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Item Open Access Establishing a cisplatin-resistant triple negative breast cancer spheroid model(University of the Free State, 2024) Ngalonkulu, Ongeziwe; Sekhoacha, Mamello; Abrahams, Beynon; Gouws, ChrisnaIn cancer research, conventional two-dimensional (2D) cell cultures and animal studies are often used in studying the disease and efficacy of anticancer agents despite their failure to accurately replicate the complex 𝘪𝘯 𝘷𝘪𝘷𝘰 tumour microenvironment. 2D cultures lack the extracellular matrix production critical for 𝘪𝘯 𝘷𝘪𝘷𝘰-like cell behaviour, thereby jeopardizing data accuracy when developing effective cancer therapies and understanding mechanisms underlying drug resistance and cancer relapse. There is, therefore, a pressing need for models that accurately recapitulate 𝘪𝘯 𝘷𝘪𝘷𝘰 systems and enhance researchers’ accuracy in predicting disease mechanisms. This study aimed to establish a cisplatin-resistant three-dimensional (3D) spheroid model using the scaffold-free technique of rotating wall bioreactors in the CelVivo ClinoStar™ system, to facilitate efficient recapitulation of 𝘪𝘯 𝘷𝘪𝘷𝘰-like conditions and higher throughput. The MDA-MB-231 cell line is classified as triple negative breast cancer (TNBC) due to the absence of the oestrogen receptor and progesterone receptor, as well as the human epidermal growth factor receptor 2. As a result, the MDA-MB-231 cell line is characterized as a highly metastatic and aggressive cell line with a poor prognosis. Cisplatin-resistant MDA-MB-231 cells were generated by exposing the cell line to IC10-IC50 (0.01μM, 0.1μM, 1μM, 5μM and 10μM) concentrations of cisplatin for nine months. The cells acquired resistance; these resistant cells were then used to develop a 3D spheroid model. Characterization of the cisplatin-resistant MDA-MB-231 spheroids over 28 days included assessing spheroid growth and viability, and identifying the optimal experimental window between days 12 and 22. The model's reactivity to cisplatin and doxorubicin treatment was evaluated for 96 hour (h) to qualify the model for treatment screening. Treatment with the clinical cisplatin dose (4,435 x 10⁻⁶ μg cisplatin/μg protein) showed pronounced reactivity after 48 h, with decreased cell viability and increased cell death. However, spheroids recovered after 72-96 h, displaying moderate to high metabolic activity, typical of resistance. The IC₅₀ (8.06 x 10⁻² μg cisplatin/μg protein) dose of cisplatin demonstrated immediate effects on the model after 48 h, however, increase in spheroid size and decrease in cell death was observed with increased metabolic activity after apparent recovery at 72-96 h suggesting resistance. Treatment with doxorubicin clinical dose (1.064 x 10⁻⁵ μg dox/μg protein) had the most pronounced effect on the model throughout the 96 h and resulted in decreased spheroid size. It was apparent that the model was not as resistant to the doxorubicin treatment as it was to the cisplatin treatment. This model has potential for preclinical research studies as it displays reactivity to commonly used chemotherapeutic drugs and could be valuable in understanding drug-resistant cancer. In conclusion, the established cisplatin-resistant MDA-MB-231 3D spheroid model offers a robust platform for studying drug effects in TNBC research.Item Open Access 𝘐𝘯 𝘷𝘪𝘵𝘳𝘰 evaluation of expression levels of ABC-transporter genes on needle biopsies of prostate cancer patients treated with chemotherapy(University of the Free State, 2023) Ngesi, Nandi; Sekhoacha, M. P.; Abrahams, B. R.𝗜𝗻𝘁𝗿𝗼𝗱𝘂𝗰𝘁𝗶𝗼𝗻: Prostate cancer (PCa) is the second-most diagnosed cancer among men worldwide, and the foremost male malignancy in South Africa. Various therapies are used in the treatment of PCa patients. However, despite advances in cancer therapy, treatment failures still lead to the advancement of the disease, relapse, and ultimately mortality. Chemotherapy is the treatment of choice for metastatic PCa, but it is challenged by multidrug resistance (MDR) of cancer cells. Various mechanisms participate in the MDR of cancer cells, including an increase in drug efflux facilitated by members of the ATPbinding cassette (ABC) transporters such as ATP-binding cassette sub-family C (ABCC1) / multidrug resistance-associated protein (MRP1), ABCC2/MRP2, and ABCC10/MRP7. Therefore, discovering drug resistance biomarkers and mechanisms is essential in both understanding and combating chemoresistance. This study identifies cellular drug transporter genes (ABCC1, ABCC2, and ABCC10) as possible targets for the prediction of docetaxel treatment outcomes in PCa patients. 𝗣𝘂𝗿𝗽𝗼𝘀𝗲: The aim of this study was to investigate whether a correlation exists between the expression levels of ABCC1, ABCC2, and ABCC10 transporter genes and good versus (vs) poor responses to chemotherapy. Methods: A total of nine Formalin-Fixed Paraffin-Embedded (FFPE) tissue biopsies of PCa patients were obtained from National Health Laboratory Service, Universitas academic hospital, and divided into two categories, good and poor responders. RNA and proteins were extracted from the FFPE tissues, quantified, and evaluated using quantitative RT-PCR and western blot. 𝗥𝗲𝘀𝘂𝗹𝘁𝘀: quantitative RT-PCR shows that ABCC1/MRP1, ABCC2/MRP2, and ABCC10/MRP7 were expressed in the good and poor responder categories of PCa patients. In the good responder category, 50 % of the patients showed elevated expression levels of ABCC1/MRP1 and ABCC10/MRP7 in the normal sections compared to their tumour sections. The expression level of ABCC2/MRP2 was elevated in 75 % of the patients’ normal sections in comparison to their tumour sections. Moreover, in the poor responder category, 80 % of the patients showed elevated expression of ABCC1 in the tumour sections compared to the normal sections, and 60 % of patients showed elevated expression levels of ABCC10 in the tumour sections compared to their normal sections. The expression levels of ABCC2 in the tumour and normal sections of patients in this category of poor responders, could not be compared because the expression levels of ABCC2 could not be determined in 50 % of the patient samples. The average expression levels of ABCC1 and ABCC10 were upregulated in the tumour sections of patients in the poor responder category but were deregulated in the tumour sections of patients in the good responder category. Additionally, ABCC1, ABCC2, and ABCC10’s average expression levels were upregulated in the normal sections of patients in the good responder category and deregulated in the normal sections of patients in the poor responder category. 𝗖𝗼𝗻𝗰𝗹𝘂𝘀𝗶𝗼𝗻𝘀: The results suggest that a correlation exists between the expression levels of ABCC1, ABCC2, and ABCC10 and drug resistance reported in the poor responder category. Also, the detection of these transporter genes could potentially be used as indicators of possible docetaxel treatment outcomes.Item Open Access The role of CYP3A in nevirapine induced hepatotoxicity(University of the Free State, 2003-11) Barr, Shera; Walubo, A.Nevirapine is a potent non-nucleoside reverse transcriptase inhibitor with favourable pharmacokinetics that are characterised by rapid absorption and distribution with a long elimination half-life. Nevirapine is effective against HIV-1 when used in combination with other anti-retroviral agents and as a monotherapy for the prevention of mother-to-child-transmission. Unfortunately, its adverse effects, mainly hypersensitivity skin reactions and hepatotoxicity, hamper the wide use of nevirapine. Since nevirapine-induced hepatotoxicity commonly occurs between 2 -12 weeks of treatment, and nevirapine is a known inducer of CYP3A isozyme, it was envisaged that the hepatotoxicity was due to activation of nevirapine to toxic metabolites by the induced enzyme. Therefore, the aim of this study was to determine the role of CYP3A in nevirapine-induced hepatotoxicity. Fifteen male SO rats were pre-treated with either dexamethasone (50 mg/kg) or nevirapine (20 mg/kg) for 3 days. On the fourth day, the control group (n=5) was administered with the vehicle, while the 'nevirapine only' group (n = 5) was given an overdose of nevirapine (1340 mg/kg, orally) and the 'ketoconazole plus nevirapine' group (n = 5) was