The use of over-the-counter products (OTCs) has increased over the years. This is evident from the wide range of products available and also the easy availability of these OTCs in pharmacies, health shops and even supermarkets in South Africa and elsewhere. Therefore the procurement of these products is easy and they are used by many consumers for self-treatment of numerous conditions. This has given rise to the problem of irresponsible use of these products by consumers. Pain is the most common pharmacological challenge encountered by the medical practitioner and therefore treatment for pain is frequently prescribed. Narcotic analgesics and non-steroidal antiinflammatory drugs (NSAIDs) are drugs used to relieve pain. The use and misuse of drugs and medications by competitors in sport has been recognized as an important problem. Ethical aspects of competing under non-equal opportunities are of concern. Elite athletes who turn to doping take the greatest risk to satisfy their burning desire for gold regardless of the health risks involved. Deaths under the influence of drugs and combinations thereof are not uncommon in sport. Athletes might use substances to eliminate any obstacle they might encounter during their training. One of the obstacles they might encounter is pain felt from injury or excessive training. The misuse of drugs for sport enhancement is a health risk because of the kinds of drugs used and the large doses given. The narcotic analgesics and the NSAIDs are some of the drugs abused by competitors to overcome the effects of pain. The aim of this study was to develop a screening method for the simultaneous detection of an extended list of narcotic analgesics and NSAIDs in human urine using high performance liquid chromatography â tandem mass spectrometry. An extended list of some of the narcotic analgesics and NSAIDs available in South Africa was compiled. Literature research was done on these reference substances and their metabolites. It was found that information from the literature was limited. The mass spectrometry properties of each reference standard were determined using a LCMS/ MS system in positive electron ionization mode. A new method was successfully optimised using these mass spectrometric properties. Information regarding extraction procedures for these compounds was collected. The extraction procedure included a hydrolysis step with β-glucuronidase/arylsulfatase followed by liquid-liquid extraction with diethyl ether at pH 7. No derivatization steps were necessary during sample preparation, which shortens the preparation time. This extraction procedure was successfully combined with the LC-MS/MS method for the screening of the list of narcotic analgesics and NSAIDs. The final chromatographic conditions for this study are as follows: Autosampler properties: Agilent 1100 Autosampler Injection volume: 10 μl Runtime: 20 minutes Pump method properties: Agilent 1100 Binary Pump Mobile phase: Solvent A: 0.01% formic acid Solvent B: acetonitrile Step Time (min) Flow rate (μl/min) A (%) B (%) 0 0.00 200 90.0 10.0 1 2.00 200 90.0 10.0 2 8.00 200 10.0 90.0 3 9.00 200 90.0 10.0 4 20.0 200 90.0 10.0 The final extraction procedure is as follows: · 2 ml urine · 1 ml acetate buffer pH = 5.2 · 25 μl Ã-glucuronidase/arylsulfatase enzyme · Hydrolyse at 50 °C for 2 hours · Add 1 ml phosphate buffer pH = 7 · Add 5 ml diethyl ether · Shake horizontally for 5 minutes on a mechanical shaker · Centrifuge for 5 minutes · Transfer organic phase to an ampoule containing 50 ml internal standard (Apomorphine) · Evaporate to dryness · Dissolve in 100 μl mobile phase. Good results were obtained during the validation. Good specificity was obtained for each compound without the interference of the background or co-extracted compounds. Good repeatability was obtained for the listed compounds. The calculated CV% for all the compounds was under 15% for low, medium and high concentrations. The calculated LOD values was relatively low for most of the compounds, with exception of paracetamol, which has a high back-ground and piroxicam which could not be extracted effectively from the urine. The freeze and thaw stability of the listed compounds was satisfactory with only salicylic acid which was outside the range of 20% after 3 cycles of freeze and thaw. For long term stability the results were different. The results for the analysis of the control sample after 3 months of storage at -20oC were below 80% for 18 compounds when compared to the sample analysed immediately before storage. From these, 15 compounds were between 70 and 80% and 3 compounds were between 60 and 70%. The method was applied to obtain the excretion profiles for diclofenac, its metabolite, 4â- hydroxydiclofenac, and indomethacin. For diclofenac the peak excretion was determined at 6 hours after administration with a sharp decrease of excretion until 24 hours after administration. For its metabolite, 4â-hydroxydiclofenac, the peak excretion was also determined at 6 hours after administration, but with a slower decrease from 6 hours and can still be detected after 48 hours. For indomethacin the peak excretion was determined at 6 hours after administration with a slow decrease from 6 hours and can still be detected after 48 hours. A further application of this screening method was done by analyzing a series of 137 real urine samples collected from competitors from different sporting events. The results showed that there is a large number of these substances used by competitors in sport. The number of positive samples was 37% of the total number of samples tested. Rugby with 39% of the positive samples was identified as the sporting event with the highest usage of these substances, with paracetamol identified as the substance that is the most frequently used. It can be concluded that this method is effective in detection of the listed narcotic analgesics and NSAIDs in urine from human.

