Not going with the flow – human tissue-based pharmacology for urology research

The last decade has seen a vast change in the clinical management of urological problems, with a switch from the majority of patients undergoing surgery to being treated through the introduction of effective drugs. Commitment and research into urological disorders is still a key focus area for several pharmaceutical and biotechnology companies, with overactive bladder (OAB) syndrome and stress urinary incontinence (SUI) being the primary areas of interest. OAB has been estimated to occur in 16% of the population over 40 years of age [1], whereas SUI affects an average of ~50% of incontinent women between the ages of 18-90 years, with no globally accepted pharmacotherapy [2]. Despite considerable progress in this field over the last few years there is still significant investment from pharmaceutical companies to discover new drugs.

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 access to a wide range of high quality human tissues, ethically consented for research. Our human tissue network provides us with regular access to a comprehensive range of fresh urology tissues including bladder, urethra, from both male and female donors, and prostate. Using these tissues, with our PhaseZERO^® human tissue-based services platform, we are able to generate data for the pharmaceutical industry to support progression of their urology R&D programmes.

The majority of urology tissues obtained for our PhaseZERO^® pharmacology studies come from non-diseased 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 urology 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^® pharmacology studies at Asterand: segment of human, full thickness bladder (left) and partially dissected bladder showing bladder muscle bundles (right).

There are a number of experimental approaches to evaluate test compounds for the treatment of urological disorders. Using ‘classical’ pharmacological techniques, our portfolio of pharmacology-based assays can be used for profiling compounds at human native targets to determine mechanism of action, agonist and antagonist potency, magnitude of effect and onset and duration of action

There are documented species differences in expression of the targets most commonly pursued for therapeutic intervention in OAB. For example, in all species examined density of muscarinic M₂-receptors is greater than that of muscarinic M₃-receptors but the ratio can vary significantly. In rat bladder, the ratio of M₂:M₃ receptors is 9:1 compared to 3:1 in human bladder [3]. In addition, there are reported differences in nerve-mediated responses in the bladder between species. Human bladder has a small non-adrenergic non-cholinergic component (less than 5%) compared to 40-50% in the rat and rabbit [4]. In contrast to rabbits and dogs, no α₂-adrenoceptor mediated contraction was observed in human urethra in vitro [5]. The distribution of β-adrenoceptor subtypes mediating detrusor smooth muscle relaxation is species dependent [6], making extrapolation of data generated in various animal models to humans difficult. Therefore, human tissue-based data is essential for understanding species differences in pharmacology and for validating (or otherwise) animal or recombinant target-based approaches. The examples below show how human tissue can be used to understand pharmacology at human native receptors in bladder and urethra, which may enhance progression of novel compounds for the treatment of OAB and SUI.

Overactive bladder syndrome

There are currently three key therapeutic approaches for OAB, each of which can be investigated using human tissue-based approaches.

Muscarinic receptor antagonists

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 arising from actions on both the salivary glands, causing dry-mouth, and on the GI-tract, causing constipation, through the activation of muscarinic M₃- receptors. A recent study showed that <20% of patients are still on their prescribed therapy at the end of 12 months [7]. Therefore, there is still a drive both to find both novel targets for the management of OAB and to design better anti-muscarinic compounds with better patient tolerability.

The bladder receives a parasympathetic innervation, which plays a key role during voiding. Human isolated bladder can be used to assess drug effects on smooth muscle and nerve-induced contraction or relaxation. Below shows typical human tissue data, generated with the non-selective muscarinic antagonist, atropine.

This original trace shows concentration-dependent inhibition of atropine on electrical-field stimulation-induced contractions of human isolated bladder detrusor smooth muscle (concentrations are logM).

The graphs show that atropine (1x10^-8M to 1x10^-6M) 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 [8].

β-adrenoceptor agonists

There is evidence for β₃-adrenoceptors mediating relaxation in human detrusor smooth muscle [9], although their functional importance remains to be established. There is speculation that in detrusor overactivity there is a lack of β-adrenoceptor mediated inhibitory responses. Therefore, an alternative approach to anti-muscarinics for treating OAB could be to enhance the relaxation (via the stimulation of β-adrenoceptors) of the bladder during the filling phase.

Effects of novel β₃-adrenoceptor agonists can be compared to that of isoprenaline, which causes reproducible relaxation in human detrusor smooth muscle. Example data generated by Asterand is shown below.

The figure above shows the concentration-dependent relaxation of KCl pre-contracted human isolated detrusor smooth muscle with either the non-selective β-adrenoceptor agonist, isoprenaline (pEC₅₀ = 6.3-6.8, both graphs) or the L-type calcium channel blocker, nifedipine (pIC₅₀ = 8.0, right hand graph only). Comparable data are 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 [10].

