Histamine is a biogenic amine which acts as a mediator of immune responses as well as acting as a neurotransmitter in the CNS. Histamine plays a role in a variety of physiological processes including allergic reactions, gastric acid secretion, bronchoconstriction and neurotransmission. Four histamine receptors, termed H1, H2, H3, and H4, have been identified. The H1 receptor mediates most of the pro-inflammatory effects of histamine whereas the anti-inflammatory and immunosuppressive activities of histamine are mainly dependent on stimulation of the H2 receptor. The H4 receptor is expressed predominantly on haematopoietic cells and agonists of this receptor induce chemotaxis of mast cells and eosinophils as well as production of IL-16 by T-cells. H4 stimulation has also recently been shown to cause inhibition of airway resistance and inflammation in a murine model of allergic asthma. Unlike the other histamine receptors, H3 receptors are expressed mainly on neurons of the peripheral and central nervous system where they control the synthesis and release of histamine and also influence the release of other neurotransmitters including dopamine, γ-aminobutyric acid, noradrenaline, acetylcholine, serotonin and tachykinins.

In human infection with Plasmodium falciparum, as well as in murine models of malaria, increased levels of histamine have been shown to be associated with severity of infection. Histamine signalling through H1 and H2 receptors increases the susceptibility of mice to infection with lethal strains of Plasmodium berghei and mice genetically deficient in the histidine decarboxylase gene – and thus lacking histamine – are highly resistant to severe malaria whether infected by mosquito bites or via injection of infected erythrocytes. A study recently published in the journal PLoS ONE has now investigated the role of the H3 receptor in the inflammatory response in the brain during P. berghei infection in mice. Compared with wild type mice, mice deficient in the H3 receptor showed an accelerated onset of cerebral malaria, increased brain pathology and more pronounced loss of blood brain barrier integrity associated with earlier death. H3 receptors tightly regulate release of histamine and other neurotransmitters and neuronal histamine activity was found to be significantly higher in naive knockout mice than in wild type mice.

In further studies, the H3 receptor agonist, (R)-alpha-methylhistamine, was found to be effective in reducing progression to cerebral malaria. In wild type mice, both the H1 receptor antagonist, levocetirizine, and the H2 receptor antagonist, cimetidine, were found to be effective in reducing clinical symptoms and mortality caused by cerebral malaria. This protection was attributed, in part at least, to down-regulation of inflammatory response-associated genes such as IFN-γ and TNF-α. The beneficial role of the H3 receptor in controlling histamine levels and limiting disease was demonstrated by the higher effectiveness of cimetidine and, to a lesser extent, levocetirizine in wild type compared with H3 receptor knockout mice. The authors propose that H1 or H2 receptor antagonists, either alone, or together with an H3 receptor agonist if one becomes available, might be used alongside anti-malaria medicines as preventative therapies against the development of cerebral malaria, especially in areas where malaria transmission is seasonal.

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Although mutations in the gene for leucine-rich repeat kinase 2 (LRRK2) have been linked to both familial and sporadic cases of Parkinson’s disease, the exact cellular function of LRRK2 remains unclear. The mechanisms by which mutated LRRK2 variants contribute to Parkinson’s disease are also unclear but it is believed that they lead, directly or indirectly, to increased kinase activity and promote inclusion formation leading to neurotoxicity. Researchers at the University of Texas Southwestern Medical Center have now identified a strong interaction between LRRK2 and CHIP (C-terminus of Hsp70-Interacting Protein), an E3 ubiquitin ligase which is crucial for the ubiquitination of several heat shock protein (Hsp70/Hsp90) client proteins involved in neurodegenerative disease. The screen also confirmed a robust interaction between LRRK2 and Hsp90, which had previously been identified as a LRRK2 binding protein. CHIP was further found to be able to significantly reduce cellular levels of LLRK2 in a dose-dependent fashion, most likely by ubiquitination and subsequent proteasome-dependent degradation. CHIP-mediated degradation of both wild type and mutant LRRK2 was found to be similar.

CHIP was found to be able to bind to LRRK2 in at least two different ways: directly or indirectly, but independent of Hsp90, to the ROC (Ras of complex) domain of LRRK2 via CHIP’s charged domain; indirectly, via Hsp90, to the N-terminal domain of LRRK2 via the TPR (tetratricopeptide repeat) domain of CHIP. Hsp90 was shown to block CHIP-mediated degradation of LRRK2, which could be overcome using the Hsp90 inhibitor, geldanamycin.

The authors hope that the discovery of cellular mechanisms that regulate LRRK2 will provide new therapeutic targets for the treatment of familial and sporadic Parkinson’s disease.

The study is published in the journal PloS ONE.

