Theses and Dissertations (Pharmacology)

Permanent URI for this collectionhttp://hdl.handle.net/2263/32240

Browse

Now showing 1 - 20 of 103

Assessing the acetylcholinesterase inhibitory potential of novel pharmacophores for potential treatment of Alzheimer’s disease
(University of Pretoria, 2023-08) Steenkamp, Vanessa; Theron, Anjo; Panayides, Jenny-Lee; Cordier, Werner; lerato.maboko@gmail.com; Maboko, Lerato Mamabote
Alzheimer’s disease (AD) is the predominant form of dementia which primarily affects the elderly. To date the aetiology of AD has not been fully elucidated, though may include: loss of cholinergic transmission; excessive accumulation of amyloid-beta (Aβ) aggregates extracellularly in the brain; accumulation of tau protein intracellularly; glutamate excitotoxicity and oxidative stress. Current AD therapeutic approaches rely on the administration of acetylcholinesterase inhibitors (AChEIs), memantine and monoclonal antibodies. However, these drugs provide only symptomatic relief and do not prevent progressive neurodegeneration. Among the above-mentioned approved drugs, AChEIs are suggested to have the ability to inhibit the synthesis, deposition and aggregation of toxic Aβ proteins and tau-protein phosphorylation. For these reasons, AChE has received much attention as the prime target in drug discovery and development for the treatment of AD. However, an overall attrition rate of more than 95% has been noted for novel drugs targeting neurodegenerative diseases, and this is mainly due to a lack of efficacy, blood-brain barrier (BBB) permeability, multifactorial nature and/or toxicity. This reinforces the rationale for assessing a broad spectrum of effects of pharmacophores that show potential for therapeutic use at a preclinical level to reduce the high attrition rate. The aim of the study was to identify novel pharmacophores with the potential to be developed further as treatments for AD. An in-house miniaturised AChE assay was used to assess the AChEI activity of 1,453 synthetic compounds from a commercial library (Charles River Laboratory/BioFocus, UK) housed at the Council for Scientific and Industrial Research (CSIR). The selected compounds had been previously identified through in silico screening of the compound library within the active site of the electric eel and human AChE enzyme. The Swiss Institute for Bioinformatics pharmacokinetic prediction tool, SwissADME, was used to assess the compound’s BBB permeability and pharmacokinetics. Combinational AChEI effects of active pharmacophores and donepezil were assessed using a checkerboard assay. The sulforhodamine B assay was employed to determine the active compounds and synergistic combinations cytotoxic potential after 72 h in SH-SY5Y neuroblastoma and brain endothelial (bEnd.5) cells. Michaelis-Menten kinetics of active in silico BBB-permeable compounds were determined using the miniaturised AChE assay to elucidate its inhibitory mechanism. Ultra-performance liquid chromatography (UPLC) coupled to a mass spectrometry (MS) was used for quality control analysis to ensure the chemical integrity of compounds. Cellular AChEI activity of active in silico BBB-permeable predicted compounds was determined using an SH-SY5Y AChE-based assay. An in vitro BBB model was used to assess the effect of compounds on the integrity of the bEnd.5 monolayer, as well as the permeation of compounds using UPLC-MS. The miniaturised assay’s validation parameters were within acceptable ranges (coefficient of variation ≤ 20%, Z’-factor ≥ 0.5 but < 1, and signal window ≥ 2). Only six compounds (A8, A51, A73, A136, A175 and A176) from the primary screen of subset A (n = 400) were regarded as possessing AChEI activity (≥ 60% inhibition at 5 µM). For the secondary screen, two subsets of structurally similar compounds, subset B (n = 508) and subset C (n = 545) were selected and evaluated. The AChEI activity for a further six compounds (C33, C43, C53, C82, C129 and C189) were regarded as positive and ranged between 62 and 72%. Of the 12 active compounds, compound A51 had the lowest half maximal inhibitory concentration (IC50) of 0.20 µM, albeit less potent than donepezil (IC50 = 0.03 µM). The SwissADME pharmacokinetic tool predicted that compounds A8, A73, A136, C53 and C129 were BBB-permeable. Synergism (combination index [CI] < 1) was observed between: ¼IC50 donepezil and ½IC50 A136 (CI = 0.61); ¼IC50 donepezil and IC50 A136 (CI = 0.81); ½IC50 donepezil and ½IC50 A136 (CI = 0.81); ½IC50 donepezil and IC50 A136 (CI = 0.69); ¼IC50 donepezil and ¼IC50 C53 (CI = 0.82). Compounds A8 and A73 were not cytotoxic (IC50 >100 µM), whereas A136 (IC50 = 4.99 µM) and C129 (IC50 = 13.64 µM) possessed greater cytotoxicity than donepezil and C53 (IC50 = 43.00 and 40.45 µM, respectively). The combination of A136 and donepezil eradicated all cell growth, while moderate cytotoxicity (20% cell density reduction) was observed for the combination of C53 and donepezil. Furthermore, no compounds were found to induce cytotoxicity in the bEnd.5 cells (IC50 >100 µM). Donepezil displayed mixed competitive and non-competitive inhibition, while A8 indicated uncompetitive inhibition, and A73 and C53 mixed inhibition. No decomposition was observed for the active in silico BBB-permeable non-cytotoxic compounds. Compound A73 exhibited dose-dependent AChEI activity in SH-SY5Y neuroblastoma cells, whereas A8 and C53 did not. At the IC50, 61% AChEI activity was observed for C53, with the combination of C53 and donepezil indicating 69% AChEI activity. The latter possessed the highest in situ activity. All compounds, including donepezil, increased transendothelial electrical resistance (TEER) over 48 h exposure, suggesting decreased BBB-permeability of the bEnd.5 monolayer. Furthermore, all compounds were detected in both the apical and basolateral chambers of the in vitro BBB model. This suggests in vitro BBB permeation of the compounds and further supporting the in silico BBB permeability predictions. Factors such as efficacy, BBB permeability, and/or toxicity contribute to the high attrition rate noted for central nervous system targeting drugs. In this study 1,453 synthetic compounds were assessed for these aspects in search for potential treatment for AD. Among the 12 compounds that displayed AChEI activity, compound A51 had the greatest AChEI activity. However, A51 was predicted in silico to be impermeable to the BBB and would most likely not be further developed as it may not cross the BBB. Compound A136 was cytotoxic as monotherapy and in combination with donepezil, indicating its capability to kill neuronal cells. Due to the low in situ AChEI activity displayed by compounds A8, A73 and C53, these compounds might not inhibit AChE efficiently in vivo. Therefore, compound C53 alone (at the IC50 concentration) and combined with donepezil indicated the potential for further investigation as AChEIs given their promising acellular and cellular AChEI activity, in silico BBB-permeability, absence of cytotoxicity, and ability to penetrate the in vitro BBB monolayer. Keywords: acetylcholinesterase, acetylcholinesterase inhibitors, Alzheimer’s disease, blood-brain barrier, cytotoxicity.
Characterising select hepatocyte cultures for improved hepatotoxicity testing
(University of Pretoria, 2023) Cromarty, Allan Duncan; Stander, Barend Andre; Ellero, Andrea Antonio; u14168244@tuks.co.za; Mashaba, Clement Vutivi
Drug-induced hepatotoxicity is a major contributor towards post-marketed drug withdrawals. Most of these liver injuries can be associated with drug metabolism, which is primarily performed by hepatic cytochrome P450 enzymes, a superfamily of drug metabolising haem enzymes. Pre-clinical in vitro models are commonly used in an attempt to predict the toxicity profile of lead drug compounds during the early development phases. These in vitro models need to accurately resemble the human liver functionality to be able to predict drug hepatotoxicity. Primary human hepatocytes are the ‘gold standard’ for mimicking liver function. However, due to their expensive culturing requirements, limited time in active culture, and limited availability, other commercially available, transformed hepatic cell lines are commonly used as substitutes for primary hepatocyte cultures. The HepG2 cell line is one of the most commonly used because they are readily available, and the culturing methods are relatively inexpensive. Conventional monolayer culturing of human-derived cell lines has shown limitations when used for drug toxicity tests and lacks sufficient resemblance to human liver tissue, compared to three-dimensional cultured cells. This has led to three-dimensional cell cultures being recommended over monolayer cultures to predict potential in vivo toxicity. The aim of this study was to characterise HepG2 cells differentially cultured as monolayer or three-dimensional spheroids, in the presence and absence of chronic enzyme-inducing drug cocktail exposure. Also, to evaluate the models’ feasibility over an extended culture time and compare their metabolic capabilities as candidates for hepatotoxicity screening platforms. This was done by generating HepG2 spheroids using the liquid overlay method for up to 21 days and culturing same origin HepG2 monolayers for 17 days. Cells were evaluated for morphology, viability, protein content, monolayer cell cycle profile, proteins mass profile differences, and the presence of hepatic markers in spheroids. The metabolic activity was assessed using liquid chromatography tandem mass spectrometry. The monolayer and drug cocktail exposed monolayer cells were viable for up to 17 days while the spheroids and drug cocktail exposed spheroids were viable up to 21 days. CYP1A2 activity was detected in all cultures with slightly more acetaminophen (μmol/μgprotein) detected in monolayer cultures. The activity confirms CYP1A2 expression detected in all spheroid cultures from Day 7 to Day 21. Furthermore, minimal hydroxybupropion, dextrorphan and hydroxymidazolam was detected in all cultures suggesting low CYP2B6, CYP2D6 and CYP3A4 activity, respectively. The metabolite levels were similar between induction and non-induction cultures suggesting that the induction drug cocktail had no significant effect on the metabolic capacity of the HepG2 cells under either of the culturing conditions used. Therefore, the metabolic activity may be due to accumulated innate metabolic capability and/or long-term culture. Furthermore, the growth plateau observed in spheroid cultures’ protein levels after Day 4 and the hepatic markers (AFP, HNF-4α, CK18 and albumin) expression observed from Day 7, would be desirable for repeated hepatotoxicity testing. Other studies have shown HepG2 spheroid cell viability and increased hepatic marker expression for more than 21 days, with a relatively consistent metabolic profile after 21 days, suggesting a stable differentiated phenotype. This is beneficial for repeated, long-term drug exposure for acute and chronic hepatotoxicity screening.
