Thesis (M.Pharm.)--University of the Witwatersrand, Faculty of Health Sciences, 2014. / Traditional medicine plays a vital role in the cultural heritage of many South Africans, with at least 80% of the population relying on medicinal plants for their primary source of healthcare. It has been acknowledged that even in some of the finest hospitals in South Africa, people are often found to be using traditional medicine in combination with conventional treatment regimens. Despite the substantial use of medicinal plants in South Africa, limited information is available on the interactive properties between commercially relevant, southern African medicinal plants and conventional drugs. Furthermore, the potential for toxicity of these combinations has been sorely neglected. In orthodox medicine, antimicrobials such as antibiotics and antifungals are amongst the most commonly prescribed group of drugs. Therefore, there is a high probability for the concurrent use of these two forms of healthcare. The aim of this study was to evaluate the interactive antimicrobial and toxicity profiles, when seven conventional antimicrobial agents (amphotericin B, ciprofloxacin, erythromycin, gentamicin, nystatin, penicillin G and tetracycline) were combined with the essential oils, aqueous and organic extracts of seven medicinal plants (Agathosma betulina, Aloe ferox, Artemisia afra, Aspalathus linearis, Lippia javanica, Pelargonium sidoides and Sutherlandia frutescens). The antimicrobial activity of the plant samples and conventional antimicrobials were evaluated, alone and in combination, using the minimum inhibitory concentration (MIC) assay against two yeasts, three Gram-positive and three Gram-negative bacteria. The combinations were further evaluated using the fractional inhibitory concentration (ΣFIC) assessment. Combinations demonstrating notable synergistic or antagonistic interactions were studied in various ratios (isobolograms). Toxicity of the antimicrobials and plant samples were assessed, individually and in combination, using the brine-shrimp lethality assay (BSLA) and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay on human kidney epithelial cells (Graham or HEK-293 cell line). A total of 476 combinations were assessed for interactive antimicrobial potential. Of these combinations, 14.29% were synergistic, 7.56% antagonistic, 35.71% additive and 42.44% indifferent in nature. Some notable interactions were identified, such as the combination of A. linearis (aqueous and organic extract) with penicillin G, where a synergistic profile was most often seen against the three tested Gram-positive micro-organisms (Staphylococcus aureus, Bacillus cereus and Enterococcus faecalis), with ΣFIC values ranging from 0.01 (synergistic) to 0.94 (additive). Further notable interactions included A. betulina and S. frutescens, when combined with ciprofloxacin and tested against E. coli, which demonstrated a favourable synergistic profile, and could be of importance in the treatment of urinary tract infections. In the BSLA, the notable interactions that were tested demonstrated no toxic effect. In the MTT cellular viability assay, the only combination demonstrating possible toxicity was that of A. linearis (aqueous and organic extract) in combination with nystatin (inhibitory effects of 73.76 ± 3.36% and 56.88 ± 6.61%, respectively). Therefore, concurrent use should be cautioned and further in vivo studies warranted. In conclusion, most combinations were found to be non-interactive, alleviating some of the concern related to the concurrent use of the two forms of healthcare. However, some notable combinations were identified, which could possibly have an impact on conventional treatment regimens. Therefore, further in vivo testing is warranted to support the in vitro findings.

