Rheumatoid arthritis (RA) is a chronic inflammatory joint disease associated with high levels of tumour necrosis factor-alpha (TNF-a) in synovial fluid and synovial tissue (Saxne et al., 1989). Thalidomide is a proven inhibitor of the biological synthesis of TNF-a (Sampaio et al., 1991) and is believed to rely on this action for its suppression of the wasting of tissue which accompanies RA. Oral administration of thalidomide has proven to be effective in RA, but unacceptable side effects are easily provoked (Gutierrez-Rodriguez, 1984). Administration of thalidomide via the dermal route can down-regulate TNF-a production in and around the affected joint, and this without raising the systemic blood level to a problematical level. Based on thalidomide's physicochemical properties, it is unlikely that it can be delivered percutaneously at a dose required for RA. Therefore, we have embraced the idea of using N-alkyl analogues of thalidomide. The most important feature that an analogue of this compound might contribute is decreased crystallinity and increased lipophilicity. Ordinarily both these parameters should favour percutaneous delivery. The current study was primarily aimed at exploring the feasibility of percutaneous delivery of thalidomide and subsequently, three of its odd chain IV-alkyl analogues (methyl, propyl and pentyl) via physicochemical characterization and assessment of their innate abilities to diffuse through skin as an initial step towards developing a topical dosage form for the best compound. The biological activities, more specifically their potential to inhibit the production of TNF-a was determined for thalidomide and its N-alkyl analogues. In order to achieve the objectives, the study was undertaken by synthesizing and determining the physicochemical parameters of thalidomide and its N-alkyl analogues. A high level of crystallinity is expressed in the form of a high melting point and heat of fusion. This limits solubility itself, and thus also sets a limit on mass transfer across the skin. Generally, the greater a drug's innate tendency to dissolve, the more likely it is that the drug can be delivered at an appropriate rate across the skin (Ostrenga et al., 1971). Therefore, the melting points and heats of fusion were determined by differential scanning calorimetry. Aqueous solubility and the partition coefficient (relative solubility) are major determinants of a drug's dissolution, distribution and availability. N-octanollwater partition coefficients were determined at pH 6.4. Solubilities in water, a series of n-alcohols and mixed solvents were obtained, as well as the solubility parameters of the compounds in study. Secondly, in vitro permeation studies were performed from these solvents and vehicles using vertical Franz diffusion cells with human epidermal membranes. Thirdly, tumour necrosis factor-alpha (TNF-a) inhibition activities were assessed for thalidomide and its N-alkyl analogues. By adding a methyl group to the thalidomide structure, the melting point drops by over 100°C and, in this particular instance upon increasing the alkyl chain length to five -CH2- units the melting points decrease linearly. Heats of fusion decreased dramatically upon thalidomide's alkylation as well. Methylation of the thalidomide molecule enhanced the aqueous solubility 6-fold, but as the alkyl chain length is further extended from methyl to pentyl, the aqueous solubility decreased exponentially. The destabilization of the crystalline structure with increasing alkyl chain length led to an increase in lipophilicity and consequently an increase in solubility in nonpolar media. Log partition coefficients increased linearly with increasing alkyl chain length. Solubilities in a series of n-alcohols, methanol through dodecanol, were found to be in the order of pentyl > propyl > methyl > thalidomide. The N-alkyl analogues have more favourable physicochemical properties than thalidomide to be delivered percutaneously. The in vitro skin permeation data proved that the analogues can be delivered far easier than thalidomide itself. N-methyl thalidomide showed the highest steady-state flux through human skin from water, n-alcohols and combination vehicles. Thalidomide and its N-alkyl analogues were all active as TNF-a inhibitors. Finally, active as a TNF-a inhibitor, N-methyl thalidomide is the most promising candidate to be delivered percutaneously for treatment of rheumatoid arthritis, of those studied. / Thesis (PhD (Pharmaceutics))--PU for CHE, 1999.

Thesis (PhD (Pharmaceutics))--PU for CHE, 1999.