Assessing the feasibility of quick response codes for patient information delivery in the Tshwane district
(University of Pretoria, 2023) Steenkamp, Vanessa; Brand, Sarel; githasingh@gmail.com; Singh, Githa
Introduction: The inclusion of a patient information leaflet (PIL) in medicine packaging is a legal requirement in most countries and ensures the patient has the latest product information. The advancement in technology has led to many countries implementing electronic patient information leaflets (ePILs) via quick response (QR) codes on medicine packaging. This study aimed to assess the feasibility of QR codes for patient information delivery. Method: A mixed method involving two cross sectional surveys carried out amongst 333 patients as well as 17 pharmacists and pharmacists’ assistants at Tshwane District Hospital and one focus group study amongst 18 regulatory affairs pharmacists. Ethics approval was received from the University of Pretoria Faculty of Health Sciences, Research Ethics Committee (444/2022). Patients older than 18 years who speak English as a primary or secondary language were included in the study. Results: The majority of patients were willing (85%), and able (80%) to scan the QR code presented to them. Among the patients who scanned the QR code, over 96% found it easier to read the ePIL (C=0.487, p <0.001) and locate the information they needed (C=0.521, p <0.001). Patients reported a positive sentiment towards the ePIL with 80% of the population preferring either the ePIL (35%) or ePIL with a hardcopy (45%). Pharmacists and pharmacist assistants, 56% were willing and able to scan the QR code, whereas 69% preferred the provision of the ePIL with a hardcopy. Of the pharmacists and pharmacist assistants who scanned the QR code, 89% found it easy to read the ePIL (C=0.746, p <0.05), 78% confirmed they could locate the information on the ePIL (C=0.630, p <0.05) and would utilise the ePIL to counsel patients. All the regulatory affairs pharmacists in the focus group preferred the ePIL and indicated that it was an easy process to create a QR code for an ePIL. Conclusion: Despite the positive sentiment toward the inclusion of the ePIL, neither patients nor pharmacy staff are ready to fully transition to ePIL only format. They preferred a dual system including both a QR coded PIL and hardcopy PIL. In contrast regulatory affairs pharmacists advocated for ePILs only given the efficiency of managing safety updates on PILs.
Effect of glioblastoma cells on brain endothelial cell growth, migration, permeability, and tight junction proteins in vitro
(University of Pretoria, 2023) Flepisi, Brian; Cordier, Werner; Mabeta, Peace; xolisilemokoena@gmail.com; Mokoena, Xolisile
Introduction: Invasion of cancer cells within the central nervous system (CNS) can impact the blood-brain barrier (BBB) directly through cell invasion or indirectly through the cancer secretome. The BBB, formed mainly by endothelial cells, is a crucial protective barrier of the CNS. Glioblastoma (GBM) is the most aggressive and malignant tumour of the CNS, with high mortality and an average survival rate of 12 to 15 months with treatment. The underlying mechanisms of BBB disruption due to GBM remain unclear. To elucidate the overall effect GBM has on the BBB, this study aimed to investigate the effect of GBM-conditioned media and cells on endothelial cell growth, migration, permeability, and the expression of tight junctions. Methods: The brain endothelial cell line (bEnd.3) was used as a model of the BBB. The GBM (U87) cell line was used to collect conditioned media (CM) at 48 hours (h) and for the indirect co-culture with End.3 cells. The study used two endothelial models: mono-culture of bEnd.3 cells treated with U87-CM and an indirect co-culture of bEnd.3 cells with U87 cells. Both models were compared to a mono-culture of bEnd.3 cells untreated (negative control). The endothelial cell morphology (by phase-contrast microscopy), and the cytotoxicity of CM (using the sulforhodamine B assay), were assessed on the untreated bEnd.3 mono-culture and CM treated mono-culture of bEnd.3 cells. The following was conducted in the two models: endothelial cell migration rate (scratch migration assay), cell growth (xCELLigence real-time cell analyser system), barrier integrity (sodium fluorescein permeability), and tight junction protein expression (Western blotting). Results: The cytotoxic effects of CM on bEnd.3 cells were observed only in the U87-CM collected at 96 h. The U87-CM suppressed the growth of bEnd.3 cells. Conversely the indirect co-culture significantly increased cell growth (p<0.05). The U87-CM increased the migration of bEnd.3 cells from 10% at 24 h to 33% at 48 h compared to the negative control. The U87-CM did not alter permeability of bEnd.3 cells, whereas the indirect co-culture weakened the permeability of the barrier from 15.97 × 10-5 cm/s (negative control) to 20.96 × 10-5 cm/s (treated bEnd.3 cells). The U87-CM increased the expression levels of occludin and ZO-1 in bEnd.3 cells following 48 h, compared to the negative control. The indirect co-culture increased the expression of ZO-1 by 1.2 fold following 48 h exposure. Conclusion: The chosen U87-CM did not alter the morphology of endothelial cells and had minimal cytotoxic effect on the bEnd.3 cells. However, a real-time analysis of the bEnd.3 cell growth revealed that U87-CM suppressed cell growth, and an indirect co-culture with the U87 cells promoted cell growth. The CM maintained the integrity of the BBB and the co-culture caused BBB instability. Both U87-CM and U87 indirect co-culture can influence the growth, migration, permeability, and tight junction expression process in bEnd.3 cells. It is evident that the secreted factors play a role in BBB modulation. However, further investigations are required to understand the signalling pathways activated by the secretome in CM and indirect co-culture on bEnd.3 cells.
Investigation of non-prostatic in vitro prostate-specific membrane antigen expression in MCF-7 and MDA-MB-231 breast tumour cells
(University of Pretoria, 2020) Cromarty, Allan Duncan; Serem, June Cheptoo; Ebenhan, Thomas; mtshabalala7t@gmail.com; Tshabalala, Malvin Thabani
Introduction Breast and prostate cancer mutually represent the most commonly occurring malignancies worldwide in women and men, respectively. The mutative state, recurrence capacity, resistance to conventional chemotherapy, low success rate of surgery and risks associated with radiotherapy confound the management of both these malignancies. There are several similarities between breast and prostate cancer, like growth hormone dependence and similar chemotherapeutic interventions. Therapy based on radiopharmaceuticals targeting the prostate-specific membrane antigen (PSMA) is proving to be a cutting-edge theranostics intervention for prostate cancer. Clinical positron emission tomography (PET) scans have located anti-PMSA binding sites in breast cancer in vivo. This indicates possible non-prostatic expression of PSMA, therefore research focused on understanding the cellular kinetics, PSMA expression profiles using two breast cancer adenocarcinoma cell lines as breast cancer models. This approach was to assess PSMA as a biomarker molecule that can aid in development of more selective, effective and safe diagnostic and therapeutic alternatives for breast cancer. This study was aimed at evaluating PSMA expression of MCF-7 or MDA-MB-231 mammary adenocarcinoma cell lines in comparison to a known high PSMA expressing LNCaP prostate carcinoma and EA.hy926 hybrid vascular endothelial cell line. Methods In vitro cultures of LNCaP’s , a prostatic adenocarcinoma cell line, MCF-7 and MDA-MB-231 breast adenocarcinoma cell lines and endothelial EA.hy926 cells were tested for expression of PSMA by flow cytometry. The LNCaP cells were used a positive control. Cellular localisation of PSMA was achieved utilising confocal microscopy and fluorescently-tagged antibodies in all the cell lines tested. PSMA was quantified in all the cell lines utilising ELISA. Prior to experimentation, a pilot study was undertaken to optimise cell detachment methods. Trypsinisation was compared to mechanical scraping to evaluate a cell detachment method that allowed optimal downline experimentation. Results Findings from three supporting and complementary techniques demonstrate positive PSMA identification, localisation and quantification in all the probed cell lines despite three cell types not having a prostrate origin. Quantitatively, LNCaP cells reported the highest concentration of PSMA followed by the malignant MDA-MB-231 cells, then the MCF-7 cell line and least in EA.hy926 cells. The difference in fluorescence between LNCaP cells and all three investigational cell lines was statistically significant however the difference in fluorescence between the three investigational cell lines was not statistically significant. The PSMA antigen was localised on the cell membrane and diffused within the cytosol in LNCaP cells. The MDA-MB-231, MCF-7 and EA.hy926 cells all exhibited a differential expression pattern of PSMA. These cells showed diffuse cytosolic accumulation and intense circular region accumulation apparently bordering the cell membrane and the cell nucleus. The quantification of PSMA reported the highest concentration as being in LNCaP cells. The MDA-MB-231 cells were second, then the MCF-7 cells and the lowest concentration. Significant differences were seen between the positive control and the investigation cell lines. The difference in concentration between the investigational cell lines was not significant. Finally, cryotome sections of biopsies of tumours from two breast cancer patients were found to show detectable PSMA presence. Discussion Fluorescence is directly proportional to concentration. The high fluorescence of PSMA exhibited by LNCaP cells in the flow cytometry results can be equated to concentration. A fundamental point of departure from which PSMA expression in the breast carcinoma cell lines could be investigated was established. Expression of PSMA is associated with cancer aggression, metastatic progression and increased malignancy. These clinicopathological