Melinda Wuest

Propiverine and metabolites: differences in binding to muscarinic receptors and in functional models of detrusor contraction

Propiverine is a commonly used antimuscarinic drug used as therapy for symptoms of an overactive bladder. Propiverine is extensively biotransformed into several metabolites that could contribute to its spasmolytic action. In fact, three propiverine metabolites (M-5, M-6 and M-14) have been shown to affect various detrusor functions, including contractile responses and L-type calcium-currents, in humans, pigs and mice, albeit with different potency. The aim of our study was to provide experimental evidence for the relationship between the binding of propiverine and its metabolites to human muscarinic receptor subtypes (hM1–hM5) expressed in chinese hamster ovary cells, and to examine the effects of these compounds on muscarinic receptor-mediated detrusor function. Propiverine, M-5, M-6 and M-14 bound to hM1–hM5 receptors with the same order of affinity for all five subtypes: M-6 > propiverine > M-14 > M-5. In HEK-293 cells expressing hM3, carbachol-induced release of intracellular Ca2+ ([Ca2+]i) was suppressed by propiverine and its metabolites; the respective concentration-response curves for carbachol-induced Ca2+-responses were shifted to the right. At higher concentrations, propiverine and M-14, but not M-5 and M-6, directly elevated [Ca2+]i. These results were confirmed for propiverine in human detrusor smooth muscle cells (hDSMC). Propiverine and the three metabolites decreased detrusor contractions evoked by electric field stimulation in a concentration-dependent manner, the order of potency being the same as the order of binding affinity. We conclude that, in comparison with the parent compound, loss of the aliphatic side chain in propiverine metabolites is associated with higher binding affinity to hM1–hM5 receptors and higher functional potency. Change from a tertiary to a secondary amine (M-14) results in lower binding affinity and reduced potency. Oxidation of the nitrogen (M-5) further lowers binding affinity as well as functional potency.

Pharmacodynamics of propiverine and three of its main metabolites on detrusor contraction

1 Besides its antimuscarinic effects, propiverine may possess an additional mode of action. We compared the effects of propiverine, three of its metabolites (M‐5, M‐6, M‐14) and atropine in human, pig and mouse urinary bladder preparations in order to elucidate the nature of a possible additional mode of action. 2 Like the parent compound, M‐5, M‐6 and M‐14 reduced to variable degrees the contractions elicited by electric field stimulation (EFS) of isolated, urothelium‐denuded detrusor strips. In mouse the atropine‐resistant and therefore the nonadrenergic, noncholinergic component of contractile response to EFS was reduced by M‐5, M‐14 and propiverine, but was hardly affected by M‐6. 3 Atropine, propiverine and M‐6 significantly shifted the cumulative concentration–response curves for carbachol (CCh) to higher concentrations. Atropine and M‐6 did not affect the maximum tension induced by CCh. Propiverine, M‐5 and M‐14 reduced the maximum CCh effect, suggesting at least one additional mode of action. This pattern of response was observed in all the three species, albeit with some differences in sensitivity to the various agents. 4 In freshly isolated human detrusor smooth muscle cells, propiverine and M‐14 inhibited the nifedipine‐sensitive L‐type calcium current (ICa) in a concentration‐dependent manner. In contrast, the effects of M‐5 and M‐6 on ICa were insignificant in the concentration range examined. 5 The investigated responses to propiverine and its metabolites suggest that impairment of maximum CCh‐induced contractions is due to strong effect on ICa and that this may be associated with the presence of the aliphatic side chain.

Effect of rilmakalim on detrusor contraction in the presence and absence of urothelium.

Openers of KATP channels are known to inhibit KCl-, carbachol- and also electrically induced contractions in detrusor muscle strips from various species. Contractions of isolated strips of urinary bladder are usually of higher amplitude when the urothelium has been removed. This has been explained by the release of an urothelium-derived relaxing factor. In this study we examined whether intact urothelium may modulate the effect of the selective KATP channel opener rilmakalim. Contractile responses to 85xa0mM KCl and 10xa0μM carbachol were measured in detrusor strips from mouse, pig and man. In the presence of an intact urothelium, contractions were significantly reduced in strips from all three species investigated. In preparations with urothelium rilmakalim reduced KCl contractions with similar potency and efficacy [−logIC50 (M) 4.6 to 5.1; Effmax reduction to 14-30% of control]. However, in urothelium-denuded strips rilmakalim was more potent in pig (−logIC50 5.5) than in mouse and man (−logIC50 4.7 and 4.4, respectively). The order of potency for rilmakalim to suppress carbachol-induced contractions was pig (—logIC50 6.7) > man (5.8) > mouse (4.7); contractions were significantly more reduced in pig (Effmax reduction to 11±2%, n=10) and in mouse (21±2%, n=8) than in human detrusor (55±5%, n=5). The presence of urothelium did not affect the concentration–response curves for rilmakalim, with the exception of KCl-induced contractions in pig. Only the rilmakalim-induced relaxation of carbachol-mediated contractions in pig were prevented by the KATP channel blocker glibenclamide. We conclude that with this one exception, the responses to rilmakalim in detrusor contractions were not mediated by KATP channel opening.