The Rho family small GTPases are key regulators of the cytoskeleton, through which they impact and control many vital cellular functions, including growth, vesicle trafficking, intercellular junctions, transepithelial transport, migration, and gene transcription. Activation of Rho GTPases is induced by Guanine Nucleotide Exchange Factors (GEFs). We have previously shown that Tumour Necrosis Factor-α (TNF), plasma membrane depolarization, and immunosuppressive drugs activate RhoA through a specific exchange factor, GEF-H1. However, the question of whether other stimuli, such as hyperosmolarity, that activate RhoA, act through GEF-H1 and whether GEF-H1 activates other RhoGTPases was not known. The overall objective of this research project has been to gain insights into the complex mechanism through which the Rho GTPases, Rac and RhoA, are regulated in tubular cells. Specifically, we wished to explore the role and pathway-specific regulation of GEF-H1 in hyperosmotic stress- and TNF-induced signalling in tubular cells. In order to accomplish our goals, we optimized and used affinity precipitation assays to detect GEF-H1 activation (RhoA(G17A) and Rac(G15A)). We found that 1) GEF-H1 is activated by hyperosmotic stress and mediates the hyperosmolarity-induced RhoA activation, as well as nuclear translocation of the Myocardin-Related Transcription Factor (MRTF); 2) TNF induces activation of both Rac and RhoA through GEF-H1, but via different mechanisms. Epidermal Growth Factor Receptor (EGFR)- and Extracellular signal Regulated Kinase (ERK)-dependent phosphorylation at the Thr678 site of GEF-H1 is a prerequisite for RhoA activation only, while both Rac and RhoA activation require GEF-H1 phosphorylation on Ser885. Interestingly, Rac is required for TNF-induced RhoA activation. Together these findings highlight a role for GEF-H1 as an osmosensitive molecule that regulates cellular reprogramming through MRTF. Importantly, we have also uncovered a novel mechanism explaining hierarchical activation of Rac and RhoA by TNF. Such a mechanism could be key in coordinating GEF function and fine-tuning Rac and RhoA activation.

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease associated with high levels of tumour necrosis factor-alpha (TNF-a) in synovial fluid and synovial tissue (Saxne et al., 1989). Thalidomide is a proven inhibitor of the biological synthesis of TNF-a (Sampaio et al., 1991) and is believed to rely on this action for its suppression of the wasting of tissue which accompanies RA. Oral administration of thalidomide has proven to be effective in RA, but unacceptable side effects are easily provoked (Gutierrez-Rodriguez, 1984). Administration of thalidomide via the dermal route can down-regulate TNF-a production in and around the affected joint, and this without raising the systemic blood level to a problematical level. Based on thalidomide's physicochemical properties, it is unlikely that it can be delivered percutaneously at a dose required for RA. Therefore, we have embraced the idea of using N-alkyl analogues of thalidomide. The most important feature that an analogue of this compound might contribute is decreased crystallinity and increased lipophilicity. Ordinarily both these parameters should favour percutaneous delivery. The current study was primarily aimed at exploring the feasibility of percutaneous delivery of thalidomide and subsequently, three of its odd chain IV-alkyl analogues (methyl, propyl and pentyl) via physicochemical characterization and assessment of their innate abilities to diffuse through skin as an initial step towards developing a topical dosage form for the best compound. The biological activities, more specifically their potential to inhibit the production of TNF-a was determined for thalidomide and its N-alkyl analogues. In order to achieve the objectives, the study was undertaken by synthesizing and determining the physicochemical parameters of thalidomide and its N-alkyl analogues. A high level of crystallinity is expressed in the form of a high melting point and heat of fusion. This limits solubility itself, and thus also sets a limit on mass transfer across the skin. Generally, the greater a drug's innate tendency to dissolve, the more likely it is that the drug can be delivered at an appropriate rate across the skin (Ostrenga et al., 1971). Therefore, the melting points and heats of fusion were determined by differential scanning calorimetry. Aqueous solubility and the partition coefficient (relative solubility) are major determinants of a drug's dissolution, distribution and availability. N-octanollwater partition coefficients were determined at pH 6.4. Solubilities in water, a series of n-alcohols and mixed solvents were obtained, as well as the solubility parameters of the compounds in study. Secondly, in vitro permeation studies were performed from these solvents and vehicles using vertical Franz diffusion cells with human epidermal membranes. Thirdly, tumour necrosis factor-alpha (TNF-a) inhibition activities were assessed for thalidomide and its N-alkyl analogues. By adding a methyl group to the thalidomide structure, the melting point drops by over 100°C and, in this particular instance upon increasing the alkyl chain length to five -CH2- units the melting points decrease linearly. Heats of fusion decreased dramatically upon thalidomide's alkylation as well. Methylation of the thalidomide molecule enhanced the aqueous solubility 6-fold, but as the alkyl chain length is further extended from methyl to pentyl, the aqueous solubility decreased exponentially. The destabilization of the crystalline structure with increasing alkyl chain length led to an increase in lipophilicity and consequently an increase in solubility in nonpolar media. Log partition coefficients increased linearly with increasing alkyl chain length. Solubilities in a series of n-alcohols, methanol through dodecanol, were found to be in the order of pentyl > propyl > methyl > thalidomide. The N-alkyl analogues have more favourable physicochemical properties than thalidomide to be delivered percutaneously. The in vitro skin permeation data proved that the analogues can be delivered far easier than thalidomide itself. N-methyl thalidomide showed the highest steady-state flux through human skin from water, n-alcohols and combination vehicles. Thalidomide and its N-alkyl analogues were all active as TNF-a inhibitors. Finally, active as a TNF-a inhibitor, N-methyl thalidomide is the most promising candidate to be delivered percutaneously for treatment of rheumatoid arthritis, of those studied. / Thesis (PhD (Pharmaceutics))--PU for CHE, 1999.