characteristics support the expression of PSMA seen in MDA-MB-231. Contrastingly, the same characteristics aren’t seen in MCF-7 cells but expression of PSMA was observed. The expression is not entirely dismissible as other luminal A cell lines have also been shown to express PSMA. The EA.hy926 cells are somatic hybrids that are made up of lung A549 cells and HUVEC’s. Lung cancer has been shown to also express PSMA when probed utilising histology. The expression of PSMA in EA.hy926 is the first of its kind but may be attributable to its lung carcinoma makeup. The pattern of expression in the LNCaP confocal microscopy images can be expected. The PSMA antigen is a transmembrane receptor and as such intense fluorescence was seen on the membrane. Expression of PSMA in the cytoplasm has been reported and was equally observed in the LNCaP cells. The investigation cell line showed accumulation of green fluorescence in vesicular bodies bordering the cell membrane and in juxtanuclear positions. The expression of PSMA has been reported in the mitochondria and the green fluorescence at the nucleus could be mitochondrial. The Golgi apparatus and endoplasmic reticulum have also been recognised as potential location of PSMA expression. The localisation of both these organelles at the nucleus along with the expression of PSMA seen close to the nucleus could be associated. Worth noting is the internalisation properties of PSMA. The antigen has an internalisation signal that can be induced by ligand binding or in the absence of a ligand. Upon internalisation the receptors are vesicled and transported either for degradation of for recycling. The vesicular expression seen close to the membrane in the investigational cell lines could be PSMA that is being trafficked for recycling or degradation upon internalisation. The ELISA quantification revealed the levels of PSMA in the positive control are 100-fold greater than those in the investigation cell lines. The ability to translate PSMA targeting in clinical settings is questionable when considering the difference in concentration values. The probing of PSMA in histological slices was positive and showed patterns that are similar to those seen in the monolayer cultures. This shows continuity between two-dimensional cultures and heterogeneous tissue samples. The premise for investigation of PSMA as a potential theranostic target was established through positive identification, localisation and quantification across three independents methods. Conclusion This study is the first of its kind to report reproducible expression of PSMA in the two-dimensional cultures of breast adenocarcinoma MDA-MB-231 and MCF-7 cell lines as well as in the hybrid endothelial EA.hy926 cell line. The results were confirmed by three different techniques where different antibodies were used for the ELISA showing reproducibility in the findings. Moreover, the generated results support the apparent localisation of PSMA in breast cancer patients utilising PSMA targeting radionuclides in PET imaging in a clinical setting. The potential application of this study’s result is stimulating. The success being realised in prostate cancer theranostics through PSMA targeting, may conceivably be realised in other carcinomas, particularly breast carcinoma theranostics.
Mechanisms facilitating uptake of carboxyl-polyethylene glycol-functionalised gold nanoparticles into multicellular spheroids
(University of Pretoria, 2021) Cordier, Werner; Steenkamp, Vanessa; Gulumian, Mary; sethfobian@gmail.com; Fobian, Seth-Frerich
The drug discovery pipeline is hindered by confounding and non-representative in vitro cellular models. Traditionally, monolayer cell cultures have been used to evaluate drug toxicity and efficacy; however, are not sufficiently representative of the in vivo milieu. More advanced culture methods, including three-dimensional (3D) multicellular tumour spheroids, offer a solution by presenting a more appropriate physiological state for the cellular system. Equally damaging to the drug discovery pipeline, however, are candidate drug compounds with little potential. Nanomedicines have shown promise in drug delivery and various other theragnostic applications, and increasingly continue to do so. Generally, medicines are required to permeate structures, especially solid tumours, into the intracellular environment to exert activity. As such, there is a need for both uptake mechanisms and intracellular trafficking pathways to be well characterised. Further to the potential for cancer research, the National Institute for Occupational Health (South Africa) researches the Health, Safety, and Environment (HSE) of engineered nanomaterials. This study aimed to establish an A549 alveolar carcinoma spheroidal drug discovery and toxicity testing platform for the elucidation of uptake mechanisms employed for the pilot nanoparticles (NPs) in this study: 14 nm carboxyl-polyethylene glycol-functionalised gold nanoparticles (PCOOH-AuNPs). The PCOOH-AuNPs were manufactured and characterised in terms of size, surface charge, spectral activity, and concentration by Mintek (South Africa). The A549 alveolar carcinoma cell line was used in conjunction with the liquid overlay technique, allowing for efficient and reproducible spheroid formation. Phase contrast microscopy was used alongside ImageJ analysis to monitor morphological aspects of spheroid growth, as well as cell lysis and lactate dehydrogenase (LDH) release for relative enumeration of cells. Live/dead staining was used to visualise areas of metabolic activity (viability) and compromised membranes (cell death) within a spheroid. Cytotoxicities of the PCOOH-AuNPs and pharmacological uptake inhibitors were assessed by monitoring LDH release from spheroids after exposure. Uptake mechanisms were assessed via CytoViva® hyperspectral imaging of 5 µm cryotomed spheroid sections after exposure to the pharmacological uptake inhibitors; sodium azide, dynasore, 5-(N-ethyl-N-isopropyl) amiloride (EIPA), genistein, and chlorpromazine. Cells formed compact and reproducible spheroids within seven days after seeding, showing an average diameter and circularity index of 702.11 μm and 0.80, respectively. Zones of rapid metabolism and viability were evident from live/dead staining towards the superficial layers of a spheroid, as were zones of cell death towards the core of the spheroid. The Day 7 spheroids exhibited no greater LDH release than negative controls when exposed to either the AuNPs (2 and 24 h exposures) or pharmacological inhibitors (3 h exposures). Internalised AuNPs were counted in the presence or absence of uptake inhibitors to deduce employment of endocytic mechanisms. Counts were obtained and the following proportions of uptake mechanisms were calculated to have been employed for PCOOH-AuNP uptake: 9.2% passive diffusion, 6.6% macropinocytosis, 17.1% clathrin- and caveolae-independent pathways, 33.5% to 54.8% clathrin-mediated endocytosis, and 3.1% to 24.4% caveolae-mediated endocytosis. Penetration of AuNPs into spheroids was, on average, 4.5 μm, which is low, and indicates low levels of transcytosis, as well as intracellular retention of the PCOOH-AuNPs. Uptake mechanisms employed by A549 spheroids for PCOOH-AuNPs were found to be diverse; however, primarily occurred via clathrin-mediated endocytosis. This means that PCOOH-AuNPs are likely to be trafficked towards a degradative fate in the lysosome. Such a destination largely invalidates their use for intracellular drug delivery, unless drugs or NPs are to induce lysosomal membrane permeabilization, exerting action in that way. Findings made in this study promise to inform intelligent design of future AuNP renditions having the goal of greater safety, efficacy, and achievement of desired theragnostic purpose. Further, the lack of cytotoxicity of both the PCOOH-AuNPs and all the uptake inhibitors, validates the methods in this study as reliable. A reproducible, representative 3D in vitro NP testing model has been established, using the A549 alveolar carcinoma cell line, which can be used to assess uptake strategies employed by NPs.
Prevalence of SLCO1B1 single nucleotide variations, and their association with statin intolerance in hypercholesterolaemic patients in Gauteng, South Africa
(University of Pretoria, 2020) Soma, Prashilla; Phulukdaree, Alisa; Outhoff, Kim; renedbr1@gmail.com; De Beer, René
Statins, the standard treatment for hypercholesterolaemia, have been associated with side effects, including statin intolerance. This study determined the prevalence of SLCO1B1 single nucleotide variations (SNVs) and possible associations between SLCO1B1 SNVs, statin intolerance and creatine kinase (CK) in hypercholesterolemic patients on statin therapy. One hundred and eighty one healthy controls and 100 hypercholesterolaemic patients receiving either simvastatin or atorvastatin were recruited. A questionnaire was used to assess the risk of statin intolerance. Polymerase Chain Reaction - Restriction Fragment Length Polymorphism (PCR-RFLP) was used to identify the presence of SLCO1B1 SNVs (rs4149056, rs2306283 and rs4363657) and enzyme-linked immunosorbent assay (ELISA) was used to quantify serum creatine kinase (CK) levels. Of the 100 hypercholesterolaemic patients, 15% presented with high risk, 49% with moderate risk and 36% with low risk to statin intolerance. The prevalence of the rs4149056 variant was 16% for the control group and 20% for the test group, while the rs2306283 variant present in 31.5% of the control group compared to only 10.5% in the test group. The prevalence of rs4363657 variant was similar in each group. A comparison of genotype frequencies based on calculated statin intolerance risk, i.e. low risk versus moderate to high risk, showed no significant association between any of the SNVs and the either low risk or moderate to high risk statin intolerant presentation. CK levels in patients on simvastatin were significantly higher compared to patients on atorvastatin. The prevalence of the SLCO1B1 SNVs in this population is a novel finding. No association between the presence of any one of the SNVs and the statin intolerance severity risk score or CK elevation was found.