treated with a CYP3A inhibitor (ketoconazole) 1 hour before the overdose of nevirapine was given. The animals were sacrificed 24 hrs later and plasma was sent for liver function tests, the liver was excised for microsomal extraction and histopathology studies. Microsomal CYP3A activity was measured using the erythromycin demethylation test. Results of these animals were compared with results obtained in rats that were not pre-treated with an inducer before the toxic dose of nevirapine was administered. Treatment with dexamethasone or nevirapine lead to increased CYP3A activity. CYP3A activity in the untreated, dexamethasone treated and nevirapine treated rats was 0.59 ± OA8, 10.39 ± 3.59 and 7.28 ± 2.65 nmol/min/mg protein, respectively. In the dexamethasone pre-treated groups, the histopathological findings as well as the elevated liver enzymes of the 'nevirapine only' treated group (AST 239.25 ± 50.7 UIL and ALT ± 386.00 ± 154.3 UIL) were indicative of hepatotoxicity as opposed to the control group (AST 146AO ± 10A UIL and ALT 149.60 ± 39.7 UIL). However, the corresponding group pre-treated with nevirapine did not show significant elevations in liver enzymes (AST 150.20 ± 19.1 UIL and ALT 67.20 ± 8.6 UIL) but the histopathological findings exhibited hepatotoxicity that was similar to the dexamethasone group. Nevirapine-induced hepatotoxicity was not prevented in the groups treated with ketoconazole before the overdose of nevirapine was given. Interestingly, there was no hepatotoxicity when the overdose of nevirapine was administered to animals that were not pre-treated with nevirapine or dexamethasone. In conclusion, nevirapine-induced hepatotoxicity was associated with enzyme induction by dexamethasone or nevirapine, and the use of ketoconazole did not prevent the hepatotoxicity. Therefore, CYP3A may not be involved in the pathogenesis of nevirapine-induced hepatotoxicity, suggesting that a different enzyme may be responsible. As the liver function tests did not correlate well with the histopathological findings in the nevirapine pre-treated groups, it was suggested that liver function tests alone might not be good markers for determining nevirapineinduced hepatotoxicity.Item Open Access The effect of liposomal charge on the distrubution of liposomes to the liver, brain, lungs and kidneys in a rat model(University of the Free State, 2003-01) Abraham, Aju Mary; Walubo, A.English:Gentamicin was selected out of three drugs as the most appropriate liposomal marker based on its properties There after, a simple method for preparation of charged Iiposomes by rotary evaporation and hydration was adopted. Surface charge was induced by varying the lipid composition whereby neutral liposomes were prepared using phosphatidyl choline and cholesterol (9.7:6.9, molar ratio), negative and positive liposomes were prepared by addition of dieetyl phosphate (5: I :0.5, molar ratio) and stearylamine (5: I :0.5, molar ratio) to the neutral liposomes, respectively. The distribution of the encapsulated gentamicin to the specified organs in liposome treated groups was compared to a control group treated with free gentamicin at the following intervals: 1, 2, 4, 6 and 8 hours post injection. Gentamicin (60 mg/kg) free and liposome entrapped was administered intraperitoneally and five rats of each group were utilised at each time interval. Under ether anaesthesia, a blood sample was drawn and the relevant organs were harvested. The sodium hydroxide digestion method was used to extract gentamicin from the organs, and gentamicin in plasma and organ extracts was measured by fluorescence polarisation immunoassay. Liposomal characterisation revealed multilammelar Iiposomes with a mean internal diameter of 3.17 ± 1.9 urn, and encapsulation efficiency greater than 15 %. In the animal studies, liposomes delayed elimination of the encapsulated drug. The half life was 2.02 ± 0.5, 1.76 ± 0.1 and 2.04 ± 0.3 hours for the negative, positive and neutral liposome treated groups, respectively, . versus 1.53 ± 0.02 hours for the control group. Peak plasma gentamicin concentrations were higher with positive liposomes than negative and neutral liposomes at 1 hour, while the negative Iiposomes depicted a sustained release pattern between 4 and 8 hours. Distribution of liposomes to the brain and liver was dependent on liposomal surface charge. Liposomes improved gentamicin concentrations in the brain with positive liposomes highest in this regard. A biphasic pattern of distribution to the brain, with lowest gentamicin concentration at 4 hours was observed in the three liposome groups, and this was more marked in the negative liposome group. Generally, hepatic gentamicin concentrations were higher with liposomes than the control. Although, the average hepatic gentamicin concentrations were highest for positive liposomes, the negative liposomes were preferred for the liver because the concentrations were more consistent and increased with time. Uptake of gentamicin by the lungs was not enhanced by liposomes and was independent of surface charge of the liposomes. Renal concentrations of gentamicin were lower (3 to 5 folds) with liposomes, and uptake was not charge dependent. In conclusion, a simple method for preparation of liposomes was adopted. The distribution studies suggested that positively charged liposomes had highest affinity for the brain and the negative liposomes for the liver. Also, liposomes irrespective of charge exhibited reduced renal concentration of gentamicin.Item Open Access The pharmacokinetic interactions between valproic acid and acyclovir assessed in vitro and in a rabbit model(University of the Free State, 2008-03) Van Jaarsveld, Magdalena Francina Petronella Catharina; Walubo, A.English: Valproic acid is an antiepileptic drug that is widely used for treatment of epilepsy, while acyclovir is an antiviral drug indicated for treatment of infections caused by herpes simplex type I & II and varicella-zoster viruses. Given the high prevalence of people with conditions for which chronic use of valproic acid is indicated, and the notion that valproic acid increases the antiviral activity of acyclovir, it is not uncommon for the two drugs to be used concomitantly. As such, recent reports on the interaction between valproic acid and acyclovir with break through convulsions were a cause for concern. Since understanding the mechanism of this interaction is vital to the establishment of concrete guidelines on the use of the two drugs in patients, the aim of this study was to investigate the possible pharmacokinetic interaction between acyclovir and valproic acid. First, a high performance liquid chromatography (HPlC) method for analysis of acyclovir in plasma was developed. It involved simple protein precipitation of 200 IJl of plasma with perchloric acid, followed by centrifugation after which 20 IJl of the supernatant was injected in the HPlC. The sample was eluted with acetonitrile: octanesulfonic acid: ammonium acetate-citrate (vol.lvol.; 5%:11.88%:83.12%) at 1.5 ml/min over a luna C18 (4.60 x 150 mm) 51Janalytical column. Gancyclovir was used as the internal standard. Under these conditions, gancyclovir eluted at 3.4 min and acyclovir at 4.5 min. Over the calibration range of 10 - 100 IJg/ml, linearity was demonstrated by a linear regression equation of y = 0.03196 - 3.207x with a regression coefficient r² = 0.995, and accuracy by a percentage coefficient of variation (CV%) of less than 15%. The method was successfully used to analyze acyclovir in a rabbit treated with acyclovir single dose. Thereafter, the possibility of a direct interaction between acyclovir and valproic acid in vitro was investigated by monitoring the concentrations of valproic acid and acyclovir at different pH (pH 7.4 or pH 3 or pH 10) and temperatures (25°C and 37°C) when mixed in a 1:1 molar ratio or prepared separately in phosphate buffer. The samples were incubated at 25°C for 2 hours and a further 1 hour at 370C, and aliquots were drawn at 10 min., 2 and 3 hours to measure the concentration of valproic acid and acyclovir (n=3). The average concentrations of valproic acid and acyclovir from the samples containing the single drug were not different (P > 0.05) from those in the mixture of both drugs at the different