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December, 2007

Improving The Flow – Asterand’s PhaseZERO® Pharmacology Services For Urology Research Historically, normal urological function and associated pathologies have been hard to characterise in human tissue in vitro due to poor availability and quality of tissue. Over the last 11 years, Asterand has established an extensive global network that allows access to a wide range of high quality human tissues, ethically consented for research. This provides us with regular access to a comprehensive range of urinary tissues including bladder, prostate and urethra from both male and female donors. The majority of urinary tissues obtained for our PhaseZERO® pharmacological studies come from non-diseased bladder samples, which are obtained from a wide age range of donors. Our access to such tissue allows compounds to be assessed in ‘normal’ human tissue. As soon as fresh human urinary tissue becomes available, studies are performed by our experienced pharmacology team based in Royston, near Cambridge, in the UK.     Typical examples of fresh human bladder tissue available for PhaseZERO® pharmacological studies at Asterand: segment of human, full thickness bladder (left) and partially dissected bladder showing bladder muscle bundles (right). Asterand offers a number of experimental approaches to evaluate test compounds for the treatment of urinary incontinence. Using ‘classical’ pharmacological techniques, our portfolio of urological-based assays can be used for profiling compounds at human native targets to determine: Agonist and antagonist potency Magnitude of effect Onset and duration of action Mechanism of action These human tissue-based data can be essential for understanding species differences in pharmacology and for validating (or otherwise) animal or recombinant target-based approaches. Overactive bladder (OAB) syndrome has been estimated to occur in ~17% of the population in the United States and Europe. Therefore there is still considerable investment from pharmaceutical companies to discover new drugs in this field and it is an area in which Asterand, with its range of assays, can help accelerate the progression of potential new pharmacotherapies. Below are some examples of data that Asterand has generated using human urological tissue. Muscarinic receptor function in human bladder Over the last three decades, there has been widespread use of anti-muscarinic compounds, both subtype-selective (e.g. darifenacin) and non-selective (e.g. oxybutinin), for the management of OAB. Anti-muscarinic compounds have been associated with poor tolerance due to the side effects seen on both the salivary glands, causing dry-mouth, and on the GI-tract, causing constipation, through the activation of M3 muscarinic receptors. Therefore, there is still a drive to find both novel targets for the management of OAB and design anti-muscarinic compounds that will have better patient tolerability. Human isolated urological tissue can also be used to assess drug-induced smooth muscle contraction or relaxation and to assess the drug effects on nerve-induced contractions. The bladder dome receives a parasympathetic innervation, which plays a key role during voiding. Below shows some of our data with the non-selective muscarinic antagonist, atropine. This original trace shows stable, electrical-field stimulation induced contractions of human isolated detrusor, with concentration-dependent inhibition in the presence of atropine, a non-selective muscarinic receptor antagonist (concentrations are logM). Here we have shown that atropine (10nM to 1µM) causes concentration-related antagonism of carbachol-induced contractions in human isolated detrusor smooth muscle. The affinity values generated agree with published literature values for atropine in human bladder. ß-adrenoceptor function in human bladder The functional importance of ß-adrenoceptors in the human detrusor remains to be established. There is speculation that in detrusor overactivity there is a lack of ß-adrenoceptor mediated inhibitory responses. Therefore, an alternative approach to anti-muscarinics for treating overactive bladder could be to enhance the relaxation (via the stimulation of ß-adrenoceptors) of the bladder during the filling phase. The distribution of ß-adrenoceptor subtypes mediating detrusor smooth muscle relaxation is species dependent, making extrapolation of data generated in various animal models to humans difficult. Therefore the use of human tissue can bridge the gap between animal or cell models and the clinic. The figure above shows the concentration-dependent relaxation of KCl pre-contracted human isolated detrusor with either the non-selective ß-adrenoceptor agonist, isoprenaline (pEC50 = 6.5, both graphs) or the L-type calcium channel blocker, nifedipine (pIC50 = 8.0, right hand graph only). Comparable data is produced with isoprenaline in human detrusor obtained either through a surgical sample or from a whole bladder donor (left hand graph only). The potency of isoprenaline and nifedipine concurs with literature values. Another original trace of human isolated detrusor to show the effects of isoprenaline on electrical field stimulation (EFS) induced contraction. Isoprenaline inhibited EFS-induced contraction but also markedly decreased basal tone. The remaining EFS-response was abolished by atropine (concentrations are logM). Role of the urothelium in human bladder function Another area of interest as a potential target for the management of overactive bladder is the role of the bladder urothelium. Classically, this was thought to be only a passive barrier to ions and solutes, but a number of novel properties have been attributed to urothelial cells. Further discovery of mechanisms that influence urothelial function might provide insights into the pathology in bladder dysfunction. As Asterand regularly receives whole bladders, we have access to intact human urothelium, which provides an ideal platform for basic discovery work and to screen novel compounds at any exciting new urothelial targets. This figure shows the contractile responses to the muscarinic agonist carbachol on human detrusor smooth muscle with the urothelium either intact or denuded. Maximum responses to carbachol were reduced by ~40% in tissues with the urothelium intact. In contrast, the potency of carbachol was not affected by the presence of the urothelium. These findings are in agreement with similar studies performed by others using human tissues. Stress incontinence – human urethral studies Stress urinary incontinence (SUI) affects an average of ~50% of incontinent women between the ages of 18-90 years, with no globally accepted pharmacotherapy. Deficient urethral closure may contribute to SUI. The degree of sympathetic innervation is high in the urethra and contraction is mediated predominantly through a1-adrenoceptors. Several classes of drug have been investigated for their efficacy in treating SUI by trying to mimic or prolong the action of endogenously released noradrenaline. Below shows some of our data with noradrenaline (non-selective a-adrenoceptor agonist), phenylephrine (a1-adrenoceptor agonist) and carbachol (muscarinic receptor agonist) in human proximal urethra. Whole length of human female urethra still attached to the base of the bladder received at Asterand (upper left). The upper right graph and original trace below show the concentration-dependent contractile effects of noradrenaline (NA, pEC50 = 5.6), phenylephrine (PE, pEC50 = 5.0) and carbachol (pEC50 = 4.8, in top graph only) in human circular, proximal urethral smooth muscle isolated from a female donor. The larger responses to noradrenaline and phenylephrine may reflect the fact that adrenergic neurotransmission is important to urethral contraction. In summary, Asterand’s regular supply of high quality, fresh urological tissues from both men and women allows our customers to use our PhaseZERO® pharmacology research services for the discovery of new therapies for the large number of urological pathologies. For further information, please e-mail: advantage@asterand.com. ASTERAND SUPPORTS HUMAN DRUG DISCOVERY AND TRANSLATIONAL MEDICINE THROUGH THE PROVISION OF HIGH QUALITY WELL CHARACTERIZED HUMAN BIOMATERIALS AND PRECLINICAL RESEARCH SERVICES Visit us at www.Asterand.com