Erythropoietin treatment in anaemic patients at the Nephrology Unit of the Steve Biko Academic Hospital - a retrospective, cross-sectional study
(University of Pretoria, 2020) Marais, Andrè; Steenkamp, Vanessa; elandre.kok@gmail.com; Kok, Elandre
Anaemia in chronic kidney disease (CKD) mostly results from a decrease in the production of erythropoietin (EPO) by the failing kidney. CKD progression requires treatment with erythropoiesis-stimulating agents and iron supplementation to ensure sufficient erythrocyte production. Best clinical practice guidelines should be adhered to in managing CKD to reduce morbidity and mortality related to anaemia associated cardiovascular disease. Likewise, guideline deviations create an increased strain on the resources of the treatment facility. It is uncertain to which extent these guidelines are followed by Nephrology Units in the public healthcare sector, or whether the documented international trends are prevalent locally due to the paucity of local data, and therefore further investigation is warranted. This study aimed to assess treatment trends in managing anaemia in CKD patients at the Steve Biko Academic Hospital (SBAH). Files of patients receiving treatment at the SBAH Nephrology Unit between 2 January 2018 - 31 August 2018 were reviewed. Only individuals with stage 5 CKD receiving either haemodialysis, or peritoneal dialysis were included, while those with less than three months’ treatment were excluded. Measured variables included demographical information, current EPO treatment and/or iron supplementation regimens versus serum haemoglobin/iron levels and quantity of administered blood products. Ninety-seven patients met the inclusion criteria. Haemodialysis accounted for 43% (n = 42), and peritoneal dialysis 57% (n = 55). Intergroup comparison between the number of results where both haemoglobin and iron were within the target range versus the number of results where both parameters fell outside the target range yielded a significant difference (p = 0.0031). Patients receiving peritoneal dialysis reached serum haemoglobin and iron levels closer to normal target values compared to those receiving haemodialysis. Managing anaemia in CKD is a complex process. More stringent iron control, especially for patients receiving haemodialysis, including the administration of long-acting EPO preparations once a month, is proposed. The latter will contribute to the improvement of clinical outcomes of patients with CKD. Keywords: Chronic kidney disease, anaemia, erythropoiesis stimulating agent, haemoglobin, iron
Assessing modified chitosan wound dressings to enhance wound healing in the porcine model
(University of Pretoria, 2020-08) Cromarty, Allan Duncan; Balogun, Mohammed; bonghie14@gmail.com; Khathide, Bongai
Dressings enhancing wound healing can improve the outcome of wounds where tissue replacement is required, like for burns and ulcers. Treatment of these wounds is complex due to their depth and excessive tissue loss. Replacement of the lost tissue and delivery of growth factors could enhance healing and reduce scarring. The natural biomaterial; chitosan is reported to bind growth factors, with reduced wound healing times when used in dressings. This study aimed to modify chitosan into a wound dressing filler that would optimise growth factor delivery to full-thickness wounds and overall reduce healing times with minimum scarring. Lipophilic modified chitosan was chemically synthesised by addition of different percentages (10%, 20%, and 34%) of lauric acid residues into three lauroyl chitosan (LCs) derivatives (LCs10, LCs20, LCs34). Lauric acid was the fatty acid of choice due to its superior antimicrobial properties among the saturated fatty acids.1 The loading densities selected were based on commonly used concentration ranges as found in literature. The three derivatives were then characterised using Nuclear magnetic resonance (NMR) and Fourier-transform infrared (FT-IR) spectroscopy. Thereafter, swelling tests and water drop shape analysis followed to assess the physical characteristics of the derivatives. Cytotoxicity/proliferation assays using primary fibroblasts and sulphorhodamine-B for cell enumeration were performed followed by a preliminary skin sensitivity test. The acid phophatase assay was used to measure platelet adhesion while the enzyme-linked immunosorbent assay (ELISA) measured the release profile of platelet derived growth factor AB (PDGF-AB) over 24 hr. These assays assisted with determining which derivative had the optimum lauric acid loading density for wound healing. After determining the derivative with the optimum loading density, porcine collagen was extracted from skin and added to the selected LCs derivative at the ratio 1:4 to make a wound filler paste that would increase cellular ingrowth. Wound healing studies using LCs10 enriched with collagen fibres (Co/LCs10) alone and with platelet-rich plasma (Co/LCs10/PRP) as dressing material were performed using the porcine full skin thickness wound healing protocol. Finally, histological analysis of the cellular events taking place in the wounds at different stages of healing were done using the Haematoxylin and Eosin and the Masson’s Trichrome stains. Evidently, the FT-IR and NMR, displayed successful modification of chitosan with the lauric acid side chains with a visible aliphatic group in both spectra. Comparison of the LCs derivatives to the underivatized chitosan using the drop shape analysis, showed increased contact angles with increased hydrophobicity. It appeared that as the molar concentration of lauric acid increased, the contact angle also increased. In the swelling tests, LCs34 had the highest swelling capacity. Results from the in vitro assays showed that hydrophobic modification of chitosan reduced the adhesion capacity of platelets to chitosan as the lauric acid density on the underivatized chitosan increased. Cytotoxicity assays indicated that neither LCs nor chitosan were toxic to primary fibroblast cells, with the LCs34 significantly (43%) promoting fibroblast proliferation compared to the control. A preliminary skin sensitivity test comparing LCs34 to chitosan showed that LCs34 was compatible with human skin. From the ELISA study the LCs10 sample exhibited a sustained release of growth factors over 24 hr compared to both chitosan and collagen. Consequently, the LCs10 derivative was then selected for further analysis and for final analysis in the wound study. Sixteen full-thickness skin wounds were thereafter made along the dorsum of each of four pigs with two treatments and a control (Jelonet®) randomly applied as dressing material: Co/LCs10, Co/LCs10/PRP and the Jelonet® treatment. The differences in wound healing were observed with biopsies taken at 3-day intervals over 21 days. By the 12th day, all wounds had completely healed with little scarring. The Co/LCs10/PRP dressing significantly induced haemostasis, wound contraction and accelerated wound closure and healing from the wound bed. Results from histological examinations demonstrated advanced granulation tissue formation, collagen deposition and epithelialisation in the wounds treated with Co/LCs10/PRP. This study therefore revealed that hydrophobically modified chitosan at 10% loading density provided a wound dressing material that allowed sustained growth factor release. The Co/LCs10/PRP dressing also demonstrated that it was an improved wound dressing due to acceleration of wound healing, promotion of fibroblast proliferation with increased collagen deposition and minimal scarring. These materials may significantly reduce healing times of full-thickness wounds and should be studied further in in vivo models.
Assessing cardiotonic steroids involvement in hypertensive rat models with Helicobacter pylori infections
(University of Pretoria, 2020-07-31) Cromarty, Allan Duncan; Geoffrey, Geoffrey P.; Leuschner, Machel; flaviennemasso@gmail.com; Masso, Zelie Flavienne
Introduction: Hypertension is an important public health challenge worldwide, being the leading cause of cardiovascular disease, morbidity and mortality. It is particularly prevalent in people in sub-Saharan Africa, especially in urban areas. There is an urgent need to develop strategies to prevent, detect, treat, and control hypertension effectively in the African region. Helicobacter pylori, a gram-negative bacterium responsible for many gastric disorders worldwide, has been associated with hypertension in some previous studies; where blood pressure of patients with Helicobacter pylori infection did not subside after hypertensive treatment, when compared to patients without Helicobacter pylori infections. This effect was suggested to be due to Helicobacter pylori produced and modified cardiotonic steroids that are found in elevated concentrations in hypertensive patients. Cardiotonic steroids are positive inotropic agents which are known to increase blood pressure. A sensitive analytical method is needed to detect and quantify the low concentrations of cardiotonic steroids in biological samples. Materials and Methods: An extraction method was optimised using reversed phase Solid Phase Extraction. A targeted liquid chromatography tandem mass spectrometry method using an Agilent binary series 1100/1200 LC system with a Kinetex C18 RP column (100 x 2.1 mm, 2.6 µm) coupled to a Sciex 4000QTRAP tandem mass spectrometer was developed and validated for the detection and quantitation of 9 different cardiotonic steroids in both solvent and whole blood. The method was validated according to the International Conference on Harmonization guidelines with regards to precision, accuracy, sensitivity, selectivity, linearity, range, limit of detection, limit of quantification, reproducibility, recovery, carry-over and stability. Media from Helicobacter pylori cultures and faecal samples from human and different normo- and hypertensive rat strains were analysed. Data analysis was performed with Analyst® Software (version 1.5.2) and multiple t-test and Kruskal Wallis test using GraphPad Prism 8 software. Results and Discussion: The calibration curves of tested cardiotonic steroids were linear over a concentration range of 0.1-40 ng/mL with coefficients of determination greater than 0.990 except for telocinobufagin. The analytical method was selective with an estimated limit of detection and limit of quantification between 0.02-0.5 ng/mL and 0.1-2 ng/mL respectively. All tested cardiotonic steroids showed good recovery of over 70%. Accuracy and precision were found to be within acceptable limits of 15% and 20% at lowest limit of quantification for almost all the analytes and their stability in blood and solvent at room temperature, 4°C, -20°C and -80°C was tested for a month. Cardiotonic steroids were detected in Helicobacter pylori cultures and faecal samples with the exception of ouabain and proscillaridin A which were not detected at all. Although Helicobacter pylori were shown to produce cardiotonic steroids in vitro, no evidence of the effect of Helicobacter pylori on cardiotonic steroids production was detected in different normo- and hypertensive rat groups. Conclusion: The quantitative analytical method was successfully validated, over expected in vivo concentration ranges for 8 different cardiotonic steroids. The extraction and analytical methods were both successfully applied to Helicobacter pylori cultures and faecal rat samples where cardiotonic steroids were detected.
