Amino acid variation at VP1-145 of enterovirus A71 determines the viral infectivity and receptor usage in a primary human intestinal model

Enterovirus A71 (EV-A71) can elicit a wide variety of human diseases such as hand, foot, and mouth disease and severe or fatal neurological complications. It is not clearly understood what determines the virulence and fitness of EV-A71. It has been observed that amino acid changes in the receptor binding protein, VP1, resulting in viral binding to heparan sulfate proteoglycans (HSPGs) may be important for the ability of EV-A71 to infect neuronal tissue. In this study, we identified that the presence of glutamine, as opposed to glutamic acid, at VP1-145 is key for viral infection in a 2D human fetal intestinal model, consistent with previous findings in an airway organoid model. Moreover, pre-treatment of EV-A71 particles with low molecular weight heparin to block HSPG-binding significantly reduced the infectivity of two clinical EV-A71 isolates and viral mutants carrying glutamine at VP1-145. Our data indicates that mutations in VP1 leading to HSPG-binding enhances viral replication in the human gut. These mutations resulting in increased production of viral particles at the primary replication site could lead to a higher risk of subsequent neuroinfection.ImportanceWith the near eradication of polio worldwide, polio-like illness (as is increasingly caused by EV-A71 infections) is of emerging concern. EV-A71 is indeed the most neurotropic enterovirus that poses a major threat globally to public health and specifically in infants and young children. Our findings will contribute to the understanding of the virulence and the pathogenicity of this virus. Further, our data also supports the identification of potential therapeutic targets against severe EV-A71 infection especially among infants and young children. Furthermore, our work highlights the key role of HSPG-binding mutations in the disease outcome of EV-A71. Additionally, EV-A71 is not able to infect the gut (the primary replication site in humans) in traditionally used animal models. Thus, our research highlights the need for human-based models to study human viral infections.Graphical Abstrac

Impact of selected herbal products on intestinal epithelial permeation and metabolism of indinavir

MSc (Pharmaceutics), North-West University, Potchefstroom Campus, 2015Patients on anti-retroviral (ARV) drug treatment are sometimes simultaneously taking other prescribed drugs and/or over-the-counter drugs and/or herbal remedies. Pharmacokinetic drug-drug or herb-drug interactions can occur in these patients, which might be synergistic or antagonistic in nature leading to increased or decreased bioavailability of the ARV. Consequences of bioavailability changes may either be adverse effects due to increased plasma levels, or lack of pharmacological responses due to decreased plasma levels. The aim of this study is to determine if pharmacokinetic interactions exist between selected commercially available herbal products, namely Linctagon Forte®, Viral Choice® and Canova® and the ARV, indinavir, in terms of transport and metabolism in cell culture models. Bi-directional transport of indinavir was evaluated across Caco-2 cell monolayers in four experimental groups, namely indinavir alone (200 μM, negative control group), indinavir in combination with Linctagon Forte®, indinavir in combination with Viral Choice® and indinavir in combination with Canova® at three different concentrations. Verapamil (100 μM), a known P-gp inhibitor, was combined with indinavir in the positive control group. Samples obtained from the transport studies were analysed by means of a validated high performance liquid chromatography (HPLC) method. The apparent permeability coefficient (Papp) values were calculated from the transport results in both directions and the efflux ratio (ER) values were calculated from these Papp values. The metabolism of indinavir was determined in LS180 cells in the same groups as mentioned for the transport study but with ketoconazole (40 μM), a known CYP3A4 inhibitor, as the positive control group. Indinavir and its predominant metabolite (M6) were analysed in the metabolism samples by means of liquid chromatography linked to mass spectroscopy (LC/MS/MS) to determine the effect of the herbal products on the biotransformation of indinavir. The BL-AP transport of indinavir increased in a concentration dependent way in the presence of Linctagon Forte® and Viral Choice® when compared to that of indinavir alone (control group). Canova® only slightly affected the efflux of indinavir compared to that of the control group. Noticeable increases in the efflux ratio values of indinavir were found for Linctagon Forte® and Viral Choice®, whilst the effect of Canova® on the efflux ratio value was negligible. There was a pronounced inhibition of the metabolism of indinavir in LS180 cells over the entire concentration range for all the herbal products investigated in this study. These in vitro pharmacokinetic interactions indicate the selected herbal products may affect indinavir’s bioavailability, but the clinical significance needs to be confirmed with in vivo studies before final conclusions can be made.Master

Establishing three-dimensional cell culture models to measure biotransformation and toxicity