Phela is a herbal traditional medicine product prepared using well defined parts of four African medicinal plants. The aim of the study was to determine the mechanism of action by which phela boosted the immune system of a rat model. Unfortunately, subsequent literature research revealed that very little is known about phela. As such chromatographic methods were developed by which to identify phela and for quality control purposes. Of note, the developed methods complemented each other; they were compiled into a comprehensive method for phela fingerprinting. It involved sample extraction of the phela by either acidic extraction or a simple âsaltingoutâ method, followed by Thin Layer Chromatography (TLC), and/or preparative Column Chromatography (CC) that were supported by High Performance Liquid Chromatography with UV-detector (HPLC_UV), HPLC with fluorescence detector (HPLC_FL), HPLC with photo diode array detector (HPLC_PDA) and Gas Chromatography-Mass selective detector (GC_MSD) spectrometry. The method was successfully used to differentiate phela from another herbal product made from Hypericum perforatum (St Johnâs wort) illustrating its high potential for application to other herbal medicines in development. Thereafter a HPLC_UV method for monitoring phela in plasma was developed and validated. However, due to its pitfalls, a HPLC_FL method was developed as well. The HPLC_UV method had a linear regression equation of y = 0.02x â 0.59, correlation coefficient of r2 = 0.9983 and accuracy within 15 %. For HPLC_FL three marker peaks were selected and standardized by the retention time. The developed HPLC_UV and HPLC_FL methods were tested by analysing blood collected from rats treated with phela after 1, 2, 4, 6 and 8 hours respectively. Unfortunately, analysis by HPLC_UV method had no peaks whereas, during HPLC_FL method analysis, a new peak was observed at 9.2 minutes. The peak was depicted as a metabolite for phela and was then utilized as the plasma marker. Since the metabolite is unknown and therefore no standard exists by which to express its plasma concentration in conventional units, the changes in peak area per unit volume (L) of plasma (peak-area/L) were used to derive the appropriate pharmacokinetic parameters hence peak-kinetics. The predictive parameters show that concentration of the metabolite at steady state would be 48 peak-area/ml, hence no accumulation of the drug. The final part of the study was undertaken to understand the mechanism of action of phela using a rat model by observing for changes in the levels of TH1 and TH2 cytokines. Rats were divided into six groups. The first group was not treated with anything. The four groups were treated daily with either normal-saline, orally (control); cyclosporine in olive oil, subcutaneously (negative control); Phela, orally (test-group 1) and phela+cyclosporine (test-group 2). The last group was inoculated once with influenza vaccine. The treatment period was 14 days and in each group rats were sacrificed after 7 and 14 days. Haematology tests, biochemistry tests, and cyclosporine level analysis were done. Serum cytokine analysis for TH1 (IL-2, IFN-γ and TNF-α) and TH2 (IL-4 and IL-10) cytokines was measured by ELISA. On days 7 and 14, the concentrations of TH1 cytokines in the Phela-only treated group were similar to the control. However, the TH1 cytokines were higher in the Phela+cyclosporine-A treated group than in the cyclosporine-A group, and cyclosporine- A concentrations were similar in both groups. These results show that Phela did not affect TH1 cytokines of a normal immune system but stimulated them when the immune system was suppressed by cyclosporine-A. This implies that Phela is a cyclosporine-A antagonist that may stimulate a compromised immune system. In conclusion, Phela is an immune stimulant.

