Huff and puff: Predicting the effects of new respiratory medicines need not be so difficult. By Lee Christie
As a number of the big Pharma companies move out of the respiratory and allergy fields, you would perhaps think that the interest in this market is starting to dwindle. In our experience it is pretty much the opposite. More and more we are being asked to develop new and innovative human tissue-based tests for our clients to assess the immunological or functional aspects of human airways and pulmonary blood vessels, for diseases as common as asthma, to the rarer conditions such as primary pulmonary hypertension that affects only 1 in 10,000 women each year. So how do we do it?
Obtaining Fresh Tissues for Research
First of all we need to obtain the fresh lung samples. We could spend the rest of this article on the complexities of tissue access, but it all really boils down to the creation of effective partnerships with clinical teams. We have frequent access to lung tissue from a variety of patient sub-sets including those with asthma and COPD. At Biopta, our work really starts once a sample has been donated. Often the hardest part is identifying and dissecting the airways or blood vessels within a tissue sample as there can be a lot of variation in the location and size of the sample, which depends on the pathology of the tissue (e.g. the type of surgery it is retrieved from), and also the hospital which is retrieving the sample (transplant obviously yields a greater volume of tissue as opposed to surgical operations, where the smallest section possible is removed).
Next, we have to maintain the tissue quality; this means getting the tissue sample to our labs as quickly as possible so that all normal functions are still intact. This is extremely important when examining the immunological response of a tissue sample as this is the first point of degradation, and often means a night shift for our lab staff (of which I am familiar!). Other functional aspects of the tissue samples are maintained longer, such as responses of airways to known bronchoconstrictors or dilators, however our aim is always to use a sample as soon as possible so that it is fresh and therefore any intervention in our experiments reflects as much as possible the usual response that would take place in the human body.
Investigations in Human Lung
Currently we use organ culture and organ bath systems, which are two methods that allow a wide range of end-points to be measured. As each client study is tailor-made, we build each protocol around the specific drug-induced effects we wish to detect.
Organ culture or organoculture (the term is really a bit of a misnomer as it is intact tissues that are being cultured) is a technique we commonly use to assess inflammation within a tissue and the method can be flexibly applied to a number of different organs/tissues including lung. Cytokines released by airways during inflammation are critical parts of the pathophysiology of disorders such as asthma and COPD. The aim of our assays is often to up-regulate these inflammatory mediators and then assess whether or not they can they be dampened by anti-inflammatory drugs. We culture these tissues over hours or days in order to see the long-term effects of the compounds.
In our organ bath systems, currently the most popular assay is one that reflects duration of action of asthma drugs. Today’s target for pharma companies is to create long-lasting compounds that dissociate from their binding site very slowly. For the patient this can mean a once a day dose, which can greatly improve patient compliance. To test these compounds we first incubate the airway segment with the compound; this allows the drug to bind to its target receptors. We then repeatedly challenge the airway with a bronchoconstrictor over a defined time period, washing the fluid surrounding the airway between each challenge. As the drug compound slowly dissociates from the receptor, the response to the bronchoconstrictor increases and thus those that keep the constrictor response at bay for a longer time have a longer duration of action and are potentially the more worthy compounds.
Another popular assay type is to assess bronchodilatory responses. Test and/or reference compounds are added cumulatively to bronchial rings following pre-constriction with a bronchoconstrictor such as carbachol. For example, see the figure below where the phosphodiesterase inhibitor theophylline (3 mM) was added at the end of each cumulative concentration response curve to induce full relaxation. All reference compound responses shown as % of maximum response to theophylline.
In addition to this we are able to monitor airway bronchoconstriction to antibodies, vascular dilators to relieve pulmonary hypertension, transportation of drugs across the airway epithelium…who knew you could get so much information from one lung sample?!
And on to the next step the compounds proceed. Fingers crossed they pass the next pre-clinical challenge!