PhD (Pharmaceutics), North-West University, Potchefstroom Campus, 2018.A great proportion of new chemical entities will be terminated from the clinical drug development pipeline as a result of deficiencies in drug absorption, distribution, biotransformation and elimination as well as potential to cause toxicity. Exposure of the liver (and other organs) to hepatotoxins, may potentially interact with cellular constituents, causing toxicity and various lesions. The pre-clinical assessment of hepatotoxic potential of new drug entities and herbal compounds are investigated on a tissue, cellular and molecular level by employing various in vitro and in vivo techniques. The in vitro models currently available mainly involve traditional two-dimensional (2D) cell culture techniques; however, these models lack various tissue specific properties found in the in vivo environment. As a result, pre-clinical assessment of drug hepatotoxicity and biotransformation still rely predominantly on in vivo animal models. To reduce the use of animal models, more reliable and readily available in vitro models are needed, capable of bridging the gap between the existing models and the in vivo situation. Three-dimensional (3D) spheroid cell cultures offer higher physiological relevance than traditional 2D cell cultures, overcoming many of the shortcomings associated with traditional 2D cell cultures. Specifically, the dynamic micro-tissue 3D spheroid cell culture system produced in micro-gravity bioreactors has attracted attention, although several other types of multi-cellular spheroid systems are also currently under investigation. This study investigated the potential of the 3D HepG2/C3A spheroid model to evaluate the acute and sub-chronic hepatotoxic potential of a crude aqueous Xysmalobium undulatum (Uzara) extract. Acute hepatotoxic effects were investigated in 2D and 3D HepG2/C3A cell cultures at concentrations of 200, 350, 500, and 750 mg/kg. Parameters evaluated included cell proliferation, glucose uptake, intracellular adenosine triphosphate (ATP) levels and adenylate kinase (AK) release. Furthermore, sub-chronic hepatotoxicity of crude Uzara aqueous extract was investigated during a sub-chronic 21-day study in the 3D HepG2/C3A spheroid model as well as in Sprague Dawley rats. The results from the in vitro study clearly indicated hepatotoxic effects and possible liver damage following treatment with valproic acid (the positive control group) as indicated by the growth inhibition observed, the loss of cell viability and the increased cytotoxicity as indicated by the reduced intracellular ATP levels and increased AK levels. The results also indicated that crude Uzara water extract had dose-dependent hepatotoxic potential, although the effects appeared to be exaggerated in the 2D cell cultures compared to the 3D spheroid cultures. The results was also supported by the increased in vivo levels of AST, ALT and LDH and the slight increase in triglycerides, following treatment of the Sprague Dawley rats with valproic acid. This is indicative of hepatic cellular damage, possibly resulting in hepatotoxicity. Similarly, following treatment with the crude Uzara aqueous extract, results obtained from the in vivo Sprague Dawley model indicated moderate hepatotoxic potential. The results confirmed the potential of the 3D HepG2/C3A spheroid model to effectively and reliably predict the long-term outcomes of possible hepatotoxicity. A novel 3D spheroid model for biotransformation applications was also developed, employing the LS180 cell line and micro-gravity bioreactors. The human colon carcinoma cell line, LS180, is often used as a biotransformation model to study inhibition and induction of CYP450 enzymes in vitro. The new three-dimensional cell culture model combined the dynamic rotating micro-gravity bioreactor technique with the micro-encapsulation technique, using sodium alginate. These encapsulated LS180 spheroids have the potential to be employed as a novel long-term culturing model for future in vitro biotransformation studies.NRF (National Research Foundation)Doctora

Herb-drug pharmacokinetic interactions: transport and metabolism of indinavir in the presence of selected herbal products

Patients receiving anti-retroviral drug treatment are sometimes simultaneously taking herbal remedies, which may result in pharmacokinetic herb-drug interactions. This study aimed to determine if pharmacokinetic interactions exist between selected commercially available herbal products (i.e., Linctagon Forte®, Viral Choice® and Canova®) and indinavir in terms of in vitro transport and metabolism. Bi-directional transport of indinavir was evaluated across Caco-2 cell monolayers in the presence and absence of the selected herbal products and verapamil (positive control). Metabolism of indinavir was determined in LS180 cells in the presence and absence of the selected herbal products as well as ketoconazole (positive control). The secretory transport of indinavir increased in a concentration dependent way in the presence of Linctagon Forte® and Viral Choice® when compared to that of indinavir alone. Canova® only slightly affected the efflux of indinavir compared to that of the control group. There was a pronounced inhibition of the metabolism of indinavir in LS180 cells over the entire concentration range for all the herbal products investigated in this study. These in vitro pharmacokinetic interactions indicate the selected herbal products may affect indinavir’s bioavailability, but the clinical significance needs to be confirmed with in vivo studies before final conclusions can be madeMedical Research Council (MRC) of South Afric

Recent advances in three-dimensional cell culturing to assess liver function and dysfunction: from a drug biotransformation and toxicity perspective