Background: Historically characterized by a high prevalence of alcohol addiction and dependency, South Africa has in recent years experienced an unprecedented increase in illicit drug use, linked to organized criminal activities. While internationally, the role of pharmacotherapy in the multi-disciplinary treatment of addiction/dependency becomes more important based on an increasing body of evidence revealing the biological nature of the condition, major transformation in the Health and Social delivery systems are taking place locally. Aim: The study aims to provide a critical analysis of current treatment practices regarding pharmacotherapy for drug addiction/dependency in the Free State against the background of the biological processes involved in the addiction/dependency state as well as aspects of health service delivery that may influence the use of pharmacotherapy. The analysis forms the basis for the development of a framework for the treatment of substance addiction and dependence regarding pharmacotherapy, taking into account the findings of the literature study and local context. Material and Methods: Both quantitative and qualitative methods were used. A questionnaire and structured interview were conducted with 121 health care professionals that could reasonably be expected to be confronted by patients with addiction and dependency. The population included a randomized sample of general practitioners selected from regional, district and basic environments in the Free State; purposely selected representatives of state hospitals and private treatment centres, as well as private psychiatrists and therapists in the corresponding towns. Results: Help-seeking for addiction occurs in a distinguishable pattern across the various professional groups. Private general medical practitioners are an important conduit into treatment for alcohol addiction and dependency. Depending on the local organization of services, they are also actively involved in the medical treatment of addiction and dependency cases. Private psychiatrists exclusively deal with dual diagnosis patients and are exposed to a wider range of addiction/dependency cases. State hospital service delivery varies from comprehensive services to no services. Perceptions regarding access to state hospitals and the quality of services in state hospitals are poor, while private services are generally regarded as costly, yet effective. Medical Scheme policies play an important role in determining access to facilities and services and dictate the individual prescriberâs approach to pharmacotherapy. Respondents regarded the role of pharmacotherapy as essential in withdrawal and neuropsychological support, yet less important in relapse prevention. Convention mainly determines the withdrawal regimens used by respondents, with a number of area-dependent exceptions. Recognition of the neurotoxic nature of the withdrawal state is not universally reflected in the selection of pharmacotherapeutic agents in withdrawal regimens. Only disulfiram is commonly used for relapse prevention and its use is limited by high cost. Besides financial status, the decision to prescribe these drugs is based on the patientâs motivation or willpower. Conclusion: A basic lack of recognition of the biological basis of addiction and dependency exists in the current legislation, in the organization of services and in the management of addiction/dependency. Medical intervention in addiction/dependency typically occurs late and follows an intermittent course with short-term goals. Recommendations: An integrated framework was developed and needs to be considered for implementation at both organizational and treatment practice levels in the region with the primary objective to improve treatment outcomes. Rational prescribing of pharmacotherapy requires an expansion of medication options and improved screening methods to allow individualized treatment, a biological imperative for successful treatment. At the same time standardization of evidence-based best treatment practices should be implemented. The role of private general practitioners as primary gatekeepers of the health system should be restored to provide a platform for accessible medical treatment of addiction and dependency.

Since the discovery of the anti-epileptic action of diphenylhydantoin, it has been repeatedly observed that some patients undergoing treatment develop gingival changes. These changes appear superficially like a low grade inflammatory reaction, showing irregularity of the gingival surface, but progressing slowly to a firm, painless overgrowth of the gums. [...]

The concept of antagonism has proved to be a fruitful approach in efforts to gather information on many physiological and pharmacological problems. In the case of the sympathetic system, ergot was used as early as 1906 by Dale, who then supplied an accurate description of what we know as 'adrenergic blockade'. This pharmacological effect may be defined as the blockade of responses to adrenaline, nor-adrenaline and other sympathomimetic amines and to sympathetic nerve activity. [...]

The essential roles of glutamatergic signaling in both normal and impaired cognitive functioning have caused metabotropic glutamate (mGlu) receptors to become targets of interest for the treatment of psychiatric disorders. The eight mGlu receptors are G-protein coupled receptors, which have been placed into three groups based on their sequence homology and preferred signaling pathway. Studies of the group II receptors, mGlu2 and mGlu3, have been limited by a lack of selective ligands. We used iterative, parallel, multidimensional synthesis along with pharmacologic and pharmacokinetic analyses to develop novel allosteric modulators for these targets, which act at a distinct site from where the endogenous ligand glutamate binds. These efforts led to the development of the first reported selective mGlu3 negative allosteric modulator (NAM), a series of dual mGlu2/3 NAMs, and provided the first characterization of a selective mGlu2 NAM. These compounds were then used in a series of electrophysiological and behavioral studies to reveal an unexpected role for postsynaptic mGlu3 receptors in the development of long term depression (LTD) of synaptic firing in the medial prefrontal cortex and implicate mGlu3 dysfunction as a potential cause for persistence of cued memories. Second generation mGlu2 and mGlu3 selective NAMs were then used in rodent models of depression and anxiety, reveal a potential antidepressant effect of mGlu3 NAMs. Finally, we used a candidate gene approach to look at the distribution of variants in the genes encoding for mGlu receptors in substance dependent patients and in matched controls, which uncovered a possible correlation of structural variance in mGlu1, mGlu2, and mGlu3 receptors with the development of substance dependence.