The high prevalence of metabolic syndrome in children is mainly due to the adoption of an unhealthy diet and a sedentary lifestyle. The disorder is also associated with obesity, insulin resistance, hypertension and increased blood levels of triglycerides, free fatty acids and glucose. Although metabolic syndrome and its metabolic complications can be managed by the use of conventional medicines such as fenofibrate and metformin, there is a growing use of plants with medicinal properties. Aloe vera has been used for several medicinal purposes such as wound and burn healing, treatment of diabetes and treatment of cancer. Aloe vera also has biological activities that include anti-inflammatory, anti-microbial and laxative effects. Previous studies exploring the metabolic effects of Aloe vera have been in adult animals where metabolic syndrome was induced by the use of pharmacological agents. However, its beneficial effects in growing children who are at risk of developing metabolic syndrome have not been fully explored. The objectives of this study were to evaluate the effects of a crude Aloe vera leaf extract on circulating metabolic substrates, the morphometry and morphology of the gastrointestinal tract and the liver function of growing rats. Fifty-nine male Sprague-Dawley rats of 21 days old were randomly divided into one of six treatment groups. Group I (control) was fed normal rat chow (NRC) with plain gelatine cubes (vehicle). Group II received a high carbohydrate diet (HCD) with plain gelatine cubes; Group III received normal rat chow and fenofibrate at 100mg.kg-1; Group IV received a HCD and fenofibrate at 100mg.kg-1; Group V received a normal rat chow and Aloe vera at 300mg.kg-1; Group VI received a HCD and Aloe vera at 300mg.kg-1. The Aloe vera and fenofibrate were suspended in gelatine cubes and administered daily. After 20 weeks of feeding, the rats were fasted over night and an oral glucose tolerance test (OGTT) was performed. The rats were then euthanazed after 48 hours of re-feeding and tissues were collectedfor further analysis. The data was expressed as mean ± SEM and analyzed by a one-way ANOVA. A repeated measures ANOVA was used for statistical analysis of the data from the oral glucose tolerance test. The values were considered statistically significant when p < 0.05 followed by a Bonferroni Post hoc test. After 20 weeks, the growing rats fed a high carbohydrate diet had a significantly higher body mass than the other groups (p < 0.05, ANOVA), however the administration of fenofibrate prevented the high carbohydrate-induced increase in body mass whilst Aloe vera was not effective. Linear growth as measured by the tibial length was not significantly different between the groups (p >0.05, ANOVA). There was no significant difference in the mass and relative density of the tibia bones of the rats between the groups. Feeding rats a HCD resulted in a higher (p< 0.05, ANOVA) visceral fat mass in the rats. Fenofibrate administration prevented the HCD-induced visceral fat mass gain whilst Aloe vera administration had no effect. Whilst the treatments did not result in any significant differences in the lengths and mass of the small intestine, the mass of the large intestine was significantly lower in the rats that received the HCD alone (p < 0.05, ANOVA). Fenofibrate administration resulted in a significantly increased liver mass compared to the other groups (p < 0.05, ANOVA). However there was no significant difference in the lipid and glycogen content in the liver. Fasting concentrations of metabolic substrates (glucose, triglycerides and free fatty acids) were not significantly different between the groups and no significant differences were observed in the circulating concentrations of insulin and the homeostasis model assessment of insulin resistance (HOMA-IR) (ANOVA; p>0.05). The OGTT did not show any abnormalities in the ability of the rats to handle a glucose load between the groups. An indirect assessment of liver function was performed by measurements of the blood concentrations of alkaline phosphatase (ALP), total bilirubin (TBIL), alkaline transaminase (ALT) and gamma-glutamyl transferase (GGT). There were significantly increased (p<0.05) ALP levels in rats fed NRC + FENO compared to the others. TBIL levels were significantly lower in rats fed a NRC + Av (p< 0.05, ANOVA) however the TBIL levels were within the normal range. The alkaline transaminase (ALT) levels were not significantly different between the groups (p < 0.05, ANOVA) and GGT was not detectable in any of the groups. Weaning rats onto a high carbohydrate diet and feeding them the diet for 20 weeks resulted in the development of visceral obesity without altering the glucose tolerance and metabolic substrates. The treatment with fenofibrate prevented the high carbohydrate diet-induced visceral adiposity however compared to fenofibrate, treatment with the Aloe vera leaf preparation was not significantly effective.

