Acute crush injury to the spinal cord is immediately followed by secondary tissue damage, linked to massive release of ATP and activation of high-affinity P2X₇ receptors. Researchers at the University of Rochester Medical Center have previously shown that intraspinal injection of the P2X₇ antagonist, adenosine 5′-triphosphate-2′,3′-dialdehyde (OxATP), improves outcomes of spinal injury in rats, but intraspinal injection – together with cardiovascular toxicity – makes this treatment unattractive for human trauma victims. In a new study, the team have shown that outcomes in rats can also be improved by systemic treatment with a different P2X₇ antagonist, the Coomassie dye, Brilliant Blue G.

Writing in the journal PNAS, they show that iv administration of Brilliant Blue G (10 or 50 mg/kg) 15 minutes after injury, and for three consecutive days, protected spinal cord neurons from purinergic excitotoxicity and also reduced local inflammatory responses, resulting in reduced spinal cord anatomic damage and improved motor recovery. After 6 weeks, treated animals recovered sufficiently to walk with a limp whereas untreated animals did not walk again. Although it seems unlikely that Brilliant Blue G would efficiently cross the intact blood brain barrier, the dye was found to accumulate in the lesions in the injured animals.

There is currently no effective treatment to prevent secondary damage in patients with acute spinal cord injury and the team hope that their work will lead to safe practical treatments that could be administered soon after injury to improve outcomes for spinal injury victims.

Brilliant Blue G is a noncompetitive inhibitor of rat and human P2X₇ receptors with IC₅₀ values of 10 and 200 nM respectively.

The team chose to use Brilliant Blue G for their experiments because they saw structural and functional similarities with a food additive, FD&C blue dye No 1 (E133), used in a variety of processed foods and generally considered to be safe. A number of groups have now designed selective P2X₇ antagonists – some of which have entered the clinic – and it would be interesting to see the effect of these newer compounds in the rat spinal injury model. Because of the differing affinities of antagonists for rat and human receptors, care will be needed in the choice of appropriate molecules for study, and in extrapolation of results from rodents to humans.

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Mast cells are best known for their role in allergic responses but a new study by researchers at Brigham and Women’s Hospital and colleagues has now shown a link with diet-induced obesity and type 2 diabetes. Writing in the July edition of Nature Medicine, they show that mast cells are far more abundant in white adipose tissue from obese humans and mice than in tissue from normal weight individuals.

In mice on a high calorie diet, treatment for two months with either of the allergy treatments, ketotifen fumarate or cromolyn, led to significant weight loss and improvement in diabetic markers compared with control animals. More dramatic improvements were seen if the animals were also switched to a reduced fat diet.

In further studies, the team showed that mice which lack mature mast cells neither became obese nor developed diabetes over a three month period, despite being fed a Western diet rich in sugars and fats. As a next step towards possible testing in humans, the researchers plan to study the effect of the compounds on obese and diabetic non-human primates.

Ketotifen fumarate and cromolyn are both used in anti-allergy eye drops, and to prevent asthma attacks. Although both stabilise mast cells, the exact mechanisms by which they achieve this are somewhat different.

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A number of organs, including the heart, have limited regenerative powers, but US scientists have now shown that fully differentiated cardiac muscle cells can be induced to proliferate and regenerate. Writing in the journal Cell, they show that the growth factor neuregulin 1, which plays a role in early development of the heart and nervous system, induces mononucleated, but not binucleated, cardiomyocytes to divide in vitro by acting on the receptor tyrosine kinase, ErbB4. In mice, genetic inactivation of ErbB4 was shown to reduce cardiomyocyte proliferation, whereas increasing ErbB4 expression enhanced proliferation. Following heart attack in adult mice, daily intraperitoneal injection of neuregulin 1 for 12 weeks led to regeneration of the heart muscle and improved function. Unlike the control mice, the treated animals showed reduced signs of heart failure such as left-ventricular dilation and cardiac hypertrophy. If the neuregulin/ErbB4 signalling pathway plays the same role in human heart muscle, stimulating proliferation of differentiated cardiomyocytes by activation of this pathway may provide an alternative to stem cell therapy to regenerate damaged heart muscle in patients with heart failure or children with congenital heart defects. Since ErbB receptor tyrosine kinases and neuregulins have oncogenic potential and may cause proliferation of other tissues, a full safety assessment would be needed before any clinical studies.

