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The use of a cystine binding protein assay to establish the diagnosis of cystinosis was investigated. The assay has a lower limit of detection of 0.03μmol/l, is linear to a concentration of 1.5μmol/l and has a coefficient of variation of 1.7 and 15.0% between 0.05 and 0.6μmol/l. In 32 patients the mean ± SD pre-treatment leucocyte cystine concentration was 5.78 ± 2.49 nmol 1/2 cystine per mg protein (controls 0.10 ± 0.05). The median polymorphonuclear leucocyte cystine concentration in a group of 24 obligate heterozygotes was 0.55 (range 0.23 - 1.79) nmol 1/2 cystine per mg protein (controls 0.10, range 0.04 - 0.38). The effects of single doses of phosphocysteamine solution, rectal cysteamine gel, intravenous cysteamine and a cysteamine capsule were studied in 10 patients with cystinosis. No significant diurnal variation in leucocyte cystine was found. Compared with the intravenous dose, cysteamine was poorly absorbed from rectal gel (21% bioavailability) but well absorbed after administration of either oral phosphocysteamine solution (73% bioavailability) or a cysteamine capsule (50% bioavailability). Oral phosphocysteamine (10mg/kg cysteamine base), intravenous cysteamine (5mg/kg) and cysteamine capsule (15mg/kg) significantly reduced the mean leucocyte cystine with maximal depletion 1-3 hours after the dose. At 12 hours the mean leucocyte cystine was significantly lower than the pre-treatment level in each of these studies. Rectal cysteamine did not significantly reduce the mean leucocyte cystine concentration. In conclusion, phosphocysteamine suspension may be administered 12 hourly. Rectal cysteamine administration is feasible but higher doses are required before efficacy can be judged. A cysteamine capsule may prove to be a viable alternative to oral phosphocysteamine. 59 patients have received cysteamine and/or phosphocysteamine in the UK up to May 1990. In the 44 pre-transplant patients, cysteamine did not prevent a decline in glomerular renal function but a normal growth rate was maintained.

This study is an evaluation of the process of fluidised bed granulation with the objectives of elucidating the mechanism of granule formation and identifying the fundamental parameters affecting granule quality thereby enabling the true benefits of the process to be realised. Initially the effect of seven process variables upon granule quality was quantified using a factorially designed experiment. During granule assessment the torque generated by a paddle revolving in a granule mass was shown to be a useful measure of granule quality. The variables found to exert a significant effect upon granule quality were those which affected the wetting of the powder mix and rate of evaporation during granulation. A closer examination of those factors influencing bed wettability was indicated. This was achieved by studying the spray characteristics and droplet size of the atomised granulating solution, and investigating powder bed hydrophobicity. Spray characteristics of the atomised granulating solution were measured using a Malvern ST 1800 Particle and Droplet Size Analyser. A direct correlation was shown between droplet size and granule siize. Coarse, free flowing granules were obtained from sprays with large droplets, wide droplet size distributions and high granulating solution addition rates. A linear relationship was shown between the cosine of the solid/liquid contact angle and granule size. The addition of surfactant {sodium lauryl sulphate} to a model hydrophobic system was shown to improve granulation. This was related to improved powder/liquid affinity. Surfactant dissolved in the granulating solution gave slightly coarser granules with improved flow properties than when added directly to the powder mix. This was attributed to changes in spray characteristics and improved wetting by the atomised granulating solution. The growth mechanism and granule structure were subsequently investigated in a number of model powder systems using sieve analysis, scanning electron microscopy, a specially developed fluorescent technique to monitor binder distribution and a solvent extraction procedure which left a network of binder. Close examination of the data enabled a growth mechanism to be proposed for lactose and modifications to this were discussed for the other materials investigated. Thus, based on the changes occurring in the microenvironment during powder particle/droplet collision, those factors which significantly influence granule formation have been identified. These were principally the physico-chemical properties of the starting materials and the properties of the granulating droplets.

