Effect of the cardiac glycoside, digoxin, on neuronal viability, serotonin production and brain development in the embryo

Digoxin has been known as a treatment for chronic heart failure for over 200 years. Its effect on the heart itself has been extensively studied and its inotropic effect well established. The inotropic effect of digoxin is the result of its inhibition of the membrane sodium pump or Na+/K+-ATPase, which plays an important role in maintaining the resting membrane potential across the plasma membrane through constantly pumping Na+ and K+ across the plasma membrane. Na+/K+-ATPase is not found exclusively in heart muscle. It is also found extensively throughout the brain. As digoxin is the drug of choice for pregnant woman with chronic heart failure, this study aimed to examine how digoxin affects brain development and neurons in culture. The well established chicken embryo animal model was used in this study. To probe for deviations from normal brain development, chicken embryos were exposed in ovo. Brains were examined using both transmission and scanning electron microscopy. Microscopy indicated significant damage to the neurons, specifically membranes and mitochondria, as well as cellular death by means of aponecrosis. An unexpected result was premature myelinogenesis in the brain. Chick embryo neurons (CEN) were exposed to digoxin in vitro and cell viability was assessed by performing crystal violet (CV) assays. Results showed that cell number increased over time. This is however, impossible as CEN are non-dividing cells and results were therefore interpreted as an increase in protein synthesis over time, correlating with the myelinogenesis results seen with electron microscopy. To assess membrane integrity, fluorescence microscopy was performed using propidium iodide as stain. Results from this experiment showed a sharp increase in propidium iodide uptake in exposed cells indicative of the membrane damage caused by digoxin. These results also correlated with the aponecrosis seen with electron microscopy, as the nuclei indicated apoptosis while propidium iodide is normally only absorbed by cells undergoing necrosis. Finally, a literature search was conducted to shed some light on the role that digoxin plays in serotonin production and levels in the brain. From the literature it seems that digoxin could increase serotonin production and elevate serotonin levels in the brain, which may influence normal brain development and may therefore play a role in myelinogenesis in the brain.Dissertation (MSc (Anatomy))--University of Pretoria, 2008.Anatomyunrestricte

Development of an in vitro mechanistic toxicity screening model using cultured hepatocytes

In vitro testing includes both cell-based and cell-free systems that can be used to detect toxicity induced by xenobiotics. In vitro methods are especially useful in rapidly gathering intelligence regarding the toxicity of compounds for which none is available such as new chemical entities developed in the pharmaceutical industry. In addition to this, in vitro investigations are invaluable in providing information concerning mechanisms of toxicity of xenobiotics. This type of toxicity testing has gained popularity among the research and development community because of a number of advantages such as scalability to high throughput screening, cost-effectiveness and predictive power. Hepatotoxicity is one of the major causes of drug attrition and the high cost associated with drug development poses a heavy burden on the development of new chemical entities. Early detection of hepatotoxic agents by in vitro methods will improve lead optimisation and decrease the cost of drug development and reduce drug-induced liver injury. Literature highlights the need for a cellbased in vitro model that is capable of assessing multiple toxicity parameters, which assesses a wider scope of toxicity and would be able to detect subtle types of hepatotoxicity. The present study was aimed at developing an in vitro procedure capable of mechanistically profiling the effects of known hepatotoxin dichlorodiphenyl trichloroethane (DDT) and its metabolites, dichlorodiphenyl dichloroethylene (DDE) and dichlorodiphenyl dichloroethane (DDD) on an established liver-derived cell line, HepG2, by evaluating several different aspects of cellular function using a number of simultaneous in vitro assays on a single 96 well microplate. Examined parameters have been suggested by the European Medicines Agency and include: cell viability, phase I metabolism, oxidative stress, mitochondrial toxicity and mode of cell death (apoptosis vs. necrosis). To further assess whether the developed method was capable of detecting hepatoprotection, the effect of the known hepatoprotectant, N-acetylcysteine, was determined. Viability decreased in a dose-dependent manner yielding IC50 values of 54 μM, 64 μM and 44 μM for DDT, DDE and DDD, respectively. Evaluation of phase I metabolism showed that cytochrome P4501A1 activity was dose-dependently induced. Test compounds decreasedlevels of reactive oxygen species, and significantly hyperpolarised the mitochondrialmembrane potential. Assessment of the mode of cell death revealed a significant elevation of caspase-3 activity, with DDD proving to be most potent. DDT alone induced dosedependent loss of membrane integrity. These results suggest that the tested compounds produce apoptotic death likely due to mitochondrial toxicity with subsequent caspase-3 activation and apoptotic cell death. The developed in vitro assay method reduces the time it would take to assess the tested parameters separately, produces results from multiple endpoints that broadens the scope of toxicity compared to single-endpoint methods. In addition to this the method provides results that are truly comparable as all of the assays utilise the same batch of cells and are conducted on the same plate under the exact same conditions, which eliminates a considerable amount of variability that would be unavoidable otherwise. The present study laid a solid foundation for further development of this method by highlighting the unforeseen shortcomings that can be adjusted to improve scalability and predictive power.Thesis (PhD)--University of Pretoria, 2011.Pharmacologyunrestricte