The liver is a vital organ fulfilling a central role in over 500 major metabolic functions, including serving as the most essential site for drug biotransformation. Dysfunction of the drug biotransformation processes may result in the exposure of the liver (and other organs) to hepatotoxins, potentially interacting with cellular constituents and causing toxicity and various lesions. Hepatotoxicity can be investigated on a tissue, cellular and molecular level by employing various in vivo and in vitro techniques, including novel three-dimensional (3 D) cell culturing methods. This paper reflects on the liver and its myriad of functions and the influence of drug biotransformation on liver dysfunction. Current in vivo and in vitro models used to study liver function and dysfunction is outlined, emphasizing their advantages and disadvantages. The advantages of novel in vitro 3 D cell culture models are discussed and the possibility of novel models to bridge the gap between in vitro and in vivo models is explained. Progression made in the field of cell culturing methods such as 3 D cell culturing techniques over the last decade promises to reduce the use of in vivo animal models in biotransformation and toxicological studies of the live

Anticancer Potential of Sutherlandia frutescens and Xysmalobium undulatum in LS180 Colorectal Cancer Mini-Tumors

Colorectal cancer remains to be one of the leading causes of death worldwide, with millions of patients diagnosed each year. Although chemotherapeutic drugs are routinely used to treat cancer, these treatments have severe side effects. As a result, the use of herbal medicines has gained increasing popularity as a treatment for cancer. In this study, two South African medicinal plants widely used to treat various diseases, Sutherlandia frutescens and Xysmalobium undulatum, were evaluated for potential activity against colorectal cancer. This potential activity for the treatment of colorectal cancer was assessed relative to the known chemotherapeutic drug, paclitaxel. The cytotoxic activity was considered in an advanced three-dimensional (3D) sodium alginate encapsulated LS180 colorectal cancer functional spheroid model, cultured in clinostat-based rotating bioreactors. The LS180 cell mini-tumors were treated for 96 h with two concentrations of each of the crude aqueous extracts or paclitaxel. S. frutescens extract markedly decreased the soluble protein content, while decreasing ATP and AK per protein content to below detectable limits after only 24 h exposure. X. undulatum extract also decreased the soluble protein content, cell viability, and glucose consumption. The results suggested that the two phytomedicines have potential to become a source of new treatments against colorectal cancer

Toxicity and anti-prolific properties of Xysmalobium undulatum water extract during short-term exposure to two-dimensional and three-dimensional spheroid cell cultures

Xysmalobium undulatum (Uzara) is one of the most widely used indigenous traditional herbal remedies in Southern Africa. Commercially available Uzara plant material was used to prepare a crude aqueous extract, of which the toxicity potential was investigated in the hepatic HepG2/C3A cell line in both traditional two-dimensional (2D) and rotating three-dimensional (3D) spheroid cell cultures. These cultures were treated over a period of 4 days at concentrations of 200, 350, 500, and 750 mg/kg plant extract to protein content. Basic physiological parameters of the cell cultures were measured during exposure, including cell proliferation, glucose uptake, intracellular adenosine triphosphate levels, and adenylate kinase release. The results indicated that all physiological parameters monitored were affected in a dose dependent manner, with the highest concentration of Uzara crude water extract (750 mg/kg) resulting in toxicity. Anti-proliferating effects of Uzara crude water extract were observed in both the 2D and 3D cell cultures, with the most pronounced effects at concentrations of 350, 500, and 750 mg/kg. Discrepancies between results obtained from the 2D and 3D cell culture models may be attributed to the type of repair system that is initiated upon exposure, depending on where cells are within the cell cycle. DNA repair systems differ in cells within the G1 phase and non-diving cells, (i.e. cells found predominantly in in vitro 3D and the in vivo situation

Anthracyclins Increase PUFAs : Potential Implications in ER Stress and Cell Death

Metabolic and personalized interventions in cancer treatment require a better understanding of the relationship between the induction of cell death and metabolism. Consequently, we treated three primary liver cancer cell lines with two anthracyclins (doxorubicin and idarubin) and studied the changes in the lipidome. We found that both anthracyclins in the three cell lines increased the levels of polyunsaturated fatty acids (PUFAs) and alkylacylglycerophosphoethanolamines (etherPEs) with PUFAs. As PUFAs and alkylacylglycerophospholipids with PUFAs are fundamental in lipid peroxidation during ferroptotic cell death, our results suggest supplementation with PUFAs and/or etherPEs with PUFAs as a potential general adjuvant of anthracyclins. In contrast, neither the markers of de novo lipogenesis nor cholesterol lipids presented the same trend in all cell lines and treatments. In agreement with previous research, this suggests that modulation of the metabolism of cholesterol could be considered a specific adjuvant of anthracyclins depending on the type of tumor and the individual. Finally, in agreement with previous research, we found a relationship across the different cell types between: (i) the change in endoplasmic reticulum (ER) stress, and (ii) the imbalance between PUFAs and cholesterol and saturated lipids. In the light of previous research, this imbalance partially explains the sensitivity to anthracyclins of the different cells. In conclusion, our results suggest that the modulation of different lipid metabolic pathways may be considered for generalized and personalized metabochemotherapies