The main aim of this research is to investigate a novel emulsification device and its application to the production of biodegradable particles for controlled release drug encapsulation. The emulsification method chosen was a non crossflow membrane technique. The membrane is a flat thin layer with very regular array of pores and it is treated to produce oil-in-water or water-in-oil emulsions. Initially, a range of tests were conducted in order to link the operating conditions with the droplet size and size distribution. For this part a simple system of sunflower oil in water was used. Applied shear, injection rate, pore size and pore distance all had an effect on the resulting droplets. Sometimes these factors are not independent from each other leading to different overall effects. A model based on the force balance was proposed. lt includes the Capillary force acting against the Drag Force and a novel Push-off force originated by the interaction of neighbouring droplets in the absence of coalescence. The knowledge of the system was then applied to particle production. There is the requirement of a production method for very uniform particles with a diameter ranging between 50 and 100 μm to be used for subcutaneous (under the skin) administrations. The main benefit of making uniform particles is that it enables the engineering (i.e. mixing) of the monosized particles to give the required size distribution hence the required release pattern. The particles were produced by membrane emulsification followed by solvent evaporation. lt was of interest to encapsulate a water soluble drug, as it is more challenging to maintain high encapsulation efficiency in this case. Hence a double emulsification, W/O/W was performed. lt is shown that by changing the operating conditions it is possible to vary the size and size distribution, while by controlling the solvent evaporation rate it is possible to optimize the encapsulation efficiency. Particles of exactly 50 and 100 μm in diameter were produced, with a best span of 0.29 and encapsulation efficiency as high as 100% when encapsulating a hydrophilic. The obtained particles were used to study the release of a model hydrophilic drug and the changes in size and size morphology were followed over time too. Previously, PLGA was believed to undergo bulk erosion due to hydrolysis once in body-like conditions. The data gathered regarding the changes in size suggests that together with bulk erosion, when a hydrophilic phase is present inside the particles, surface erosion takes place too. A model for the release has been proposed based on diffusion and considering the variation in size of the particles.

The assessment of drug formulations delivered by the pressurised metered dose inhaler and used in the treatment of Asthma are assessed commercially using cascade impactors which are the preferred instruments for the assessment of particle size and respirable mass or fraction delivered by inhalation devices. The fundamental principle underpinning the design of cascade impactors is particle motion defined by Stokes theory. The analysis of impactor data raises a number of functional issues as calibration curves have long tails, which are not easily explained by a simplistic application of Stokes law. The atomisation process, propellant flashing, evaporation and aerodynamic properties of the residual drug particle detennine the distribution of the drug particles within the lung and resultant therapeutic effect. The research uses mathematical modelling and computational fluid dynamics (CFD) to evaluate the flow and inertial deposition in the USP throat and the plates of the ACI which is the most widely used cascade impactor. The CFD analysis shows the flow in the outlet section of the USP throat to be unstable for the basic design, when coupled to an outlet extension and when coupled to the ACI via the standard coupler and first jet stage. The modelling also provides insight as to why the calibration curves of the ACI have long tails and reveals a number of issues with the design of the ACI coupler and the fundamental design of impactor jet arrays as well as the position and functional response of upper impactor plates. Additional particle sizing methodologies were used to assess the lognonnal characteristics of the atomised droplets and residual drug particles. The experimental data was compared to current atomisation model and modification recommended and a proposed alternative model with improved fit to the data.

The use of polar anti-solvents for the crystallisation of lactose from 10% (w/v) aqueous solutions has been investigated. Crystal growth was observed at 50-65% antisolvent content and showed a morphological transition from polyhedral to needle-like habit with increasing antisolvent content, which coincided with a polymorphic transition from alpha lactose monohydrate (t:a. H20) to beta lactose (L~). Where anhydrous dehydrating antisolvents were employed such a~ methanol and ethanol, evidence of La. H20 dehydration to form stable anhydrous alpha lactose (Las) was also observed at 95% antisolvent content. Powder X-ray diffraction (PXRD) analysis of the samples highlighted the preferred orientation effects exhibited by large crystals of this kind, indicating the difficulties experienced by the non-specialist when performing phase identification of lactose polymorphs by PXRD. Application of the same crystallisation procedures to a racemic mixture of the active pharmaceutical ingredient (API) salbutamol sulfate (SS) indicated that some conditions can promote the formation of solvated SS. Ethanolic suspensions of spray dried and micronised La. H20, with average particle size between 3 and 200~m, were prepared by static and reflux methods and compared with sub-micron lactose (SML) suspension prepared by a high pressure homogenisation approach. Dehydration behaviour as a function of time was investigated by 13C CP MAS NMR spectroscopy and in all cases, indicated that suspensions contained Las. Several approaches were employed to remove ethanol from these suspensions and the products were analysed by PXRD and scanning electron microscopy (SEM). For samples with mean particle size greater than one micron, Las was retained on removal of the ethanol, although differences in the morphology and particle size of the crystals were apparent. These data imply that while SML is stable under dry conditions it is more susceptible to rehydration than standard Las with particle size between 3 and 200~m. in-situ 13C CP MAS NMR spectroscopy, employing hand-made glass inserts, was used to investigate the dehydration behaviour of La. H20 to Las in ethanolic suspension. Strong ethanol 13C resonances were observed for some samples, indicating a liquid-solid interaction between the ethanol and lactose surface. Replacement of ethanol with anhydrous methanol, n-butanol and 3-methylbutan- 2-01 implied that the solvent mediated dehydration is sterically controlled. 13C CP MAS NMR studies of SS, fluticasone propionate (FP) and salmeterol xinafoate (SX) showed that both SS and FP exhibit very short relaxation times for a solid material (-2s). This liquid-like behaviour is particularly beneficial pharmaceutically, as these APls are often mixed with an excess of lactose in inhaled formulations. Lactose behaves as a typical crystalline solid, and therefore experiences significantly longer relaxation times (-360s). Signals from SS and FP were successfully isolated from lactose/API blends (98%/2%) simply by reducing the delay times used within the 13c CP MAS NMR experiment.

