Abdominal cramping pain is a common symptom experienced by many people. The correlation between spasmodic abdominal pain and colonic motility often stems from disturbances in the normal rhythmic contractions of the colon (Sarna, 2010). Antispasmodic medications are frequently utilized to target the relaxation of gastrointestinal (GI) smooth muscle, helping alleviate this discomfort. Drugs such as hyoscine butylbromide (HBB), pinaverium, and propinox are commonly prescribed for managing abdominal pain associated with dysfunction in the digestive and urinary tract.

Propinox, also known as pargeverine (2-(dimethylamino)ethyl diphenyl(2-propynyloxy)acetate), is an antispasmodic indicated for the treatment of spasmodic states of the digestive, hepatobiliary, and urinary tracts (Sweetman, 2007; Zheng et al., 2016). In the human gallbladder, propinox has been reported to have a dual mechanism of action, acting directly on the smooth muscle cells as a calcium channel blocker and blocking muscarinic receptors in a moderate and non-selective way (Baistrocchi et al., 1999a). However, the mode of action of propinox in the human colon has not been characterized.

Pinaverium bromide is a quaternary ammonium derivate that is also commonly used as an antispasmodic on the GI smooth muscle and indicated for the treatment of irritable bowel syndrome symptoms (Bor et al., 2021; Liu et al., 2023). Pinaverium bromide reduced contractility linked to slow wave activity in canine tissue, and it has been postulated that it acts as an L-type calcium channel blocker (Malysz et al., 1997), reducing smooth muscle contraction by preventing the calcium influx into intestinal smooth muscle cells. Patch-clamp experiments demonstrated that pinaverium reduced the voltage-dependent inward currents with an IC₅₀ of 1.5 µM in the intestinal smooth muscle cells from the rabbit small intestine (Beech et al., 1990).

Hyoscine butylbromide, also referred to as scopolamine butylbromide, is a tropane alkaloid and a compound derived from the tertiary ammonium compound hyoscine. HBB is an antispasmodic drug that is globally used for relieving abdominal pain linked to muscle contractions in the GI and genitourinary systems (Corsetti et al., 2023). HBB is recognized for its action on the muscarinic receptors situated on the smooth muscle cells within the GI tract (Krueger et al., 2013; Zhang et al., 2016; Traserra et al., 2024a), leading to a relaxing and spasmolytic effect (Corsetti et al., 2023; Tytgat, 2007; Tytgat, 2008). Recent findings indicate that HBB exhibits antimuscarinic effects, inhibiting acetylcholine-induced contractions, irrespective of the origin of the mediator triggering the spasm (Traserra et al., 2024a).

Colonic tissue from macroscopically normal marginal areas, obtained from colon cancer patients, serves as a valuable biological tool for testing the effects of drugs targeting neuromuscular interactions. While variabilities related to disease, region, age, or gender may exist, ex vivo studies using human tissue are essential for characterizing the mechanism of action of drugs, thus eliminating the need to extrapolate from animal studies (Sanger et al., 2013). No previous studies have evaluated the modes of action of pinaverium bromide and propinox in human colonic smooth muscle tissue. Furthermore, the potential complementary effects of antispasmodics with different mechanisms of action have not been systematically explored. Our study was aimed at closing this gap by evaluating the effects of both drugs on the human colon alone and in combination with HBB.

Human tissue samples of the colon from macroscopically normal regions were obtained from patients (n = 33; 11 women and 22 men, aged 38–93 years) during colon resections for neoplasms (Table 1).

The present work is the first study that provides a comprehensive assessment of the effects of pinaverium bromide and propinox, alone and in combination with HBB, on the motility of both circular and longitudinal colonic tissue muscles ex vivo. Moreover, selective pharmacological conditions were used to properly study inhibitory and excitatory neuromuscular transmissions.