Neurofibromatosis type 1 (NF1) is the most common autosomal dominant genetic disorder occurring in 1 of every 3500 live births. NF1 is caused by loss-of-function mutations in NF1, the gene encoding the Ras-GAP neurofibromin. Forty percent of NF1 patients will develop orthopedic complications which often includes unilateral bowing of the extremities, fracture, and subsequent fracture healing deficits (pseudarthrosis). Because the molecular and cellular aspects mechanisms of fracture healing largely recapitulate the processes of bone development, the goal of this dissertation is to characterize the function of neurofibromin in growth plate chondrocytes and the signaling pathways it regulates during endochondral ossification. Using conditional mouse knockout models of NF1, we found that neurofibromin regulates growth plate chondrocyte proliferation, hypertrophic maturation, and matrix catabolism at the osteochondral border. Furthermore, we found that neurofibromin in prehypertrophic chondrocytes likely attenuates FGFR1 and FGFR3 signaling to inhibit chondrocyte proliferation, and neurofibromin in hypertrophic chondrocytes attenuates FGFR1 signaling to inhibit matrix catabolism as the osteochondral border. Finally, in a series of pharmacological proof-of-principle experiments, we identified C-type natriuretic peptide and the pan-FGFR inhibitor BGJ-398 as potential therapeutic agents for the treatment of NF1 pseudarthroses via their action on Nf1-/- chondrocytes. Further investigation of these agents in NF1 fracture healing models is warranted.

Basement membranes are a distinct form of extracellular matrix responsible for signal transduction and mechanical integrity throughout development, in mature tissues, and during wound healing. The collagen IV scaffold of basement membranes relies on a sulfilimine crosslink (S=N) between methionine and lysine for its essential function of maintaining basement membrane architecture. The sulfilimine crosslink is formed by the heme peroxidase, peroxidasin. The precise mechanism by which peroxidasin forms sulfilimine crosslinks, or if this crosslinking process is involved in disease, remains largely unknown. Biochemical investigation of peroxidasin-catalyzed formation of the crosslink found that bromide (Br^-) appeared to be the preferred enzymatic cofactor through its conversion to hypobromous acid (HOBr). Through my development of Br-free salts, purified proteins, and in vitro cell culture models of basement membranes, Br^- was shown to be essential to the formation physiologically observed levels of sulfilimine crosslink. I further investigated the underlying mechanism of sulfilimine formation with chemical crosslinking, mass spectrometry analysis, and modeling. These approaches cumulatively supported the presence of a S-Br⁺ (bromosulfonium-ion) intermediate by the crosslinked methionine of the NC1 domain of collagen IV as the key reaction intermediate and energetic basis for bromines role in sulfilimine crosslinking. The essentiality of Br was therefore tested in vivo in Drosophila by developing novel Br-free culture techniques. I found that dietary Br-deficiency is lethal in Drosophila while Br-replenishment restores viability, demonstrating a physiologic Br^- requirement. Importantly, through electron and fluorescence microscopy, I was also able to show that Br-deficient flies phenocopy the developmental and basement membrane defects observed in peroxidasin mutants which indicates a functional connection between Br^-, collagen IV, and peroxidasin. These data collectively established that Br^- is required for sulfilimine crosslinking of collagen IV, an event critical for basement membrane assembly and tissue development. Thus, bromine is an essential trace element for animals through the enzymatic activity of peroxidasin, and Br-deficiency may be relevant to basement membrane alterations observed in patients undergoing dialysis, receiving total parenteral nutrition, and in some smoking related disease. I also sought to test the hypothesis that anti-peroxidasin autoantibodies occur in a specific rapidly progressive glomerulonephritis known as Goodpastures disease (GP). Goodpastures Disease is characterized by anti-collagen IV NC1 antibodies and the sulfilimine crosslink is thought to modulate immunogenicity of the collagen IV epitopes. Many GP patients have concurrent autoantibodies which recognize myeloperoxidase (MPO), a structurally related heme peroxidase to peroxidasin. Through testing multiple independent patient cohorts by immunoassay, I found anti-peroxidasin autoantibodies in GP patient sera, both before and at the time of clinical presentation. Unexpectedly, the anti-peroxidasin specific antibodies cross-react with coated, but not native MPO, accounting for a subset of the historically characterized dual-positive (anti- collagen IV and anti-MPO) patients. I also found that anti-peroxidasin antibodies inhibited HOBr production, suggesting a possible contribution of these antibodies in GP pathogenesis within this subset of anti-peroxidasin positive patients. These studies demonstrate chemical, biochemical, and tissue level evidence for the role of peroxidasin and Br^- in the assembly of sulfilimine-crosslinked collagen IV scaffolds in basement membranes and peroxidasins potential role in disease, including as a novel autoantigen in a subset Goodpastures disease patients.