Thesis (M.Pharm.)--University of the Witwatersrand, Faculty of Health Sciences, 2013. / Essential oils are not only used singularly but have been used in combination for many years. There is, however, very little scientific evidence to support the claims made for combined antimicrobial efficacy. With this in mind, a study was designed to investigate the antimicrobial activity of Lavender (Lavandula angustifolia) essential oil, in combination with other essential oils with antimicrobial relevance. The micro-dilution minimum inhibitory concentration (MIC) assay was undertaken, whereby the fractional inhibitory concentration (ƩFIC) was calculated for 54 oil combinations. When lavender oil was assayed in 1:1 ratios with other oils, synergistic (23.5%), additive (52.5%), non-interactive (23.5%) and antagonistic (0.5%) interactions were observed. Four 1:1 combinations were synergistic against Candida albicans and Staphylococcus aureus (Lavandula angustifolia in combination with Daucus carota (ΣFIC 0.50 and 0.50); Juniperus virginiana (ΣFIC 0.50 and 0.50); Cinnamomum zeylanicum (ΣFIC 0.40 and 0.50) and Citrus sinensis (ΣFIC 0.42 and 0.38)). In order to understand the antimicrobial potential of these synergistic essential oil combinations, further antimicrobial analysis was undertaken whereby the oils were placed in varying ratios. Two of the four combinations (Lavandula angustifolia in combination with either Cinnamomum zeylanicum or Citrus sinensis), were identified as the most promising, demonstrating synergy at varying ratios, and thus the major chemical constituents of the essential oils were investigated further. The major chemical constituents identified in Lavandula angustifolia (GC-MS) were linalyl acetate (36.7%), linalool (31.4%) and terpinen-4-ol (14.9%). The GC-MS profiles for all other oils in the study were also confirmed. The major chemical constituents of the most promising essential oil combinations were investigated in equal and varying ratios to determine the effect of chemistry on antimicrobial outcome. When one of the major essential oil constituents (linalyl acetate) of Lavandula angustifolia was combined with limonene found in Citrus sinensis, synergistic interactions were noted for all nine combinations against C. albicans; including the ratio at which the two major constituents would be mixed should the two oils be combined. Lavandula angustifolia essential oil was placed in combination with four conventional antimicrobial agents (ciprofloxacin, chloramphenicol, fusidic acid and nystatin) to determine which of these agents in combination with Lavandula angustifolia would demonstrate the best antimicrobial effect. Synergy was determined for Lavandula angustifolia in combination with ciprofloxacin against S. aureus (ΣFIC of 0.49) and Lavandula angustifolia in combination with chloramphenicol against P. aeruginosa (ΣFIC of 0.29). No antagonism was noted for the combinations investigated. When placed in variable ratios it was identified that Lavandula angustifolia provided the pivotal role in the synergistic interactions observed against C. albicans and S. aureus, with ratios higher in Lavandula angustifolia essential oil concentration showing considerably better antimicrobial effects. In order to determine the antimicrobial effects of Lavandula angustifolia in triple essential oil combinations, the method of MODDE® Design of Experiments was employed. The Design of Experiments (MODDE 9.1®) software identified that Lavandula angustifolia (from the combination of Lavandula angustifolia: Citrus sinensis: Cedrus atlantica) and Thymus vulgaris (from the combination of Lavandula angustifolia: Daucus carota: Thymus vulgaris) were the essential oils with the greater antimicrobial effect in the combinations analysed. Lavender remains one of the most sought after essential oils. This comprehensive study on the antimicrobial effects of Lavender (Lavandula angustifolia) combinations demonstrates promising in vitro effects and lends some credibility for combined use in aromatherapy for the treatment of infections

Dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Master of Science in Medicine. Johannesburg, 2012 / Oral candidiasis is an infection prevalent in immunocompromised individuals. The main causative agent is Candida albicans. Many antifungal agents are available and are effectively used. However, due to the development of drug resistance, toxicity and poor solubility resulting in poor absorption; medicinal plants have been investigated. Dodonaea viscosa var. angustifolia, an indigenous South African plant has shown to have an antifungal effect including inhibition of adherence of C. albicans to oral epithelial cells; which is the crucial first step of infection. This study investigated the effect of the crude extract on the ultrastructure of C. albicans cell wall, which might be responsible for the reduced adherence to oral epithelial cells. The effect of the plant extract on C. albicans germ tube and biofilm formation was also studied since biofilm structure allows for high resistance to antifungal agents and host defense mechanisms. Crude plant extracts were prepared using dried leaves and acetone. Three C. albicans strains were used throughout the study. Minimal fungicidal concentrations of plant extract were determined using a microdilution technique. Three subinhibitory concentrations 3.125, 1.562 and 0.781 mg/ml were selected for further studies. The effect of these subinhibitory concentrations of plant extract on the C. albicans cell wall structure, cell membrane, germ tube formation, biofilm formation and cell wall proteins were studied using transmission electron microscopy, light microscopy, scanning electron microscopy and SDS-PAGE respectively. v The subinhibitory concentrations of crude plant extract rendered C. albicans cell wall thinner and at some places caused cell wall breakage and disruption. This effect increased with a decrease in plant extract concentration. The cell membrane was also damaged by the plant extract showing increased undulation. This effect was not concentration dependent. The subinhibitory concentrations decreased C. albicans germ tube formation and the effect increased with an increase in concentration. Biofilm formation was reduced by the plant extract and in addition, hyphal formation by cells within the biofilm was also reduced. However, SDS-PAGE showed that on a molecular level, the plant extract did not remove any specific adhesin proteins from the cell wall. The crude plant extract of D. viscosa var. angustifolia at high concentrations, kills C. albicans and at low concentrations, renders the surviving cells avirulent. Therefore it has the potential to be developed into an effective therapeutic agent to treat and prevent oral candidiasis. However, further research is required to identify the mode of action of the extract, the specific chemicals responsible for the effect, and the cytotoxicity.

