Dynamic analysis of drug-induced cytotoxicity using chip-based dielectrophoretic cell immobilization technology

Quantification of programmed and accidental cell death provides useful end-points for the anticancer drug efficacy assessment. Cell death is, however, a stochastic process. Therefore, the opportunity to dynamically quantify individual cellular states is advantageous over the commonly employed static, end-point assays. In this work, we describe the development and application of a microfabricated, dielectrophoretic (DEP) cell immobilization platform for the realtime analysis of cancer drug-induced cytotoxicity. Microelectrode arrays were designed to generate weak electro-thermal vortices that support efficient drug mixing and rapid cell immobilization at the delta-shape regions of strong electric field formed between the opposite microelectrodes. We applied this technology to the dynamic analysis of hematopoietic tumor cells that represent a particular challenge for real-time imaging due to their dislodgement during image acquisition. The present study was designed to provide a comprehensive mechanistic rationale for accelerated cell-based assays on DEP chips using real-time labeling with cell permeability markers. In this context, we provide data on the complex behavior of viable vs dying cells in the DEP fields and probe the effects of DEP fields upon cell responses to anticancer drugs and overall bioassay performance. Results indicate that simple DEP cell immobilization technology can be readily applied for the dynamic analysis of investigational drugs in hematopoietic cancer cells. This ability is of particular importance in studying the outcome of patient derived cancer cells, when exposed to therapeutic drugs, as these cells are often rare and difficult to collect, purify and immobilize.

Efficacy of nefazodone in the treatment of neuroleptic induced extrapyramidal side effects: a double-blind randomised parallel group placebo-controlled trial.

Many atypical antipsychotics show antagonism at both serotonergic and dopaminergic neurones and show fewer extrapyramidal side effects (EPS). Nefazodone blocks postsynaptic 5HT2A receptors and weakly inhibits serotonin reuptake. This study aimed to elucidate the role of nefazodone in the treatment of antipsychotic-induced EPS. The trial was a double-blind, randomised, placebo-controlled trial of patients requiring antipsychotic treatment with haloperidol 10mg daily; from which a subgroup of patients who developed EPS were selected for the study. Patients were randomised to add-on therapy with either placebo (n=24) or nefazodone (n=25) 100mg bd. EPS were measured on days 0, 3 and 7 using the Simpson Angus, Barnes akathisia, abnormal involuntary movement and Chouinard scales. Nefazodone significantly reduced EPS as measured by both the Simpson Angus scale and CGI (p=0.007 and 0.0247, respectively). Akathisia and tardive dyskinesia did not differ between the two groups (p=0.601; p=0.507, respectively). These results suggest the role of 5HT2 antagonism in the mechanism of action of atypical antipsychotics with respect to lowering rates of drug-induced EPS. In addition, a therapeutic role for nefazodone is suggested in the treatment of antipsychotic-induced EPS.

Clinical trials of N-acetylcysteine in psychiatry and neurology: A systematic review

N-acetylcysteine (NAC) is recognized for its role in acetaminophen overdose and as a mucolytic. Over the past decade, there has been growing evidence for the use of NAC in treating psychiatric and neurological disorders, considering its role in attenuating pathophysiological processes associated with these disorders, including oxidative stress, apoptosis, mitochondrial dysfunction, neuroinflammation and glutamate and dopamine dysregulation. In this systematic review we find favorable evidence for the use of NAC in several psychiatric and neurological disorders, particularly autism, Alzheimer's disease, cocaine and cannabis addiction, bipolar disorder, depression, trichotillomania, nail biting, skin picking, obsessive-compulsive disorder, schizophrenia, drug-induced neuropathy and progressive myoclonic epilepsy. Disorders such as anxiety, attention deficit hyperactivity disorder and mild traumatic brain injury have preliminary evidence and require larger confirmatory studies while current evidence does not support the use of NAC in gambling, methamphetamine and nicotine addictions and amyotrophic lateral sclerosis. Overall, NAC treatment appears to be safe and tolerable. Further well designed, larger controlled trials are needed for specific psychiatric and neurological disorders where the evidence is favorable.