Neuregulin 1 has previously been associated with susceptibility to schizophrenia and has also been shown to protect neurones following stroke.

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The polypeptide hormone precursor, pro-opiomelanocortin (POMC) undergoes extensive, tissue-specific, post-translational modification to yield a number of peptides with diverse biological activities. POMC neurons in the hypothalamus convert POMC via a multi-enzyme process into the 13 residue α-melanocyte-stimulating hormone (α-MSH_1-13). As well as stimulating production and release of melanin in skin and hair, α-MSH_1-13 acts on postsynaptic melanocortin receptors in the brain to suppress appetite. In the late 1990s, mutations in MCR4 that prevent α-MSH_1-13 signalling were linked to inherited human obesity but, although the pathways leading to activation of MCR4 are relatively well understood, little was known about how signalling is terminated.

Writing in the Journal of Clinical Investigation, researchers at Yale University School of Medicine and the University of California Davis now show that, in mice, α-MSH_1-13 is inactivated by removal of the amidated C-terminal valine residue by the enzyme prolylcarboxypeptidase (PRCP). PRCP is a serine protease that cleaves the C-terminal residue from peptides with a penultimate proline residue that also converts the vasopressor peptide, angiotensin II, into the inactive angiotensin 1-7. Mice lacking PRCP had increased levels of α-MSH_1-13 in the hypothalamus, were leaner and shorter than normal mice, and did not become obese when fed a high-fat diet. Additionally, icv administration of a PRCP inhibitor, Boc-prolylprolinal, reduced food intake in rats, as did systemic administration to obese, leptin-deficient mice.

On the basis of their studies, the authors propose that regulation of PRCP activity in the hypothalamus may be a target for the treatment of obesity and related disorders but an accompanying commentary cautions that brain-penetrating drugs would need to be developed to be useful for the treatment of obesity. Skin and hair colour could be affected by inhibiting PRCP, as well as levels of the vasoactive peptides, angiotensin II and bradykinin. Since PRCP is widely expressed, there may be other – not yet unidentified – substrates, and inhibitors of PRCP could have other, unforeseen, effects.

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The term amyloidosis encompasses a wide variety of diseases characterised by extracellular deposition of insoluble fibrils of abnormally folded proteins in organs and tissues. Transthyretin amyloidosis (ATTR) is a disorder in which a serum protein, transthyretin (TTR), accumulates in tissues such as heart, kidneys, nerves, and intestine. TTR is a 127 amino acid transport protein for thyroxine and retinol which circulates as a tetramer. It is believed that the disease is caused by TTR tetramer dissociation into monomers which misfold and form amyloid deposits. The inherited form of the disease is caused by autosomal dominant mutations in the TTR gene. The only specific treatment at present for this progressive disorder is liver transplantation. TTR is synthesised primarily in the liver and, if an individual receives a new liver, they will no longer produce the mutated version of the protein. Although the procedure has benefitted some patients, for others disease progression continues after the transplant, suggesting that abnormal amyloid deposits may act as seeds, even for normal protein.

Scientists at the Scripps Research Institute investigating new treatments for ATTR have focussed on inhibition of tetramer dissociation, the rate-limiting step in amyloid formation, and discovered tafamidis (Fx-1006A), a small molecule that acts as a pharmacological chaperone for TTR and prevents misfolding. The drug stabilizes both wild-type and variant TTR, prevents misfolding of the protein by preventing tetramer dissociation, and inhibits the formation of TTR amyloid fibrils. Tafamidis is being developed by FoldRx Pharmaceuticals who announced last week that the new drug significantly halts disease progression for patients with transthyretin amyloid polyneuropathy (ATTR-PN), a form of ATTR in which the amyloid fibrils are deposited in peripheral nerve tissues. Results from a randomised, controlled Phase II/III clinical study showed that once daily oral treatment with tafamidis was safe and well-tolerated and, compared to placebo, significantly halted disease progression after 18 months (no progression in 60% of tafamidis patients compared with 38% of placebo patients). Placebo-treated patients also experienced a significant deterioration in quality of life compared with tafamidis-treated patients.