Prescription patterns and drug duplication in specialist outpatient clinics at a tertiary hospital in the greater Tshwane metropolitan area
(University of Pretoria, 2020) Marais, Andre; Grobler, Gerhard P.; genius.musae@yahoo.co.uk; Ncube, Musawenkosi Genius
Background: Tertiary hospitals have multiple specialist outpatient clinics attended by patients suffering from various comorbid diseases. This results in individuals attending more than one clinic per month, since dedicated clinic days are seldom on the same day. As patients attend discrete clinics, they have separate encounters with various prescribers, increasing the potential for irrational drug use. In addition, multiple clinic visits have a negative socio-economic impact on health care users from poorer communities where financial resources are limited due to transport expenses and days of work missed. The aim of this study was to determine the prescribing pattern of drugs to chronic disease outpatients, and find possible solutions to provide a system that would reduce overprescribing of chronic medication at Steve Biko Academic Hospital (SBAH) in one measure namely drug duplication. Methods: A retrospective descriptive cross-sectional study with the use of convenience sampling was employed to determine the medication prescribing practises to comorbid chronic disease patients attending multiple specialist clinics at SBAH from February 1, 2018-May 31, 2018. Participants were selected according to their appearance in the hospital records, with sample saturation reached when each participant had visited all the different clinics. Chronic disease outpatients attending the SBAH clinics had reviews every three months. The reviews were controlled by issuing patients with medication for a three-month period, where after a follow up visit was mandatory in order to ensure prescription and medication renewal. Therefore, each patient visited all the clinics rendering a service relating to a specific chronic condition within a four-month period that determined the study period chosen. Hospital records of patients attending the most frequently visited clinics as reported by the SBAH Pharmacy and Therapeutics committee (PTC) were evaluated. These clinics included outpatient departments of diabetes, haematology, internal medicine, neurology, oncology and psychiatry. Each drug prescription observed was evaluated using guidelines of World Health Organization (WHO) titled, “How to investigate drug use in health facilities: selected drug use indicators.” Prescribing indicators relevant to this study were used from the WHO guidelines. Results: One hundred and six patients were multiple clinic-attendees during the study period. Of the 106 patients retained, 103 (97.17%) patients attended two clinics and three (2.83%) patients attended three clinics. Regarding the WHO prescribing indicators, the average number of visits to SBAH by the comorbid chronic disease outpatients observed was 3.03 visits during the four-month study period. Prescription analysis included 80 (75.47%) patients out of 106 patients attending multiple clinics at the same time. The average number of drugs prescribed per encounter was 4.97. The results also showed that 45.45% of the 187 prescriptions observed contained five or more drugs. Most frequently prescribed drugs were tramadol 51 (5.49%), followed by simvastatin 48 (5.17%) and enalapril 45 (4.84%). Drug duplication occurred in 68 individual cases in the 80 patients observed. In total, drug duplication affected 39 patients (48.75%) [95% CI = 37.80%: 59.70%]. The most duplicated drug classes were analgesics 18 (26.47%), followed by anti-depressants 14 (20.59%) cases recorded. Conclusion: The results from this study support findings from similar studies at different institutions. The study confirmed multiple clinic visits are prevalent in the medical disciplines, often prescribing drugs from the same class. Clinical implications from these frequent and separate encounters may result in irrational prescribing, adverse drug events, drug-drug interactions and polypharmacy. The establishment of polypharmacy to comorbid chronic disease patients indicates the high risk of drug-drug interactions and adverse drug events. A prospective study would have provided more data for analysis to determine the level of polypharmacy and drug duplication. Thus, supplementation of this study with further studies could provide conclusions on whether the patients suffered from problematic or had appropriate polypharmacy. Physicians treating multiple clinicattendees should be equipped to monitor rationality of prescribing encounters. Installation of an advanced electronic Hospital Information System (HIS) could aid in improving drug prescribing in tertiary hospitals. Use of electronic prescribing tools as shown in previous studies is a requirement to improve tertiary hospitals in developing countries such as SBAH. The incidence of drug duplication at SBAH builds on existing evidence of unnecessary healthcare costs because of medication errors.
Assessing inter-method agreement of drug-based phenotyping metrics between dried blood spot and plasma sampling
(University of Pretoria, 2019) Cromarty, Allan Duncan; machel.leuschner@gmail.com; Leuschner, Machel
Introduction: Pharmacokinetic variability in response to pharmacotherapy contribute to adverse drug reactions, drug-drug interaction and therapeutic failure seen in clinical practice. Poor therapeutic response to medication has been attributed to inter-individual and interethnic variability in cytochrome P450 (CYP450)-dependent metabolism and altered drug absorption via expressed transport channels such as P-glycoprotein (P-gp). An individualised approach in therapeutic management would be beneficial in a South-African population considering the country’s large genetic diversity. A single time point, non-invasive capillary sampling, combined with a low dose probe drug cocktail, to simultaneously quantify in vivo drug and metabolite concentrations, would enhance the feasibility and cost-effectiveness of routine phenotyping in clinical practice and guide personalised prescribing to individual patients. A recent development in dried blood spot sampling is the Mitra™ device, using Volumetric Absorptive Micro Sampling (VAMS™) technology to collect an accurate volume (10-30 µL) of whole blood onto a hydrophilic polymeric tip as an alternative to plasma sampling. Small volume blood sampling however presents bioanalytical challenges in terms of the reproducibility and sensitivity of the quantitative method and the agreement between quantitative measurement from a dried blood spot (DBS) and that from plasma sampling. The physicochemical diversity of the structurally related aromatic probe drugs, used together in a drug cocktail, further require optimised analytical procedures for simultaneous quantification. Phenotyping cocktails are compounded from commercially available dosage forms and introduce challenges with regards to dosage homogeneity, chemical interference or degradation and possible incompatibilities of drugs when used in combination. Aim and objectives: The purpose of this study was to compound the validated “Geneva phenotyping drug cocktail”, from available API sources and develop a validated, targeted, analytical LC-MS/MS method to quantify the seven probe drugs and six respective metabolites in dried blood spots when using the Mitra™ volumetric absorptive micro-sampling device for blood collection. The aim was to assess inter-method agreement of the measured probe drug and metabolite concentrations between the low sample volume, from a dried blood spot, and conventional plasma sampling. Methods: An Agilent binary series LC system coupled to a Sciex 4000 QTRAP triple quadrupole tandem mass spectrometer was used for method optimisation and validation. Targeted LC-MS/MS methods, in both negative and positive ESI mode, were validated according to ICH guidelines for matrix effects, recovery, linearity, limits of quantitation and detection, carry-over, inter and intraday precision and accuracy and analyte stability. The selectivity of the structurally related ionisable analytes was compared between a Kinetex C18 and Kinetex Biphenyl column and the influence of changes in the analytical conditions (involving mobile phase pH and solvent mixture composition as well as the solvent type) studied. An initial assessment of statistical in vitro agreement between plasma and DBS sampling were carried out. USP assays were performed to determine the weight and content uniformity of the compounded phenotyping cocktail containing six of the seven probe drugs. Content uniformity was evaluated with an Acquity UPLC system coupled to a Synapt G2 QTOF mass spectrometer. Results and discussion: A biphenyl stationary phase in combination with methanol as the organic eluent, provided improved resolution and analyte selectivity of the structurally related aromatic compounds. Results from the robustness experiment further confirmed the importance of controlling analytical conditions to ensure reproducibility and reliability of the quantitative method. Separation selectivity and higher throughput were prioritised over optimised ionisation efficiency, although the sensitivity of the analytical method for individual analytes were still within the expected in vivo concentration ranges to infer metabolic and transport phenotypes. This study successfully validated the use of DBS, collected with the volumetrically controlled absorptive microsampling device Mitra™, to measure expected probe drug and metabolite concentrations using the “Geneva phenotyping cocktail”. The validated method met all the required standards accepted in bioanalytical chemistry for specificity, sensitivity, linearity, accuracy, precision, carry-over and stability. From the initial in vitro assessment of agreement, it was concluded that blood cell distribution kinetics are regulated by the blood-to-plasma concentration ratio and time dependent equilibrium between different blood compartments, the physicochemical properties of the analytes, temperature during extraction, analyte concentration and stability. A conclusive confounding factor was the extent to which the extraction procedure liberated bound drug from either plasma proteins or erythrocytes. It was further concluded that the compounded low dose phenotyping cocktail capsules could be used successfully to assess inter-method agreement of drug-based metabolic ratios and drug transport between plasma and DBS collected with the Mitra™ device. Conclusion: To our knowledge, this is the first DBS validation study using the Mitra™ device for the purpose of simultaneous phenotyping of the in vivo P-gp transport and CYP450 metabolic activity of the CYP1A2, -2B6, -2C9, -2C19, -2D6 and -3A4 enzymes and activity.