temperatures and pH. However, when the temperature and pH were evaluated separately, there was a trend whereby, at high temperature (37°C), the concentrations of acyclovir (percentage detected) tended to be higher in the mixture (87%) than when it was alone (84%), while those of valproic acid tended to be lower in the mixture (89%) than when it was alone (92%). This same trend was observed at acid or alkaline pH. In conclusion, although temperature and pH did not induce significant effects on the concentrations of both acyclovir and valproic acid, increased concentrations of acyclovir were associated with reduced concentration of valproic acid when the two drugs were mixed under constrained conditions. These observations suggested a possible direct interaction between the two drugs This final part of the study was undertaken to investigate the effect of coadministration of valproic acid and acyclovir on the pharmacokinetic parameters of each other in a rabbit model. Fifteen white New Zealand rabbits were divided into 3 groups A, Band C whereby group A received acyclovir only, group B received valproic acid only, and group C received a combination of acyclovir and valproic acid. In a cross-over design, the intravenous route was studied first, followed by the oral route after a two-week wash out period. Blood samples were drawn over a 10 hr period and the pharmacokinetic parameters were derived from the concentrations. After intravenous administration, the area under the plasma concentration time curve (AUC) and plasma concentrations of acyciovir in group C were higher than in group A, while the volume of distribution (Vd) and plasma clearance (CLp) of acyciovir in group C were only 12.8% and 10.36% of those of group A, respectively. A similar trend was observed after oral administration. However, the bioavailability (F) of acyclovir was 8.4% in group A versus 1.5% in group C. Of note, the concentrations and kinetic parameters of valproic acid between the two groups after oral and intravenous administration were not different. In conclusion, co-administration of single doses of acyclovir and valproic acid led to reduced oral bioavailability of acyclovir, but increased concentrations of acyclovir due to reduced volume of distribution and clearance and this was most probably due to inhibition of the membrane transport proteins for acyclovir by valproic acid. Overall, a simple and accurate HPLC method for analysis of acyclovir in plasma was successfully developed, and a possibility of direct interaction between the two drugs was observed both in vitro and in vivo. These observations call for a cautious approach to the concomitant use of the two drugs until human studies are done.Item Open Access Development and validation of assay methods for the quantitative determination of drugs and their metabolites in biological specimens (piroxicam and nabumetone)(University of the Free State, 2001-05) De Jager, Andrew David; Hundt, H. K. L.; Swart, K. J.English: The development and validation of bioanalytical assay methods suitable for the quantitation of drugs in biological matrices is discussed, as well as general principles applicable to this particular aspect of drug development. Relevant literature is consulted, with a view to exemplifying what constitutes good assay method development strategy, as well as to reflect current international policy in this field, with particular reference to bioequivalence studies. Comparisons are made between international practices and those in place at FARMOVSPAREXEL Bioanalytical Services Division®. Attention is given inter alia to detector selection, chromatographic optimisation, extraction procedures and method validation, with reference to new assay methods for two drugs in particular that have been developed and validated according internationally acceptable standards. In the first instance, a highperformance liquid chromatographic method with ultraviolet detection was developed for the determination of 6-methoxy-2-naphthylacetic acid (6-MNA, the active metabolite of nabumetone ). The sample preparation involved a simple but effective protein precipitation procedure. Reversed-phase liquid chromatography was optimised, and full resolution between the analyte and endogenous matrix peaks achieved in a chromatographic runtime of five minutes. The assay method was validated over a range of plasma concentrations between 0.070 and 145 µg/ml. 1242 Plasma samples generated during a comparative bioequivalence study were then assayed and the performance of the assay method shown to be well within accepted international norms. The coefficient of variation for quality control standards over the range of 0.18 to 39 µg/ml processed during the assaying of the study samples, varied between 3.6 and 8.1 %. In the second instance, a novel method for the determination of piroxicam in four biological matrices (sub-cutaneous tissue (SCT), synovial fluid (SF), synovial capsule (SC) and plasma) was developed. A double back-extraction procedure was followed by reversed-phase liquid chromatography and electrochemical detection (ECD). Extracts from all four biological matrices were injected onto a single HPLC system. Ratios between plasma and the three remaining matrices were used to characterise transdermal absorption of two topical preparations of piroxicam when applied to the knee. Low systemic levels associated with topical formulations necessitated the development and validation of a highly sensitive assay method. Plasma was used as a surrogate matrix for all the processed tissue samples and the assay method was validated over a range of plasma concentrations between 1.24 and 600 ng/ml. 168 Samples generated during a multi-centre study involving knee replacement surgery, were assayed and the performance of the assay method shown to be well within accepted international norms. The coefficient of variation for quality control standards over the range of 1.74 to 300 ng/ml processed during the assaying of the study samples, varied between 7.7 and 13.5 % which can be considered excellent in the light of the complexity of the sample preparation process. Analytical data generated during the above-mentioned two research projects are discussed, with novelties and improvements to existing assay methods being elucidated. Both assay methods were presented and accepted for publication in peer reviewed scientific journals. Both full-length publications are included in an appendix in this dissertation, together with the correspondence entered into with journal editors and referees. Furthermore, a section containing copies of the slides used to present the latter HPLC assay method as an oral presentation at the 1998 Annual Congress of the South African Pharmacological Society, is included.Item Open Access Clozapine: the correlation between clinical improvement and laboratory parameters(University of the Free State, 1977) Gosling, John Albert; Muller, F. O.English: In this study, fifteen Black patients suffering from acute schizophrenia were treated with clozapine for a period of 40 days in order to ascertain whether certain laboratory parameters could be utilized to give an indication of the clinical efficacy of clozapine treatment in these patients. S.l.l Therapeutic efficacy Utilizing the B.P.R.S. and F.C. rating scale as indication of the clinical improvement of the patients, it was found that significant clinical improvement occurred upto day 30 whereafter clinical improvement was only slight. Clozapine was well tolerated by all the patients while more than half the patients (53,3%) were fit for unmndhional discharge on completion .of the study. On completion of the study the working capacity of the majority (80%) was satisfactory. 5.1.2 Side effects The most common side effects encountered were daytime sedation which was especially prominent during the early stages of the study and hypersalivation which occurred with equal frequency throughout the study period. Other side effects encountered in descending order of frequency were nausea and vomiting, dizziness, headache, disturbance of visual accomodation, constipation and diarrhoea, disturbed sleep, sweating, inhibition of micturition, and collapse. 