Crystallization is a widely used purification and separation technique in the pharmaceutical industry. More than 90 % of the active pharmaceutical ingredients are produced in the crystalline form. The quality of the crystalline product greatly affects the downstream processing and bioavailability of the drug. The Food and Drug Administration (FDA) initiated in 2004 the use and implementation of process analytical technology (PAT) in the pharmaceutical development and production and encourages the pharmaceutical industry to adopt quality by design (QBD) approaches. The prime objective of this initiative has been to optimize the drug development and manufacturing process by reducing cost, improving product quality and reducing the number of failed batches. The work presented in this thesis focuses on expanding the use of two PAT tools, namely attenuated total reflection ultra violet/visible spectroscopy (ATR-UV/Vis spectroscopy) and focused beam reflectance measurement (FBRM). ATR-UV/Vis spectroscopy and FRBM are mostly used for process monitoring. The aim here was to develop sophisticated control approaches using these in situ tools for enhancing the product quality. Chemometrics is an integral part of PAT, and can provide valuable information about the system. This tool has also been used in this study for calibration model development and monitoring the cooling and antisolvent crystallization processes for single and muticomponent crystallisations. The development of an accurate and robust calibration model is necessary for qualitative and quantitative analysis of a system using spectroscopy. A systematic methodology was therefore presented for the selection of a suitable calibration model for ATR-UV/Vis spectroscopy. The developed model was then used as part of supersaturation control approach (SSC). SSC uses information from ATR-UV/Vis spectroscopy in a feedback control loop to keep the system at desired supersaturation. The developed approach resulted in the production of crystals of uniform size and can represent the bases for a model-free direct design approach for crystallization systems.