Electrophysiological profile of propiverine : relationship to cardiac risk

Drugs that prolong the QT interval by blocking human ether-a-go-go (HERG) channels may enhance the risk of ventricular arrhythmia. The spasmolytic drug propiverine is widely used for the therapy of overactive bladder (OAB). Here, we have investigated the effects of propiverine on cardiac ion channels and action potentials as well as on contractile properties of cardiac tissue, in order to estimate its cardiac safety profile, because other drugs used in this indication had to be withdrawn due to safety reasons. Whole-cell patch clamp technique was used to record the following cardiac ion currents: rapidly and slowly activating delayed rectifier K+ current (IKr, IKs), ultra rapidly activating delayed rectifier K+ current (IKur), inwardly rectifying K+ current IK1, transient outward K+ current (Ito), and L-type Ca2+ current (ICa,L). Action potentials in cardiac tissue biopsies were recorded with conventional microelectrodes. The torsade de pointes screening assay (TDPScreenTM) was used for drug scoring. Propiverine blocked in a concentration-dependent manner HERG channels expressed in HEK293 cells, as well as native IKr current in ventricular myocytes of guinea pig (IC50 values: 10xa0μM and 1.8xa0μM respectively). At high concentrations (100xa0μM), propiverine suppressed IKs. IK1 and the transient outward current Ito and IKur were not affected. In guinea-pig ventricular and human atrial myocytes, propiverine also blocked ICa,L (IC50 values: 34.7xa0μM and 41.7xa0μM, respectively) and reduced force of contraction. Despite block of IKr, action potential duration was not prolonged in guinea-pig and human ventricular tissue, but decreased progressively until excitation failed altogether. Similar effects were observed in dog Purkinje fibers. Propiverine obtained a low score in the TDPScreenTM. In conclusion, in vitro and in vivo studies of propiverine do not provide evidence for an enhanced cardiovascular safety risk. We propose that lack of torsadogenic risk of propiverine is related to enhancement of repolarization reserve by block of ICa,L.

Mucosa of human detrusor impairs contraction and β‐adrenoceptor‐mediated relaxation

To elucidate the impact of the mucosa on detrusor muscle function by investigating force of contraction under various stimulatory conditions and during subsequent relaxation using catecholamines.

Muscarinic receptor expression and receptor-mediated detrusor contraction: comparison of juvenile and adult porcine tissue

Urinary bladder function is known to mature during fetal and postnatal development, including changes in neurotransmitter regulation of detrusor contraction. However, only few experimental data are available about muscarinic receptor antagonist function in the urinary bladder from young animals. In the present study, we compare the muscarinic receptor-mediated contractions in juvenile and adult porcine detrusor and the effects of antimuscarinic compounds. Urinary bladders from young (8–12xa0weeks; 12- to 35-kg body weight) and mature pigs (>40xa0weeks; >100xa0kg) were compared. Muscarinic receptor expression was assessed by real time polymerase chain reaction and radioligand binding. Muscle contraction was measured with a force transducer; L-type Ca2+ currents (ICa,L) of isolated detrusor myocytes were recorded with standard voltage clamp technique. Juvenile and adult detrusor expressed similar quantities of the messenger RNA of M2 and M3 receptors. The number of [3H]QNB-binding sites and their affinity for the radioligand were also similar between juvenile and adult detrusor. In contrast, maximum contractile responses to the muscarinic receptor agonist carbachol were slightly larger in juvenile than adult bladders. On the other hand, carbachol was slightly less potent in juvenile than in adult tissue. The M3 antagonist DAU 5884 and the spasmolytic drug propiverine inhibited contractile responses with comparable efficacies and potencies in juvenile and adult tissue. ICa,L was somewhat smaller in juvenile than in adult cells. Taken together, these data suggest that expression and function of M2 and M3 receptors are similar in the detrusor of juvenile and mature pigs. Therefore, similar responses to antimuscarinic compounds could be expected in young and adult patients.

Effects of three metabolites of propiverine on voltage-dependent L-type calcium currents in human atrial myocytes.

The non-selective muscarinic receptor antagonist propiverine impairs L-type Ca(2+) currents (I(Ca,L)) in human detrusor smooth muscle cells and atrial cardiomyocytes. Here, we have investigated the effects of three metabolites of propiverine on human cardiac I(Ca,L). Propiverine reduced I(Ca)(,L) with a -logIC(50) [M] value of 4.1, M-5 only showed minor effect on I(Ca)(,L) at high concentrations, M-6 did not influence I(Ca)(,L) at all. Like the parent compound M-14 also reduced I(Ca)(,L) (-logIC(50) [M]=4.6). We conclude, that propiverine and M-14 reduce cardiac I(Ca)(,L) at higher concentrations than in detrusor cells and therefore preferentially reduce I(Ca)(,L) in the urinary bladder than in the heart.