Turning a Drug Target into a Drug Candidate: A New Paradigm for Neurological Drug Discovery?

The conventional paradigm for developing new treatments for disease mainly involves either the discovery of new drug targets, or finding new, improved drugs for old targets. However, an ion channel found only in invertebrates offers the potential of a completely new paradigm in which an established drug target can be re-engineered to serve as a new candidate therapeutic agent. The L-glutamate-gated chloride channels (GluCls) of invertebrates are absent from vertebrate genomes, offering the opportunity to introduce this exogenous, inhibitory, L-glutamate receptor into vertebrate neuronal circuits either as a tool with which to study neural networks, or a candidate therapy. Epileptic seizures can involve L-glutamate-induced hyper-excitation and toxicity. Variant GluCls, with their inhibitory responses to L-glutamate, when engineered into human neurons, might counter the excitotoxic effects of excess L-glutamate. In reviewing recent studies on model organisms, it appears that this approach might offer a new paradigm for the development of candidate therapeutics for epilepsy.     

Molecular and Cellular Effects of C-peptide—New Perspectives on an Old Peptide

New results present C-peptide as a biologically active peptide hormone in its own right. Although C-peptide is formed from proinsulin and cosecreted with insulin, it is a separate entity with biochemical and physiological characteristics that differ from those of insulin. There is direct evidence of stereospecific binding of C-peptide to a cell surface receptor, which is different from those for insulin and other related hormones. The C-peptide binding site is most likely a G–protein–coupled receptor. The association constant for C-peptide binding is approximately 3 × 10⁹M^-1. Saturation of the binding occurs already at a concentration of about 1 nM, which explains why C-peptide effects are not observed in healthy subjects. Binding of C-peptide results in activation of Ca²⁺ and MAPK-dependent pathways and stimulation of Na⁺,K⁺-ATPase and eNOS activities. The latter 2 enzymes are both deficient in several tissues in type 1 diabetes. There is some evidence that C-peptide, and insulin may interact synergistically on the insulin signaling pathway. Clinical evidence suggests that replacement of C-peptide, together with regular insulin therapy, may be beneficial in patients with type 1 diabetes and serve to retard or prevent the development of long-term complications.     

Loss of Control of HIV Viremia With OTC Weight‐Loss Drugs: A Call for Caution?

Improved survival achieved by HIV‐infected patients has complicated their medical care, as increasing numbers of comorbidities have led to polypharmacy and a higher risk of drug–drug interactions. Here, evidence is provided that weight‐loss drugs should be used with caution in HIV‐infected patients treated with lipophilic antiretroviral drugs because of the risk of virologic failure. This is particularly relevant considering that these agents are available on the market as over‐the‐counter medications, thus escaping the control of the physician.     

Physiology-Based Pharmacokinetic Modeling—Promise for Pediatric Drug Development?

Physiologically based modeling has gained much interest from pharmaceutical industry. Prediction of pharmacokinetic properties based on physicochemical properties and in vitro data appears possible. Two applications are of high interest: the prediction of the pharmacokinetics before conducting first in man studies based on preclinical data, and the prediction of pharmacokinetics in children based on the pharmacokinetics in adults. To date, only a few investigations describing the prediction of the pharmacokinetics in children have been published. Some of these investigations showed data on the precision of these predictions by comparing the model with experimental pharmacokinetic data form clinical investigations in children. However, the method holds the promise of speeding up drug development in children, by rationalizing study planning and thus avoiding unnecessary clinical studies. This would ultimately save costs and more importantly, reduce the risks for children in clinical studies. Unfortunately, most of the work done in this field is not published, as investigations are conducted by the pharmaceutical industry during drug development. Therefore, it is difficult to assess the success rate of this approach. However, as practical experience is gained and knowledge on drug-metabolizing enzymes and drug transporters increases, the value of this approach will probably increase in the next few years.     

Folate Deficiency after Anticonvulsant Drugs: An Effect of Hepatic Enzyme Induction?

Serum and red cell folate levels were reduced in 59% and 58% respectively of 75 children with epilepsy attending a residential school. The degree of folate deficiency was significantly related to increased hepatic microsomal enzyme activity, assessed from increased urinary excretion of D-glucaric acid and also correlated with the daily dose of anticonvulsant taken. Anticonvulsant drugs are known to have inducing properties, and since folate is required as a cofactor in drug hydroxylations it is suggested that folate depletion results from increased demand for the cofactor after induction of drug-metabolizing enzymes. As folate deficiency may ultimately limit drug metabolism this hypothesis would explain why blood phenytoin levels decrease and fit control may worsen after correction of folate deficiency in epileptic patients.     