Our study indicated that pinaverium bromide reduced myogenic contractions and partially reduced carbachol-induced contractions in both muscle layers as well as EFS-induced contractions only in the circular muscle. Pinaverium bromide exhibited a slight reduction in the EFS-induced off-response, without altering the EFS-induced relaxation. Similar results have been obtained in tissues from animals where pinaverium-inhibited acetylcholine-triggered contractions in dog colonic smooth muscles (Malysz et al., 1997). In the rat colon, it decreased contractile responses that induced an increase in intraluminal pressure ex vivo. The reduction was observed after stimulation with acetylcholine or barium chloride or EFS, showing that the mode of action was probably associated with L-type calcium channel inhibition (Baumgartner et al., 1985). According to this hypothesis, the effect of pinaverium bromide has been characterized in smooth muscle cells isolated from the rabbit jejunum, where it decreased currents (IC₅₀ = 1.5 µM) driven by calcium channels (Beech et al., 1990). Similar results have been reported on L-type calcium channels expressed in Chinese hamster ovary cells (Morel et al., 1998). The pharmacodynamic responses of pinaverium bromide in our study occurred within a concentration range of 10^–6 to 10^–5 M. These concentrations are similar to previous studies where the mode of action was assessed but higher compared to the therapeutic levels detected in plasma following the oral administration of 100 mg of pinaverium bromide (Patiño-Rodriguez et al., 2015). Similar plasma concentrations were reported after the oral administration of pinaverium bromide in Chinese healthy volunteers (Ren et al., 2011). However, it is important to note that pinaverium bromide is a quaternary ammonium derivative, and the drug can accumulate in tissues. Accordingly, the concentration found in plasma might not be the same as the concentration found in tissues (Traserra et al., 2024b) In the present study, pinaverium bromide exhibited only partial efficacy in reducing contractions induced by carbachol and the EFS. The involvement of M₃ muscarinic receptors is pivotal in the initiation of muscle contraction, as these receptors are linked to Gq proteins, which activate phospholipase C and inositol trisphosphate, culminating in the release of calcium from intracellular stores. The stimulation of M₃ receptors on the muscle cell membrane results in smooth muscle depolarization and the opening of L-type calcium channels. Consequently, pinaverium bromide specifically attenuates the contraction associated with the activation of voltage-gated calcium channels but is unlikely to influence the pathway linked to the release of calcium from intracellular stores. As a result, it only partially mitigates the contractile response. Accordingly, the effect of HBB was examined alone (Traserra et al., 2024a) and in combination with pinaverium bromide. In these conditions, HBB alone strongly (90%–95%) reduced the responses induced by carbachol and the EFS, confirming the involvement of muscarinic receptors in this response (Traserra et al., 2024a). The association of HBB and pinaverium bromide was more effective in reducing contractions than HBB alone in both muscle layers in carbachol-induced contractions, whereas a synergistic effect was observed just in the circular muscle after the EFS-induced contractions. The differences between the EFS-induced contractions and carbachol responses might be due to the activation of junctional receptors when neurons are stimulated after an EFS and both junctional and extra junctional receptors are stimulated with carbachol (Drumm et al., 2020). Moreover, it is important to note that pinaverium bromide up to 10^–5 M was ineffective in the reduction of EFS responses in the longitudinal muscle, whereas at the same concentration, HBB abolished the EFS-induced responses, suggesting a minor role of L-type calcium channels in the contractile responses in this muscle layer.

In our study, the off-contraction, which is unrelated to muscarinic receptors, was observed in the presence of 10^–6 M atropine in an ex vivo setting, indicating its independence from cholinergic inhibition. Despite atropine, the EFS induced an inhibitory response followed by an off-contraction, demonstrating that the off-contraction persists under NANC conditions. Previous research from our group delineated the co-transmission involving nitric oxide (NO) and a purine acting on P2Y₁ receptors (Gallego et al., 2006; Gallego et al., 2011), where NO activates cytosolic guanylyl cyclase, leading to increased cyclic guanosine 3′5′-monophosphate. Purines activate P2Y₁ receptors, releasing calcium and activating small-conductance calcium-activated potassium channels. P2Y₁-receptor activation contributes to the fast inhibitory junction potential (IJP), while NO induces the slow IJPs (Gallego et al., 2008), collectively forming the electrophysiological basis for smooth muscle relaxation (Mañé et al., 2014). Following stimulus cessation, smooth muscle repolarization and the opening of L-type calcium channels trigger an off-contraction. Consistent with this hypothesis, pinaverium bromide and nifedipine both partially reduced the off-contraction.

It has been previously reported that otilonium bromide, another quaternary ammonium derivate, inhibits the current associated with L-type calcium channels in isolated cells (Martin et al., 2004) and decreases the rise of intracellular calcium after several stimuli (Martínez-Cutillas et al., 2013). Also, in the human sigmoid colon, otilonium bromide partially decreased on- and off-contractions, although pan-neural stimulation was performed, and therefore, it was difficult to distinguish between both responses (Gallego et al., 2010). In the present study, through the selective stimulation of inhibitory and excitatory motor neurons, we were able to define the impact of pinaverium bromide more precisely in comparison with previous studies.

It has been reported that the administration of pinaverium bromide alone or in combination with an intramuscular injection of HBB 1 day before a nonanesthetic colonoscopy was beneficial in relieving symptoms of abdominal pain during the exploration (Wang et al., 2017). This may align with the synergistic mechanism of action of pinaverium bromide with HBB, consistent with a spasmolytic effect due to inhibition of L-type calcium channels and muscarinic receptors, respectively.