5.1.3 Blood pressure and pulse rate Clozapine had no significant' prolongated effect on blood pressure while a significant and sustained rise in pulse rate during the treatment period was noted. It is suggested that this rise in pulse rate could be utilized as a convenient clinical aid in checking patient compliance in patients being treated with clozapine. 5.1.4 Serum concentration of clozapine, clozapine plus metabolites, and metabolites only. 5.1.4.1 No correlation was found between serum levels of clozapine, clozapine plus its metabolites, or its metabolites only and clinical improvement. 5.1.4.2 It was found that on treatment day 5 steady state serum levels of clozapine and of clozapine plus it metabolites had been reached. 5.1.4.3 No auto-induction of the metabolism of clozapine appeared to occur during the treatment period. 5.1.4.4 No accumulation of clozapine or its metabolites appeared to occur during the treatment period. 5.1.4.5 It can be concluded that a certain period of exposure to a more or less constant serum level of clozapine and/or its metabolites is necessary to effect clinical improvement. 5.1.4.6 A significant correlation was found between the lying pulse rate and serum levels of clozapine plus its metabolites. The lying pulse rate can thus offer a reasonable indication of the expected serum levels of clozapine plus its metabolites. 5.1.5 Prolactin serum levels No rise in serum prolactin levels occurred in these patients after institution of treatment with clozapine. Therefore no correlation between clinical improvement and prolactin serum levels could be ascertained. 5.1.6 5-hydroxytryptamine-induced platelet aggregation No enhancement of 5-HT-induced platelet aggregation could be determined in these patients undergoing treatment with clozapine. Therefore no correlation could be established between clinical improvement and enhancement of 5-HT-induced platelet aggregation. 5.1.7 Plasma cholinesterase and red blood cell ace~- cholinesterase activitX' Both the plasma cholinesterase and red blood cell acetylcholinesterase activity fell within the normal range prior to the institution of treatment with clozapine. These parameters can therefore not be used as diagnostic aids in the diagnosis of schizophrenia. The activity of both parameters also fell within the normal range on conclusion of the study. It would thus appear that treatment with clozapine did not significantly affect these·.parameters.Item Open Access Physical activity and lifestyle aspects of female students at a tertiary institution(University of the Free State, 2013-07) Losper, T'Neil Sarelle; Opperman, M. M.; Coetzee, F. F.; Bloemhoff, H. J.English: BACKGROUND AND RATIONALE: It is generally believed that a sharp rise in chronic diseases and unhealthy living has occurred. Researchers believe that the modern lifestyle and a lack in physical activity (PA) are the main reasons for this problem (McGinnis, 1992:S196). Chronic diseases and obesity are factors that can be prevented or reduced with physical activity and a healthy way of living. The way in which physical activity can have an indirect influence on conserving health can be explained in two ways: Firstly physical activity can be used as trigger mechanism to change other destructive lifestyle habits (Weinstein, 1987:8; Eddy & Beltz, 1989: 168). Secondly, participation in PA can have an indirect effect on the reduction of coronary diseases because of its reducing effect on depression, anxiety and tension, to name a few (Willis & Campbell, 1992:47). According to Bray and Born, (2004:181) there is an increasing need for physical activity among young adults. Young adults attending universities gain increased control over their lifestyles. However, they may not necessarily develop positive behaviors like regular PA. The lifestyle that students live is questionable. Whether their activity levels are adequate and whether they generally lead to healthy lifestyles is unknown as little research is available on this matter, especially in South Africa. Keating, Guan, Pinero and Bridges (2005:116) stated that it is well known that students' PA as a research topic has been seriously neglected. Young adulthood is considered to be an important phase of life, as many lifelong health behaviour patterns are established during this phase (Timperio, Salmon & Ball, 2004:20). OBJECTIVES: The purpose of the study is twofold: 1. To identify PA levels of undergraduate female students indifferent ethnic groups on a South African university campus, and 2. To establish the lifestyle profile and body composition of female students in different ethnic groups in a South African university campus. RESEARCH METHODS: The sample constituted of female students at the University of the Free State in their 1st, 2nd and 3rd year+ of study residing on the campus. The sample consisted of 244 students (78 1st years, 98 2nd years, 68 3rd years-: 139 black, 21 coloured and 84 white students). The following three research instruments were used: • International Physical Activity Questionnaire (IPAQ) (2012) • Belloc and Breslow's 7 lifestyle habits questionnaire • The Heath and Carter anthropometrical assessment. RESULTS AND DISCUSSION: By comparing the 1st, 2nd and 3rd year groups it is evident that 40.16% of the group as a whole (all ethnic groups) did take part in some form of physical activity. Fifty five point one percent (55.13%) of 1st year female students, 42.86% of the 2nd year and 44.12% of the 3rd year female students participated in PA. The White female students had the highest physical activity participation rate (67.86%), followed by the coloured students (38.10%). The black students' physical activity participation (35.97%) was the lowest. An average of 4 out of the 7 lifestyle habits being followed by the majority of the participants. The majority of participants eat breakfast daily (51.64%) but they do not eat 3 meals per day. Eighty seven present (87.70%) of the sample are nonsmokers, with 77.05% of the respondents consuming little to no alcohol, and at least 66.80% of the group maintains a healthy body weight. Unfortunately their eating, sleeping and exercise habits are not optimal. It is evident that the lifestyle habits of the students decrease from the 1st to the s= year, but that by the time they progress to the 3rd year-, they start trying to change their lifestyles habits to a certain extent. The ethnic groups do not show a significant difference among their lifestyle habits but white female students do have a more positive profile.Item Open Access Identification and confirmation of the presence of some steroid-like growth promoters in the urine of cattle and swine(University of the Free State, 1998-11) Pieterse, Jacobus Wilhelmus; Van der Merwe, P. J.English: Anabolic steroids and/or growth promoters are used to improve growth rate and feed conversion efficiency of livestock. The residues of these anabolics, which are present in the meat, may have a pharmacological activity due to oral bioavailability, and pose a risk to the consumer. Certain anabolics can be given legally to farm animals in some countries, but are banned in most others because of their proved or alleged toxic and/or carcinogenic properties. The use of these substances is completely forbidden within the European Community (EC). Before meat products can be exported to any member state of the EC, it is compulsory for the exporting country to have a monitoring programme to test for illegal use of these anabolic substances. The necessity to test for illegal use or to determine residue levels after legal use, has led to a strong interest in developing analytical methods for the identification and confirmation of anabolic agents in biological samples. The objectives of this study were to develop suitable analytical methods with a view to identify residues of some growth-promoting veterinary drugs in the urine of cattle and/or swine, to confirm the presence of these veterinary drugs unequivocally in the urine and to examine the stability of these drugs in urine under different environmental storing conditions. The excretion of clenbuterol, diethylstilbestrol, nandrolone, trenbolone and zeranol from cattle and/or swine were studied. A thorough literature study was done on the published analytical methods as well as the metabolism and pharmacokinetics