In vitro evaluation of motilin agonism by macrolide immunosuppressive drugs

Bruno Van Vlem1,, Renaat Schoonjans2, Raymond Vanholder1, Martine De Vos2, Inge Depoortere3, Theo L. Peeters3 and Romain Lefebvre4

1 Renal Division, 2 Gastroenterology Division, Department of Internal Medicine, Ghent University Hospital, Ghent, 3 Gut Hormone Laboratory, University of Leuven, Leuven, 4 Heymans Institute of Pharmacology, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. The immunosuppressive drugs tacrolimus and sirolimus may have a stimulatory influence on gastric emptying, in view of their macrolide structure. The aim of this study was to investigate in vitro the possible interaction of tacrolimus and sirolimus with motilin receptors in the rabbit antrum and duodenum.

Methods. Rabbit duodenum strips were mounted under a load of 1 g in 10 ml organ baths containing Krebs solution. Erythromycin, tacrolimus, cyclosporin, and sirolimus were added to the bathing solution in a cumulative way; in a second series, after incubation with cyclosporin (10-7 mol/l), tacrolimus (10-8 mol/l), or sirolimus (3x10-8 mol/l), a cumulative concentration–response curve to erythromycin was obtained.

 The effect of cumulatively added tacrolimus and nle13-porcine motilin on the contractile response to electrical field stimulation was tested in strips from the rabbit gastric antrum. Displacement by tacrolimus of 125I-nle13-porcine motilin bound to its receptor was tested with crude homogenates of the smooth-muscle layer of the rabbit antrum.

Results. In rabbit duodenum strips, carbachol (10-5 mol/l) induced a stable and reproducible contraction. Erythromycin induced concentration-dependent contractions. Expressed as a percentage of the response to carbachol, the maximal attained effect was 78%; the EC50 was 4.8x10-7 mol/l. Tacrolimus (10-8 to 10-5 mol/l), cyclosporin (10-8 to 10-5 mol/l), and sirolimus (10-8 to 3x10-5 mol/l) had no influence. The response to erythromycin (10-8 to 3x10-5 mol/l) in the presence of cyclosporin (10-7 mol/l), tacrolimus (10-8 mol/l), or sirolimus (3x10-8 mol/l) did not differ from that obtained with erythromycin alone, except for a decrease of the EC50 in the presence of tacrolimus (2.2x10-7 mol/l) (P<0.05 vs erythromycin alone). Motilin enhanced the response to electrical field stimulation of rabbit antral strips, but tacrolimus had no stimulatory effect. Tacrolimus weakly displaced motilin bound to its receptor. The pIC50 was 4.97.

Conclusions. As opposed to erythromycin, neither tacrolimus nor sirolimus showed a contractile effect in the rabbit duodenum. Tacrolimus did not activate the neural motilin receptor of the rabbit gastric antrum and had low affinity for the smooth-muscle motilin receptor. It is thus unlikely that these macrolide immunosuppressive drugs possess gastroprokinetic effects via motilin agonism.

Keywords: cyclosporin; immunosuppressants; macrolide; motilin agonism; sirolimus; tacrolimus



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Patients with chronic renal failure frequently suffer from dyspeptic complaints such as nausea, vomiting, abdominal distension, early satiety, and anorexia. Gastroparesis may be a cause of dyspeptic complaints. Gastric emptying has not been studied extensively in renal-transplant recipients, except for diabetic patients undergoing simultaneous pancreas–kidney transplantation [13]. Renal transplantation is followed not only by the recovery of a considerable fraction of renal function; in addition, immunosuppressive drug therapy is started on a chronic basis. Immunosuppression might have an important influence on gastric emptying, especially since two of the drugs used for maintenance immunosuppression (tacrolimus and sirolimus) have a macrolide structure. Several macrolides have been demonstrated to improve gastric emptying [46] via stimulation of motilin receptors [7].

In a non-controlled cross-sectional study, Maes et al. [8] suggested that in stable renal transplant recipients gastric emptying of solids differed depending on the type of immunosuppressive therapy. Patients treated with cyclosporin and corticosteroids showed a slower solid emptying. Gastric emptying of solids was normal, however, in patients treated with azathioprine and corticosteroids, or with tacrolimus and corticosteroids. A gastroprokinetic effect of tacrolimus via motilin receptors was proposed as the responsible mechanism, in view of the macrolide structure of tacrolimus. In pigs, instrumented with strain gauge force transducers to measure mechanical activity along the duodenum and jejunum, tacrolimus stimulated small-bowel motility similarly to erythromycin [9].

The aim of this study was to investigate in vitro the possible interaction of the immunosuppressants with motilin receptors. The effect of tacrolimus, sirolimus, and cyclosporin was compared with that of the prototype macrolide agonist erythromycin in the rabbit duodenum, a standard preparation for characterizing agents active at motilin receptors [10,11]. Interaction of tacrolimus with smooth-muscle and neural motilin receptors was also explored in binding studies and in electrical field stimulation experiments with preparations of the rabbit gastric antrum.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Tissue preparation and general methodology
Adult male New Zealand white rabbits (2.5–3 kg) were killed by cervical dislocation and bleeding. After midline laparotomy, the stomach and the duodenum were removed.