Development of In Vitro 3D TissueFlex? Islet Model for Diabetic Drug Efficacy Testing

Increasing individuals diagnosed with type II diabetes pose a strong demand for the development of more effective anti-diabetic drugs. However, expensive, ethically controversial animal-based screening for anti-diabetic compounds is not always predictive of the human response. The use of in vitro cell-based models in research presents obviously ethical and cost advantages over in vivo models. This study was to develop an in vitro three-dimensional (3D) perfused culture model of islets (Islet TF) for maintaining viability and functionality longer for diabetic drug efficacy tests. Briefly fresh isolated rat islets were encapsulated in ultrapure alginate and the encapsulated islets were cultured in TissueFlex^®, a multiple, parallel perfused microbioreactor system for 7 days. The encapsulated islets cultured statically in cell culture plates (3D static) and islets cultured in suspension (2D) were used as the comparisons. In this study we demonstrate for the first time that Islet TF model can maintain the in vitro islet viability, and more importantly, the elevated functionality in terms of insulin release and dynamic responses over a 7-day culture period. The Islet TF displays a high sensitivity in responding to drugs and drug dosages over conventional 2D and 3D static models. Actual drug administration in clinics could be simulated using the developed Islet TF model, and the patterns of insulin release response to the tested drugs were in agreement with the data obtained in vivo. Islet TF could be a more predictive in vitro model for routine short- and long-term anti-diabetic drug efficacy testing.     

Perifosine Induces Cell Apoptosis in Human Osteosarcoma Cells: New Implication for Osteosarcoma Therapy?

Despite the advances of adjuvant chemotherapy and significant improvement of survival, the prognosis for patients with osteosarcoma is generally poor. The search for more effective anti-osteosarcoma agents is necessary and urgent. Here we report that perifosine induces cell apoptosis and growth inhibition in cultured human osteosarcoma cells. Perifosine blocks Akt/mTOR complex 1 (mTORC1) signaling, while promoting caspase-3, c-Jun N-terminal kinases (JNK), and p53 activation. Further, perifosine inhibits survivin expression probably by disrupting its association with heat shock protein-90 (HSP-90). These signaling changes together were responsible for a marked increase of osteosarcoma cell apoptosis and growth inhibition. Finally, we found that a low dose of perifosine enhanced etoposide- or doxorubicin-induced anti-OS cells activity. The results together suggest that perifosine might be used as a novel and effective anti-osteosarcoma agent.     

New Aspects of an Old Drug – Diclofenac Targets MYC and Glucose Metabolism in Tumor Cells

Non-steroidal anti-inflammatory drugs such as diclofenac exhibit potent anticancer effects. Up to now these effects were mainly attributed to its classical role as COX-inhibitor. Here we show novel COX-independent effects of diclofenac. Diclofenac significantly diminished MYC expression and modulated glucose metabolism resulting in impaired melanoma, leukemia, and carcinoma cell line proliferation in vitro and reduced melanoma growth in vivo. In contrast, the non-selective COX inhibitor aspirin and the COX-2 specific inhibitor NS-398 had no effect on MYC expression and glucose metabolism. Diclofenac significantly decreased glucose transporter 1 (GLUT1), lactate dehydrogenase A (LDHA), and monocarboxylate transporter 1 (MCT1) gene expression in line with a decrease in glucose uptake and lactate secretion. A significant intracellular accumulation of lactate by diclofenac preceded the observed effect on gene expression, suggesting a direct inhibitory effect of diclofenac on lactate efflux. While intracellular lactate accumulation impairs cellular proliferation and gene expression, it does not inhibit MYC expression as evidenced by the lack of MYC regulation by the MCT inhibitor α-cyano-4-hydroxycinnamic acid. Finally, in a cell line with a tetracycline-regulated c-MYC gene, diclofenac decreased proliferation both in the presence and absence of c-MYC. Thus, diclofenac targets tumor cell proliferation via two mechanisms, that is inhibition of MYC and lactate transport. Based on these results, diclofenac holds potential as a clinically applicable MYC and glycolysis inhibitor supporting established tumor therapies.     

Development of a Microfluidic Culture Paradigm for Ex Vivo Maintenance of Human Glioblastoma Tissue: A New Glioblastoma Model?