A practical guide to the interpretation of PK/PD profiles of longer-acting analogue insulins. Part one: The principles of glucose clamp studies

Glucose clamp studies are used to determine pharmacokinetics (PK) and  pharmacodynamics (PD) of analogue insulins. With the development of longer-acting basal analogue insulins, including glargine 300 (Gla-300) and insulin degludec (IDeg), results from numerous glucose clamp studies are readily available. However, interpreting PK/PD profiles in a scientifically  sound manner can be a challenging feat. This is the first in a series of publications that will suggest practical tips for interpreting and comparing results from glucose clamp studies. Variations in the glucose clamp  methodology, duration of clamp studies and glucose clamp targets influence the study design and results significantly. Selection of study  populations, including healthy patients or patients with Type 1 or 2 diabetes mellitus, has important implications. The dose of study insulin should  reflect that of the general treatment population, and ideally steady-state conditions should be used. During the study the plasma insulin  concentration and glucose infusion rate describe the pharmacokinetics and pharmacodynamics of the study insulin. With these practical tips in mind, results of glucose clamp studies can be interpreted in a scientifically correct manner. The next article in this series will discuss the interpretation of PK/PD profiles using two newly developed longer-acting basal analogue insulins: Gla-300 and IDeg.Keywords: analogue insulins, glucose clamp, time–action profile, glucose infusion rate, pharmacokinetic

In vitro neuroprotective potential of four medicinal plants against rotenone-induced toxicity in SH-SY5Y neuroblastoma cells

BACKGROUND: Lannea schweinfurthii, Zanthoxylum capense, Scadoxus puniceus and Crinum bulbispermum are used traditionally to treat neurological disorders. The aim of this study was to evaluate the cytoprotective potential of the four plants, after induction of toxicity using rotenone, in SH-SY5Y neuroblastoma cells. METHODS: Cytotoxicity of the plant extracts and rotenone was assessed using the sulforhodamine B (SRB) assay. Fluorometry was used to measure intracellular redox state (reactive oxygen species (ROS) and intracellular glutathione content), mitochondrial membrane potential (MMP) and caspase-3 activity, as a marker of apoptotic cell death. RESULTS : Of the tested plants, the methanol extract of Z. capense was the least cytotoxic; LC50 121.3 ± 6.97 μg/ml, while S. puniceus methanol extract was the most cytotoxic; LC50 20.75 ± 1.47 μg/ml. Rotenone reduced intracellular ROS levels after 24 h exposure. Pre-treating cells with S. puniceus and C. bulbispermum extracts reversed the effects of rotenone on intracellular ROS levels. Rotenone exposure also decreased intracellular glutathione levels, which was counteracted by pre-treatment with any one of the extracts. MMP was reduced by rotenone, which was neutralized by pre-treatment with C. bulbispermum ethyl acetate extract. All extracts inhibited rotenone-induced activation of caspase-3. CONCLUSION : The studied plants demonstrated anti-apoptotic activity and restored intracellular glutathione content following rotenone treatment, suggesting that they may possess neuroprotective properties.The National Research Foundation (NRF) and the University of Pretoria, Research Committee, Faculty of Health Sciences (RESCOM)http://www.biomedcentral.com/1472-6882/13/353am201