Tafamidis is the first disease-modifying compound that targets protein misfolding and it is hoped that it will provide a better alternative to liver transplant for ATTR-PN patients.

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Genome-wide association studies aim to identify genetic contributions to common diseases such as diabetes and atherosclerosis by comparing samples from those with the disease and healthy individuals. Because of the difficulty in sampling diseased tissue from living patients, blood samples are often used for the comparisons. The impact of such studies is potentially substantial, but scientists at McGill University have now sounded a note of caution about the use of blood samples as surrogates.

It had been assumed that DNA is essentially identical in every cell in the body, and that blood acts as a good surrogate for other tissues but, in an investigation into the underlying genetic causes of abdominal aortic aneurysms (AAA), the McGill team found differences between DNA from blood samples and diseased tissue. AAA is a localised widening and weakening of the abdominal aorta which can lead to fatal rupture of the aorta and is one of a small number of vascular diseases in which tissue samples are removed as part of therapy. Since activation of apoptosis has been linked to the development and progression of AAA, the team were examining the role of sequence variations in the BAK1 gene (BAK1) which encodes a pro-apoptotic protein. BAK1 abdominal aorta cDNA from AAA patients and healthy individuals were compared with each other as well as with BAK1 genomic sequences obtained from matching blood samples. Specific BAK1 single nucleotide polymorphism (SNP) containing alleles were found in both aneurysmic (31 cases) and healthy aortic tissue (5 cases) but not in the matching blood samples. If the mutations discovered in the tissue cells are found to predispose for AAA – or other vascular diseases – they could lead to new therapeutic targets. Although the study compared complementary DNA from aortic tissue with genomic DNA from blood, if the mismatch between blood and tissue samples turns out to be a general phenomenon when comparing like DNA, it questions the validity of the widespread practice of using blood samples to represent diseased tissue in genome-wide association studies.

The study is published in full in the July issue of the journal Human Mutation.

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Globally, around 200 million people are infected with Hepatitis C virus (HCV). Infection generally leads to chronic liver disease, albeit over a period of decades, which can lead to cirrhosis, hepatocellular carcinoma and liver failure. Current treatment, a combination of pegylated interferon-α and ribavirin, is only effective in 50-85% of patients, dependent on viral genotype. Not surprisingly, much effort is being expended to identify new therapeutic interventions.

Study of the viral lifecycle has been difficult in the past since serum-derived HCV (sHCV) replicates poorly in primary human hepatocytes and hepatoma cells in vitro. Surrogate systems have been developed, however, that have enabled reproduction of all steps of the HCV replication cycle, including cell entry (recently reviewed).

Viral cell entry is still incompletely understood, but a new study from researchers at the University of Rennes 1 has identified host kinases involved in HCV infectivity. Using a small-interfering RNA (siRNA) library, the scientists have shown that knock-down of phosphatidylinositol 4-kinase type III-α (PI4KIIIα) prevents infection by either HCV pseudoparticles (HCVpp) or by cell-culture grown JFH-1-based HCV (HCVcc). A second PI4K-family member, PI4KIIIβ, also influenced cellular susceptibility to HCVpp infection and the ability of the cells to sustain HCV replication. These kinases may therefore represent new targets for HCV therapeutics.

The study is published in the FASEB Journal.