Molecular targets in arthritis and recent trends in nanotherapy

Due to its severity and increasing epidemiology, arthritis needs no description. There are various forms of arthritis most of which are disabling, very painful, and common. In spite of breakthroughs in the field of drug discovery, there is no cure for arthritis that can eliminate the disease permanently and ease the pain. The present review focuses on some of the most successful drugs in arthritis therapy and their side effects. Potential new targets in arthritis therapy such as interleukin-1β, interleukin-17A, tumor necrosis factor alpha, osteopontin, and several others have been discussed here, which can lead to refinement of current therapeutic modalities. Mechanisms for different forms of arthritis have been discussed along with the molecules that act as potential biomarkers for arthritis. Due to the difficulty in monitoring the disease progression to detect the advanced manifestations of the diseases, drug-induced cytotoxicity, and problems with drug delivery; nanoparticle therapy has gained the attention of the researchers. The unique properties of nanoparticles make them highly attractive for the design of novel therapeutics or diagnostic agents for arthritis. The review also focuses on the recent trends in nanoformulation development used for arthritis therapy. This review is, therefore, important because it describes the relevance and need for more arthritis research, it brings forth a critical discussion of successful drugs in arthritis and analyses the key molecular targets. The review also identifies several knowledge gaps in the published research so far along with the proposal of new ideas and future directions in arthritis therapy.

Altered fast- and slow-twitch muscle fibre characteristics in female mice with a (S248F) knock-in mutation of the brain neuronal nicotinic acetylcholine receptor

We generated a mouse line with a missense mutation (S248F) in the gene (CHRNA4) encoding the α4 subunit of neuronal nicotinic acetylcholine receptor (nAChR). Mutant mice demonstrate brief nicotine induced dystonia that resembles the clinical events seen in patients with the same mutation. Drug-induced dystonia is more pronounced in female mice, thus our aim was to determine if the S248F mutation changed the properties of fast- and slow-twitch muscle fibres from female mutant mice. Reverse transcriptase-PCR confirmed CHRNA4 gene expression in the brain but not skeletal muscles in normal and mutant mice. Ca²⁺ and Sr²⁺ force activation curves were obtained using skinned muscle fibres prepared from slow-twitch (soleus) and fast-twitch (EDL) muscles. Two significant results were found: (1) the (pCa₅₀ - pSr₅₀) value from EDL fibres was smaller in mutant mice than in wild type (1.01 vs. 1.30), (2) the percentage force produced at pSr 5.5 was larger in mutants than in wild type (5.76 vs. 0.24%). Both results indicate a shift to slow-twitch characteristics in the mutant. This conclusion is supported by the identification of the myosin heavy chain (MHC) isoforms. Mutant EDL fibres expressed MHC I (usually only found in slow-twitch fibres) as well as MHC IIa. Despite the lack of spontaneous dystonic events, our findings suggest that mutant mice may be having subclinical events or the mutation results in a chronic alteration to muscle neural input.

Restless leg syndrome caused by olanzapine: a case series

Restless leg syndrome (RLS) is a common disorder associated with significant distress. We report three cases of drug induced RLS caused by olanzapine. In each case, RLS commenced after initiation of treatment with olanzapine and resolved after ceasing olanzapine. All three patients were subsequently treated with other atypical antipsychotics, risperidone, quetiapine or aripiprazole, without re-emergence of RLS. RLS is associated with central dopaminergic dysfunction. Dopamine agonists and l-dopa reduce the symptoms of RLS, and some agents that block the dopaminergic system aggravate RLS. Greater awareness of potential causes of RLS, and its differentiation from akathisia and illness related agitation might help in reducing the distress associated with it and improving patient compliance.

A review of clozapine safety

Clozapine is a distinctive antipsychotic agent, having a unique clinical profile and an idiosyncratic safety profile. More so than with other agents, the weighting of its adverse event profile is critical, in order to counterbalance its clear clinical advantages. The safety issues with clozapine are in a number of areas, some of which are considered medical emergencies and potentially life-threatening. These include haematological (neutropenia and agranulocytosis), CNS (seizures), cardiovascular (myocarditis and cardiomyopathy), metabolic (diabetes), gastrointestinal and neuromuscular. Understanding the safety profile of clozapine allows an informed use of the agent that can maximise its clear clinical benefit and minimise the known risks.