Tumour necrosis factor-stimulated gene-6 (TSG-6) is a protein expressed in a wide range of cell types and tissues, predominantly in response to inflammatory stimuli. The expression of TSG-6 is believed to be associated with the protection of tissues from the damaging effects of inflammation. In animal models treatment with TSG-6 protein has been found to reduce inflammatory damage in myocardial infarction, corneal injury and arthritis. Endogenous TSG-6 production has been suggested to play a protective role in inflammatory arthritis and has been implicated in bone homeostasis. The expression of TSG-6 is also essential in the process of cumulus matrix formation that occurs around the oocyte in the periovulatory period and is necessary for successful ovulation and fertilisation. In many cases the mechanism underlying a particular TSG-6 function is not fully understood. TSG-6 has numerous binding partners including the serum glycoprotein inter-alpha-inhibitor (IαI), the growth factor bone morphogenetic protein-2 (BMP-2) and the extracellular matrix protein fibronectin, as well as glycosaminoglycans (GAGs) such as hyaluronan and heparan sulphate (HS). The TSG-6 protein is mostly composed of contiguous Link and CUB domains, with the majority of ligand binding sites identified within the Link module. The CUB domain of TSG-6 has been less extensively studied. Here biophysical techniques have been used to investigate the TSG-6 interactome including both the Link module and CUB domain. Intrinsic fluorescence spectroscopy was used to establish the metal-ion binding properties of the CUB domain, which was established to have a high affinity Ca2+-binding site. Using surface plasmon resonance (SPR), a novel metal-ion dependent interaction was found for the CUB domain of TSG-6 and the heavy chains (HCs) of IαI. Investigation using mutants of both the CUB domain of TSG-6 and HC of IαI established that the metal-ion binding sites within each protein are involved in the interaction. SPR analysis was also used to define the affinities and binding sites for TSG-6 interactions with fibronectin and BMP-2. High affinity interactions between TSG-6 ligands were also revealed (e.g. BMP-2 and HC, fibronectin and HC) and their binding sites defined. The discovery of the novel interactions between these TSG-6 ligands suggests crosstalk within the TSG-6 interactome, with the potential for ternary complex formation or indeed hierarchical orders of binding. Thus work was undertaken to develop a passivated lipid bilayer platform for use with surface sensitive techniques. This platform was used to investigate the hierarchy of protein and GAG interactions using quartz crystal microbalance with dissipation monitoring (QCM-D) and dual polarisation interferometry (DPI). The investigation revealed a novel role for the Link module of TSG-6 in heparin condensation, potentially via protein dimerisation and/or oligomerisation which could affect heparin/HS functions within the extracellular matrix (ECM). Thus the biophysical analysis of TSG-6 presented here has identified novel interactions and functions of TSG-6 which may provide mechanisms for the protective functioning of TSG-6 in inflammation and its ECM structuring role in ovulation.