of these drugs in cattle and/or swine. Reference standards were used to develop a GC-MS screening method for the identification of these drugs and/or their metabolites in the urine of cattle and/or swine. Recoveries of 61-99% and detection limits of 0.9-2.1 ng/ml were obtained for the different analytes with the developed analytical method. Trials were conducted in which these drugs were administered to cattle and/or swine. Urine samples were collected at regular time intervals and stored immediately at -20°C until time of analysis. Reference standards were also used for the development of GC-MS-MS analytical methods to confirm the presence of these drugs and/or their metabolites in the urine of cattle and/or swine. Confirmation of the substances in urine was done by obtaining a MS-MS spectrum of the extract and comparing this with the MS-MS spectrum of a reference standard. The MS-MS spectra was obtained by using the ion-trap technique. Urine samples are often collected at different farms and transported to the laboratory for residue analysis. Although it is standard procedure to freeze samples immediately after collection, it is not always possible. The results of this study show that the analytes contained in urine samples that were stored frozen remained stable for at least 10 days. If urine samples are stored at ambient temperature, concentrations of the analytes can decrease with as much as 30% after 10 days. Epi-nandrolone (metabolite ofnandrolone in cattle) could however not be detected in the urine samples after 4 days. If urine samples are stored in direct sunlight, concentrations of the analytes can decrease with as much as 90% after 10 days. It can be concluded that a method was developed to identify residues of some growthpromoting veterinary drugs and/or their metabolites in the urine of cattle and/or swine, and to confirm the presence of these drugs unequivocally in the urine. It can further be concluded that urine samples should be frozen as soon as possible after collection to prevent false negative results.Item Open Access Hypothalamic-pituitary-adrenal axis function and hypothalamic-pituitary-thyroid axis function in mentally retarded oatients with and without self-injurious and/or aggressive behaviour(University of the Free State, 2003-12) Van Zyl, Paulina Maria; Gagiano, C. A.; Walubo, A.; Bester, C. J.English: The etiology of aggression and self-injuring behaviour in low functioning mentally retarded patients is multi-factorial and may reflect the presence of undiagnosed psychiatric conditions, unapparent due to the degree of the patient's impairment. It may also reflect hyperactivity of the stress response. The intricacies of diagnosis in this group of patients call for the development of biological markers to aid in diagnosis, therapy selection and drug response monitoring. Measuring and determining the relative contribution of individual neurotransmitters in the problem behaviour is complex and impractical. An alternative route may be to evaluate the functions of the hypothalamic-pituitary axis, which has extensive connections with the limbic area and is relatively easy to assess. The hypothalamic-pituitary system controls the behavioural, endocrine, autonomic and immunological responses to stress. The dexamethasone suppression test (OST) as adapted by Carroll and the thyroid-releasing hormone stimulation test (TRHST) has been extensively used in research on biological markers in major depression. Stress is known to activate the hypothalamic-pituitary-adrenal (HPA) axis, reflected by elevated cortisol levels. The study is a matched control study comparing hypothalamic-pituitary-adrenal axis function and hypothalamic-pituitary-thyroid axis function in 44 institutionalised mentally retarded patients with and without self-injuring and aggressive behaviour through the measurement of baseline cortisol levels and the application of the dexamethasone suppression test and the thyroid-releasing hormone stimulation test. The groups were matched according to gender, age and level of functioning. The mean age of the aggressive group 106 was 44,1 years (±SO 9,8) and the mean age of the non-aggressive group was 44,2 years (±SO 10,5). Baseline hypercortisolaemia occurred in five of the 22 aggressive subjects (22,7 %) and in two of the 22 non-aggressive subjects (9,1 %). Cortisol nonsuppression with the OST occurred in two subjects in the aggressive group (9,1 %) and one subject in the non-aggressive group (4,5 %). The OST did not demonstrate a difference in the two groups, yet there were more individuals in the aggressive group with abnormal high baseline cortisol, as well as a tendency towards a higher baseline cortisol in the aggressive group, suggesting an abnormal or more reactive stress response. Higher baseline cortisol levels were not related to age or the type of aggression, yet subjects with more recent aggressive activity showed higher baseline cortisol levels. The TRHST was generally well tolerated by the subjects. Side effects were few and transient. There were two male subjects in the aggressive group showing a blunted TRHST. Primary hypothyroidism was demonstrated in one of the female subjects in the non-aggressive group and subclinical hypothyroidism in two subjects in the non-aggressive group, as well as in one subject in the aggressive group. Longitudinal studies are needed to determine cortisol levels in unmedicated patients, in addition to comparing cortisol levels during different kinds of treatment.Item Open Access The development and validation of quantitative methods for the determination of stavudine and alfuzosin in plasma and monic acid in urine(University of the Free State, 2003-05) Wiesner, Joachim Lubbe; Swart, K. J.; Hundt, H. K. L.English: The development and validation of bio-analytica) assay methods suitable for the quantification of stavudine in plasma, alfuzosin in plasma and monic acid in urine is discussed. A short summary of these methods are given: • A sensitive method for the determination of stavudine in plasma was developed, using highperformance liquid chromatographic separation with tandem mass spectrometric detection. The samples were extracted from plasma with Waters, Sep-Pak®Vac, 100 mg, tC18®solid phase extraction (SPE) columns. Chromatography was performed on a Supelco Discovery" C18, 5 urn, 150 x 2 mm column with a mobile phase consisting of ammonium acetate (0.01 M): acetonitrile: methanol (800:100:100, v/v/v) at a flow rate of 0.3 ml/min. Detection was achieved by an Applied Biosystems API 2000 mass spectrometer (LC-MS/MS) set at unit resolution in the multiple reaction monitoring mode (MRM). Atmospheric pressure chemical ionization (APCl) was used to obtain deprotonated ions (molecular ion m/z 223.1 to the product ion m/z 42.01). The mean recovery for stavudine was 94 % with a lower limit of quantification set at 4 ng/ml. This assay method makes use of the increased sensitivity and selectivity of mass spectrometric (MS/MS) detection to allow for a more rapid (extraction and chromatography) and selective method for the determination of stavudine in human plasma than has previously been published. The assay metod was used to quantitatively determine stavudine concentrations in plasma samples to follow the concentration vs. time profile for at least five half lives of the drug after a single 40 mg oral dose of stavudine was given to healthy adult male human subjects. • A selective, sensitive and rapid liquid chromatography-tandem mass speetrometry method for the determination of alfuzosin in plasma was developed. An Applied Biosystems API 2000 triple quadrupole mass speetrometer in multiple reaction monitoring (MRM) mode, using TurboIonSpray (TIS) with positive ionisation was used (molecular ion of alfuzosin m/z 390.2 to the product ion m/z 71.2; molecular ion of prazosin m/z 384.2 to the product ion m/z 95.0). Using prazosin as an internal standard, liquid-liquid extraction was followed by CI8 reversed phase liquid chromatography and tandem mass spectrometry. The mean recovery for alfuzosin was 82.9 % with a lower limit of quantification set at 0.298 ng/ml, the calibration range being between 0.298 and 38.1 ng/ml. This assay method makes use of the increased sensitivity and selectivity of tandem