Sputum Matrix Metalloproteinases (MMP) mRNA and Montelukast
Sputum Matrix Metalloproteinases (MMP) mRNA and Montelukast
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.
Inclusion Criteria:

 -  Male or female, aged 18 to 60 years.

 -  Diagnosed with asthma, defined as episodic chest tightness, wheezing and dyspnoea,
 cough.

 -  Non-Smoker or Ex-Smoker for at least 10 years and a smoking history of less than 5
 pack years.

 -  History of asthma symptoms for more than 10years.

 -  Receiving as required short acting bronchodilators.

 -  Post bronchodilator FEV1 50 to 100 % predicted

 -  Evidence of airway calibre reversibility within the previous 12 months: reversibility
 to salbutamol of 12% following 400mcg inhaled salbutamol, histamine PC20 < 8mg/ml,
 diurnal variation in peak expiratory flow of 20%.

 -  Able to produce sputum after induction with saline.

 Exclusion criteria:

 -  Cardiac or pulmonary disease other than asthma.

 -  Respiratory infection defined as fever, nasal/sinus congestion, fatigue, cough,
 antibiotic use or yellow/green sputum within 4 weeks prior to study.

 -  Receiving inhaled or oral corticosteroid therapy, long acting Beta2 agonist therapy or
 leukotriene modifying therapy for the previous 1 month.

 -  Severe or uncontrolled co-morbid disease.

 -  Pregnancy or breastfeeding.

 -  Unable to give written informed consent