A characteristic of the disease asthma are episodes of increased constriction of the airways musculature which causes shortness of breath, chest tightness, wheezing and rapid respiration. Constriction of the airways musculature results from contraction of the smooth muscle in the airway, inflammation of the bronchial wall and increased mucous secretion.

Smooth muscle contraction is controlled by calcium ions (Ca2+).

In relaxed muscle Ca2+ is sequestered in the sarcoplasmic reticulum by ATP driven active transporters until the Ca2+concentration in the cytosol is less than 1AM. A nerve impulse leads to the release of Ca2+ from the sarcoplasmic reticulum, which raises the cytosolic concentration to about lOgM and leads to muscle contraction.

Asthma is thought to result from a disordered imbalance of the immune system hence the influx of cells usually observed in an immune response, such as neutrophils and mast cells.

Inflammatory mediators, such as endotoxins, ozone and cigarette smoke, can induce an immune response, resulting in the release of factors which directly induce airway muscle hyper-responsiveness; TNF-a, a cytokine, is one such factor.

Several studies have now shown that asthmatic patients have up to 20 times more TNF-a present in their airways than normal subjects.

Worldwide, 150 million people suffer from asthma. In Britain, 3 million people (one third of those children) are affected by this condition. There has been a fourfold increase in asthma sufferers over the past 30 years and the death rate has more than doubled. In addition to the health risks, the

direct medical costs associated with asthma in Britain are approximately £500 million.

To date, asthma therapy is largely symptomatic, it typically involves immune suppressing agents which relax the airway via indirect mechanisms. These treatments vary in success because they do not directly counteract the cause of the hyper- responsiveness. Corticosteroids, which are used to treat patients with moderate to severe asthma, until recently were thought to have no effect on the airway smooth muscle but new research suggest that corticosteroids may block TNF.

Corticosteroid are known to decrease the number and activity of the cells involved in airway inflammation-macrophages, eosinophils and T-lymphocytes. This reduces inflammation by reversing mucosal edema, decreasing the permeability of capillaries and inhibiting the release of leukotrienes. One drawback of corticosteroid treatment is it is long term and the effect can only be seen after months of use.

Only about 20% of aerosol corticosteroid is deposited in the airways the remainder is deposited in the mouth and pharynx or is swallowed. Deposition in the oropharynx can result in oropharyngeal candidiasis, especially in immunocompromised patients. The swallowed corticosteroids are absorbed from the gut and enter the systemic circulation through the liver, which metabolises them. Thus in practise only a small amount of corticosteroids reach the systemic circulation. Although these drugs work effectively and have minimal side effects, due to the lack of a significant systemic presence, it would be advantageous to have agents which worked almost immediately and in a specific way.

In order to produce agents which directly counteract the hyper-responsiveness the mechanisms and pathways involved in the hyper-responsiveness must be examined. It is known that TNF is involved in triggering allergic asthma. Previous experiments have compared the responses of asthmatic rats and

guinea pigs when treated with a TNF-receptor fusion protein, dexamethasone (a corticosteroid) and a placebo. The researchers assessed the number of white blood cells produced and the degree to which airflow was restricted to the lungs.

The results showed that as an asthma preventative, the TNF blocker worked as effectively as dexamethasone. These experiments led scientists to the conclusion that inhibitors of TNF may be useful agents in the treatment of allergic asthma.

However, to date, the exact role of TNF-a in the pathogenesis of the disease has not been elucidated. The key to uncovering the mechanisms of TNF-a induced hyper-responsiveness in airway musculature is to examine the actions inside the muscle cells where this hyper-responsiveness occurs.

According to a first aspect of the present invention, there is provided a method for the assay of TNF-a induced airways contraction; which comprises treating isolated airways musculature with a cell permeabilization agent in a calcium controlled buffer solution to control calcium transport through a cell wall thereby to control contraction of the musculature in a concentration dependent fashion to give a first contraction value; relaxing the treated musculature in a calcium controlled buffer solution and subsequently treating the relaxed musculature with TNF-a and exposing to a calcium buffer solution to obtain a second contraction value; and comparing said contraction values.

The current inventive technique has resulted in the first evidence of a cellular mechanism whereby TNF-a produces an increased response in airway musculature. As TNF-a alone does not cause any contractions, it has been deduced that the TNF-a must be activating a biochemical pathway in the cells which leads to the potentiation of normal stimuli.

According to the second aspect of the invention, there is provided a method for the in vitro assay of the efficacy of a respiratory disease treatment; which comprises treating isolated airways musculature with a cell permeabilization agent in a calcium controlled buffer solution to control calcium transport through a cell wall thereby to control contraction of the musculature in a concentration dependent fashion to give a first contraction value; relaxing the treated musculature in a calcium controlled buffer solution and subsequently treating the relaxed musculature with TNF-a and exposing to a calcium buffer solution to obtain a second contraction value; relaxing the treated musculature in a calcium controlled buffer solution and subsequently treating the relaxed musculature with TNF-a and a potential respiratory disease treatment and exposing to a calcium buffer solution to obtain a third contraction value; and comparing said contraction values.

Using the technique in the second aspect the extent of the contraction of the muscle, due to treatment with TNF-a, can be compared before and after treatment with existing and novel compounds thought to reduce smooth muscle contraction in respiratory disease patients. Any decrease in the contraction after treatment would show that the compound had had a positive effect and might lead to the development of a novel treatment for respiratory disease. Any treatments developed from this technique would be known to treat the hyper- responsiveness directly and therefore more effectively than the previously known indirect treatments.