Original trace generated in human isolated bladder detrusor smooth muscle, showing the effects of isoprenaline on electrical field stimulation (EFS) induced contraction. Isoprenaline inhibits EFS-induced contraction but also markedly decreases basal tone. The remaining EFS-response is abolished by atropine (concentrations are logM).

Role of the urothelium

The third key area of interest as a potential target for the management of OAB is the role of the lining of the bladder. Classically, the urothelium was thought to be only a passive barrier to ions and solutes, but a number of novel properties have recently been attributed to urothelial cells [11]. Further discovery of mechanisms that influence urothelial function might provide insights into the pathology in bladder dysfunction.

Muscarinic receptors are found on the urothelium in humans, where their activation results in the release of an unidentified inhibitory factor (which inhibits contraction of the underlying detrusor smooth muscle tone [12]). Understandably, this is another exciting area of current urology research, which could potentially have a significant impact on the clinical management of OAB. Species differences are known to exist; in the rat bladder this inhibitory factor is released from the detrusor smooth muscle cells and not the urothelial cells.

As Asterand regularly receives whole bladders, we have access to intact human urothelium, which provides an ideal platform for basic discovery work to identify and validate new urothelial targets and ultimately screen novel compounds at these targets using fresh human tissue pharmacology or cell-based approaches.

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 [13].

Stress urinary incontinence

Deficient urethral closure may contribute to SUI. The degree of sympathetic innervation is high in the urethra and contraction is mediated predominantly through α₁-adrenoceptors [14,15]. 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.

The data below were generated using fresh human isolated proximal urethra and show the effects of noradrenaline (non-selective α-adrenoceptor agonist), phenylephrine (α₁-adrenoceptor agonist) and carbachol (muscarinic receptor agonist).

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 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 more important than cholinergic neurotransmission to urethral contraction.

Most drugs investigated for use in SUI are problematic, due to common side effects seen with α-adrenoceptor agonists, including elevated blood pressure, anxiety, arrhythmias and respiratory difficulties [2]. At Asterand human tissue can be used to investigate potential side effects with novel compounds e.g. effects on peripheral resistance with a range of isolated blood vessels.

Conclusions

The attrition rate for compounds entering clinical development remains high and is largely due to lack of clinical efficacy [16,17]. Therefore, a clear understanding of species differences in target expression and function in urological tissues may help to achieve the desired clinical effect in urinary disorders with novel compounds.

To date the physiology and pharmacology of the lower urinary tract has been advanced, in part, due to in vitro assays that have facilitated this exploration. We believe that confidence for clinical success can be greatly enhanced by not going with the flow of only using animal models or human recombinant assays but by also generating human tissue-based data prior to compounds entering clinical development. 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 and testing of new therapies for a range of urological pathologies.

References

1. Milsom I, Abrams P, Cardozo L, Roberts RG, Thüroff J and Wein AJ (2001). BJU Int. 87: 760-766
2. Schuessler B and Baessler K (2003). Urology 62: 31-38
   
3. Wang P, Luthin GR, Ruggieri MR (1995). J Pharmacol Exp Therap. 273: 959-966
   
4. Sibley GN (1984). J Physiol. 354:431-443
   
5. Kunisawa Y, Kawabe K, Nijima T, Honda K, Takenaka T (1985) J Urol. 134:396-398
   
6. Yamaguchi O, Chapple CR (2007) Neurourol & Urodyn. 26:752-756
7. Chui MA, Wiliamson T, Arciniega J, Thompson C, Benecke H (2004). Value Health 7:366
   
8. Fetscher C, Fleichman M, Schmidt M, Krege S, Michel MC (2002) Br. Journal Pharmacol. 136:641-643
   
9. Igawa Y, Yamazaki Y, Takeda H, Hayakawa K, Akahane M, Ajisawa Y, Yoneyama T, Nishizawa O, Andersson KE (1999) Br J Pharmacol 126:819-825
   
10. Yamanishi T, Yasuda K, Kitahara S, Nakai H, Yoshida K, Iizuka H (2006) Neurourol & Urodyn. 25:815-819
    
11. deGroat WC (2004) Urology 64:7-11
    
12. Hawthorn MH, Chapple CR, Cock M, Chess-Williams R. (2000) Br J Pharmacol. 129:416-41
    
13. Chaiyaprasithi B, Mang CF, Kilbinger H, Hohenfellner M (2003) J Urol 170:1897-1900
    
14. Brading AF, McCoy R, Dass N (1999). Eur Urol. 36(Suppl 1):74-79
    
15. Nishimatsu H, Moriyama N, Hamada K, Ukai Y, Yamazaki S, Kameyama S, Konno N, Ishida Y, Ishii Y, Murayama T, Kitamura T (1999) BJU Int 84:515-520
    
16. Kennedy T. (1997). Drug Discovery Today 2: 436-444
    
17. Kola I and Landis J. (2004). Nature Reviews Drug Discovery 3: 711-715