mass spectrometric (MS/MS) detection to allow for a more rapid (extraction and chromatography) and selective method for the determination of alfuzosin in human plasma than has previously been published. The assay method was used to quantify alfuzosin in human plasma samples generated in a multiple-dose (5 mg bd.) study at steady state. • Two selective methods for the determination of monic acid (a metabolite of mupirocin) in urine were developed, using high-performance liquid chromatographic separation with tandem mass spectrometric detection. An Applied Biosystems API 2000 triple quadrupole mass spetrometer in multiple reaction monitoring (MRM) mode, using TurboIonSpray (TIS) with positive ionisation, was used (molecular ion of monic acid m/z 345.2 to the product ion m/z 327.0) The minimal sample preparation and short chromatography time (retention time ~ 3.8 min.) makes these methods suitable for the assay of large numbers of samples per day. Linearity (weighted 1/concentratiorr²) was established from 50.1 to 1001 ng/ml for the direct injection method, and linearity (weighted 1/concentration) was established from 15.8 to 1013 ng/ml for the SPE method. The assay method was used for the determination of monic acid concentrations in human urine in order to detect and quantify the absorption of mupirocin after multiple topical applications ofO.5 g of a 2 % ointment. Analytical data that were generated during these three research projects are discussed in this dissertation, improvements and novelties to existing methods are elucidated. The methods for the determination of stavudine and alfuzosin have been published. Both full-length publications are included in this dissertation, together with correspondence between myself and the journal editors and referees. The assay methods for monic acid will soon be submitted for publication in the Journal of Chromatography B.Item Open Access The development and validation of assay methods for the quantitative determination of drugs and their metabolites in biological specimens: clarithromycin, carbamazepine and carbamazepine-10, 11-epoxide(University of the Free State, 2003-05) Van Rooyen, Gert Frederick; Swart, Ken; Hundt, HansThe projects described in this dissertation use cutting edge technology to quantitate various drugs in plasma. Highly sensitive assay methods for the quantification of clarithromycin, carbamazepine and its major metabolite, carbamazepine-l0, Il-epoxide in human plasma were developed and validated. The application of these methods to analyse samples generated during pharmacokinetic studies is also discussed where up to 230 samples were analysed per day. The methods were sensitive enough to quantitate the analytes for at least 5 half-lives of the drug after an oral dose to human volunteers. A sensitive method for the determination of clarithromycin, a macrolide antibiotic, USing high-performance liquid chromatographic separation with tandem mass spectrometry, and the method development thereof, are described in this dissertation. Samples were prepared using liquid-liquid extraction and were separated on a CI8 column with a mobile phase consisting of acetonitrile, methanol and acetic acid. Detection was performed by a mass speetrometer in the multiple reaction monitoring (MRM) mode, using TurboIonSpray ionisation. The mean recovery of clarithromycin was 87.3%, with a lower limit of quantification of 2.95 ng/ml when using 300f!1 plasma. This high-throughput method was used to quantify 230 samples per day, and is sufficiently sensitive to be employed in pharmacokinetic studies. Carbamazepine, an anticonvulsant, and its major metabolite, carbamazepine-10, l l-epóxide, were quantified using tandem liquid chromatography-mass spectrometry, with electrospray ionisation. Both these analytes were extracted from 500f!1 of human plasma, using a liquid-liquid extraction method that showed recoveries greater than 95% for both these analytes. Concentrations as low as 0.722 ng/ml for carbamazepine and 5.15 ng/ml for carbamazepine-10, 11- epoxide could be quantified by using this method. These are the first LC-MSIMS assay methods described for the quantification of carbamazepine and its metabolite, carbamazepine-10, ll-epoxideas well as clarithromycin and their applications to pharmacokinetic studies.Item Open Access The effect of the traditional medicine phela on p-glycoprotein and multidrug resistance-associated protein 2 drug transporters in the gastrointestinal tract of a rat model(University of the Free State, 2015-11) Binyane, Moleboheng Emily; Walubo, A.English: Phela is the herbal preparation of four African traditional medicinal plants, and is under the development by the Medical Research Council (MRC) as an immune stimulant for immune compromised individuals. Patients might use Phela with other medicines; therefore, the herb-drug interactions profiling of Phela is important. Membrane drug-transporters such as P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2) are considered important factors in determining the pharmacokinetic parameters of drugs such as paclitaxel (PTX) and methotrexate (MTX), respectively. Inhibition or induction of transport might result in drug interactions with other drugs transported by these respective transporters. Moreover, significant herb-drug interactions involving P-gp and MRP2 have been described. Therefore, the effect of Phela on P-gp and MRP2 in the gastrointestinal tract of a rat model was investigated here. First, a high performance liquid chromatography (HPLC) method for determination of PTX in plasma was developed. It involved liquid-liquid extraction of 100 μl plasma, spiked with PTX, extracted with diethyl ether: dichloromethane (2:1), followed by centrifugation. The supernatant was evaporated to dryness under a stream of nitrogen, reconstituted, and 100 μl was injected into the HPLC. The sample was eluted with a mobile phase of sodium phosphate buffer (pH 2): acetonitrile (60:40, v/v) over a C8 (1) (4.6 X 250 mm) 5 μ analytic column at 1 ml/min. PTX was detected by UV at 230 nm. Docetaxel (DTX) was used as the internal standard. Under these conditions, DTX and PTX eluted at retention times of 6.595 and 6.038 minutes, respectively. The average calibration curve (0-15 μg/ml) was linear with a regression equation of y = 0.1931x + 0.0705, and correlation coefficient (r) of 0.9973. The method was used successfully in animal experiments to measure PTX in the plasma of treated rats. Thereafter, a preliminary experiment was conducted in vitro to establish whether Phela has a direct/ physical effect on PTX, using a direct drug interaction testing experiment in buffer, as well as Slide-A-Lyzer® dialysis. During the direct drug interaction experiment, buffer was spiked with 10 μg/ml of PTX with or without 3.85 mg/ml Phela, and PTX concentrations were determined by HPLC. Then, using a Slide-A-Lyzer® dialysis cassette, the time of equilibrium of PTX was determined by monitoring the changes in PTX concentrations over 12 hours, in plasma containing 230 μg/ml PTX and buffer. Thereafter, the potential of an interaction was tested by adding 88.55 mg/ml Phela to the same experiment after 8 hours of incubation, and monitoring PTX concentrations after 10 and 12 hours by HPLC. In the first experiment, Phela had no direct effect on PTX concentrations, while in the second experiment the time of equilibrium of PTX was estimated at 8 hours. After Phela was added, PTX concentrations and its free fraction (fu) remained unchanged. Therefore, it was concluded that there is no interaction between Phela and PTX in vitro. This final part of the study was undertaken to investigate the effect of Phela on P-gp and MRP2 transporters. PTX and cyclosporin A (CyA) were used as the respective substrate and inhibitor of P-gp, while MTX and probenecid (PRO) were those of MRP2. Ethical approval was obtained and male Sprague-Dawley (SD) rats (200-250 g) were used. The animal experiment was divided into two parts. In Part I, three groups of 40 rats each received a one-off oral dose of PTX-only (10 mg/kg); PTX & CyA (10 mg/kg); or PTX & Phela (15.4 mg/kg), while in Part II, three groups of 40 rats each received a one-off oral dose of MTX-only (10 mg/kg); MTX & PRO (20 mg/kg); or MTX & Phela (15.4 mg/kg). For