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Vinegar can be made from almost any carbohydrate source – the action of yeast first ferments the natural sugars to alcohol which is then converted into acetic acid by acetic acid bacteria. Vinegars typically contain around 5% acetic acid together with a variety of other components including polyphenols and organic acids.

Vinegars have been used for thousands of years as food preservatives and flavourings and, over the years, have also acquired a reputation for many health benefits. The human evidence for many of these claims remains equivocal although several studies have confirmed the anti-glycaemic properties of vinegar.

Largely anecdotal evidence has pointed to positive effects on weight loss and at a recent meeting of the Japan Society of Nutrition and Food Science, scientists at the Mizkan Central Research Institute have now described reductions in waist size, abdominal fat and blood neutral fat in overweight (BMI 25-30) men and women who drank 30mL of an apple vinegar-based drink (containing 1.5g acetic acid) each day for 12 weeks. Those who drank 15mL of the vinegar-based drink also saw reductions, whilst control subjects who did not take the drink saw no changes.

Writing in the Journal of Agricultural and Food Chemistry, researchers at Mizkan have also described studies in mice that highlight the mechanisms responsible for the changes in abdominal fat. Mice on a high-fat diet were divided into three groups and treated with 0.3% or 1.5% acetic acid solutions or water for 6 weeks. The groups treated with the acetic acid solutions consumed the same amount of food as the control group but showed reduced accumulation of body fat (about 10%) and hepatic lipids, with no changes in skeletal muscle weight. Significant increases in the expression of genes for peroxisome-proliferator-activated receptor-α (PPARα) and for fatty acid oxidation- and thermogenesis-related proteins were seen in the livers of the treatment groups. Similar up-regulation of gene expression was observed in vitro on addition of acetate to HepG2 cells. The effects were not observed in cells depleted of α2 5′-AMP-activated protein kinase (AMPK) by siRNA. The authors conclude that acetic acid suppresses accumulation of body fat and liver lipids by up-regulating genes for PPARα and fatty-acid-oxidation-related proteins in the liver via an α2 AMPK mediated process.

If larger clinical studies confirm fat reduction and positive effects on metabolism in overweight humans, vinegar could be set to take a share of a very large weight-loss market.

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Although the first written descriptions of gout date back over 4000 years, it wasn’t until the mid nineteenth century that excess uric acid in the blood leading to needle-like urate deposits in joint spaces was recognised to be the cause of this painful condition. Nowadays, gout is the most common cause of inflammatory joint disease in older men, most commonly affecting men between the ages of 30 and 60. Uric acid is the end product of purine metabolism and most current treatments for gout inhibit xanthine oxidase, the enzyme responsible for oxidising xanthine to uric acid. On June 16^th, Savient Pharmaceuticals announced that an FDA advisory committee had recommended that Krystexxa™ (pegloticase), a PEGylated form of recombinant porcine uricase, be granted marketing approval by the FDA for the treatment of chronic refractory gout.

Although uricase (urate oxidase) is present in many species, the gene is non-functional in humans so that uric acid is the endpoint of purine catabolism. Most other non-primates are able to convert uric acid to the more soluble allantoin, which is more easily excreted by the kidneys. A recombinant uricase, Elitek® (rasburicase) is already used to treat some patients undergoing cancer chemotherapy where tumour lysis is expected to lead to elevated levels of plasma uric acid.

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Respiratory depression is a leading cause of death from overdose of some classes of abused drugs but can also arise during medically supervised procedures such as perisurgical anaesthesia. Fentanyl, a µ-opioid agonist which is approximately 100 times more potent than morphine, is widely used to treat acute, postoperative, and chronic pain. Activation of opiate receptors can, however, lead to significant respiratory depression in a subset (10-15%) of patients. This has commonly been reversed by administration of a µ-opioid antagonist such as naloxone, albeit with the undesirable effect of blocking pain relief.

Writing in the journal Anesthesiology, researchers at the University of Alberta have now described the use of an Ampakine compound, CX717, for treating fentanyl-induced respiratory depression without interfering with analgesia. Ampakines bind allosterically to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors and are undergoing clinical trials as potential treatments for cognitive disorders and as enhancers of memory and attentiveness. AMPA receptors mediate fast synaptic transmission in the CNS and play a central role in maintaining respiratory rhythm. In the present study, pre-administration of CX717 to rats was found to markedly attenuate fentanyl-induced respiratory depression and post-administration of CX717 rescued the animals from a lethal dose of fentanyl. The effective doses of CX717 were within the range considered to be safe from clinical study data (ca 10mg/kg bid) and, importantly, did not reverse the analgesia, suggesting that co-administration of Ampakines with fentanyl could improve pain management in a variety of clinical settings.