Cancer targeted nanoparticles specifically induce apoptosis in cancer cells and spare normal cells

Curing cancer is the greatest challenge for modern medicine and finding ways to minimize the adverse effects caused by chemotherapeutic agents is of importance in improving patient’s physical conditions. Traditionally, chemotherapy can induce various adverse effects, and these effects are mostly caused by the non-target specific properties of the chemotherapeutic compounds. Recently, the use of nanoparticles has been found to be capable of minimizing these drug-induced adverse effects in animals and in patients during cancer treatment. The use of nanoparticles allows various chemotherapeutic drugs to be targeted to cancer cells with lower dosages. In addition to this, the use of nanoparticles also allows various drugs to be administered to the subjects by an oral route. Here, locked nucleic acid (LNA)-modified epithelial cell adhesion molecules (EpCAM), aptamers (RNA nucleotide), and nucleolin (DNA nucleotide) aptamers have been developed and conjugated on anti-cancer drug-loaded nanocarriers for specific delivery to cancer cells and spare normal cells. Significant amounts of the drug loaded nanocarriers (92 ± 6 %) were found to distribute to the cancer cells at the tumour site and more interestingly, normal cells were unaffected in vitro and in vivo. In this review, the benefits of using nanoparticle-coated drugs in various cancer treatments are discussed. Various nanoparticles that have been tried in improving the target specificity and potency of chemotherapeutic compounds are also described.

Dielectrophoresis of micro/nano particles using curved microelectrodes

Dielectrophoresis, the induced motion of polarisable particles in non-homogenous electric field, has been proven as a versatile mechanism to transport, immobilise, sort and characterise micro/nano scale particle in microfluidic platforms. The performance of dielectrophoretic (DEP) systems depend on two parameters: the configuration of microelectrodes designed to produce the DEP force and the operating strategies devised to employ this force in such processes. This work summarises the unique features of curved microelectrodes for the DEP manipulation of target particles in microfluidic systems. The curved microelectrodes demonstrate exceptional capabilities including (i) creating strong electric fields over a large portion of their structure, (ii) minimising electro-thermal vortices and undesired disturbances at their tips, (iii) covering the entire width of the microchannel influencing all passing particles, and (iv) providing a large trapping area at their entrance region, as evidenced by extensive numerical and experimental analyses. These microelectrodes have been successfully applied for a variety of engineering and biomedical applications including (i) sorting and trapping model polystyrene particles based on their dimensions, (ii) patterning carbon nanotubes to trap low-conductive particles, (iii) sorting live and dead cells based on their dielectric properties, (iv) real-time analysis of drug-induced cell death, and (v) interfacing tumour cells with environmental scanning electron microscopy to study their morphological properties. The DEP systems based on curved microelectrodes have a great potential to be integrated with the future lab-on-a-chip systems.

The effect of oral administration of iron saturated-bovine lactoferrin encapsulated chitosan-nanocarriers on osteoarthritis

In this study, the therapeutic potentials of 100% iron saturated-bovine lactoferrin encapsulated in alginate-chitosan polymeric nanocarriers (AEC-CP-Fe-bLf-NCs) were examined in in vitro inflammatory OA model and in collagen-induced arthritis (CIA) mice. Oral administration of nanocarriers in mice were non-toxic and significantly induced disease modifying activity by reducing joint inflammation and downregulating the expression of catabolic genes, IL-1β, NO, JNK and MAPK. In addition, up-regulation of type II collagen, aggrecan and inflammation depleted iron and calcium metabolisms via inhibition of miRNA of iron transporting receptors was shown in AEC-CP-Fe-bLf-NCs treated mice.

Novel protein and atenolol co-encapsulated nanocapsules to impede cardiomyocyte apoptosis

 The research conducted was based on regenerating the dying heart cells (cardiomyocytes) by employing novel therapeutic proteins and their respective co-encapsulated nanoformulation with an antihypertensive drug. This promising therapeutic strategy to revive the heart can help in the treatment of several cardiac pathologies such as myocardial infarction and drug induced cardiotoxicity.

Role of dopamine D3 and serotonin 5-HT1A receptors in l-DOPA-induced dyskinesias and effects of sarizotan in the 6-hydroxydopamine-lesioned rat model of Parkinson's disease