Identification of single nucleotide polymorphisms in inflammatory bowel disease patients on azathioprine therapy
(University of Pretoria, 2019) Soma, Prashilla; Cromarty, Allan Duncan; Phulukdaree, Alisa; u13020740@tuks.co.za; Adam, Lyla
Azathioprine, an immunosuppressant used for many years in the medical sector to maintain long term disease remission in inflammatory bowel disease has a side effect profile that has raised many concerns over the years with numerous studies into the risk, cause and prevention of these adverse events. It is estimated that 18 in every 100 000 South Africans suffer from Crohn's disease, while 5 in every 100 000 suffer from ulcerative colitis. Of these inflammatory bowel disease patients at least 20% will suffer from leukopenia and eventually bone marrow suppression. Much of the side effect profile of Azathioprine can be linked to a single nucleotide polymorphism in the thiopurine methyltransferase gene which ensures the breakdown and efficacy of Azathioprine. The thiopurine methyltransferase single nucleotide polymorphism profiles of various American, Asian and European populations have been studied, but little literature is available for the South African population. The aim of this study was to determine if it is essential to include "early warning" single nucleotide polymorphism testing in South African health care before treatment with Azathioprine is initiated. This was performed by evaluating 40 patients suffering from inflammatory bowel disease who met the inclusion/ exclusion criteria and who were on continuous Azathioprine therapy. The prevalence of *3A, *3B or *3C thiopurine methyltransferase gene SNP’s were determined to assess the efficacy of Azathioprine in reducing therapeutic markers and to compare the frequency of SNP’s within the Azathioprine dosing groups. This study showed an increase in the allelic frequency of thiopurine methyltransferase *3B and a statistically significant presence (p<0.001) of homozygous thiopurine methyltransferase *3B alleles in South African inflammatory bowel disease patients when compared to healthy South African individuals- this has not previously been reported in published literature. Thus, in future, the enzymatic effect of thiopurine methyltransferase *3B/*3B should be studied in a larger sample size prior to recommending early warning single nucleotide polymorphism testing in inflammatory bowel disease patient using Azathioprine as these results cannot be ignored.
Comparison of metabalome attenuation in monolayer and three dimensional hepatocyte cultures
(University of Pretoria, 2019) Cromarty, Allan Duncan; Hurter, Tineil; gerbendk@gmail.com; De Koker, Gerben
Introduction: Drugs have been removed from the market for years due to toxicity screening. The highest attrition from the market can be seen by drugs that cause hepatotoxicity. Most drugs are metabolised by phase I cytochrome enzymes with five cytochromes metabolising approximately 95% of drugs. One of the most used screening platforms is the immortalised hepatocyte cancer cell line: HepG2. As the common model displays almost no phase I metabolomic competency, HepG2 cells are under investigation for prediction of hepatotoxicity via phase I enzymes using both an affordable and reliable methodology. Multicellular spheroid cell models have been shown to be closer to physiological conditions than monolayer cell cultures. Thus, the aim of the study was to determine the degree to which long term exposure to a tailored cytochrome inducing drug cocktail could enhance cytochrome P450 activity, in both traditional monolayer and spheroid cell cultures, at the level of the metabolome Methods and materials A suspension spheroid model was used with an agarose mould which, post-optimization, formed 81 spheroids per well. Protein quantification, live-dead microscopy and cell cycle analysis was used to determine the cell viability and culture time. The sulfurhodamine B assay was used to determine the IC50 of each drug individually as well as the combined IC50 of the cocktail for induction. Cells were cultured in presence of an induction cocktail, containing five prototypical cytochrome inducers, continuously for three weeks in monolayers. Cells were then cultured in monolayer and spheroid formats for a further six days. The liquid chromatography mass spectrometry method was optimised by first optimising the chromatography then the mass spectrometer parameters. A single method was developed for liquid chromatography tandem mass spectrometry analysis of 10 analytes in one consecutive run. Cell culture supernatant was collected, and the developed method used to determine the change in parent drug versus metabolites. Results and discussion During the optimisation of the spheroids, cells were shown to be compromised by day seven. Therefore, the cells were only cultured for a total time of six days within the spheroid format, to ensure viability. Only two of the drugs appeared to be metabolised. Dextromethorphan was metabolised more in the monolayer format and midazolam was metabolised more in the spheroid format. The overall phase I metabolic capacity of the HepG2 cells were not increased by long-term culture in the presence of a drug cocktail when cultured in either the monolayer or the three-dimensional spheroid format. Conclusion Exposure to the drug cocktail containing phenacetin, dextromethorphan, diclofenac, midazolam and bupropion was assessed using the validated method established during this study. Metabolism was not consistent for the enzyme-combination, as implemented in this study, as only certain drugs were metabolised sufficiently to quantitate the metabolites. It remains unclear whether or not the measured metabolism is because of actual enzyme induction, from inherent metabolism potential or from further problems regarding abnormalities of other inherent metabolism (such as possible phase III transporter inactivity) of the cells.a
Identification of mycolic acid class ratios from Mycobacterium species using liquid chromatography-mass spectrometry
(University of Pretoria, 2019) Cromarty, Allan Duncan; naickerbrendon@gmail.com; Naicker, Brendon
Alongside the recent technology advancements in liquid chromatography (LC) and mass spectrometry (MS) instrumentation there has also been steady progress made with respect to the systematic lipidomic studies of Mycobacterium tuberculosis (M. tb) where hyphenated LC and MS instrumentation were the main analytical technique employed. Although many reports have shown low limits of detection and high precision in analysing mycolic acids (MAs) and other M. tb lipids, few have reported efficient LC selectivity, even when employing high resolution mass spectrometry (HRMS) to determine the accurate mass of a lipid molecule for identification purposes. MAs are one of the most widely reported lipid classes that are found in the cell wall of M. tb and consist of a diversity of high mass branched chain of fatty acids that could potentially be used as diagnostic biomarkers for determining active M. tb infections. The chemical diversity of MAs can be used for taxonomic identification of mycobacterial species, but their analysis is complicated due to their extreme hydrophobic properties and homologous chemistry. Three dominant subclasses of MA molecules are found in mycobacteria: alpha-(α-), ketoand methoxy-MAs. When analysing MAs using MS, the product ions obtained from the infusion of a purified mixture of MAs, extracted from M. tb H37Rv strain, on a tandem mass spectrometer (MS/MS) are m/z 367.3 and 395.4, which correlate with published data but are not unique to single MA classes and therefore cannot be used for the identification of a specific MA subclass molecule. The ratios of the diverse MA class precursor ions can however be used to predict the identity of M. tb strains. Further genetic assays are however required to confirm the taxonomic identification. In this study, self-extracted MAs (M. tb H37Rv strain) were compared to that of commercially available MAs (M. bovis strain) and by integrating the chromatographic peak area of each dominant precursor ion within a MA class, a peak area ratio between the α-, keto- and methoxy- MAs were determined. The resulting percentage ratios between α-, keto- and methoxy- MA classes were found to be 53:8:38 for M. tb H37Rv and 53:15:32 for M. bovis respectively. The LC data dependent acquisition (DDA) precursor ion method, developed here specifically for the MA ratio comparison, has also shown the ability to resolve isomers within a MA class that has not previously been reported. Two other classes of mycobacterial lipids, phthiocerol dimycocerosates (DIMa) and phthiodiolone dimycocerosates (DIMb), collectively called PDIMs, have recently emerged as lipids which play a significant role in the virulence of drug resistant M. tb. Hence, a robust LC-HRMS method was developed for the analysis of extractable non-polar lipids extracted from three different drug resistant phenotypes of M. tb from clinical isolates and compared to a drug susceptible M. tb H37Rv clinical isolate. dimycocerosates (DIMa) and phthiodiolone dimycocerosates (DIMb), collectively called PDIMs, have recently emerged as lipids which play a significant role in the virulence of drug resistant M. tb. Hence, a robust LC-HRMS method was developed for the analysis of extractable non-polar lipids extracted from three different drug resistant phenotypes of M. tb from clinical isolates and compared to a drug susceptible M. tb H37Rv clinical isolate. In future, the feasibility of using LC-HRMS as a routine technique to phenotype M. tb drug resistant strains may require a mass analyser with a mass resolution in excess of 100 000, as well as chromatographic technologies that are capable of resolving non-derivatized complex mixtures of large (C60-C100) nonpolar and/or polar lipid molecules.
Mass spectrometric proteomic profiling of acute wound healing processes in the porcine model
(University of Pretoria, 2019) Cromarty, Allan Duncan; Stoychev, Stoyan; kbaron19@gmail.com; Sheva, Kim Lisa
Wounds are a common occurrence and generally heal well within a short time depending on whether tissue needs to be replaced or not. Although there is an understanding of the physiological processes taking place during wound healing, the changes taking place at the molecular level as well as the causes of chronic wounds are not well defined. The purpose of this study was a proof of concept making use of complementary current mass spectrometric techniques to better define the spatial and kinetic changes occurring during healing of well-defined acute wounds across proteomic, metabolomic and lipidomic bases. Several wound tissue preparation techniques, analytical approaches, data conversion methodologies and statistical tools were assessed to best visualise, characterise and identify molecular features in the complex and continuously changing environment of the acute wound at six pre-selected time points over 16 days of healing. Different mass spectrometric approaches to analyse snap frozen histological sections of wound tissue collected from a porcine wound healing model allowed ‘omic’ analyses to provide a better understanding of the acute wound healing process. Unidentified features found at key time points were compared to provide a foundation to develop an acute wound healing model, which could be applied to non-healing chronic wounds. An initial animal study involved full-skin thickness wounds being surgically created in the porcine model using strictly controlled wound formation and the subsequent healing thereof accurately investigated. Photographic and histological analyses of resected wound tissue were used to track the sequential yet overlapping phases of the acute wound healing pathway. The spatial resolution capabilities of matrix-assisted laser desorption ionisation imaging mass spectrometry (MALDI-IMS) was linked to histological staining and localised healthy reference tissue to successfully characterise changes in wound tissue. Optimised multivariate analyses and statistical analysis could statistically characterise the kinetic changes and revealed molecular features and key time points during acute wound healing from proteomic, metabolomic and lipidomic assay data collected individually from sequential tissue sections from the same wound. Methods for extraction of soluble protein from histological wound tissue sections were compared and further analysed using hydrophilic interaction-based bead capture with in-solution tryptic digestion followed by classic proteomic liquid chromatography tandem mass spectrometry (ESI-LC-MS/MS) analysis. The extracted proteins were also assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis where protein mass fingerprints were visualised and major protein bands excised, followed by standard in-gel digestion and LC-ESI-MS/MS analysis. All mass spectral proteomic data was subject to bioinformatic analysis using open source packages. A comparative analysis of in-solution versus on-tissue sample preparation was performed to determine optimal methodology for tissue protein extraction and identification. The optimised protocol was used to identify proteins across six time points and 16 days showing potential as predictive acute wound healing phase features. A total of 18 proteins showed potential as acute wound healing phase markers through relative quantitative change and functional healing relation. Biological functions of these proteins included involvement in adaptive and innate immunity, inflammation, cellular adhesion and various biosynthetic processes, and may be used as validating features of acute wound healing in clinical settings or as new drug targets for healing therapies. This study revealed the best time range and individual features that could be used to most accurately represent essential molecular environmental changes in the progression of healing of acute wounds across proteomic, metabolomic and lipidomic bases. These approaches may aid in comparative healing studies, such as those seeking to better define differences that can be linked to chronic wounds. Using a well-defined but reduced sample set of acute wound molecular environment data as a fixed comparable model, as well as potential markers of successful healing, may simplify the approach and aid in the identification of potential drug targets in non-healing chronic wounds.