Further mechanistic studies demonstrated that both Radix Rubiae and realgar were capable of inducing cellular apoptosis on HaCaT cells in a dose- and time-dependent manner as shown by morphological inspection, DNA fragmentation, TUNEL assay, cell cycle analysis, annexin V---PI staining and Western blot analysis. HPLC fingerprintings were constructed for quality control of the Radix Rubiae extract using mollugin as the chemical marker. Further phytochemical study found that ethyl acetate fraction of this herb possessed potent growth inhibition on HaCaT cells, with IC50 of 0.9 microg/ml. However, the chemical compounds obtained from commercial sources including mollugin, alizarin, purpurin, and quinizarin failed to induce growth inhibition. Meanwhile, arsenic trioxide, arsenic pentoxide and arsenic iodide, three arsenic salts presented in realgar, had significant anti-proliferative effect on HaCaT cells, with IC50 values of 2.4, 16 and 6.8 microM, respectively; and cellular apoptosis was found to be the underlying mechanism for the observed growth inhibitory activity. Furthermore, Radix Rubiae, realgar and arsenic compounds were also revealed to possess growth inhibition when evaluated in a PHA-activated PBMC model, and all of the substances except arsenic pentoxide significantly attenuated the release of inflammatory cytokines such as IFN-y, TNF-alpha and IL-2 in PBMC, indicating an anti-inflammatory effect. The in vivo mouse tail model experiments demonstrated that arsenic trioxide, arsenic pentoxide and arsenic iodide were able to markedly induce mouse tail keratinocyte differentiation, while such differentiation-modulating effect observed in the fraction of Radix Rubiae was only marginal. / In summary, Radix Rubiae and realgar extracts and three arsenic compounds have been identified and characterized as potential anti-psoriatic agents. The discoveries from the present PhD project not only help put the traditional use of these medicinal substances for psoriasis treatment on a scientific footing, but also open up new opportunities for their development into novel anti-psoriatic therapies. / Psoriasis, a chronic inflammatory skin disorder affecting approximately 2-3% of the population worldwide, is characterized histologically by hyperproliferation and aberrant differentiation of epidermal keratinocytes. Many conventional therapies are offered for psoriasis treatment but there exist problems such as unsatisfactory efficacy, side effects and drug resistance. Many patients therefore turn to alternative and complementary medicines for help. Traditionally, Chinese herbal medicine has been extensively used to treat psoriasis and produced promising clinical results. The present PhD study was conducted to investigate psoriasis-treating Chinese herbal medicines with an aim to identify effective anti-psoriatic agents. Sixty Chinese medicinal materials were selected for the screening project based on their ethnomedical use in psoriasis. The ethanolic extracts of these medicinal substances were evaluated for their anti-proliferative action on cultured HaCaT human keratinocytes using microplate SRB and MTT assays. Among them, the root of Rubia cordifolia L. (Radix Rubiae) and realgar were found to have significant anti-proliferative effects, with IC50 values of 1.4 and 6.6 microg/ml, respectively as measured by MTT assay, while they exerted mild significant cytotoxicity on the human fibroblast Hs-68 cell line. / Tse, Wai Pui. / Advisers: C. T. Che; Z. X. Lin. / Source: Dissertation Abstracts International, Volume: 70-09, Section: B, page: . / Thesis submitted in: October 2008. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 298-340). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

No description available.

