Pulmonary, Gastrointestinal and Urogenital Pharmacology
Pharmacological properties of TD-6301, a novel bladder selective muscarinic receptor antagonist

https://doi.org/10.1016/j.ejphar.2008.12.043Get rights and content

Abstract

Existing antimuscarinic drugs for overactive bladder have high affinity for M3/M1 muscarinic receptors and consequently produce M3/M1-mediated adverse effects including dry mouth, constipation, mydriasis and somnolence. TD-6301 is a M2/4 muscarinic receptor-selective antagonist developed for the treatment of overactive bladder. The present studies characterize the in vitro and in vivo pharmacological properties of this molecule in comparison to other marketed antimuscarinics agents. In radioligand binding studies, TD-6301 was found to possess high affinity for human M2 muscarinic receptor (Ki = 0.36 nM) and was 31, 36, 2 and 128-fold selective for the human M2 muscarinic receptor compared to the M1, M3, M4 and M5 muscarinic receptors, respectively. The in vivo bladder selectivity of TD-6301 in rats was determined to be 26, 28, > 100, 16 and 0.4-fold, respectively, assessed by comparing its potency for inhibition of volume-induced bladder contractions to that for inhibition of oxotremorine-induced salivation, inhibition of small-intestinal transit, decreases in locomotor activity, increases in pupil diameter and increases in heart rate. TD-6301 was more potent in inhibiting volume-induced bladder contractions (ID50 = 0.075 mg/kg) compared to oxotremorine-induced salivation (ID50 = 1.0 mg/kg) resulting in a bladder/salivary gland selectivity ratio greater than that observed for tolterodine, oxybutynin, darifenacin and solifenacin. The preclinical properties of TD-6301 suggest that this molecule is likely to be efficacious in overactive bladder patients with a lower propensity to cause M3 muscarinic receptor mediated adverse effects.

Introduction

Overactive bladder, which is characterized by symptoms of urinary frequency, urgency and urge incontinence, is a chronic and debilitating disease (Abrams et al., 2003). The symptoms of overactive bladder are often attributed to involuntary contractions of the detrusor muscle during bladder filling, a condition known as detrusor instability or overactivity (de Groat, 1997). Although the precise etiology of overactive bladder is unknown, it is generally accepted that activation of muscarinic receptors in the detrusor via acetylcholine released from postganglionic parasympathetic nerves is the ultimate mechanism driving detrusor overactivity in the diseased bladder (de Groat, 1993). Thus, it is not surprising that muscarinic antagonists serve as the cornerstone in the pharmacotherapy of overactive bladder. Although these antimuscarinic drugs have adequate efficacy, they fail to selectively inhibit abnormal detrusor contractions. Furthermore, existing drugs have marginal therapeutic indices owing to mechanism based dose-limiting adverse effects such as dry mouth, constipation and blurred vision. This results in poor patient compliance and limits the therapeutic dose. More than 80% of patients who initiate therapy discontinue within 3 to 6 months because they do not tolerate the side effects (Kelleher et al., 1997, Chui et al., 2004). The four leading antimuscarinic drugs for overactive bladder (tolterodine, oxybutynin, darifenacin and solifenacin) all have high affinity for M3 and/or M1 muscarinic receptors (Hegde et al., 2004). It is not surprising that these drugs have a poor tolerability profile given the key role played by the M3/M1 muscarinic receptors in the salivary gland, gastrointestinal, ocular and behavioral function (Anagnostaras et al., 2003, Caulfield, 1993, Eglen et al., 1996, Wess, 2004).

M2 and M3 are the predominant muscarinic receptor subtypes found within the bladder and are present in a ratio of about 4 to 1 (M2:M3) (Hegde and Eglen, 1999, Hegde, 2006). M3 muscarinic receptors mediate the direct contractile responses necessary for normal bladder function through a mechanism that depends on entry of extracellular calcium through L-type calcium channels and activation of a rho kinase (Schneider et al., 2004). The functional role of the M2 muscarinic receptor in detrusor contraction remains unclear. Studies suggest that M2 muscarinic receptor activation also mediate detrusor contraction but through an indirect mechanism involving inhibition of sympathetically mediated (β3 adrenoceptor evoked) relaxation via a cAMP-dependent mechanism (Hegde et al., 1997, Yamanishi et al., 2002, Ehlert et al., 2005, Ehlert et al., 2007). More importantly, since M2 muscarinic receptors do not appear to be functionally important in the salivary gland, gastrointestinal tract and eye (Bymaster et al., 2003, Eglen et al., 1996, Eglen and Harris, 1993, Gil et al., 1997, Ishizaka et al., 1998, Messer et al., 1990), antagonists which selectively target this receptor may be expected to possess a better tolerability profile. However, the potential effects of an M2 muscarinic receptor selective antagonist on heart rate and in the central nervous system (CNS) needs to be considered given the known importance of M2 muscarinic receptors in regulation of heart rate (Caulfield, 1993) and autoregulation of acetylcholine release in the brain (Zhang et al., 2002).

TD-6301 (Fig. 1) is a selective M2/M4 muscarinic receptor antagonist developed for the treatment of overactive bladder. The objective of the present studies was to characterize the in vitro and in vivo pharmacological properties of this molecule in comparison to marketed antimuscarinics agents. The in vivo bladder selectivity of the molecule was assessed in a range of animal models that evaluated the antimuscarinic potency in the bladder, salivary gland, eye, gastrointestinal tract, heart and CNS.

Section snippets

Material

TD-6301 was synthesized at Theravance, Inc. Tolterodine (tartrate salt), oxybutynin (hydrochloride salt), darifenacin (trifluoroacetate salt) and solifenacin (hydrochloride salt) were all prepared using published methods. All test compounds were dissolved in 5% dextrose in water and formulated with respect to the base weight of the compound.

In vitro pharmacological characterization of TD-6301 in human muscarinic receptor subtypes; displacement of [3H]-N-methylscopolamine

Membranes were prepared from CHO-K1 cells stably expressing the human muscarinic receptor subtypes, hM1, hM2, hM3 or hM4. Membrane fractions expressing the

Binding affinities for human muscarinic subtypes

TD-6301 binds with high affinity to human M2 and M4 muscarinic receptors (Ki = 0.36 and 0.78 nM, respectively) and was 36-fold more selective for the human M2 muscarinic receptor compared to the M3 muscarinic receptor (Ki = 13 nM). Across the five muscarinic receptor subtypes, the rank order from highest to lowest binding affinity for TD-6301 was: hM2 = hM4 > hM1 > hM3 > hM5 (Fig. 2, Table 1). In comparison, tolterodine, oxybutynin and solifenacin did not discriminate between the five muscarinic subtypes;

Discussion

Muscarinic antagonists currently remain the only effective therapeutic strategy for controlling the symptoms of overactive bladder, however the non-selective or M3 muscarinic receptor selective profile of existing drugs inevitably limit the therapeutic index of these agents due to adverse effects. Incidences of dry mouth are particularly confounding as patients increase fluid consumption thus marginalizing the benefits of controlling overactive bladder with antimuscarinic therapies.

Conclusion

In rats, the bladder-to-salivary gland selectivity of TD-6301 (14 - 26-fold) and bladder-to-ocular selectivity (> 24-fold) is superior to all competitors. Furthermore, the bladder-to-gut selectivity of TD-6301 is equivalent to that of tolterodine but exceeds oxybutynin, darifenacin and solifenacin. Lastly, the bladder-to-CNS selectivity of TD-6301 is equivalent to that of tolterodine and darifenacin but exceeds that of oxybutynin and solifenacin. Overall, TD-6301 is a highly M2-selective

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