Day: 09/24/2010

Tranexamic acid for trauma

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We were very interested in the CRASH-2 trial.1 It showed not only the efficacy of tranexamic acid, but also its excellent safety in a large cohort of patients. Although the inclusion criteria for this trial were very relevant to routine clinical practice, hypotension in a trauma patient can be related to other causes (pneumothorax, spinal cord injury, etc) and not necessarily to documented bleeding.2
The CRASH-2 trial included younger patients than in other series and therefore underestimates pre-existing occlusive lesions with their corollary of platelet antiaggregant or anticoagulant treatments that predispose to bleeding.
A high frequency of coagulopathy is established on emergency-room admission and is affected by prehospital management and the magnitude of injury (reflected by the injury severity score [ISS]). ISS is not presented in the results of the CRASH-2 trial. Organisation of the health-care system therefore has a major role.3 The CRASH-2 trial does not take into account prehospital organisation, raising the question of whether these results can be extrapolated to all health-care systems.
Since randomisation did not take into account patients in whom tranexamic acid would have been immediately indicated, this trial excluded the most critically ill patients.
Many European and North American studies on the therapeutic approach to haemorrhage have assessed the effect on mortality of the transfusion ratio of fresh frozen plasma to packed red blood cells.4 The CRASH-2 trial did not address this issue. Although this trial done on an intention-to-treat basis has a substantial statistical power, the absence of analysis of the transfusion ratio could represent a bias and a limitation to the use of tranexamic acid for the most seriously ill patients. Finally, the prevalence of thrombotic complications in the CRASH-2 trial remains relatively low compared with other studies.5
Owing to its power and rigorous methods, this trial nevertheless remains a major trial in the therapeutic approach to haemorrhage in trauma patients, showing that this inexpensive and easy-to-use molecule could be particularly useful in emergency situations without any major adverse effects.
source:Lancet

COX inhibition: Naproxen by proxy

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Naproxen is one of the original non-steroidal anti-inflammatory drugs (NSAIDs) that target the cyclooxygenase (COX) enzymes, yet the molecular basis of its interaction with COX enzymes has not been well established. Through a structure-activity-guided approach, Lawrence Marnett and colleagues uncover interactions that dictate naproxen binding to COX-2 and distinguish it from other NSAIDs that bind the COX enzymes.

There are at least four structural classes of molecules that characterize NSAIDs. Naproxen is a relatively simple molecule, containing three functional groups: an arylpropionic acid (carboxylate), a p-methoxy group and an α–methyl group on a naphthyl scaffold. As a drug, naproxen inhibits both COX-1 and COX-2 with comparable IC50s and exhibits gastrointestinal side effects, but reduced cardiovascular side effects relative to other selective as well as non-selective COX inhibitors.

To probe the interaction between naproxen and the COX enzymes, Marnett and colleagues mutated residues R120 and Y355 of murine COX-2 (mCOX-2), as these residues have been identified to mediate the canonical interaction of the arylcarboxylic acid family of NSAIDs. These mutations suggest that the carboxylate group of naproxen binds in the canonical orientation, coordinated to these two residues. To verify this and to identify other naproxen interactions with mCOX-2, the authors solved the co-crystal structure at 1.7 Å resolution.

This high resolution structure enabled visualization of solvent, ion and detergent molecules not observed in lower resolution structures of the COX enzymes, as well as subtle differences from existing co-crystal structures with COX-2. The naproxen-mCOX-2 complex also revealed a single orientation of naproxen within the COX-2 active site. Beyond the hydrogen-bonding interactions of the naproxen carboxylate group to R120 and Y355 predicted from the mutagenesis data, all other interactions are van der Waals contacts. W387 is an important residue at the top of the mCOX-2 active site. That naproxen was unable to inhibit a W387F mutant, whereas other NSAIDs like diclofenac and indomethacin could inhibit enzymatic activity suggests that an interaction between naproxen and this residue is specific to this NSAID.

Turning to mutagenesis of the naproxen molecule, the authors substituted the α–methyl group with hydrogen, ethyl or dimethyl substituents and found a loss of enzymatic potency with each of them, indicating that an (S)-methyl group at this position is critical. The co-structure shows that this group inserts into a small hydrophobic cleft below V349, which seems to anchor naproxen within the active site and reinforce the canonical binding orientation. A similar structure-activity relationship analysis of the naproxen p-methoxy group suggested the importance of this constituent at the top of the enzyme active site channel. Interestingly, p-ethyl and p-methylthio substitutions at this position still allowed inhibition of mCOX-2, but these compounds could not inhibit ovine COX-1.

