alpha-Synuclein aggregation in neurodegenerative diseases and its inhibition as a potential therapeutic strategy

There is strong evidence for the involvement of alpha-synuclein in the pathologies of several neurodegenerative disorders, including PD (Parkinson's disease). Development of disease appears to be linked to processes that increase the rate at which alpha-synuclein forms aggregates. These processes include increased protein concentration (via either increased rate of synthesis or decreased rate of degradation), and altered forms of alpha-synuclein (such as truncations, missense mutations, or chemical modifications by oxidative reactions). Aggregated forms of the protein are toxic to cells and one therapeutic strategy would be to reduce the rate at which aggregation occurs. To this end we have designed several peptides that reduce alpha-synuclein aggregation. A cell-permeable version of one such peptide was able to inhibit the DNA damage induced by Fe(II) in neuronal cells transfected with alpha-synuclein (A53T), a familial PD-associated mutation.

Inhibitors of alpha-synuclein oligomerization and toxicity: a future therapeutic strategy for Parkinson's disease and related disorders.

An abundance of genetic, histopathological, and biochemical evidence has implicated the neuronal protein, alpha-synuclein (alpha-syn) as a key player in the development of several neurodegenerative diseases, the so-called synucleinopathies, of which Parkinson's disease (PD) is the most prevalent. Development of disease appears to be linked to events that increase the intracellular concentration of alpha-syn or cause its chemical modification, either of which can accelerate the rate at which it forms aggregates. Examples of such events include increased copy number of genes, decreased rate of degradation via the proteasome or other proteases, or altered forms of alpha-syn, such as truncations, missense mutations, or chemical modifications by oxidative reactions. Aggregated forms of the protein, especially newly formed soluble aggregates, are toxic to cells, so that one therapeutic strategy would be to reduce the rate at which such oligomerization occurs. We have therefore designed several peptides and also identified small molecules that can inhibit alpha-syn oligomerization and toxicity in vitro. These compounds could serve as lead compounds for the design of new drugs for the treatment of PD and related disorders in the future.

Dried blood spot liquid chromatography assay for therapeutic drug monitoring of metformin

The use of blood spot collection cards is a simple way to obtain specimens for analysis of drugs for the purpose of therapeutic drug monitoring, assessing adherence to medications and preventing toxicity in routine clinical setting. We describe the development and validation of a microanalytical technique for the determination of metformin from dried blood spots. The method is based on reversed phase high-performance liquid chromatography with ultraviolet detection. Drug recovery in the developed method was found to be more than 84%. The limits of detection and quantification were calculated to be to be 90 and 150 ng/ml, respectively. The intraday and interday precision (measured by CV%) was always less than 9%. The accuracy (measured by relative error, %) was always less than 12%. Stability analysis showed that metformin is stable for at least 2 months when stored at -70 degrees C. The small volume of blood required (10 mu L), combined with the simplicity of the analytical technique makes this a useful procedure for monitoring metformin concentrations in routine clinical settings. The method is currently being applied to the analysis of blood spots taken from diabetic patients to assess adherence to medications and relationship between metformin level and metabolic control of diabetes. (c) 2006 Elsevier B.V. All rights reserved.

Potential improvements in the therapeutic ratio of prostate cancer irradiation: dose escalation of pathologically identified tumour nodules using intensity modulated radiotherapy.

The potential of intensity modulated radiotherapy (IMRT) to improve the therapeutic ratio in prostate cancer by dose escalation of intraprostatic tumour nodules (IPTNs) was investigated using a simultaneous integrated boost technique. The prostate and organs-at-risk were outlined on CT images from six prostate cancer patients. Positions of IPTNs were transferred onto the CT images from prostate maps derived from sequential large block sections of whole prostatectomy specimens. Inverse planned IMRT dose distributions were created to irradiate the prostate to 70 Gy and all the IPTNs to 90 Gy. A second plan was produced to escalate only the dominant IPTN (DIPTN) to 90 Gy, mimicking current imaging techniques. These plans were compared with homogeneous prostate irradiation to 70 Gy using dose–volume histograms, tumour control probability (TCP) and normal tissue complication probability (NTCP) for the rectum. The mean dose to IPTNs was increased from 69.8 Gy to 89.1 Gy if all the IPTNs were dose escalated (p=0.0003). This corresponded to a mean increase in TCP of 8.7–31.2% depending on the /ß ratio of prostate cancer (p

Development of sampling methods for Raman analysis of solid dosage forms of therapeutic and illicit drugs

The results of a study aimed at determining the most important experimental parameters for automated, quantitative analysis of solid dosage form pharmaceuticals (seized and model 'ecstasy' tablets) are reported. Data obtained with a macro-Raman spectrometer were complemented by micro-Raman measurements, which gave information on particle size and provided excellent data for developing statistical models of the sampling errors associated with collecting data as a series of grid points on the tablets' surface. Spectra recorded at single points on the surface of seized MDMA-caffeine-lactose tablets with a Raman microscope (lambda(ex) = 785 nm, 3 mum diameter spot) were typically dominated by one or other of the three components, consistent with Raman mapping data which showed the drug and caffeine microcrystals were ca 40 mum in diameter. Spectra collected with a microscope from eight points on a 200 mum grid were combined and in the resultant spectra the average value of the Raman band intensity ratio used to quantify the MDMA: caffeine ratio, mu(r), was 1.19 with an unacceptably high standard deviation, sigma(r), of 1.20. In contrast, with a conventional macro-Raman system (150 mum spot diameter), combined eight grid point data gave mu(r) = 1.47 with sigma(r) = 0.16. A simple statistical model which could be used to predict sigma(r) under the various conditions used was developed. The model showed that the decrease in sigma(r) on moving to a 150 mum spot was too large to be due entirely to the increased spot diameter but was consistent with the increased sampling volume that arose from a combination of the larger spot size and depth of focus in the macroscopic system. With the macro-Raman system, combining 64 grid points (0.5 mm spacing and 1-2 s accumulation per point) to give a single averaged spectrum for a tablet was found to be a practical balance between minimizing sampling errors and keeping overhead times at an acceptable level. The effectiveness of this sampling strategy was also tested by quantitative analysis of a set of model ecstasy tablets prepared from MDEA-sorbitol (0-30% by mass MDEA). A simple univariate calibration model of averaged 64 point data had R-2 = 0.998 and an r.m.s. standard error of prediction of 1.1% whereas data obtained by sampling just four points on the same tablet showed deviations from the calibration of up to 5%.

Proteasome inhibitors: a therapeutic strategy for haematological malignancy

The proteasome is a multicatalytic enzyme complex responsible for the regulated degradation of intracellular proteins. In recent years, inhibition of proteasome function has emerged as a novel anti-cancer therapy. Proteasome inhibition is now established as an effective treatment for relapsed and refractory multiple myeloma and offers great promise for the treatment of other haematological malignancies, when used in combination with conventional therapeutic agents. Bortezomib is the first proteasome inhibitor to be used clinically and a second generation of proteasome inhibitors with differential pharmacological properties are currently in early clinical trials. This review summarises the development of proteasome inhibitors as therapeutic agents and describes how novel assays for measuring proteasome activity and inhibition may help to further delineate the mechanisms of action of different proteasome inhibitors. This will allow for the optimized use of proteasome inhibitors in combination therapies and provide the opportunity to design more potent and therapeutically efficacious proteasome inhibitors.