Propinox reduced myogenic contractions and partially reduced carbachol- and EFS-induced contractions in both muscle layers. It also reduced the off-contraction, although no effect was observed on the EFS-induced relaxation. As mentioned previously, the off-contraction is independent of muscarinic receptors (Traserra et al., 2024a), and therefore, the mode of action of propinox is possibly attributable to L-type calcium channel inhibition. Previous studies suggested that propinox might have a dual mode of action, binding with muscarinic receptors and calcium channels. A previous study showed that propinox, with an EC₅₀ of 1.25 × 10⁻⁷ M, reduced carbachol (10^–4 M)-induced contractions in an isolated human gall bladder and showed affinity for muscarinic receptors (M1, M2, and M3) as well as L-type calcium channels (Baistrocchi et al., 1999a). The potency was compared with other antispasmodics, obtaining the following rank of order inhibiting carbachol-induced contractions: atropine (EC₅₀: 5.03 × 10⁻⁸ M) > propinox (1.25 × 10⁻⁷ M) > verapamil (6.63 × 10⁻⁶ M) > HBB (5.4 × 10⁻⁵ M). The effect of propinox has also been assessed in the Guinea pig gallbladder. In this case, less inhibition of carbachol-induced contractions was observed, with a relative potency that was 20.22-fold less than that in the human model (Baistrocchi et al., 1999b).

In the present study, we could not reproduce these results in the human colon. Propinox only partially reduced carbachol- (at 10^–5 M) and EFS-induced contractions (10⁻⁶–10⁻⁵ M), whereas the addition of HBB (10⁻⁷–10⁻⁵ M) strongly reduced both responses. Compared with the results previously obtained with atropine and HBB in the human colon (Traserra et al., 2024a), the rank of potency in this tissue is atropine > HBB > propinox, both in the EFS- and carbachol (10^–5 M)-induced contractions.

According to the aforementioned results, the effects of propinox might be attributable to L-type calcium channel inhibition causing the suppression of myogenic activity and the off-contractions associated with smooth muscle relaxation. The reduction observed in carbachol-induced contractions and EFS-induced responses might also be attributable to L-type calcium inhibition, with a weak effect on muscarinic receptors. The effect obtained with the association of HBB and propinox was more potent than that of HBB alone in some experiments, suggesting that the spasmolytic effects of HBB and propinox are probably synergistic inhibition of muscarinic receptors and L-type calcium channels, respectively.

For the time being, it is not possible to correlate the concentrations used in this study with plasma concentrations in vivo as there is no published information available on plasma levels of propinox.

It is important to note that the mechanism of action of these drugs can be linked to their effects on various cell types. Smooth muscles are responsible for generating the force of contraction, making them the final mediators of spasm. However, muscle contractions are strongly regulated by interstitial cells, such as ICCs and PDGFRα+ cells. Smooth muscle cells are electrically coupled to ICCs and PDGFRα+ cells, forming a SIP (Smooth muscle, PDGFRα+ cells, and ICC) syncytium (Koh et al., 2012). It is well established that ICCs serve as pacemaker cells in the gut and, along with PDGFRα+ cells, participate in neuromuscular transmission. For instance, excitatory cholinergic neuromuscular transmission has been linked to the activation of M3 receptors in intramural ICCs (Drumm et al., 2020). In this context, the spasmolytic effect of HBB may be related to the inhibition of this mechanism. Spasmolytic drugs targeting L-type calcium channels not only reduce the development of muscle contractions but may also impact slow waves originating in ICCs. Indeed, the expression of genes encoding L-type calcium channels (Cacna1c) has been observed in various subclasses of ICCs. Due to the interaction of various cell types in the development of spontaneous phasic contractions, the term “myogenic” has been questioned, and the term “SIPgenic” has been proposed instead (Sanders et al., 2024). In this manuscript, a detailed analysis of the Ca²⁺ dynamics within the SIP syncytium is provided, showing that spasmolytic agents that reduce myogenic activity may act on different cell types (Sanders et al., 2024). Other potential drug effects on the SIP syncytium, such as intracellular calcium release in various cell types or impacts on neural pathways, require further investigation.

Both pinaverium bromide and propinox reduced spontaneous myogenic contractions and partially reduced carbachol- and EFS-induced contractions in the colonic tissue. Both also slightly reduced the off-response without any impact on the EFS-induced relaxation. HBB partially decreased spontaneous myogenic contractions, and in contrast, HBB strongly reduced carbachol- and EFS-induced responses. Propinox has a pharmacological profile like pinaverium, which is consistent with an L-type calcium channel blocker and may have a synergistic mechanism of action with HBB (inhibiting muscarinic receptors) to provide smooth muscle relaxation. However, the possible antimuscarinic effect previously described is unlikely or very weak. This could carry significant clinical implications as a combined treatment approach may provide therapeutic advantages not observed with a singular therapy.