Comparative toxicity of pentachlorophenol with its metabolites tetrachloro-1,2-hydroquinone and tetrachloro-1,4-benzoquinone in HepG2 cells

The organochlorine compound, pentachlorophenol (PCP), is classified as a hazardous substance. Its metabolite, tetrachloro-1,2-hydroquinone (TCHQ), has been detected in occupationally-exposed subjects and can readily be converted to tetrachloro-1,4-benzoquinone (TCBQ) under physiological conditions. Hazard characterization has previously identified the liver as the target organ of PCP toxicity in rats and dogs and as the liver is the major site of metabolism of the parent compound, this raises concern for the effects that the metabolites of PCP may have on the liver. Although the hepatotoxic effects of PCP have been described, less is known about the effects of its metabolites on hepatocyte function. Studying the effects of these metabolites on hepatocytes may provide valuable information regarding the effects that these compounds could exert on the liver itself and allude to the clinical manifestations of toxicity that can be expected. The aim of this study was therefore to assess the effect of PCP, TCHQ and TCBQ on the following cellular parameters: cell viability, mitochondrial membrane potential and intracellular ROS formation, as indicators of hepatocyte homeostasis. Both PCP and its metabolites, TCHQ and TCBQ decreased cell viability with IC50 of 68.05, 129.40 and 144.00 μM, respectively. All three compounds caused mitochondrial depolarization, with the effect being more profound following exposure to TCHQ and TCBQ. PCP did not induce any ROS generation, whereas TCHQ and TCBQ produced extensive ROS. Findings from this study suggest that in hepatocytes the mechanism of toxicity of PCP differs from that of its metabolites, TCHQ and TCBQ.This work was supported by a grant from the National Research Foundation of South Africa [FA2007041600014].http://www.bentham.org/open/totoxijam2013ay201

In vitro effect of N-acetylcysteine on hepatocyte injury caused by dichlorodiphenyltrichloroethane and its metabolites

The organochlorine pesticide, dichlorodiphenyltrichloroethane (DDT), is still used to combat the spread of malaria in several developing countries despite its accumulation and known hepatotoxic effects that have been demonstrated both in vitro and in vivo. N-Acetylcysteine (NAC) is a recognized hepatoprotective agent that has been reported to reduce hepatotoxicity initiated by many different compounds. The aim of this study was to determine whether NAC could counter in vitro hepatocyte injury induced by DDT or its two major metabolites, dichlorodiphenyldichloroethylene and dichlorodiphenyldichloroethane. HepG2 cell cultures were used to assess the following parameters of toxicity: cellular viability, intracellular levels of reactive oxygen species (ROS), mitochondrial membrane potential and initiation of apoptosis. None of the three test compounds induced ROS generation, yet exposure to any of the three compounds produced mitochondrial hyperpolarization, which was countered by NAC pretreatment. All three test compounds also induced apoptotic cell death, which was inhibited by NAC. Despite NAC counteracting some adverse intracellular changes due to organochlorine exposure, it appeared to aggravate the cytotoxic effects of the organochlorine compounds at low test concentrations. As the same outcome may also occur in vivo, results from the present study raise concern about the use of NAC as treatment for DDT-induced hepatotoxicity.National Research Foundation of South Africa [FA2007041600014].http://het.sagepub.com/hb2013ay201

Evaluation of the phenolic and flavonoid contents and radical scavenging activity of three southern African medicinal plants

Warburgia salutaris (Bertol. F.) Chiovs, Rhoicissus tridentata (L.f.) Wild & Drum and Terminalia sericea (Burch. ex DC.), are widely used medicinal plants in southern Africa. The aim of the study was to determine the phenolic and flavonoid content and evaluate the antioxidant activity of the three medicinal plants. Total phenolic and flavonoid contents were determined spectrophotometrically as gallic acid and rutin equivalents, respectively. Individual phenolic acids were identified by means of gas chromatography-mass spectrometry. Antioxidant activities of the crude extracts were assessed using the TEAC assay. The highest phenolic content was detected in the crude methanol extract of the bark of W. salutaris and the highest flavonoid content was found in the crude methanol extract of the leaves of this plant. In all the studied plants the alkaline hydrolysable fraction yielded a greater variety of phenolic acids compared to the soluble/free phenolic acid fractions. The three medicinal plants investigated were found to be strong radical scavengers supporting the traditional use of these medicinal plants.The Gauteng Department of Health and the National Research Foundation in South Africa.http://www.academicjournals.org/AJPPam201