Sarizotan, a 5-HT1A agonist with additional affinity for D3 and D4 receptors, has been demonstrated to have anti-dyskinetic effects. The mechanism by which these effects occur is not clear. Using unilateral 6-hydroxydopamine-lesioned rats that received chronic intraperitoneal (ip) administration of L-3,4-dihydroxyphenylalanine (L-DOPA) we investigated the involvement of D3 and 5-HT1A receptors in the effects of sarizotan on contraversive circling and abnormal involuntary movements (AIMs). Before sensitization by chronic L-DOPA treatment (12.5 with 3.25 mg/kg benserazide ip, twice daily for 21 days), no effect of the selective D3 agonist, PD128907 (1 or 3 mg/kg ip), or the selectiveD3 antagonist,GR103691 (0.5 or 1.5 mg/kg ip), was observed. Treatment with sarizotan (1 or 5 mg/kg ip) dosedependently inhibited the L-DOPA-induced contraversive turning and AIMs. In co-treatment with the 5-HT1A antagonist, WAY100635 (1 mg/kg ip), sarizotan failed to affect this behaviour, confirming the prominent 5-HT1A receptor-mediated mechanism of action. In the presence of PD128907 (3 mg/kg ip), the effects of sarizotan on contraversive turning, locomotive dyskinesia and axial dystonia, but not on orolingual and forelimb dyskinesia, were blocked. On its own, PD128907 had no effect on the behavioural effects of L-DOPA except that it tended to reduce orolingual and forelimb dyskinesia. GR103691 had no effect on its own or in combination with sarizotan. These data identify an involvement of D3 receptors in the action of sarizotan on some, but not all L-DOPA-induced motor side effects. This selective involvement is in contrast to the more general involvement of 5-HT1A receptors in the anti-dyskinetic effects of sarizotan.

Tumor progression locus 2 (Tpl2) deficiency does not protect against obesity-induced metabolic disease

Obesity is associated with a state of chronic low grade inflammation that plays an important role in the development of insulin resistance. Tumor progression locus 2 (Tpl2) is a serine/threonine mitogen activated protein kinase kinase kinase (MAP3K) involved in regulating responses to specific inflammatory stimuli. Here we have used mice lacking Tpl2 to examine its role in obesity-associated insulin resistance. Wild type (wt) and tpl22/2 mice accumulated comparable amounts of fat and lean mass when fed either a standard chow diet or two different high fat (HF) diets containing either 42% or 59% of energy content derived from fat. No differences in glucose tolerance were observed between wt and tpl22/2 mice on any of these diets. Insulin tolerance was similar on both standard chow and 42% HF diets, but was slightly impaired in tpl22/2 mice fed the 59% HFD. While gene expression markers of macrophage recruitment and inflammation were increased in the white adipose tissue of HF fed mice compared with standard chow fed mice, no differences were observed between wt and tpl2 mice. Finally, a HF diet did not increase Tpl2 expression nor did it activate Extracellular Signal-Regulated Kinase 1/2 (ERK1/2), the MAPK downstream of Tpl2. These findings argue that Tpl2 does not play a non-redundant role in obesity associated metabolic dysfunction.

Cisplatin-induced formation of biocompatible and biodegradable polypeptide-based vesicles for targeted anticancer drug delivery

Novel cisplatin (CDDP)-loaded, polypeptide-based vesicles for the targeted delivery of cisplatin to cancer cells have been prepared. These vesicles were formed from biocompatible and biodegradable maleimide-poly(ethylene oxide)114-b-poly(L-glutamic acid)12 (Mal-PEG114-b-PLG12) block copolymers upon conjugation with the drug itself. CDDP conjugation forms a short, rigid, cross-linked, drug-loaded, hydrophobic block in the copolymer, and subsequently induces self-assembly into hollow vesicle structures with average hydrodynamic diameters (Dh) of ∼ 270 nm. CDDP conjugation is critical to the formation of the vesicles. The reactive maleimide-PEG moieties that form the corona and inner layer of the vesicles were protected via formation of a reversible Diels-Alder (DA) adduct throughout the block copolymer synthesis so as to maintain their integrity. Drug release studies demonstrated a low and sustained drug release profile in systemic conditions (pH = 7.4, [Cl(-)] = 140 mM) with a higher "burst-like" release rate being observed under late endosomal/lysosomal conditions (pH = 5.2, [Cl(-)] = 35 mM). Further, the peripheral maleimide functionalities on the vesicle corona were conjugated to thiol-functionalized folic acid (FA) (via in situ reduction of a novel bis-FA disulfide, FA-SS-FA) to form an active targeting drug delivery system. These targeting vesicles exhibited significantly higher cellular binding/uptake into and dose-dependent cytotoxicity toward cancer cells (HeLa) compared to noncancerous cells (NIH-3T3), which show high and low folic acid receptor (FR) expression, respectively. This work thus demonstrates a novel approach to polypeptide-based vesicle assembly and a promising strategy for targeted, effective CDDP anticancer drug delivery.