Background: There is need for judicious use of immunosuppression in patients with active lupus nephritis (LN), however this is guided by renal biopsy which is invasive and not freely available in most centres. Novel urinary biomarkers such as monocyte chemoattractant protein-1 (MCP-1) and tumour necrosis factor-like weak inducer of apoptosis (TWEAK) are secreted in the kidney and may be useful for predicting histological class, monitoring flares and assessing response to therapy. We assessed the utility of urinary MCP-1 (uMCP-1) and TWEAK (uTWEAK) in predicting renal histological findings, disease flares and treatment response 6 months following initiation of treatment for LN in newly biopsied patients. Methods: We recruited consenting patients with active LN confirmed on kidney biopsy. Relevant baseline demographic, biochemical and histological information was collected from the patients. ELISA methods were used to assess uMCP-1 and uTWEAK at baseline and at 6 months after completion of induction therapy. Results: There were 14 females and 6 male patients with a mean age of 29.8 ± 10.7 years, 60% were of mixed ancestry, 70% had proliferative LN. There was no association between uMCP-1 and uTWEAK and histological features (LN class, activity index, chronicity index and interstitial fibrosis). At 6 months, 6 patients were lost to follow-up and of the remaining 14, 12 (85%) attained remission (partial remission (n = 7) or complete remission (n = 5)). Both biomarkers were elevated in patients with active disease and significantly declined amongst those attaining remission, p = 0.018 and p = 0.015 respectively. However, for those not attaining remission, no association was found for both biomarkers (p >0.05). Conclusion: Our study did not show correlation between uMCP-1 and uTWEAK with histological features of LN. However, both biomarkers were elevated in patients with active disease and correlated with the remission status at the end of induction phase of treatment.

Pigmentation disorders occur in multiple conditions (Hakozaki et al., 2006:105). Although many modalities of treatments are available, none are completely satisfactory (Briganti et al., 2003:101). Two cytokines normally present in the skin, transforming growth factor–beta1 (TGF–81) and tumour necrosis factor–alpha (TNF–9), have been shown to inhibit melanin synthesis (Martinez–Esparza, 2001:972). The stratum corneum has been commonly accepted as the main barrier to percutaneous absorption. Many techniques have been applied to overcome this barrier properties and to enhance penetration with varying success (Pellet et al., 1997:92). The objective of this study was to investigate the topical delivery of the above mentioned peptide drugs with aid of the Pheroid drug delivery system. Pheroid technology is a delivery system that promotes the absorption and increases the efficacy of dermatological, biological and oral medicines in various pharmacological groups (Grobler et al., 2008:4). Pheroid entraps drugs with high efficiency and delivers them with remarkable speed to target sites (Grobler, 2004:4). In order to avoid degradation of these peptides, bestatin hydrochloride (an aminopeptidase inhibitor), was used (Lkhagvaa et al., 2008:386). Topical drug delivery was achieved by means of vertical Franz cell diffusion studies performed over a 6 and 12 h period. ELISA (enzyme linked immunosorbent assay) detection was used to detect cytokine concentrations. Entrapped cytokine solutions were monitored by confocal laser scanning microscopy (CLSM). Upon removal of donor and receptor compartments, skin discs were subjected to tape stripping in order to establish the amount of active present within the stratum corneum and epidermis as well as the remaining dermis (Pellet et al., 1997:92). When comparing the two studies with each other, it is evident that the diffused concentration values obtained with PBS (phosphate buffer solution, pH 7.4) was lower than that obtained with the Pheroid drug delivery system. Both cytokine concentrations were successfully delivered topically as a minimum of concentrations for both actives were detected. This positive result was confirmed as well by the amount of active detected in stratum corneum–epidermis and epidermis–dermis solutions. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2011.

Pigmentation disorders occur in multiple conditions (Hakozaki et al., 2006:105). Although many modalities of treatments are available, none are completely satisfactory (Briganti et al., 2003:101). Two cytokines normally present in the skin, transforming growth factor–beta1 (TGF–81) and tumour necrosis factor–alpha (TNF–9), have been shown to inhibit melanin synthesis (Martinez–Esparza, 2001:972). The stratum corneum has been commonly accepted as the main barrier to percutaneous absorption. Many techniques have been applied to overcome this barrier properties and to enhance penetration with varying success (Pellet et al., 1997:92). The objective of this study was to investigate the topical delivery of the above mentioned peptide drugs with aid of the Pheroid drug delivery system. Pheroid technology is a delivery system that promotes the absorption and increases the efficacy of dermatological, biological and oral medicines in various pharmacological groups (Grobler et al., 2008:4). Pheroid entraps drugs with high efficiency and delivers them with remarkable speed to target sites (Grobler, 2004:4). In order to avoid degradation of these peptides, bestatin hydrochloride (an aminopeptidase inhibitor), was used (Lkhagvaa et al., 2008:386). Topical drug delivery was achieved by means of vertical Franz cell diffusion studies performed over a 6 and 12 h period. ELISA (enzyme linked immunosorbent assay) detection was used to detect cytokine concentrations. Entrapped cytokine solutions were monitored by confocal laser scanning microscopy (CLSM). Upon removal of donor and receptor compartments, skin discs were subjected to tape stripping in order to establish the amount of active present within the stratum corneum and epidermis as well as the remaining dermis (Pellet et al., 1997:92). When comparing the two studies with each other, it is evident that the diffused concentration values obtained with PBS (phosphate buffer solution, pH 7.4) was lower than that obtained with the Pheroid drug delivery system. Both cytokine concentrations were successfully delivered topically as a minimum of concentrations for both actives were detected. This positive result was confirmed as well by the amount of active detected in stratum corneum–epidermis and epidermis–dermis solutions. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2011.