each group, 5 rats were sacrificed after 0.5, 1, 2, 4, 6, 8, 10, and 12 hours. Blood was analysed for full blood count, liver function, and PTX and MTX concentrations. CyA and PRO increased the area under the plasma concentration-time curve (AUC) of PTX and MTX, respectively, whereas Phela had no effect on the AUC of PTX or MTX. Overall, no direct interaction between PTX and Phela was observed both in vitro and in vivo, and there were also no interactions between MTX and Phela in vivo. Phela did not inhibit P-gp or MRP2. This implies that Phela will most probably not be involved in herb- drug interactions of membrane transporter origin. Therefore, the doses of drugs that are transported by P-gp and MRP2 need not be adjusted when co-administered with Phela.Item Open Access Factors influencing antibiotic use in the paediatric intensive care unit at Universitas Hospital from 1998 to 2007(University of the Free State, 2013-02) Van Wyk, Riana; Walubo, A.English: Many antibiotics have been developed and are available on the market. An increase in the use of antibiotics in hospitals was observed and antibiotics are among the medicines most commonly prescribed to paediatric patients. Resistance to antibiotics is increasing and is a major problem not only in the Paediatric Intensive Care Unit at Universitas Hospital in Bloemfontein, but in South Africa in general. The continued value and effectiveness of antibiotics depend on careful use to avoid bacterial resistance from developing. Thus, guidelines for rational antibiotic use and prevention of resistance should be developed and implemented. This requires an understanding of the factors influencing antibiotic use in a particular setting, in this case the Paediatric Intensive Care Unit at Universitas Hospital. Therefore, the aim of this study is to describe the factors that influence the use of antibiotics in the Paediatric Intensive Care Unit from 1998 to 2007. This research consisted of a retrospective study of the records of patients admitted to the Paediatric Intensive Care Unit from 1998 to 2007. Using a datasheet, the following information was captured and evaluated: patients’ demography, indication for admission, co-morbid conditions, antibiotic and other drug therapy, culture and sensitivity and other relevant parameters. Of the 1 221 patients admitted during the study period, information could only be retrieved for 967 patients, and of these 685 patients (385 males and 299 females) met the study criteria. The Paediatric Intensive Care Unit performance, measured as Intensive Care Unit utilisation, was optimal at 63%, implying that no patient needing intensive care was denied. The most common conditions on admission were respiratory (23.4%), gastro-intestinal (22%) and cardiovascular (19%) related problems. Pneumonia (8.9%) was the most common infective condition. The most common infective complications while in the Paediatric Intensive Care Unit were pneumonia (35.6%), septicaemia (11.1%) and urinary tract infection (8.8%). Broad-spectrum antibiotics were prescribed the most widely. The top ten antibiotics included cefotaxime (18.2%), amikacin (14.7%), vancomycin (9.8%), cefuroxime (8.1%) imipenem (7.5%), metronidazole (7.2%), penicillin G (6.5%), cloxacillin (4.1%), co-trimoxazole (2.7%) and gentamicin (2.4%). The top ten bacteria genera cultured were Staphylococcus (29.3%), Klebsiella (11.9%), Acinetobacter (11.7%), Pseudomonas (11.2%), Escherichia (8.5%), Enterococcus (5.9%), Streptococcus (4.1%), Enterobacter (4.1%), Stenotrophomonas (3.4%) and Haemophilus (2%). There was high resistance of the Staphylococcus genus to penicillins and penicillin-allergy substitutes (>80%, with methicillin-resistance of 85%), but no resistance to vancomycin was observed. The Klebsiella and Pseudomonas genera exhibited considerable resistance to most aminoglycosides (40–78%) and cephalosporins (70–100%), but Klebsiella remained sensitive to imipenem (1.9%), while Pseudomonas was moderately sensitive to amikacin (22.9%). The nosocomial bacteria genera Acinetobacter and Stenotrophomonas were resistant (>70%) to almost all antibiotics excluding tobramycin (25.8%) for Acinetobacter and co-trimoxazole (10.5%) for Stenotrophomonas. Lastly, the persistently challenging factors that influenced antibiotic use in the Paediatric Intensive Care Unit from 1998 to 2007 were common bacteria cultured from specific specimens, bacterial innate resistance, interaction of bacterial and host factors (multiple and severe infections), disease pattern, new antibiotics, overuse of antibiotics, length of stay, personal preferences and treatment guidelines. In conclusion, it was illustrated that bacterial resistance to antibiotics is increasing, and that antibiotic use in the Paediatric Intensive Care Unit at Universitas Hospital was greatly influenced by the efforts to contain antibiotic resistance.Item Open Access Co-morbidity of and treatment for irritable bowel syndrome, depression and anxiety in residents of retirement villages(University of the Free State, 2014-12) Tromp, Adelheit; Van Zyl, P. M.Introduction: Irritable bowel syndrome (IBS), depression and anxiety are very common and often co-occur. Data for depression prevalence in the elderly in South Africa is available, but there is no data on the prevalence of anxiety and IBS in this population. Further, the existing literature does not report on the influence of medication use on these conditions. Aim and objectives: The aim of this study was to determine the prevalence and co-morbidity of IBS, depression and anxiety in retirement village residents against the background of the pattern of medication use. Specific objectives of the study were the assessment of current symptoms of IBS, depression and anxiety and the assessment of the use of antidepressants, anxiolytics and gastrointestinal (GI) medications that might influence the symptoms of IBS. Methods: Two hundred ambulant residents older than 50 years were recruited from 2 retirement villages in an urban setting in South Africa by means of convenience sampling. A cross-sectional observational study was performed with a questionnaire. The questionnaire consisted of the Manning criteria and the Hospital Anxiety and Depression Scale (HADS), supplemented by custom designed questions to evaluate medication use. Results: The prevalence of IBS, depression and anxiety were found to be 4.5%, 3.0% and 4.5%, respectively. Sixty-nine participants (34.5%) reported antidepressant use. Selective serotonin re-uptake inhibitors (SSRis) were used by 63.8% and tricyclic antidepressants (TCAs) by 33.3% of participants reporting antidepressant medication use, fluoxetine and amitriptyline being used in the majority of such cases. Forty-one participants (20.5%) reported the current use of benzodiazepines (BZDs). Proton pump inhibitors (PPis) were used by 17.5%. The majority of participants using antidepressants, anxiolytics and PPis were taking these for one year or longer. Participants taking PPis or antidepressants were more likely to experience symptoms of IBS than those who were not taking PPis or antidepressants and these differences were statistically significant. BZDs did not have an influence on the presence of IBS symptoms. There was no association between constipation and use of the target medication groups. Conclusion: The lower than expected prevalence of IBS, depression and anxiety occurred against the background of a high level of prolonged antidepressant, anxiolytic and proton pump inhibitor use.Item Open Access The effect of Phela, a traditional medicine on the immune system of a rat model(University of the Free State, 2010-08) Lekhooa, 'Makhotso Rose; Walubo, A.; Du Plessis, J. B.; Matsabisa, M. G.English: 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 “saltingout” 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 John’s 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 – 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.Item Open Access Evaluation of grapefruit extract for the prevention of paracetamol-induced hepatotoxicity after overdose in rats(University of the Free State, 2015-05) Baleni, Refuoe; Walubo, A.English: Paracetamol is a widely used analgesic and antipyretic agent. While it is generally safe for use at