Pharmaceutical crystallisation operation is often critical because it determines product properties, such as the crystal size distribution (CSD) and polymorphic form, that can influence the subsequent downstream operations and the product therapeutic performance. Driven by the United States Food and Drug Administration s (FDA) Process Analytical Technology (PAT) initiative and the Quality-by-Design (QbD) concept, the development of control approaches, which can improve the manufacturing of products with desired properties, has become of significant interest. This thesis presents the development and application of PAT-based approaches for the monitoring and control of pharmaceutical crystallisation operations that will ensure consistent production of active pharmaceutical ingredients (APIs) with the desired size and polymorphic form. The approaches utilised Lasentec focused beam reflectance measurement (FBRM) and attenuated total reflectance ultraviolet (ATR-UV) spectroscopy as the in situ monitoring and control tools. Crystallisations of the APIs that posses multiple polymorphs are both critical and challenging. This was illustrated in this work by the crystallisations of sulfathiazole polymorphs using literature methods. The processes were monitored using FBRM and ATR-UV spectroscopy to define the design range of the process parameters. The defined range could be used as a recipe to reproduce the same quality of crystals. The obtained crystals were characterised using various techniques (optical microscopy, scanning electron microscopy (SEM), differential scanning calorimetry (DSC), thermogravimetry, hot-stage microscopy (HSM), Fourier Transform infrared spectroscopy and powder X-ray diffractometry) to assess the success of the crystallisation processes. The combined results of the techniques showed that all methods were able to produce the desired pure polymorphs. As a contribution to the technique of investigating polymorphism, a combined approach of DSC-HSM with image analysis, was introduced. Results show the capability of the approach to provide a unique insight into the polymorphic transformations and thermal behaviour exhibited by the model compound. The novel direct nucleation control (DNC) approach was introduced to control the CSD. The approach utilises information on nucleation, provided by FBRM, in a feedback control strategy that adapts the process variables, so that the desired CSD of product is achieved. It also provides in situ fines removal through the operating policy, rather than having additional equipment and external recycle loops. The approach does not require concentration measurement and has the advantage of being a model-free approach, requiring no information on nucleation or growth kinetics in order to design an operating curve; the system automatically and adaptively detects the boundary of the operating curve. Experimental results, using glycine in water-ethanol mixture as a model system, show the benefits of DNC to produce larger crystals with narrower CSD compared to uncontrolled operations. The capability of seeded cooling crystallization with temperature cycling approach to control crystal size uniformity and polymorphic purity was evaluated. Using sulfathiazole in n-propanol and in water as model systems, the method was found to accelerate the growth and enhance the size uniformity of the crystals, in comparison with runs using a linear temperature profile, by promoting Ostwald ripening. Although the approach is conceptually capable of controlling polymorphic purity of a system, the effect of solvent-mediated nucleation/growth can be more dominant, as shown by the results of the experiments. The insights into this behaviour of sulfathiazole crystals were captured very well by the FBRM. The study also demonstrated the successful use of a simple non-linear function as a calibration model to relate temperature and absorbance data, obtained using the ATR-UV spectroscopy, to solute concentration during the crystallisation process. The effect of temperature cycling, performed during seeded cooling crystallisation, on the surface features of sulfathiazole crystals was investigated using FBRM and ex situ optical microscopy, SEM and atomic force microscopy. It was observed during the initial stage of the process, the heating phases produced crystals with smooth surfaces, whilst the cooling phases promoted growth of features on the surfaces. These changes detected by the FBRM as an increase in the number of coarse counts during heating and a drop during cooling. Laser beam spreading caused by the surface roughness, and signal/chord splitting due to sharp edges are offered as an explanation for the FBRM results. This shows the capability of the FBRM to provide useful information about the changes on the surface of the crystalline products. The information can be used to avoid problems in the downstream operations, or in the final product property due to variations in flowability and friability, which are influenced by the surface property.

The objective of this project was to evaluate the alleviation of muscle wasting phenotypes by hepatic delivery and expression of a recombinant mutated myostatin propeptide mini gene in wild type mice. Initial work focused on using naked plasmid DNA as a vector for delivery of the therapeutic gene. Various β-galactosidase reporter plasmids were tested including pCMVβ, pCAGGSβ, pA2β and a liver specific pSV40 Albuminβ for systemic gene transfer to liver tissue. Following hydrodynamic intravenous administrations, pCAGGSβ proved to be the optimum plasmid and a cDNA encoding a mutated form of the myostatin propeptide (ProFcD/A) was subsequently cloned into a CAGG based plasmid (pAAVCAGGProFcD/A). The expression and bioactivity of the propeptide were assessed using in vitro cell proliferation experiments which showed the ProFcD/A was capable of increasing both proliferation and differentiation in a range of cell types. An initial in vivo study involved intravenous hydrodynamic injections of pAAVCAGGProFcD/A into MF-1 male mice and monitoring growth rates over time. A significant increase was observed in growth rate of the treated mice compared to controls with an accompanying increase in tibialis anterior (TA) muscle mass. Fibre analysis revealed pAAVCAGGProFcD/A treated mice to contain significantly larger fibres than controls owing to muscle hypertrophy. Transgene product levels were detected strongly 7 days post injection in the serum, which declined steadily over the following 21 days. By day 28, levels of transgene product were barely detectable. There was no indication response to either the vector or transgene product. The pAAVCAGGProFcD/A was then used to produce an AAV serotype 8 viral vector containing the ProFcD/A gene (AAV8ProFc) and assessed in initial intravenous tail vein injections of AAV8ProFc into MF-1 male mice. Growth rates were measured over four weeks with a significant increase in the AAV8ProFc treated mice compared to the pCAGG empty plasmid controls. In addition, significant weight increases in both TA and gastrocnemius muscles were seen in AAV8ProFc treated mice over the pCAGG control mice at week 4. Serum showed high levels of transgene expression after 7 days which was sustained up to 28 days post injection with no indication of immune response. Levels of the myogenic protein MyoD also remained higher in AAV8ProFc than pCAGG treated mice. Future studies may involve intravenous injection of both pAAVCAGGProFcD/A and AAV8ProFc into mdx mice, with subsequent monitoring of growth rates, creatine kinase levels, tissue necrosis and muscle strength over time.

No description available.