A prospective, observational study comparing the PK/PD relationships of generic Meropenem (Mercide®) to the innovator brand in critically ill patients

INTRODUCTION : Clinicians’ skepticism, fueled by evidence of inferiority of some multisource generic antimicrobial products, results in the underutilization of more cost-effective generics, especially in critically ill patients. The aim of this observational study was to demonstrate equivalence between the generic or comparator brand of meropenem (Mercide®) and the leading innovator brand (Meronem®) by means of an ex vivo technique whereby antimicrobial activity is used to estimate plasma concentration of the active moiety. METHODS : Patients from different high care and intensive care units were recruited for observation when prescribed either of the meropenem brands under investigation. Blood samples were collected over 6 hours after a 30 minute infusion of the different brands. Meropenem concentration curves were established against United States Pharmacopeia standard meropenem (Sigma-Aldrich) by using standard laboratory techniques for culture of Klebsiella pneumoniae. Patients’ plasma samples were tested ex vivo, using a disc diffusion assay, to confirm antimicrobial activity and estimate plasma concentrations of the two brands. RESULTS : Both brands of meropenem demonstrated similar curves in donor plasma when concentrations in vials were confirmed. Patient-specific serum concentrations were determined from zones of inhibition against a standard laboratory Klebsiella strain ex vivo, confirming at least similar in vivo concentrations as the concentration curves (90% confidence interval) overlapped; however, the upper limit of the area under the curve for the ratio comparator/innovator exceeded the 1.25-point estimate, i.e., 4% higher for comparator meropenem. CONCLUSION : This observational, in-practice study demonstrates similar ex vivo activity and in vivo plasma concentration time curves for the products under observation. Assay sensitivity is also confirmed. Current registration status of generic small molecules is in place. The products are therefore clinically interchangeable based on registration status as well as bioassay results, demonstrating sufficient overlap for clinical comfort. The slightly higher observed comparator meropenem concentration (4%) is still clinically acceptable due to the large therapeutic index and should ally fears of inferiority.Ranbaxy (S.A) (Pty) Ltdwww.dovepress.comam2016Pharmacolog

The anti-inflammatory properties of simvastatin can benefit statin-naïve rheumatoid arthritis patients with associated risks for cardiovascular disease

The anti-inflammatory properties of statins are well documented. The aim of this study was to determine if statins may offer therapeutic benefits in rheumatoid arthritis (RA) patients that are also at risk for cardiovascular disease. Methods: Patients with moderately active RA, despite being on maximum disease-modifying anti-rheumatic drugs (DMARDs) therapy, and that were at risk for cardiovascular disease, were screened for inclusion. Eligible patients were randomised into two groups. In this open-label, cross-over design patients in group 1 received simvastatin treatment (20 mg/day) for a period of 3 months in addition to their usual DMARDs, after which they stopped simvastatin treatment and were followed up for a further 3 months while on their usual DMARDs only. Those in group 2 were allowed to continue on their usual DMARDs without simvastatin treatment for the first 3 months of the study, after which they received 20 mg/day simvastatin for a period of 3 months in addition to their usual DMARDs. Results: The addition of 20 mg simvastatin to conventional DMARDs produced significant improvements in all of the evaluated parameters. These include significant improvements in DAS28 score, tender joint count, swollen joint count, CRP levels and ESR, while patients were receiving simvastatin treatment. Conclusions: The addition of 20 mg simvastatin to conventional DMARDs in statin-naïve RA patients at risk for cardiovascular disease may add benefit, apart from modifying lipid profiles, by modulating immune function and suppressing disease activity.Department of Pharmacology at the University of Pretoriahttp://medpharm.tandfonline.com/loi/ojfp20hb201