Head and neck cancer is the fifth most common cancer worldwide with an estimated annual global incidence of over 500 000 cases. These malignant tumours develop in the mucosal linings of the upper respiratory tract or in the salivary glands. The most common sites are in the oral cavity and larynx. Treatment modalities comprising surgery and chemoradiotherapy have improved significantly during the last 20 years, but not the long-term survival of patients. The aim of this thesis was to study the different factors affecting tumour growth in head and neck cancer that may have clinical implications in the future. Factors involving apoptosis, cell cycle activity, inflammation, and enzyme activity were of special interest. The results of the thesis indicate that patients with malignant salivary gland tumours having the lowest level of actively replicating cells have the best prognosis. The largest amount of replicating cells in tongue cancer specimens was found in the peripheral areas of tumour nests. Metallothionein, a protein that can hinder apoptosis, was found in excess in the same areas, whereas apoptosis activity was considerably lower. Taken together, these results indicate that the most aggressive cancer cells are found in the peripheral areas of tumours where apoptosis may be hindered. The expression of the death receptor Fas was higher in tongue cancer specimens than in normal mucosa. The expression of this receptor was studied further in two cell lines established from oral cancers. When a low dose of cisplatin was added to cell cultures, the Fas expression was enhanced in both cell lines and, furthermore, the Fas-induced apoptosis was increased in one of the cell lines. The results show that a common chemotherapeutic drug given in a low, less toxic dose may enhance receptor-mediated apoptosis of cancer cells. Malignant solid tumours are often distinguished by an increased proteolytic activity resulting in invasive growth, neo-angiogenesis, and metastases. This activity is conducted by enzymes that are secreted from tumour cells, or from normal cells in the tumour microenvironment. The regulation of enzyme secretion may be mediated by cytokines, small signalling molecules also present in cancer tissue. The results of this thesis show that two cytokines can synergistically induce enzyme secretion (matrix metalloproteinase-1 and -9) from oral cancer cells. Cytokine tumour necrosis factor-alpha and hepatocyte growth factor added alone to cell cultures strongly stimulated secretion of these enzymes. Thus, the tested cytokines, which are commonly secreted by fibroblasts and immune cells, may promote tumour growth. This thesis has contributed to an increased understanding of factors affecting tumour growth in head and neck cancer. The upcoming cancer therapies will be based on the increasing knowledge of these and other aberrant cellular mechanisms that may vary between different cancer forms.

Optimization of biologic therapy in children with inflammatory bowel disease (IBD) using modern biomarkers Abstract to thesis Study programme: Biochemistry and Pathobiochemistry Introduction: In adults, infliximab (IFX) levels correlate with disease activity and antibodies to IFX (ATIs) predict treatment failure. We aimed to determine the association of IFX levels and ATIs with disease activity in paediatric population. Methods: This study was performed as a prospective observational study. We prospectively collected blood, stool, and clinical data from 65 patients (age 10.5-15.1 years) with Crohn's disease (CD) before IFX administration, and measured IFX trough levels, ATIs, and faecal calprotectin levels (CPT). We used multivariate analysis to identify the predictors of IFX levels. IFX and ATIs levels were meassured using ELISA. Results: Lower levels of IFX were associated with ATIs positivity (OR [odds ratio] 0.027, CI [confidence interval] 0.009-0.077). Higher C-reactive protein (CRP) level, erythrocyte sedimentation rate (ESR) and CPT levels were found in patients with lower IFX levels. The optimal combination of specificity (50%) and sensitivity (74%) for disease activity was calculated for IFX levels ≥ 1.1 µg/ml using CRP level < 5 mg/l as a marker of laboratory remission. In a model that used CPT ≤...