Spatial characterization of the BT-20 triple negative breast cancer spheroidal model
(University of Pretoria, 2019) Cordier, Werner; Steenkamp, Vanessa; Cromarty, Allan Duncan; Van den Bout, Jan Iman; keith07ncube@gmail.com; Ncube, Keith Ntokozo
Breast cancer is a commonly diagnosed cancer in women, with increasing diagnosis and mortality rates. The triple-negative sub-type of the disease is characterised by lack of hormone-receptor overexpression, exhibition of poorly characterised molecular aberrations, and treatment failure. Chemotherapy is the current mainstay; however, it fails to slow tumor progression, and this is partially attributed to the lack of characterisation of biological features that drive treatment failure. There is, therefore, a need to characterise the biological features of triple-negative breast cancer, in order to develop effective therapies against the disease. Researchers widely use monolayer cell culture in pre-clinical screening of anticancer drugs. However, the development of effective anti-cancer drugs is hampered by limitations inherent to these culture systems, as they insufficiently mimic the physiological characteristics of tumours in vivo. Spheroids have been suggested as a bridge to the gap between monolayers and animal models, as they combine the flexibility and cost-effectiveness of cell culture with the spatial and molecular attributes of tissue. This study aimed to grow and characterise a spheroid model of triple-negative breast cancer with regards to growth, morphology, and drug sensitivity. The hanging drop and liquid overlay techniques were compared to select a method for growing spheroids. Spheroid growth was assessed using phase contrast microscopy and the bicinchoninic acid assay. Viability was assessed using the fluorescein diacetate (FDA)/propidium iodide (PI) assay. Haematoxylin and eosin staining were used for morphological evaluation. An iridium complex was used to investigate the induction of hypoxia. The sulphorodamine B and acid phosphatase assays, FDA/PI staining and phase contrast microscopy were used to assess the 72-h cytotoxicity effects of doxorubicin in monolayers and spheroids. Immunostaining and optical clearing were used to visualise the spatial distribution of the Ki-67 antigen and cadherins in spheroids.
The combined effects or arsenic cadmium and mercury on hepatocarcinoma and neuroblastoma cells in vitro
(University of Pretoria, 2018) Steenkamp, Vanessa; Cordier, Werner; u16136022@tuks.co.za; Yousaf, Muhammad
Among several heavy metallic elements, arsenic, cadmium and mercury are most toxic in the environment. These metals are acquired by humans through food and water, which result in severe health related issues. One of the most toxic metals is arsenic and it is derived from the natural environment. The main source of arsenic toxicity is due to contamination of drinking water from natural geological sources rather than from mining, smelting, or agricultural sources. Another naturally occurring highly toxic heavy metal is cadmium and possesses considerable toxicity with destructive impact on most organ systems. To date cadmium has no physiological function in the human body. Mercury is another important toxic metal found in nature and noted for inducing public health disasters. The current study has been considered to investigate individual and combined toxic effects in HepG2 hepatocarcinoma and SH-SY5Y neuroblastoma cell lines as a measure of hepatotoxicity and neurotoxicity, respectively. The aim was achieved through investigation of individual metals and their combinations on cell density, mitochondrial membrane potential, adenosine triphosphate levels, reactive oxygen species generation, glutathione levels and caspase-3/7 activity. Arsenic displayed gradual, dose-dependent cytotoxicity in both cell lines. In the HepG2 cells, IC50 of arsenic was determined as 6.71 mg/L and in the SH-SY5Y cells, IC50 of 1.19 mg/L. Arsenic had minimal effects on HepG2 mitochondrial membrane potential at IC50 (~12%). A gradual, tapered reduction (25% to 62%) was observed in the SH-SY5Y cell line from 0.94 mg/L to 2.78 mg/L. This decline parallels the reduction in cell density in the SH-SY5Y cell line. Arsenic profoundly decreased adenosine triphosphate levels at IC75 (9.61 mg/L for HepG2 and 2.78 mg/L for SH-SY5Y cells) for both cells indicating mitochondrial toxicity. A decline in reactive oxygen species level was noted for both cells after 24 h incubation with arsenic. The glutathione was reduced by 35%, 64% and 94% in HepG2 cells and 47%, 67% and 96% in SH-SY5Y cells which paralleled the results obtained for cell density, adenosine triphosphate levels and mitochondrial membrane potential. Mitochondrial toxicity lead to an increase in caspase-3/7 in both cell lines. Greater cytotoxicity was displayed in the HepG2 cells after cadmium exposure (IC50 = 0.43 mg/L) than the SH-SY5Y cells (IC50 = 1.47 mg/L). The dose dependent mitochondrial depolarization leads to mitochondrial toxicity and there is a direct correlation to the reduction in cell density. Cadmium decreased adenosine triphosphate levels when exposed to the IC25 (0.22 mg/L) by 39%, by 62% at IC50 (0.43 mg/L) and by 88% at IC75 (1.11 mg/L). The trend was similar to that detected for arsenic, albeit being less toxic. In SH-SY5Y cells, there was complete reduction in adenosine triphosphate levels as was found by arsenic. A gradual decrease in reactive oxygen species was noted in both cells after exposure to cadmium. The reduction in glutathione levels related to cell density as well as mitochondrial membrane potential reduction. The increase in caspase 3/7 activity in SH-SY5Y cells was nearly double that observed in HepG2 cells at the same concentrations tested indicating apoptosis. Mercury was more cytotoxic towards SH-SY5Y cells (IC50 = 11.99 mg/L) than HepG2 cells (IC50 = 26.23 mg/L). Mercury was found to be the least toxic among all three metals tested. Mercury decreased mitochondrial membrane potential and reactive oxygen species in both cells in a dose dependent manner. The adenosine triphosphate concentration was almost completely inhibited in HepG2 cells while a more gradual decrease in the SH-SY5Y cells of 30%, 77% and 99% when exposed to IC25 (8.45 mg/L), IC50 (11.99 mg/L) and IC75 (14.36 mg/L) concentrations of mercury, was noted. The glutathione reduction in both cell lines was dose dependent, with virtually total inhibition (99%) of glutathione when treated with the IC75 concentration of mercury. This reduction in glutathione levels correlates with the reduction in cell density, reduction in adenosine triphosphate level as well as the loss in mitochondrial membrane potential. Mercury decreased caspase-3/7 in HepG2 cells from basal level at all concentrations tested. In contrast caspase-3/7 activity was increased by 320%, 181% and 327% in SH-SY5Y cells when exposed to IC25 (8.45 mg/L), IC50 (11.99 mg/L) and IC75 (14.36 mg/L) concentrations of mercury. After exposure to the IC and EPA mixtures, the reduction in cell density was far greater than that observed for any of the single metals alone in HepG2 cells, which may imply additive or synergistic activity. Combinations of metal mixtures displayed greater mitochondrial toxicity than the metals alone. This was most prominent in the HepG2 cell line. The combination mixtures in both the cell lines almost completely inhibited adenosine triphosphate levels indicating cell death from apoptotic to necrotic pathways. Reactive oxygen species was reduced in a dose dependent manner in both cell lines. The IC combinations abolished glutathione levels completely in hepatoma and neuronal cells. All the EPA combinations reduced glutathione levels in a dose dependent manner which paralleled what was noted for cell density, mitochondrial membrane potential and adenosine triphosphate levels. An increase in caspase 3/7 activity was noted when the cells were exposed to 4mg/L EPA mixture concentration with respect to arsenic (505%) indicating additive or synergistic effect. Understanding how metal mixtures affect health is critical to decide on treatment strategies. The results indicate the potential mechanistic routes of cytotoxicity incurred by the heavy metals and their combinations. Cytotoxicity of metal mixtures was more pronounced than when cells were exposed to individual metals.