Preferably in the first and second aspects of the invention, the cell permeabilization agent is a bacterial toxin, for <BR> <BR> <BR> example a-toxin from Staphylococcus aureus. Although any suitable alternative may be used. The toxin or permeation agent allows small ions and proteins to access the inside of

the cells thus allowing the intracellular ion concentration to be carefully regulated.

In preferred embodiments of the first and second aspect of the invention, the isolated airways musculature pre-treated with the cell permeabilization agent is placed in a calcium controlled buffer solution for 1.5 hours at 28°C. The calcium controlled buffer solution may be methane sulphonate based and contain a calcium chelator, such as EGTA (Ethylene glycol bis (P-aminoethyl ether)-N, N, N', N'-tetraacetate). In this environment the isolated airways musculature is fully relaxed and readings taken in this state give a base line value of smooth muscle cell tension.

Conveniently in the first and second aspects of the invention, the airways musculature is incubated with TNF-a for 20-45 minutes. Calcium sensitisation is observed in isolated airways musculature treated with TNF-a after 20 minutes reaching maximum sensitisation after 45 minutes. As the calcium concentration is a given value this potentiation is not due to an increase in calcium ions. Therefore, it must be a TNF-a activated pathway which acts to achieve enhanced muscle contraction.

Preferably in the first and second aspects of the invention, the isolated airways musculature is placed in a calcium buffer to give a reading corresponding to a maximum contraction.

From this value the calcium concentration of another calcium buffer can be deduced so that when the musculature is placed in a further derived calcium buffer the contraction will be 20-35% of the maximum contraction. It follows that by controlling the calcium concentration, any increase in potentiation must be as a result of another factor, such as the addition of TNF-a.

According to the third aspect of the invention a potential drug target has been identified. By blocking the TNF

receptors the degree of hyper-responsiveness of the airways musculature may be reduced and therefore any asthma attack will be less severe. Further studies, using the technique described in the first aspect of the invention, have revealed that TNF-a does not have the same effect on blood vessels, which are composed of a similar type of muscle cell to airway musculature. This suggests that the TNF-a induced mechanism may be specific to the airways musculature so any drugs are targeted to this area. Thus a low dosage of drugs can be used and any side effects caused by the presence of the drug in parts of the body other than the targeted area, are thereby reduced.

The invention will now be described, by way of illustration only, with reference to the following example and figures accompanying the specification.

Figure 1 is a graphical representation of the tension record of the paired control permeabilized airway smooth muscle strip.

Figure 2 is a graphical representation of the tension record of the TNF-a treated permeabilized airway smooth muscle strip.

Figure 3 shows the scale used in Figures 1 and 2. Tension, on the y axis, is measured in milliNewtons (mN) and time, on the x axis, is measured in minutes.

EXAMPLE A strip of bronchial smooth muscle (200Am wide) was dissected from below the bronchi bifurcation in guinea pigs. The strip was attached to a Sensonor transducer to record tension and incubated initially in HEPES buffered Kreb's solution. The strip was transferred to a first calcium controlled buffer, containing 1mM EGTA, the first calcium controlled buffer being methane sulphonate based, and permeabilized in purified

Staphylococcus aureas a-toxin (2500 units of activity) for 1.5 hours at 28°C. Following permeabilization, the strip was washed in a first calcium controlled buffer for 5 mins and exposed to a second calcium buffer at pCa4.5 (buffered with lOmM EGTA). Once a maximal calcium-activated contraction was obtained and recorded, the strip was relaxed by incubating it in the first calcium controlled buffer. The strip was then placed in a third calcium buffer at pCa6.3 and steady state calcium activated contraction of the strip was recorded.

After being relaxed in the first calcium controlled buffer for 45 mins the strip was again transferred to the third calcium buffer at pCa6.3 and the resultant calcium-induced contraction was measured. This generated the control values as seen in Fig. 1.

Another strip of isolated airways musculature, pre-incubated in HEPES buffered Kreb's solution, was transferred to a first calcium controlled buffer, containing lmM EGTA, the first calcium controlled buffer solution being methane sulphonate based and permeabilized in purified Staphlococcus aureas a- toxin (2500 units of activity) for 1.5 hours at 28°C.

Following permeabilization, the strip was washed in the first calcium controlled buffer for 5 mins and exposed to a second calcium buffer at pCa4.5 (buffered with lOmM EGTA). This resulted in a maximal calcium-activated contraction.

Thereafter the strip was relaxed by incubation in the first calcium controlled buffer and placed in a third calcium buffer at pCa6.3 and the steady state calcium activated contraction was recorded. The strip was then relaxed in the first calcium controlled buffer whereupon recombinant TNF-a (1/g/ml) was added and both the strip and the TNF-a were incubated for 45 mins. After incubation the strip was placed in the third calcium buffer at pCa6.3 and the resultant contraction of the strip was recorded. The values obtained are represented in Fig. 2.

A comparison of Figures 1 and 2 shows the dramatic effect that

TNF-a has on the contraction of airways musculature. In Fig.

1 the control trace shows that after incubation in the calcium controlled buffer the contraction is comparable to that before incubation. In Fig. 2, which represents the values before and after incubation with TNF-a, there is almost a four-fol increase in the second peak compared to the first peak. As TNF-a does not itself cause any contraction it is deduced that TNF-a must be activating biochemical pathways inside the cell which do not produce contraction but lead to a potentiation of normal stimuli.