Protease activity

The use of a bioadhesive, polymeric dosage form for sustained dehvery raises questions about swallowing or aspirating the device. The surface area is small, and patient comfort should be addressed by designing a small (less than 2 cm ), thin (less than 0.1 mm (4 mil) thick) device that conforms to the mucosal surface. The buccal route may prove useful for peptide or protein dehvery because of the absence of protease activity in the sahva. However, the epithelium is relatively tight, based on its electrophysiological properties. An average conductance in the dog is 1 mS/cm (57) as compared to conductances of about 27 and 10 mS/cm in the small intestine and nasal mucosa, respectively (58,59) these may be classified as leaky epitheha. 

An example of a direct spectrophotometrical assay is the use of synthetic peptide -nitroanilide substrates to determine protease activity. The /)-nitroani1ine group Hberated from the substrates by the protease can be determined spectrophotometricaHy at 410 nm. An example of an indirect (coupled) spectrophotometric assay is the determination of a-amylase using -nitrophenyLmaltoheptaoside. Initially, the substrate is cleaved by the a-amylase and subsequentiy one of the reaction products, -nitrophenyLmaltotrioside, is cleaved by a-glucosidase, hberating -nitrophenyl, a chromophore... 

Protease-activated receptors, PAR-1, PAR-2, PAR-3, PAR-4 Thrombin receptors Thrombin-related receptors, Trypsin receptor... 

Barry GD, Le GT, Fairlie DP (2006) Agonists and antagonists of protease-activated receptors (PARs). Curr MedChem 13 243-265... 

Leger AJ, Covic L, Kuliopulos A (2006) Protease-activated receptors in cardiovascular disease. Circulation 114 1070-1077... 

Ossovskaya VS, Bunnett NW (2003) Protease-activated receptors contribution to physiology and disease. Physiol Rev 84 579-621... 

Tetanus is a disease caused by the release of neurotoxins from the anaerobic, spore-forming rod Clostridium tetani. The clostridial protein, tetanus toxin, possesses a protease activity which selectively degrades the pre-synaptic vesicle protein synaptobrevin, resulting in a block of glycine and y-aminobutyric acid (GABA) release from presynaptic terminals. Consistent with the loss of neurogenic motor inhibition, symptoms of tetanus include muscular rigidity and hyperreflexia. The clinical course is characterized by increased muscle tone and spasms, which first affect the masseter muscle and the muscles of the throat, neck and shoulders. Death occurs by respiratory failure or heart failure. 

Most GPCRs interact with and activate more than one G-protein subfamily, e.g., with Gs plus Gq/n (histamine H2, parathyroid hormone and calcitonin recqrtors), Gs plus G (luteinising hormone receptor, 32-adrenoceptor) or Gq/11 plus G12/13 (thromboxane A2, angiotensin ATb endothelin ETA receptors). Some receptors show even broader G-protein coupling, e.g., to Gi, Gq/n plus Gi n ( protease-activated receptors, lysophosphatidate and sphingosine-1-phosphate receptors) or even to all four G-protein subfamilies (thyrotropin receptor). This multiple coupling results in multiple signaling via different pathways and in a concerted reaction of the cell to the stimulus. 

One exciting approach is the development of short sequences of RNA that bind specifically to HCV helicase and/or the protease activity found in the same hepatitis C virus-encoded non-structural protein, NS3, and inhibit helicase at sub-micromolar concenttations (Umehara et al. 2005). These molecules could provide the basis for developing potent helicase inhibitors with improved pharmacotherapeutic properties. 

YU R, mandlekar s, HARVEY K J, UCKER D s and KONG A N (1998) Chemopreveutive isothiocyanates induce apoptosis and caspase-3-like protease activity . Cancer Res, 58 402-8. 

A protease-specific model has also been reported in which a replication-defective adenovirus encoding an NS3 protease-SEAP fusion protein is injected into mouse tail veins, resulting in expression of the fusion protein in the liver [82, 83]. Protease activity can be detected both by measuring activity of liberated SEAP or by protease-induced liver damage. Protease activity was found to be reduced by administration of protease inhibitors. This model can be used to show that candidate inhibitors have adequate pharmacokinetic properties in mice to function in the intended target organ, but it is not a true disease model. 

TRPVl also plays a central role in intercellular pro-inflammatory feedback loops. An important example is mast cells and sensory nerves. Mast cells release tryptase that, in turn, activates the protease-activated receptor PAR-2 activation of PAR-2 then opens TRPVl via PKC [50]. In keeping with this, PAR-2 agonists reduce the heat activation threshold of TRPVl from 42 °C to below body temperature [51]. Excited nerve endings release SP that, as a positive feedback, binds to neurokinin NKl receptors on mast cells. Mast cells also express TRPVl [52]. Consequently, endovanilloids can act in concert to stimulate mast cells and activate capsaicin-sensitive nerve endings. Of relevance is the finding that PAR-2 is up-regulated in the bladder during experimental cystitis [53]. 

Inhibitors that block the protease activity of F-XIIa and kallikrein. Plasma contains two protease inhibitors that regulate contact activation Cl inhibitor (Cl-Inh) and a2-macroglobulin (a2-M). 

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