Also surprising was that both of these analogs inhibited the W387F COX-2 mutant as effectively as they inhibited the wild-type enzyme.

To follow up on these observations of COX-2 selectivity, the authors solved a second co-structure, this time between the p-methylthio naproxen derivative and mCOX-2. Compared to the naproxen co-structure, there was a shift of the carboxylate tails of the compounds and a new interaction between the naphthyl backbone of the p-methylthio naproxen, while many of the other interactions remain the same in the two co-structures.

These results define the contribution of the COX protein and naproxen atoms to the affinity and suggest that there is little tolerance for mutation in either component. This could tend to complicate any attempts to modify naproxen to eliminate the unwanted gastrointestinal side effects of the drug.

Gene-therapy hope for β-thalassaemia patients

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The genetic disorder β-thalassaemia demands blood transfusions throughout life.Andrey Prokhorov/iStockphoto

Gene therapy for a form of β-thalassaemia, a genetic disorder whose sufferers require frequent blood transfusions because they cannot properly produce red blood cells, seems to have been successful in a patient who, three years after treatment, no longer requires transfusions1. Doubts remain, however, over whether a set of lucky circumstances is behind the success.

Patients with β-thalassaemia carry faulty copies of the genes needed to produce the β-globin chain of haemoglobin, sometimes lacking the genes altogether. This leads to a shortage of red blood cells, the body’s oxygen carriers.

Sufferers must have regular blood transfusions throughout their lives, an inconvenient and debilitating regime that ultimately shortens life expectancy. The only known cure is stem-cell transplantation, but few patients are able to find a suitable donor.

Because of the gruelling nature of this treatment, the development of gene therapies for β-thalassaemia is seen by many as an exciting prospect. The subject of the latest trial was an 18-year-old man with βE/β0-thalassaemia — in this form of the disease, one copy of the β–globin gene produces unstable β-globin and the other copy is non-functional.

Around half of the patients with this form of β-thalassaemia are dependent on transfusions, and the patient concerned had received blood transfusions since the age of three.

Philippe Leboulch of Harvard Medical School, part of the team that carried out the study, described the treatment as “life-changing”. “Before this treatment, the patient had to be transfused every month. Now he has a full-time job as a cook,” he says.

Unrepeatable?

However, Michael Antoniou of King’s College London, suggests that this case was “an extremely fortuitous event”, and that the positive outcome seen is unlikely to be repeatable in other patients.

The procedure was carried out as follows. In 2007, an international team led by Marina Cavazzana-Calvo of University Paris-Descartes extracted haematopoietic stem cells (HSCs) from the patient’s bone marrow. These cells give rise to all blood cell types, including the haemoglobin-containing red cells. The researchers cultured these cells, and mixed them with vectors based on the lentiviruses — a retrovirus subgroup with a long incubation period — into which a functional copy of the β-globin gene had been introduced. These vectors were shown in preclinical trials to be safer than those derived from the retroviruses — which are also replicated in a host cell — that have been used in previous gene-therapy procedures.

Chemotherapy was used to eliminate as many of the patient’s faulty HSCs as possible, to prevent dilution of the genetically corrected cells, which were then transplanted. Levels of healthy red blood cells and normal β-globin in the subject’s body gradually rose until, around a year after the treatment, he no longer required transfusions. After 33 months he remains mildly anaemic, but the fact that he remains transfusion-free has been hailed as a success.

However, that achievement is tempered by a cautionary note. The researchers have detected overexpression of a protein called HMGA2, which has been linked to cancers, in a high proportion of the genetically modified cells.

Overexpression occurred because the lentivirus vector can randomly integrate into chromosomes. By chance, one transplanted haematopoietic cell clone contains a vector insertion in the HMGA2 gene. A year after the transplant, the researchers noticed that the proportion of genetically modified cells that originated from this particular cell clone was rising until it reached a plateau at around 50%.