Older people, those with a history of sleep apnea, obese individuals and those with a history of chronic obstructive pulmonary disease are particularly at risk for fentanyl-induced respiratory depression during surgery. In a small Phase IIa study in alfentanil-induced respiratory depression, a single dose of 1500mg of CX717 achieved statistical significance over placebo on the primary endpoint measure of spontaneous basal respiration without affecting analgesia.

Another ampakine compound, CX546, has previously been shown to reverse respiratory distress in rats treated with fentanyl or the barbiturate, phenobarbitol, without compromising the analgesic effect.

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Evaluate Pharma predict that the six best selling drugs in 2014 will all be biologics. Despite some recent clinical set-backs, Roche’s anticancer drug, Avastin® (bevacizumab), is tipped to be the number one best seller, with 2014 revenues of more than $9 billion. Just behind Avastin® – and rapidly closing the gap – is Abbott’s anti-rheumatic drug, Humira® ( adalimumab). The next four best sellers are predicted to be Rituxan® (rituximab, Roche), Enbrel® (etanercept, Wyeth/Amgen), Lantus® (insulin glargine, Sanofi-Aventis), and Herceptin® (trastuzumab, Roche). Five of these six drugs are antibodies, with three specifically designed to treat cancer.

As the so called patent cliff looms (where many big-selling drugs go off-patent between 2011 and 2013), Evaluate Pharma also predict a large hike in sales of generics. Out of total pharmaceutical sales in 2008 of $643.6 billion, 30% were derived from patented products that are ‘at risk’ from generic competition within 5 years. In 2011, Pfizer’s blockbuster, Lipitor® (atorvastatin calcium) will go generic, together with five further products with annual sales in excess of $4 billion. As a way of maintaining revenues, many large pharma companies are diversifying into the generics market.

Although the patent life of most of the biologics extends well beyond the current patent cliff for small molecule drugs, the present situation highlights the importance of the ongoing debate, particularly in the United States, over a regulatory pathway for biosimilars. The purity and activity of biologics may be more sensitive than those of small molecules to changes in the manufacturing process, leading to concerns that generics may not be equivalent to the original branded drugs.

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T-cell acute lymphoblastic leukaemia (T-ALL) primarily affects children and adolescents, and is characterised by a high rate of infiltration into the central nervous system. Aggressive treatment using cranial irradiation and intrathecal chemotherapy to prevent CNS involvement has improved survival outcomes but at a high cost to patients – side effects can include secondary brain tumours, delayed development and mental retardation. The severe consequences of available treatments to prevent CNS involvement in T-ALL have driven a search for genes and signalling pathways involved in CNS infiltration.

CCR7 was already known as a key player in normal lymphocyte migration and adhesion, and as a binding partner of two chemokines, CCL19 and CCL21. Previous studies had also implicated these receptor-ligand interactions in the metastasis of tumours such as melanomas and breast cancers, and anti-CCR7 antibodies had been shown to block the migration of chronic lymphocytic leukaemia (CLL) cells. Scientists at NYU School of Medicine have now shown that CCR7 is also essential for infiltration of T-ALL cells into the CNS.

The Notch1 signalling pathway is essential in T-cell development and earlier research had linked mutations in Notch1 with the progression of T-ALL. In the new study, the team found that mice expressing overactive forms of Notch1 developed leukaemia and, moreover, that the leukaemia cells efficiently infiltrated the inner layer of the membrane covering the brain. Using gene-expression profiling, CCR7 gene expression was found to be controlled by Notch1 and CCR7 itself to be essential for CNS infiltration. Silencing either CCR7 or one of its cognate ligands, CCL19, in an animal model of T-ALL specifically inhibited CNS infiltration. Mouse brain infiltration by human T-ALL cells was also found to be dependent on CCR7 expression. In four out of five T-ALL cell lines derived from human patients, the gene for CCR7 was found to be upregulated, providing further evidence for its role in the disease. The identification of a single chemokine receptor as necessary and sufficient for CNS infiltration suggests a new possibility for reducing the complications associated with aggressive CNS-targeted therapy for T-ALL using small molecules, antibodies or RNAi directed against CCR7.

The study is in the June 18^th issue of the journal Nature.

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Proteasomes are abundant and ubiquitous multi-protein complexes capable of degrading almost any protein into oligopeptides. The 20S proteasome is composed of 14 subunits (seven α and seven β) arranged in four rings to form a barrel, the ends of which can open to accept substrates. Catalytic activity is confined to three of the β-subunits, all of which are N-terminal threonine proteases. The addition of 19S regulatory complexes at the ends of the 20S multimer creates the 26S proteasome and confers the ability to process ubiquitylated substrates in an ATP-dependent manner.