Aortic stiffening is an important cause of systolic hypertension and predispose to cardiovascular morbidity and mortality. The precise mechanisms underlying aortic stiffening remain undefined. Using multiple genetically modified mouse models, I performed a series of studies to investigate the roles of oxidation, inflammation and T cell-mediated adaptive immunity in the development of this pathological condition. I found that pro-inflammatory cytokines such as IL-17A released by activated T cells, in concert with increased mechanical stretch, promote the production of collagen by arterial fibroblasts in hypertension. These effects occur in a p38 MAP kinase-dependent manner and pharmacological inhibition of this enzyme lowers blood pressure and prevents aortic stiffening in angiotensin II-infused mice. To understand the mechanism of T cell activation in hypertension, I employed murine models of chronic vascular oxidative stress. Tgsmp22phox mice and SOD3loxp/loxp X Tgsmmhc/cre exhibited aged-related vascular inflammation, aortic stiffening and hypertension. Isoketal-protein adducts accumulated in aortas of tgsm/p22phox mice, forming immunogenic neoantigens. I found these are presented by dendritic cells to T cells, leading to T cell proliferation, cytokine production and infiltration of T cells to vessels, the original site of oxidant injury. Activated T cells release cytokines, including IL-17A and IFN-γ, which have previously been shown to induce the expression of chemokines. Cellular components, including vascular Sca-1+ progenitors acquire fibroblast-like phenotype and, along with collagen I+CD45+ circulating fibrocytes, become important contributors of adventitial collagen deposition and aortic stiffening. These studies demonstrate that there is interplay between chronic vascular oxidative stress, inflammation and adaptive immune cells in the pathogenesis of aortic stiffening and hypertension.

Phospholipase D (PLD) catalyzes the production of the lipid second messenger phosphatidic acid (PtdOH). PLD expression and/or enzymatic activity are both elevated in a variety of human cancers. Inhibition of PLD enzymatic activity, via genetic or biochemical methods, leads to decreased cancer cell invasion and decreased cancer cell survival. The aforementioned evidence provided the impetus for a medicinal chemistry project focused on the development of isoform-selective PLD inhibitors. A group from Novartis published a report in 2007 disclosing halopemide as a hit from a high throughput screen for PLD inhibitors. While the iterative analog synthesis campaign described in this dissertation began with halopemide a broad chemical space was explored through the synthesis of several hundred compounds. This effort yielded the most potent, isoform-selective PLD inhibitors ever described. Halopemide inhibits both PLD isoforms relatively equally; VU0359595 inhibits PLD1 1,700 times more potently than PLD2, and VU0364739 inhibits PLD2 75 times more potently than PLD1. An aryl(trifluoromethyl)diazirine containing photoprobe based on the structure of VU0359595 was prepared and was found to inhibit a truncated form of PLD missing the first 311 amino acids, PLD1c.d311. Subsequently, the ability of the probe to covalently modify PLD1c.cd311 was verified. Tandem mass spectrometry experiments to identify the covalently labeled peptide(s) and potentially amino acid(s) are ongoing. In addition to the chemical synthesis of PLD inhibitors and photolabeling experiments some molecular mechanism of action studies were conducted. The VU series of PLD inhibitors block catalysis of both lipososomal and monomeric substrates. Unfortunately, Michealis-Menten kinetics experiments were unable to be performed, because the truncated form of PLD2, PLD2.d308, displays substrate inhibition kinetics in a monomeric substrate containing enzyme activity assay. This research has provided chemical probes that are facilitating the study, both in vitro and in vivo, of how the two mammalian isoforms of PLD affect diverse physiological and pathological processes.