Many therapies prescribed by traditional healers in Southern Africa include plant combinations to treat infectious diseases. Artemisia afra is one of the most commonly used traditional medicines in African traditional medicine and most often given in combination with other plants. This plant‟s popularity coupled with its wide range of uses in combination serves as the rationale for the bases of this study. In this study, combinations of A. afra (essential oils and plant extracts), which are commonly used for the treatment of respiratory diseases were studied from an antimicrobial perspective in order to determine if a scientific basis exists for their combined use. The plants used often in double or triple combination with A. afra in the treatment of respiratory tract infections are Lippia javanica, Osmitopsis asteriscoides, Agathosma betulina, Eucalyptus globulus, Allium sativum, Leonotis randii, Tetradenia riparia and Zanthoxylum capense. Essential oils from plant samples were analysed using gas chromatography coupled to mass spectroscopy (GC-MS). Compounds found in highest concentrations were camphor (41.0%) in A. afra, linalool (70.7%) in L. javanica, 1,8-cineole (59.0%) in O. asteriscoides, isomenthone (31.4%) in A. betulina, 1,8-cineole (63.0%) in E. globulus and β-caryophyllene (32.4%) in T. riparia. Dichloromethane: methanol extracts and aqueous extracts were prepared for each plant using the dried ground plant material collected. The antimicrobial activities of each sample as well as each combination (including essential oils) were tested using the minimum inhibitory concentrations (MIC) assay against a panel of respiratory tract organisms. The highest sensitivities observed for the essential oils were that of E. globulus against Cryptococcus neoformans with a MIC value of 0.6 mg/ml. The dichloromethane: methanol extracts showed the most activity with E. globulus against Moraxella catarrhalis (MIC value of 0.01 mg/ml). The aqueous extracts showed the best activity with Z. capense against Streptococus agalactiae with a MIC value of 0.4 mg/ml. The 1:1 fractional inhibitory concentration (ΣFIC) values of the combinations of A. afra with L. javanica, A. afra with O. asteriscoides, A. afra with A. betulina, A. afra with E. globulus and A. afra with Z. capense were calculated from the MIC data. Synergy, additivity, indifference and antagonistic interactions within the combinations were then interpreted. The most significant interactions of the double combinations with synergistic ΣFIC values of 0.2 were the combination of the dichloromethane: methanol extracts of A. afra with O. asteriscoides against Streptococcus pyogenes and the combination of the aqueous extracts of A. afra with E. globulus against Streptococcus pneumoniae. Significant antagonism was noted with the combination of the dichloromethane: methanol extracts of A. afra with E. globulus against Enterococcus faecalis. The ΣFIC results of the combinations of A. afra with L. javanica, O. asteriscoides, A. betulina, E. globulus or with Z. capense were used to calculate ratios and plotted on to an isobologram. The isobolograms were interpreted with regard to any synergy, antagonism, or additive interactions present in the combination. Isobolograms revealed the most significant activity with the combination of the aqueous extracts of A. afra with E. globulus against C. neoformans with all the ratios tested being synergistic. The most prominent antagonism (five ratios) noted was in the combination (dichloromethane: methanol extracts) of A. afra with E. globulus against M. catarrhalis. The triple combinations analyzed for their antimicrobial activity were the combinations of A. afra with O. asteriscoides and E. globulus, A. afra with L. randii and E. globulus, A. afra with A. sativum and Z. capense and the combination of A. afra with T. riparia and salt. The most significant synergistic activity was noted for the combination of the essential oils A. afra with T. riparia and salt against Mycobacterium smegmatis with a ΣFIC value of 0.2. The combination of A. afra with O. asteriscoides and E. globulus of (dichloromethane: methanol extracts) displayed the most antagonistic activity against M. catarrhalis. When analysing the combinations that include A. afra, it was noted that adjuncts are an important combination ingredient in the traditional method of preparation. These were also tested for their activity. The combinations that include adjuncts i.e. honey, salt, vinegar, brandy and milk showed mainly indifferent interactions. This indifference noted supports the use of these adjuncts by traditional healers as it serves to verify that these adjuncts are at least not hindering the activity of the plant itself, which is a positive direction for future investigations. Traditional medicine, with regard to A. afra, as prescribed by traditional healers, has commonly employed the use of combinations of more than one plant to treat respiratory conditions. When the antimicrobial activities in combination were examined from a scientific viewpoint, there is evidence of some bases for their traditional use. The results obtained from the testing of the essential oils validate its traditional use as an inhalant. The dichloromethane: methanol extracts showed results varying from synergy to antagonism while the aqueous extracts showed good antimicrobial activity. It is recommended that future studies should be conducted into these interactions to determine the benefits of these combinations for possible use in the commercial and primary health-care sectors.

