Sputum Matrix Metalloproteinases (MMP) mRNA and Montelukast

Overview

Matrix metalloproteinases (MMPs) are a group of 24 zinc containing enzymes in man. These enzymes were originally described as cleaving extracellular matrix (ECM) substrates with a predominant role in ECM homeostasis, but it is now clear that they have much wider functionality. An imbalance between MMP activity and that of their inhibitors (tissue inhibitors of metalloproteinases, TIMPs) is considered to play a critical role in the synthesis or degradation of the extracellular matrix of the airway architecture which results in fixed airflow obstruction in both asthma and chronic obstructive pulmonary disease (COPD). Using quantitative real time polymerase chain reaction (RT-PCR) the investigators have identified a difference between the level of steady state mRNA for MMP-9, MMP-14 and MMP-2 in 2 patients with asthma compared to 4 healthy controls using our method. However the investigators require further refinement of the process in order to optimise RNA quality and to evaluate the effect of montelukast across the entire family of MMPs and their inhibitors (TIMPs).

The following measurements will be performed at screening: * Informed consent * Clinical examination * Spirometry * Induced sputum The following will be performed after 8 weeks of study medication: * Clinical examination * Spirometry * Induced sputum * Diary Card Spirometry: This will be performed with a Microlab spirometer (Micro Medical Ltd, Rochester, Kent, UK). The procedure will be according to American Thoracic Society specifications(13). Diary Card Data: Patients will record their symptoms on a daily basis in the morning according to "cough", "breathlessness" and "wheeze" on a 4 point scale with 0=no symptoms and 3=maximal symptoms. A total symptom score will be calculated out of 12. Patients will also measure their peak expiratory flow on a daily basis in the morning and record the highest of three measurements. They will record that they have taken their study medication. Sputum Induction \& Examination: Sputum will be obtained with hypertonic saline by the method described by Pizzichini et al(14) inhaling increasing concentrations of saline (3, 4 and 5%) each for 7 minutes, through a mouthpiece. After each period of inhalation, FEV1 will be measured for safety. Subjects will be asked to cough sputum into a sterile container. Total cell count of leukocytes will be obtained in a modified Neubauer haemocytometer. The cell viability will be determined by the trypan blue exclusion method. Four hundred non squamous cells will be counted in Wright-stained slides and the results will be expressed as a percentage and absolute number of the total non squamous count. Measurement of MMP-9, 12 TIMP-1 and TGFb will be performed in sputum supernatant. Profile of mRNA of MMP and TIMPs: Total RNA will be extracted from the cellular content of the induced sputum plug using a combination of Trizol extraction and Qiagen RNeasy spin columns in a similar way to previously described12. Quantitative RT-PCR, using previously developed primers and probes, will be used to determine the relative quantities of mRNA of MMPs and TIMPs as described12. We remain the only centre in the world to routinely profile the entire MMP and TIMP gene family in human samples. This gives an all encompassing view of the involvement of these enzymes and inhibitors in the disease process and also sheds light on potential new biomarkers. The possibility of expanding the gene profiling without the need for additional sputum collection also adds value to the research. This might include other proteinase families with roles in ECM breakdown or in inflammation.