst release was eliminated from the profile of the samples processed via supercritical CO2. The amorphous dispersion of the drug in the matrix gave a slow, sustained release throughout the duration of the drug release study. The results in this thesis have elucidated the intricate mechanisms of degradation and drug release from PEG-functionalised PLLA polymers. The overall outcome shows new ways of controlling the degradation and drug release rates of already medically established poly(-hydroxy acid) polymers extending their potential for use within temporary structural implants.

The aim of this research was to formulate, characterise and assess the feasibility of a novel drug delivery system known as the in situ gelling matrix (ISGM) where a hydrophilic polymer is suspended in a non-aqueous solvent that converts into a gel when injected subcutaneously or intramuscularly thus giving a controlled release matrix for a drug. Although the concept has been patented with claims that this kind of drug delivery is achievable in theory for a wide variety of candidate substances, actual formulation studies for making a commercially viable product for this technology are completely lacking in practice. The research embodied in this thesis addresses this lack. Initial studies involved conducting a biocompatibility study using the HET-CAM (hens egg test - chorioallantoic membrane) test on a range of possible ingredients for the delivery system. The materials deemed biocompatible were then carried through to a screening process where the physical stability of the hydrophilic polymers in non-aqueous solvents was monitored. It was found that the hydrophilic polymers tested sedimented rapidly in the non-aqueous solvents indicating such a system was not physically stable. Consequently, density-inducing or viscosity-inducing agents were added to the non-aqueous solvents to retard the sedimentation rate. The addition of polycarbophil, a viscosity-inducing agent, clearly increased the viscosity of the system. However, undesirable formation of polycarbophil globules occurred during the manufacturing process, which caused batch-to-batch variations in the viscosity of the continuous phase. Various manufacturing methods were tested before arriving at the optimum procedure to prevent globule formation using a high speed dispersion tool. A final physical sedimentation analysis of candidate continuous phases and hydrophilic polymers was conducted for determining the ideal combination of ingredients to use in the system. These investigations finally led to the adoption of an optimum mix of components consisting of 10% (w/w) hydroxypropyl methylcellulose (HPMC) (the hydrophilic polymer) suspended in a continuous phase of propylene glycol (the non-aqueous solvent) containing 0.67% (w/w) polycarbophil (the viscosity inducing agent). Using this mix of components, the in situ gelling matrix system was then subjected to various characterisation studies including infrared (IR), differential scanning calorimetry (DSC), ultraviolet-visible (UV-Vis) spectrophotometry and redispersion studies. The chemical stability of the hydrophilic polymer and the continuous phase (the non-aqueous solvent and polycarbophil) was monitored and were found to be chemically stable over a 9 month period. The feasibility of the in situ gelling matrix technology as a controlled release device was assessed using the drug propranolol. In vitro drug release studies were conducted using a custom-built dissolution apparatus. The effect of various parameters such as the concentration of the hydrophilic gelling agent on the drug release rate was investigated. Increasing the concentration of the gelling agent in the formulation resulted in a slower rate of release. The drug release data were modelled using the Higuchi relationship and a power law relationship to compare the effects of the various parameters on the release rate Stability studies on the drug in the in situ gelling matrix system were carried out by storing samples in accelerated ageing conditions of 40 C / 75% relative humidity for 4 weeks. During this time, the samples were analysed each week by high performance liquid chromatography (HPLC). These demonstrated that no apparent drug degradation had occurred over the 4-week period. This indicates that the drug propranolol in the in situ gelling matrix system is stable under ambient conditions for at least 4 weeks. The results of this study demonstrated that the in situ gelling matrix technology is potentially viable as a drug delivery system and provide a practical methodology for the commercial development of such systems.

Graduation date: 1995

Thesis (Ph. D.)--University of Washington, 1983. / Vita. Bibliography: leaves [202]-216.

Gastrointestinal (GI) transit data were collected using pigs as animal models. Density and size effects of non-disintegrating dosage forms on GI transit were investigated. Total GI transit times range from 2 to 33 days for 22 administrations of these nondisintegrating dosage forms. Pigs are found to not be an appropriate animal model for studying bioavailability or GI transit of non-disintegrating, non-erodible oral release dosage forms. Development of controlled release dosage forms where the mechanism of drug release is diffusion through polymeric membrane formed via film coating utilizing fluid-bed technology requires optimization of several processing and formulation variables. The influence of a processing variable (nozzle orifice opening) and a few formulation variables (individual vs. combination plasticizer, or a water-insoluble additive) on dissolution of a model drug (acetaminophen) spray coated with Aquacoat® were studied. Pharmacodynamic and pharmacokinetic information for a model drug (acetaminophen) and computer simulation were used to develop a dosage form with a 12 hour sustained release for oral administration to children and adults for maximum analgesic and antipyretic effect. Simulated plasma acetaminophen concentration-time curves were similar to observed bioavailability study profiles. In vitro and preliminary in vivo results from an adult human volunteer indicate that sustained therapeutic saliva acetaminophen concentration is possible using the newly developed acetaminophen molded tablet dosage form. The bioavailability of the new, oral controlled release acetaminophen molded tablet relative to a commercially available product (Extra-Strength Tylenol® caplet) was evaluated in 8 healthy, adult volunteers. Multiple doses of these two products were administered in a two-way cross-over design. Bioavailability of the new sustained release molded tablet is comparable to that of the immediate release product. Polymer coated acetaminophen beads were effective in maintaining saliva acetaminophen concentrations of 5 μ/ml over a 12 hour dosing interval. / Graduation date: 1991