recommended doses, acute overdose of paracetamol can cause potentially fatal liver damage. Despite the understanding that some cytochrome P450 isoforms are responsible for activation of paracetamol to the hepatotoxic metabolite, N-acetyl-p-benzoquinine-imine (NAPQI), the use of enzyme inhibitors for prevention and/or treatment of paracetamol hepatotoxicity is still not well researched. Therefore grapefruit juice was evaluated for prevention of paracetamol-induced hepatotoxicity. A high performance liquid chromatography (HPLC) method for the determination of paracetamol in plasma was developed. It involved protein precipitation of 50 μl of paracetamol spiked plasma with zinc sulphate followed by centrifugation. The supernatant was directly injected into the HPLC. The sample was eluted with a mobile phase of 0.01% trifluoroacetic acid in distilled water: acetonitrile (75: 25, v/v) over a Phenomenex C18 (4.60 x 250 mm) 5 μ analytical column at 1 ml/min. 4’Aminoacetophenone was used as the internal standard. Under these conditions paracetamol and 4’aminoacetophenone eluted at retention times of 4.2 minutes and 6.2 minutes, respectively. The average calibration curve (0 - 20 μg/ml) was linear with a regression equation of y = 0.0603x + 0.089, and regression coefficient of r2 = 0.9957. The method was used to measure paracetamol concentrations in rat plasma. Evaluation of grapefruit juice for the prevention of paracetamol-induced hepatotoxicity was performed with Sprague Dawley rats. The rats were treated with one-off oral dose of saline, paracetamol only, paracetamol + grapefruit juice low dose and paracetamol + grapefruit juice high dose. A commercially available grapefruit derivative, bergamottin, was also evaluated. Thereafter, 5 rats from each group were sacrificed after 24, 48 and 72 hours. After the treatment period the blood samples were collected for liver function tests, full blood count and paracetamol concentration. A piece of liver was sent for histopathology. Administration of a toxic dose of paracetamol (1725 mg/kg) elevated the liver enzymes (ALT and AST) significantly when compared to the control group. Upon physical observation of the liver during surgery, the livers of the rats at 48 hours exhibited moderate to severe hepatic injury. The haematology results, especially platelet count, revealed a very low amount of platelets at 48 hours, which is indicative of thrombocytopenia. Paracetamol plasma concentrations of rats treated with paracetamol only were higher than at 24 hours and there was a slight decrease at 48 and 72 hours due to the drug metabolism. However, the hepatic injuries of the rats treated with paracetamol only were antagonised by co-administration of paracetamol with grapefruit juice and also co-administration with bergamottin. The significant decrease in liver enzymes (ALT and AST) and the slight increase in platelet count at 72 hours revealed that grapefruit juice and bergamottin may have hepatoprotective effects against paracetamol-induced hepatotoxicity. Similarly, the concentration of paracetamol in the plasma of rats treated with paracetamol + grapefruit juice/bergamottin were lowered even further as opposed to when paracetamol was administered alone. The results of this study imply that both grapefruit juice and bergamottin have enzyme inhibition ability and hence were able to play a role in inhibiting the enzymes which are involved in the production of a toxic metabolite known as NAPQI, which is produced in high quantity during overdose of paracetamol and is a major cause of liver injury.Item Open Access Cytochrome P450 and the immune response to prolonged administration of isoniazid, nevirapine and paracetamol in a rat model(University of the Free State, 2015-02) Bekker, Zanelle; Walubo, A.English: Drug-induced liver injury is a major adverse drug reaction that presents during isoniazid (INH) and nevirapine (NVP) treatment, and after paracetamol (PAR) overdose. It was postulated that after these drugs are metabolised, reactive metabolites are formed which attack cellular proteins and result in the formation of antigenic metabolite-protein adducts. Subsequently, the immune system starts to eliminate hepatocytes expressing these adducts, and this leads to the development of overt drug-induced liver injury. As such, the effect of prolonged administration of INH, NVP and PAR on the cytochrome P450 (CYP450) and immune response was investigated here. A high performance liquid chromatography (HPLC) method for the simultaneous determination of INH, NVP and PAR in plasma was developed. Sample preparation involved protein precipitation with zinc sulphate and methanol, followed by solid phase extraction. The mobile phase was 0.06% trifluoroacetic acid (A) and acetonitrile (B) and run by a gradient programmer over a C18 (4.60 x 250 mm) 5 μ analytical column at 1 ml/min, while the eluent was detected by UV at 260 nm. INH, PAR, internal standard and NVP eluted at retention times of 3.1, 9.9, 10.6 and 11.6 minutes, respectively. The average 5 day calibration curves of INH, NVP and PAR were linear with regression equations and correlation coefficients of y = 0.029x + 0.025 (r2 = 0.9954), y = 0.043x + 0.127 (r2 = 0.9968) and y = 0.097x + 0.070 (r2 = 0.9997), respectively. The method was used successfully to monitor INH, NVP and PAR in rat plasma. The CYP450 and immune response to prolonged administration of INH, NVP and PAR were investigated using an SPD rat model. For each drug, the animal experiment was divided into three phases. In phase I, rats were orally administered saline solution (S), INH (20 mg/kg), NVP (200 mg/kg) or PAR (500 mg/kg), while for phase II, rats received S, INH, NVP or PAR in combination with an immune stimulant, levamisole (LMS; 2.5 mg/kg), and lastly, during phase III, rats received S, INH, NVP or PAR along with a CYP450 inducer, carbamazepine (CBZ; 60 mg/kg). In each phase, five rats per group were sacrificed after 2, 7, 14, 28 and 42 days. Blood was analysed for full blood count, CD4 and CD8 counts, liver function, renal function, IL-2, IL-10, IgG, IgM and drug concentrations. A piece of liver was sent for histopathology testing, and the activity of rat CYP1A2, CYP2E1 and CYP3A2 were analysed. During administration of the test drugs alone, both INH and NVP triggered an early Th1 immune response that was associated with liver injury, and counteracted by a later Th2 immune response which was associated with healing. Overall, the liver injury correlated with low concentrations of NVP, but high INH concentrations. That said, INH increased CYP2E1 activity, while NVP increased that of CYP3A2. When LMS (immune stimulant) was co-administered, INH liver injury was exacerbated, while for NVP it was the same. Again, INH and NVP provoked a Th1 response (injury) that was counteracted by a Th2 response (healing). Here, the liver injury was also associated with low NVP concentrations, and high INH concentrations. During co-administration of CBZ (CYP450 inducer), INH and NVP caused the same immune response, and resulted in improvement of the liver injury. Again, the liver injury was associated with low NVP concentrations, and high INH concentrations. Also, INH increased CYP2E1 activity and NVP increased CYP3A2 activity, but not to the same extent as the test drugs alone. PAR did not exhibit a distinct pattern of immune response by which to associate it with the liver injury, most probably because the concentrations were too low for generation of toxic metabolites. In conclusion, the pattern of immune response to prolonged administration of INH and NVP shows that the immune system is involved in the drug-induced liver injury, probably as a protective buffer to prevent further drug toxicity.Item Open Access The simultaneous detection of narcotic analgesics and non-steroidal anti-inflammatory drugs in human urine using high performance liquid chromatography - tandem mass spectrometry(University of the Free State, 2009) Coetzee, André; Van der Merwe, P. J.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 – 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 ß-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, 4’- 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, 4’-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.