Proteasomes chomp their way through a huge number of proteins including defective ribosomal products that arise from imperfections in the conversion of genetic information into proteins. The oligopeptides that result are then rapidly processed to constituent amino acids by endo- and amino-peptidases. Some oligopeptides, however, escape further processing and are presented on the cell surface by MHC Class I molecules. This provides a mechanism for the immune system to monitor the gene products that a particular cell is processing.

A variant of the proteasome, the immunoproteasome, is constitutively expressed in immune tissues and can be induced in other cell types in response to cytokines such as γ-interferon and TNF-α. The immunoproteasome differs from the proteasome at the level of the catalytic β-subunits and generates alternate peptides that modify the antigens presented by MHC class I.

The proteasome has been validated as a target in oncology and bortezomib was the first proteasome inhibitor approved in the US (2003). Until now, however, utility of proteasome inhibitors in auto-immune diseases has been hampered by the higher doses of conventional inhibitors required and the consequently smaller therapeutic window. This situation has potentially changed with results from Proteolix using their immunoproteasome-selective inhibitor, PR-957.

PR-957 is a peptide epoxy-ketone that selectively inhibits the LMP7 catalytic subunit of the immunoproteasome. Inhibition of this subunit modulates immune cell signalling and blocks production of cytokines associated with autoimmune inflammation, without affecting proteasome function in non-immune cells. In mouse models of rheumatoid arthritis, PR-957 treatment reversed signs of disease and reduced cellular infiltration, cytokine production and autoantibody levels. The study is published in the journal Nature Medicine.

Proteolix plans to file an Investigational New Drug Application for PR-957 in mid-2010.

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Although the ubiquitination pathway in eukaryotes was characterised in the early 1980s, it has only recently been recognised that bacteria also tag proteins to determine their fate. Ubiquitination controls a variety of cellular processes, but one of its major roles is to label proteins for degradation by the proteasome. In Mycobacterium tuberculosis (Mtb), the small ubiquitin-like protein that targets other proteins for proteasomal degradation is called prokaryotic ubiquitin-like protein (Pup) and, because proteasome function is essential for the Mtb virulence, interruption of the Pup pathway could potentially be a target for anti-tuberculosis drugs. Writing in Nature Structural & Molecular Biology, researchers at the Institute of Molecular Biology & Biophysics of ETH Zurich have now described an enzyme called Pup deaminase (Dop) that is involved in the Pupylation of proteins in Mtb. Dop modifies Pup by deaminating the C-terminal glutamine to glutamate and the Pup ligase, proteasome accessory factor A (PafA), then couples the modified Pup to the ε-amino group of lysine residues in the target proteins. This formation of an isopeptide bond requires hydrolysis of ATP to ADP, suggesting that the C-terminal glutamate of the modified Pup is activated for attachment via phosphorylation.

Since the Pupylation pathway differs from the ubiquitination pathway, drugs targeting the bacterial Pup pathway may offer the potential for safe and effective treatments for Mtb.

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Scientists have identified a new target for the diagnosis and treatment of age-related macular degeneration (AMD). AMD most commonly affects people over the age of fifty and is the leading cause of vision loss in those over sixty. The rarer, but more serious form of the disease, wet AMD, is caused by abnormal blood vessel growth under the macula (centre of the retina) and can develop very quickly. Although the condition is painless, the new blood vessels leak blood and fluid which lift the macula and destroy sharp central vision. The condition can be treated with laser surgery, photodynamic therapy or anti-vascular endothelial growth factor (anti-VEGF) injections into the eye, although none of these treatments provides a complete cure and loss of vision may continue despite treatment.

The new study, published online on Jun 14^th in Nature, shows that blocking the eosinophil/mast cell chemokine receptor, CCR3, can reduce the abnormal blood vessel growth that leads to AMD. The researchers detected CCR3 protein in eye tissue from people with AMD but not in eye tissue from people of the same age who did not have the disease. In studies in mice, blocking CCR3, either by genetic engineering or using antibodies, reduced the number of abnormal blood vessels. In the mice, targeting CCR3 was shown to be more effective than targeting VEGF (70% vs 60%), suggesting that CCR3 blockers could also provide an effective treatment for patients with AMD. The team injected anti-CCR3 antibodies attached to semiconductor nanocrystals into mice and, using conventional ocular angiography techniques, were able to visualise the abnormal blood vessels even before they had penetrated the retina. Since CCR3 was detected at an early stage in the development of the disease, the researchers hope that the new imaging technology could be used diagnostically, and that early detection will provide better opportunities to prevent structural damage and preserve vision.

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