BACKGROUND: One way to overcome the genetic and molecular variations within glioblastoma is to treat each tumour on an individual basis. To facilitate this, we have developed a microfluidic culture paradigm that maintains human glioblastoma tissue ex vivo. METHODS: The assembled device, fabricated using a photolithographic process, is composed of two layers of glass bonded together to contain a tissue chamber and a network of microchannels that allow continued tissue perfusion. RESULTS: A total of 128 tissue biopsies (from 33 patients) were maintained in microfluidic devices for an average of 72 hours. Tissue viability (measured with Annexin V and propidium iodide) was 61.1% in tissue maintained on chip compared with 68.9% for fresh tissue analysed at commencement of the experiments. Other biomarkers, including lactate dehydrogenase absorbance and trypan blue exclusion, supported the viability of the tissue maintained on chip. Histological appearances remained unchanged during the tissue maintenance period, and immunohistochemical analysis of Ki67 and caspase 3 showed no significant differences when compared with fresh tissues. A trend showed that tumours associated with poorer outcomes (recurrent tumours and Isocitrate Dehydrogenase - IDH wildtype) displayed higher viability on chip than tumours linked with improved outcomes (low-grade gliomas, IDH mutants and primary tumours). conclusions: This work has demonstrated for the first time that human glioblastoma tissue can be successfully maintained within a microfluidic device and has the potential to be developed as a new platform for studying the biology of brain tumours, with the long-term aim of replacing current preclinical GBM models and facilitating personalised treatments.     

What Is New for an Old Molecule? Systematic Review and Recommendations on the Use of Resveratrol

Background

Resveratrol is a natural compound suggested to have beneficial health effects. However, people are consuming resveratrol for this reason without having the adequate scientific evidence for its effects in humans. Therefore, scientific valid recommendations concerning the human intake of resveratrol based on available published scientific data are necessary. Such recommendations were formulated after the Resveratrol 2010 conference, held in September 2010 in Helsingør, Denmark.

Methodology

Literature search in databases as PubMed and ISI Web of Science in combination with manual search was used to answer the following five questions: ¹Can resveratrol be recommended in the prevention or treatment of human diseases?; ²Are there observed “side effects” caused by the intake of resveratrol in humans?; ³What is the relevant dose of resveratrol?; ⁴What valid data are available regarding an effect in various species of experimental animals?; ⁵Which relevant (overall) mechanisms of action of resveratrol have been documented?

Conclusions/Significance

The overall conclusion is that the published evidence is not sufficiently strong to justify a recommendation for the administration of resveratrol to humans, beyond the dose which can be obtained from dietary sources. On the other hand, animal data are promising in prevention of various cancer types, coronary heart diseases and diabetes which strongly indicate the need for human clinical trials. Finally, we suggest directions for future research in resveratrol regarding its mechanism of action and its safety and toxicology in human subjects.     

Development and Evaluation of an In Vitro Vaginal Model for Assessment of Drug’s Biopharmaceutical Properties: Curcumin

Vaginal administration is a promising alternative to the per-oral route in achieving systemic or local therapeutic effects, when intestinal drug absorption is hindered by problematic biopharmaceutical drug properties. The aim of this study was to establish an in vitro vaginal model and use it to characterize biopharmaceutical properties of liposomally associated curcumin destined for vaginal delivery. The in vitro permeability, metabolism, and tissue retention of high/low permeable compounds were assessed on cow vaginal mucosa and compared to the permeabilities determined through Caco-2 cells and rat jejunum in vitro. The results showed that the intestinal mucosa was superior to the vaginal one in categorizing drugs based on their permeabilities in high/low permeable classes. Passive diffusion was found to be the main mechanism of drug penetration through vaginal mucosa and it was not affected by transporter–enzyme alliance, as their expression/activity was significantly reduced compared to the intestinal tract. Curcumin permeability from the solution form was the lowest of all tested substances due to its significant tissue retention and curcumin–mucus interactions. The permeability of liposomally associated curcumin was even lower but the binding of liposomally associated curcumin to the vaginal tissue was significantly higher. The permeability and tissue retention of liposomal curcumin were vesicle size dependent. Vaginal application of liposomally associated curcumin provides relatively high levels of curcumin in vaginal tissue, with limited systemic absorption.KEY WORDS: curcumin, intestinal models, liposomes, permeability, vaginal mucosa