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Although not without opponents – and still unproven – the theory that reducing levels of β-amyloid peptides will lessen neuronal damage and cognitive deficits remains a central tenet of Alzheimer’s disease research and the focus of many pharmaceutical companies. Last week, however, it was reported at the Alzheimer’s Association annual meeting in Vienna that Dimebon^® (latrepirdine), a drug which has shown clinical benefit in people with mild to moderate Alzheimer’s disease, increases levels of β-amyloid in the releasate from isolated nerve terminals and in the interstitial fluid from the brains of transgenic mice bred to model Alzheimer’s disease. This unexpected result raises questions about how Dimebon^® works and also, perhaps, about the validity of the ‘amyloid hypothesis’. One possibility is that the neuroprotective effects of Dimebon^® are sufficient to outweigh the adverse effects of increased concentrations of β-amyloid. Preclinical studies have suggested that Dimebon^® could prevent neuronal damage and dysfunction by stabilising or improving mitochondrial function. In addition, biochemical studies have shown effects on α-adrenergic receptors, histamine receptors and serotonin receptors as well as on Ca²⁺ flux and apoptosis. Alternatively, Dimebon^® could cause an acute increase in amyloid levels, but lower levels on chronic dosing, or it could play a role in amyloid transport. Whatever the explanation for the present results, further investigations into the mechanism of action of Dimebon® and the relevance of animal models of acute amyloid lowering to human disease are of key importance.

Dimebon^®, which has been used in Russia to treat hayfever since the 1980s, is being jointly developed by Medivation and Pfizer and is currently in phase III clinical trials.

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First marketed in the US in 1953, paracetamol (acetaminophen) is one of the most widely used drugs in Western society, both in over-the-counter (OTC) products and as a component of prescription medicines. Effective in relieving pain and fever, paracetamol is noted for the absence of gastro-intestinal side-effects at therapeutic doses in contrast to the non-steroidal anti-inflammatory drugs. Exceeding the recommended dose of paracetamol (typically 4000mg daily for adults), however, can cause liver damage. Adverse events range from minor changes in liver enzymes to acute liver failure and, in some cases, death.

The toxicity is not due to paracetamol itself, but a reactive metabolite, NAPQI. Normally, NAPQI is rapidly de-toxified by conjugation with glutathione, but the pathway can become saturated as a result of overdose, combination with alcohol, or in individuals with polymorphisms in the P450 metabolizing enzymes. Inadvertent overdose can occur through combination of OTC products with prescription medicines.

In recent years, analogues of paracetamol with reduced potential for hepatic toxicity, such as the saccharin derivative, SCP-1, have been described. SCP-1 is rapidly metabolized to SCP-123, which is believed to be responsible for efficacy. Development of such analogues has been hampered by the lack of a cost-effective synthesis, but Louisiana chemists have now described a viable route to SCP-123. The synthesis comprises three steps from commercially available starting materials, requires no chromatographic purification and is amenable to large-scale processing. Full details are published in Organic Process Research & Development.

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The complement system is a complex cascade of reactions forming a central component of the innate immune system which assists in the removal of invading pathogens and cellular debris, and in the processing of immune complexes. There is substantial evidence that complement activation is associated with amyloid plaques in the brains of Alzheimer’s disease patients, although whether this is beneficial or detrimental has been unclear.

Writing in the Journal of Immunology, US and Australian scientists have now described the effect of administration of an antagonist of the receptor for the complement activation product, C5a, in animal models of Alzheimer’s disease. Oral administration of PMX205 in drinking water for 2-3 months resulted in substantial reductions in disease markers such as fibrillar amyloid deposits and activated glia in two mouse models of Alzheimer’s disease. The reduction in pathological markers correlated with improved performance in a passive avoidance task in Tg2576 mice. In 3xTg mice, PMX205 also significantly reduced hyperphosphorylated tau.

PMX205 is a cyclic hexapeptide derivative that was developed by Promics as a second generation C5a receptor antagonist for the treatment of inflammatory disorders, including inflammatory bowel disease. An earlier compound, PMX53, was found to be well tolerated in phase I clinical trials for rheumatoid arthritis and psoriasis.

The new study shows for the first time that antagonists of the C5a receptor interfere with inflammation and neurodegeneration in mouse models of Alzheimer’s disease and could, one day, lead to new treatments for human patients.

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