The khat plant, catha edulis, has been abused for some time in the Middle East and the African horn for its short-term stimulant effects. However, it was not until 1975 when cathinone, β-ketoamphetamine, was identified as the major stimulant component of khat. Structural analogues of cathinone, synthetic cathinones, are new psychoactive substances available on the clandestine market of numerous countries including the USA. Abuse of these new illicit stimulants is a worldwide growing health concern which necessitates the investigation of the pharmacological properties of these new drugs of abuse. The abuse liabilities of these compounds seem to be related to the three major monoamine transporters (MATs): the dopamine, norepinephrine, and serotonin transporters (DAT, NET, and SERT, respectively). Synthetic cathinones act as either releasing agents by stimulating the release of the presynaptic neuronal content of neurotransmitters, or as reuptake inhibitors by inhibiting normal physiological reuptake of neurotransmitters from the synaptic cleft. Bupropion (DAT/NET reuptake inhibitor) is clinically prescribed for the treatment of depression and smoking cessation, whereas its closely related cousin, cathinone (DAT/NET releasing agent), is a drug of abuse. Deconstruction of bupropion (i.e., a stepwise conversion – or structural transition – of bupropion to cathinone) and investigation of the actions of the deconstructed analogues at the three major MATs showed that the steric bulk at the terminal amine controls the molecular mechanisms of these compounds at MATs (i.e. reuptake inhibition versus substrate-induced release). This study also concluded that bupropion is abused, because it is a cathinone derivative. Methcathinone (MCAT), N-methylcathinone, (DAT/NET releasing agent) is a recreational street drug and a US Schedule I substance; however, new MCAT analogues are continually appearing on the clandestine market to circumvent prosecution under the Controlled Substance Analog Enforcement Act. We investigated the actions and structure-activity relationships of a series of 3-substituted MCAT analogues at MATs and their quantitative structure-activity relationships to determine the physicochemical properties of the 3-position substituents important for the releasing actions of these compounds. This study indicated that the steric bulk of the 3-position substituents controls the selectivity of these compounds at MATs.

Atherosclerotic disease is an almost universal phenomenon and increases in severity and frequency with increasing age. Atherosclerosis may contribute to several disorders including bursting of an artery, blockage of an artery, or induction of arterial clotting. The culmination of these diseases is usually premature since at the time of death the unaffected organs are in reasonably satisfactory condition and could have operated for several more years. Among the many factors which act in concert to produce the disease, serum cholesterol levels play a central role. It has been suggested that the lowering of cholesterol levels will provide an effective means of treatment and prophylaxis of atherosclerosis. The purpose of this investigation was to rationally design, synthesize, and evaluate agents to lower serum cholesterol levels by the inhibition of cholesterol biosynthesis at the site of the reduction of β-hydroxy-β-methylglutaryl coenzyme A (HMG CoA) to mevalonic acid. This reaction, mediated by HMG CoA reductase, was chosen as the inhibition target because it is the first irreversible reaction, the rate limiting step for the pathway, and the site of physiological regulation of cholesterol biosynthesis. Rat liver HMG CoA reductase provided a convenient test system for these agents. Using the previously reported compound, 1-(4-biphenylyl)-n-pentyl hydrogen succinate as parent inhibitor, the present study accomplished two goals: first, a contribution toward elucidation of reversible binding sites for these inhibitors and second, probing of the suggested nonpolar n-pentyl binding area of the enzyme by introduction of a functional group capable of alkylating the enzyme and providing irreversible inhibition. The first objective was approached by replacing the ester of the parent inhibitor with an amide functional group. The resulting glutarimide exhibited inhibition comparable to that of the parent inhibitor. This may be taken as evidence that isosteric replacement of the parent ester group with the amide N-H did not seriously alter the ability of the inhibitor to bind to the enzyme and that an additional binding site in this region is not available. The data also support the suggestion that the ester group of the parent agent is not necessary for binding. A similar inhibition study was made possible by synthesis of 1-(4-biphenylyl)-n-pentyl hydrogen 