The effect of three Ghanaian plants on fibroblast migration, inflammation and bacterial growth in vitro
(University of Pretoria, 2018) Steenkamp, Vanessa; Cordier, Werner; u16396929@tuks.co.za; Yahaya, Ewura Seidu
Medical conditions which cause morbidity and mortality, such as chronic wounds and infection, lead to significant medical costs. Much of the world's population is dependent on alternative medicine, of which herbal medicine forms a crucial part. In Ghana and other countries around the world, an estimated 70% of the population rely on alternative treatments like herbs for management of various forms of disease. However, even though medicinal plants are widely used for treatment, most have not been scientifically proven to be safe and efficacious. Hence there is need to assess the biological activity of these plants which may be a potential lead in drug development. This study assessed the wound-related biological activities of three commonly used medicinal plants in Ghana (Aspilia africana, Boerhavia diffusa, and Erythrina senegalensis). Sequential extracts were prepared from the three plants using hexane, ethyl acetate, methanol, and water as solvents, in increasing polarity. Also, ethnomedicinal extracts were obtained with water as solvent, in accordance with the method used by traditional healers. Extracts were screened for phytochemical components using thin layer chromatography (TLC), and phytochemical fingerprinting performed with ultra performance liquid chromatography in tandem with time-of-flight mass spectrometry (UPLC-qTOF-MS). Cytotoxic potential of the extracts in SC-1 fibroblasts, C2C12-myoblasts, and differentiated THP-1-macrophages was determined using the sulforhodamine B staining assay, and cells morphologically assessed with phase contrast, PlasDIC, and live/dead staining microscopy. Acellular antioxidant activity was conducted by exploring the 2,2'-azino-bis[3-ethylbenzothiazoline-6-sulphonic acid] (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, whilst ability to protect against cellular oxidative stress was assessed using 2',7'-dichlorofluorescin diacetate as marker. Anti-inflammatory potential of extracts was evaluated using xanthine oxidase activity. Also, the ability of extracts to alter closure of artificially generated wounds in fibroblast and myoblast monocultures was determined using the scratch assay. The extracts were also assessed for their antibacterial effect using the disk diffusion and microdilution assays. Extracts with a minimum inhibitory concentration (MIC) above or equal to 1 mg/mL were evaluated for their ability to inhibit bacterial biofilms. Analysis using TLC indicated that alkaloids, flavonoids, and phenols were the major groups present. The UPLC-qTOF-MS analysis led to the verification of previously identified, as well as tentative identification of already-described phytochemical compounds or their derivatives in the plants. In addition to the already reported ascorbic acid, quercetin was also identified in A. africana. Furthermore, kaempferol, quercetin, and rutin were identified in the extracts of B. diffusa, the latter compound being identified for the first time in this plant. Also, for the first time, kaempferol, rotenone and rutin, were identified in extracts of E. senegalensis. Apart from these three compounds, neobavaisoflavone was also detected. All the extracts recorded a half maximal inhibitory concentration (IC50) above 100 _g/mL in all three cell-lines. The most cytotoxic extracts to the myoblasts, fibroblasts, and macrophages, were the methanol and ethnomedicinal extracts of A. africana, and the hexane extract of E. senegalensis, with a maximum of 38.8% reduction in cell density respectively. The minimal cytotoxic potential of the extracts was further confirmed by the absence of morphological differences between treated and untreated cells. Most of the extracts exhibited good ABTS radical scavenging activity (IC50 < 100 _g/mL). The strongest effect against the free radical was observed with the ethyl acetate extract of B. diffusa (IC50 = 21.23 _g/mL). On the contrary, most of the extracts recorded poor ability to scavenge the DPPH free radical. Only the methanol extracts of A. africana (IC50 = 278 _g/mL) and E. senegalensis (IC50 = 291 _g/mL) yielded IC50 values below the maximum tested concentration (320 _g/mL). This could possibly be ascribed to the differences in the stereoselectivity between the two free radicals, and the poor DPPH scavenging ability of hydrophilic antioxidants. The effect of the extracts against AAPH-induced oxidation in the cells correlated with the antioxidant potential of the extracts. Whilst most of the extracts with good antioxidant potential suppressed AAPH-induced oxidative stress, the most profound effect was observed with pre-treatment of macrophages with the ethyl acetate extract of A. africana. The extract caused a 1.74-fold decrease in intracellular reactive oxygen species (ROS) concentration after 120 min following pre-treatment with 100 _g/mL, when compared with the AAPH control. This was comparable to the 1.89-fold reduction caused by the positive control compound, 5 _g/mL Trolox. The ethnomedicinal extracts of B. diffusa and E. senegalensis exhibited a dose-dependent increase in intracellular ROS in fibroblasts, with intracellular ROS concentration upon treatment with the extracts at 100 _g/mL being at least 23% higher than the negative controls. This suggest that the extracts could exhibit a possible pro-oxidant effect at higher concentrations. Quercetin, a compound with pro-oxidant effects at higher concentrations, was detected in the ethyl acetate extract of B. diffusa, which may describe this effect. However, none of the extracts used in the current study demonstrated the ability to significantly inhibit xanthine oxidase activity. The strongest activity against the enzyme (maximum of 15% inhibition) was exhibited by extracts of E. senegalensis. The hexane extract of A. africana and the water extracts of B. diffusa increased migration of myoblast cells by 44.4% and 39.4%, respectively. This indicates a possible role of the extracts in enhancing collagen deposition and wound remodelling, two processes with myoblast involvement. On the other hand, six of the extracts decreased fibroblast migration, and therefore could have negative effects on wound healing processes such as collagen and matrix metalloproteinase synthesis. Further analysis would be required to ascertain the extent to which the extracts could impact activity of the cells. Also, the methanol extract of E. senegalensis (MIC = 0.5 mg/mL in E. coli) was the most effective against the micro-organisms tested. All the other extracts had MICs above 1 mg/mL. None of the extracts showed activity against Pseudomonas aeruginosa, Staphylococcus epidermidis and bacterial biofilms. In conclusion, this study has scientifically demonstrated that the three plants may assist wound healing at different stages in the healing process. This could be achieved through their antioxidant effects, ability to suppress oxidative stress, antibacterial activity, and ability to enhance activity of fibroblasts and myoblasts. Practitioners should be cautioned against using high concentrations because of possible cytotoxicity.
Comparative analysis of a two-dimensional and three-dimensional model of BT-20 triple negative breast carcinoma cells in response to an antiproliferative agent
(University of Pretoria, 2018) Cordier, Werner; Cromarty, Allan Duncan; Van den Bout, Jan Iman; WANGJIEGIDEON@GMAIL.COM; Wang, Jie
Triple-negative breast cancer (TNBC) lacks the expression of estrogen receptor-alpha, progesterone receptor and human epidermal growth factor receptor 2 (HER2). The lack of dependence on estrogen by TNBC cells makes anti-estrogen chemotherapy ineffective. Compounding this, within solid tumors, differential blood supply creates an oxygen and nutrient gradient, providing cells close to the vasculature with a more hospitable environment, while those in the core are deprived. In the search for treatments that may display efficacy against such tumours, it is necessary to make use of in vitro systems that accurately depict the clinical setting. Traditional two-dimensional (2D) culturing fails to replicate this environment, however, they are commonly used when assessing biological activity of new chemical entities. Three-dimensional (3D) cultures, in the form of spheroids, should enact a similar gradient which includes the proliferative outer layer, a quiescent inner zone and a necrotic center. The aim of the study is to compare the growth characteristics of BT-20 triple-negative breast carcinoma cells in a traditional 2D culture to a 3D model established by the Department of Physiology, University of Pretoria. BT-20 spheroids (40 000 cells/well) were grown using traditional culturing and the liquid overlay method for monolayer (2D) and spheroid (3D) cultures, respectively. Spheroid volume was assessed using light microscopy, while viability was visualized by live-dead staining. Metabolic capacity was determined using the resazurin cleavage assay. Protein content was determined using the bicinchoninic acid assay. Cytotoxicity of doxorubicin was determined in monolayers by sulforhodamine B staining after 72 h. Monolayer cultures and spheroids (day 4) were exposed to the IC25, IC50 and IC75 of doxorubicin for 72 h, after which protein content and acid phosphatase (APH) activity were determined using spectrophotometry, cellular kinetics by flow cytometry, and p53 expression detected by Western blot analysis. BT-20 spheroids displayed structural integrity and viability over the growth period, with decreasing size and increasing numbers of membrane compromised cells (suggestive of necrosis) at Day 4. No necrosis was observed at Days 7 or 10. Due to spheroids compaction and lack of resorufin formation, metabolic activity could not be assessed accurately, highlighting the density of the spheroid as a potential contributor to reduced drug susceptibility. Neither spheroid protein content nor APH activity changed throughout the culturing period, while the monolayer cultures presented with higher values. Doxorubicin displayed an IC25, IC50 and IC75 of 1.4 _M, 3.6 _M and 11.75 _M respectively in monolayer cultures. Spheroid size, protein content and APH activity was affected only at the IC75, accompanied by an increase in the percentage of sub-G1-phase cells linked to a reduction in G1-phase cells. Lower doxorubicin concentrations resulted in increased spheroid size, protein content and APH activity. Expression of p53 was non-significantly increased after exposure to the IC25 of doxorubicin in both models, however, expression was lower in spheroids than in monolayers. Non-significant alterations to cell cycle kinetics was evident, with decreased G0/G1-phase cells, increased G2/M-phase cells and increased p53 expression, which suggest that a late cell cycle blockade was induced. In addition, the non-significant lower expression of p53 in treated spheroids suggests that the 3D-conformation exhibited reduced chemosensitivity to doxorubicin. Cultured 3D spheroids presented with higher resistance to doxorubicin compared to monolayer cultures. Given the nature of in vivo tumours, a 3D model as platform for drug screening may present as a more representative model during drug development studies.

Browse

Recent Submissions