The reasons for the over-representation of that particular clone remain unclear, but that could be down to the fact that the patient’s haematopoietic system was reconstituted from just a few modified HSCs. Luigi Naldini, a gene-therapy researcher at San Raffaele Telethon Institute for Gene Therapy in Milan, Italy, says that successfully grafting a larger initial population of modified HSCs could potentially prevent the problem from developing.

source: nature

Multiple sclerosis: Putting a dampener on inflammation

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Multiple sclerosis is a chronic, demyelinating disease of the central nervous system that is thought to have an inflammatory aetiology. High levels of glutamate are found in the brains of patients with multiple sclerosis, but the link between this excitatory neurotransmitter and the immunopathology is not clear. Now, Fallarino et al. suggest a role for metabotropic glutamate receptor 4 (mGluR4) signalling in dampening the immune response during neuroinflammation, highlighting a pathway that could be exploited therapeutically.

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Previous work in a rodent model of multiple sclerosis — experimental autoimmune encephalomyelitis (EAE) — had shown that activation of specific mGluRs, including mGluR4, could help to mediate recovery from disease. The authors therefore assessed the clinical symptoms of mice with EAE that lacked the gene encoding mGluR4 (Grm4−/− mice) and discovered that they had more severe symptoms and an earlier onset of disease than wild-type mice.

mRNA profiling of CD4+ T cells, key effector immune cells, from the brain and lymph nodes of Grm4−/− mice indicated that differentiation of these cells into pro-inflammatory T helper 17 (TH17) cells was favoured over differentiation into regulatory T (TReg) cells, which was in contrast to wild-type mice. Furthermore, EAE-induced inflammation was greater in the knockout mice, with higher levels of pro-inflammatory cytokines and lower levels of inflammation-suppressing cytokines than in wild-type mice with EAE.

Reverse transcription PCR on various cell types from wild-type mice showed that mGluR4 expression was highest in CD4+ T cells and dendritic cells — immune cells that can promote T cell differentiation into a regulatory or an inflammatory phenotype depending on signals from the local environment. Cultures of activated T cells from Grm4−/− mice had the same cytokine profile and rate of proliferation as those from wild-type mice. By contrast, activated dendritic cells from Grm4−/− mice, but not wild-type mice, had a pro-inflammatory cytokine profile. In particular, these cells secreted high levels of interleukin-6 (IL-6) and low levels of IL-27, a profile that is known to promote the differentiation of naive T cells into TH17 rather than TReg cells. Therefore, the effect of mGluR4 signalling on the T cell phenotype seems to be mediated through dendritic cells.

The intracellular signalling pathway that links dendritic cell activation with the production of TH17-type cytokines involves increased cyclic AMP formation. Administration of a positive allosteric modulator of mGluR4 known as PHCCC attenuated this cAMP increase in activated wild-type dendritic cells. In vivo, administration of PHCCC to mice with EAE attenuated disease in a statistically significant, dose-dependent manner and was associated with an increase in TReg cells in the lymph nodes. Thus, allosteric modulators of mGluR4, which are currently under investigation for Parkinson’s disease, might provide an opportunity to harness what seems to be a protective effect of glutamate in patients with multiple sclerosis.

source: Natrure

orphan drug

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To determine whether a general societal preference for prioritising treatment of rare diseases over common ones exists and could provide a justification for accepting higher cost effectiveness thresholds for orphan drugs.

Design Cross sectional survey using a web based questionnaire.

Setting Norway.

Participants Random sample of 1547 Norwegians aged 40-67.

Main outcome measure Choice between funding treatment for a rare disease versus a common disease and how funds should be allocated if it were not possible to treat all patients, for each of two scenarios: identical treatment costs per patient and higher costs for the rare disease. Respondents rated five statements concerning attitudes to equity on a five point Likert scale (5=completely agree).

Results For the equal cost scenario, 11.2% (9.6% to 12.8%) of respondents favoured treating the rare disease, 24.9% (21.7% to 26.0%) the common disease, and 64.9% (62.6% to 67.3%) were indifferent. When the rare disease was four times more costly to treat, the results were, respectively, 7.4% (6.1% to 8.7%), 45.3% (42.8% to 47.8%), and 47.3% (44.8% to 49.8%). Rankings for attitude on a Likert scale indicated strong support for the statements “rare disease patients should have the right to treatment even if more expensive” (mean score 4.5, SD 0.86) and “resources should be used to provide the greatest possible health benefits” (3.9, 1.23).

Conclusions Despite strong general support for statements expressing a desire for equal treatment rights for patients with rare diseases, there was little evidence that a societal preference for rarity exists if treatment of patients with rare diseases is at the expense of treatment of those with common diseases.

source:BM J