3-methyl-3-methoxyglutarate. With respect to the corresponding 3-methyl-3-hydroxyglutarate this compound showed an eleven fold decrease of activity. This considerable activity loss indicates that the 3-methoxy group interferes with reversible binding of inhibitor to the enzyme. The inhibition data indicate that the 3-hydroxy group of the 3-methyl-3-hydroxy compound contributes to reversible binding by participating as a hydrogen donor in hydrogen bonding with the enzyme. The major portion of this investigation was designed to probe a region of the enzyme which is nonpolar and binds the n-alkyl moiety of the parent inhibitor. The purpose was to determine the feasibility of incorporating a functional group into this region of the inhibitor which could act as an acceptor for an enzymic nucleophile located in proximity to the reversible binding area. If successful, this could provide irreversible inhibition of the enzyme. A series of compounds was synthesized which bore a terminal alkenyl group two to four carbon atoms removed from the ester moiety. Testing showed that, with respect to the parent inhibitor, no appreciable loss of binding took place. Similarly, a series of epoxyalkyl esters was prepared, the epoxide group being the portion of the inhibitor susceptible to nucleophilic attack. Reversible binding of these compounds was found to be equal to that of the parent inhibitor and it was therefore concluded that the enzyme does accommodate this alkylating group with no loss of reversible binding. This provided the necessary preliminary work upon which subsequent irreversible binding studies will be based.

Neurodegenerative diseases are a class of conditions that lead to progressive atrophy of different parts of the central nervous system (CNS). These diseases lead to devastating clinical outcomes to patients and give rise to an enormous socio-economical burden on society.1 One commonality among some of the most well-known neurodegenerative disorders, e.g. Alzheimer’s disease (AD), Parkinson’s disease (PD), and multiple sclerosis (MS), is neuroinflammation.2-4 Neuroinflammation stems from interactions of the innate immune system with toxins and insults to the central nervous system. In the case of irremovable or chronic insults and toxins, this leads to chronic damaging inflammation that hastens neuronal degeneration and exacerbates disease pathology.5,6 Recently, inflammasomes of the innate immune system have been indicated in playing essential roles in the observed inflammatory responses. The most studied inflammasome is the nod-like receptor pyrin containing 3 (NLRP3) inflammasome.7–9 Recently our research group has successfully developed sulfonamide-based small molecule inhibitors of the NLRP3 inflammasome, such as JC-21 and JC-171, as potential therapeutics for AD and MS. Our studies established that JC-21 is a selective inhibitor of the NLRP3 inflammasome.10,11 Structural modifications led to the development of JC-171 with improved pharmacokinetic properties. More importantly, our studies demonstrated the in vivo activity of JC-171 to effectively ameliorate the experimental autoimmune encephalomyelitis (EAE), a mouse model of MS.12 Our data also strongly suggested that inhibitors based on this chemical scaffold may directly target the NLRP3 inflammasome.10–12 In this dissertation, we conducted biophysical, biochemical, and modeling studies to further elucidate the mechanistic information of these compounds as inhibitors of the NLRP3 inflammasome. In order to conduct further mechanistic studies, the NLRP3 protein was produced via transfection of HEK 293 cells with a modified plasmid of full-length human NLRP3 protein.13 Furthermore, LC-MS studies were conducted to confirm the blood-brain barrier penetration (BBB) of JC-171. Our studies established that JC-171 directly binds to the NLRP3 protein. The results also suggested that JC-171 may bind to the NACHT domain of NLRP3 while in a site that is distinct from the ATP binding site. This notion is supported by the fact that our compounds do not interfere with the ATPase activity of NLRP3. Docking studies of JC-171 to the homology model of the NACHT domain of NLRP3 also supported this assertion by showing the interaction of JC-171 with residues that are not overlapping with the ATP binding pocket. BBB penetration studies in combination with LC-MS analysis confirmed that JC-171 shows better BBB penetration when compared to MCC950. Collectively, our results strongly support that our compounds function as NLRP3 inflammasome inhibitors by directly binding to the NLRP3 protein, a novel and distinct mechanism of action when compared to the known inhibitors that target the NLRP3 inflammasome pathway. These results strongly encourage further development of such inhibitors as potential therapeutics for neurodegenerative diseases.