Contraction studies, duodenum
The proximal 5 cm of the duodenum was discarded and the distal segment was mounted on a glass rod. The longitudinal muscle was gently wiped off with cotton wool soaked in Krebs solution and four longitudinal muscle strips of 15 mm were prepared. The strips were mounted under a load of 1 g in 10 ml organ baths containing Krebs solution (composition in mmol/l: NaCl 118.5, KCl 4.8, KH2PO4 1.2, MgSO4 1.2, CaCl2 1.9, NaHCO3 25.0, and glucose 10.1).

The Krebs solution was maintained at 37°C and bubbled with 95% O2/5% CO2. Tension was recorded auxotonically (via a Grass force displacement transducer FT03 coupled in series with a 1 g cm-1 spring) on a Graphtec linearcorder F WR3701. The tissues were equilibrated for 1 h with rinsing every 15 min.

Tissues were contracted twice with 10-5 mol/l carbachol at the beginning of the experiment. In a first series, erythromycin, tacrolimus, cyclosporin or sirolimus was added separately to the bathing solution cumulatively, either in the absence or in the presence of 3x10-6 mol/l tetrodotoxin (incubation for 10 min). In a second series, a cumulative concentration-response curve to erythromycin was obtained in the presence of 10-7 mol/l cyclosporin, 10-8 mol/l tacrolimus or 3x10-8 mol/l sirolimus. The duodenal strips were incubated for 30 min with one of the immunosuppressants before erythromycin was added.

Contractions were expressed as percentages of the contractile response to the second administration of carbachol. The EC50 for the concentration-response curve to erythromycin was determined by linear interpolation from each concentration-response curve as the concentration responsible for 50% of the maximal contractile response.

Binding studies, antrum
Smooth-muscle tissue from the rabbit gastric antrum was freed from mucosa and serosa and was finely minced and homogenized in sucrose buffer with inhibitors. Binding of 125I-nle13-porcine motilin was studied in washed 1000-g fractions of the tissue homogenates, and displacement curves were obtained by adding increasing amounts of nle13-porcine motilin or tacrolimus. The concentration required to displace 50% of the tracer was determined and expressed as a negative logarithm (pIC50). The methodology has been described in detail [12].

Electrical field stimulation, antrum
Circular strips, freed from mucosa (0.2x2.5 cm) were cut and suspended along their circular axis in a tissue bath filled with Krebs buffer (NaCl 120.9 mmol/l, NaH2PO4 2.0 mmol/l, NaHCO3 15.5 mmol/l, KCl 5.9 mmol/l, CaCl2 1.25 mmol/l, MgCl2 1.2 mmol/l and glucose 11.5 mmol/l) gassed with 95% O2/5% CO2. Resting tension was set at 1.0 g. After equilibration, electrical field stimulation was applied via two parallel platinum rod electrodes using a Grass S88 stimulator (Grass, Quincy, MA, USA). The strips were stimulated with pulse trains of 10 s (4 Hz, 4 V, pulse width 1 ms). Voltage was kept constant using a Med Lab Stimu-Splitter II (Med Lab, Loveland, CO, USA). Each consecutive pulse train was followed by a 90-s interval. Once a stable contraction was obtained, nle13-porcine motilin or tacrolimus was added cumulatively. Contractions in the presence of motilin or tacrolimus were expressed as percentages of the control response obtained before their addition. Contractions were measured using an isometric force transducer/amplifier (Harvard Apparatus, South Natick, MA, USA), recorded on a multicorder and sampled for digital analysis using the Windaq data acquisition system and a DI-200 PGH card (Dataq Instruments, Akron, Ohio, USA).

Drugs
The following drugs were evaluated: carbamoylcholine chloride (Fluka, Buchs, Switzerland), cyclosporin (Novartis Pharma, Basel, Switzerland), erythromycin lactobionate (Abbott, Saint Rémy-sur-Avre, France), tacrolimus (Fujisawa, Münich, Germany), sirolimus (Wyeth-Ayerst, Princeton, NJ, USA), norleucin13-porcine motilin (Novabiochem, Laüfelfingen, Switzerland) and tetrodotoxin (Alomone, Jerusalem, Israel). 125I-nle13-porcine motilin was prepared as previously described [12]. For cyclosporin and tacrolimus, ampoules available for intravenous administration were used. All drugs were dissolved or diluted with deionized water, except for sirolimus, which was dissolved in ethanol.

Data analysis
Data are given as mean±SEM values, n referring to the number of experiments performed on tissues obtained from different animals. Results between tissues were compared by the unpaired t-test; a P value <=0.05 was considered to be statistically significant.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Contractile responses of duodenal strips
Carbachol (10-5 mol/l) induced a stable and reproducible contraction. The responses to erythromycin and to the immunosuppressants are summarized in Table 1Go. Erythromycin (10-8 to 3x10-5 mol/l) induced concentration-dependent contractions (Figure 1Go). Expressed as a percentage of the response to the second administration of carbachol, the maximal effect attained was 78.2±4.5% (n=6, Figure 1Go), and the EC50 was 4.8±0.9x10-7 mol/l (n=6). Tetrodotoxin (3x10-6 mol/l) had no influence on the response to erythromycin (maximal effect 75.2±4.7%, EC50 4.0±0.8x10-7 mol/l; n=6; data not shown). Tacrolimus (10-8 to 10-5 mol/l), cyclosporin (10-8 to 10-5 mol/l), and sirolimus (10-8 to 3x10-5 mol/l) had no influence on the tone of the tissues (n=at least 4 for each substance).


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Table 1.  Contractile effect on longitudinal muscle strips of the rabbit duodenum

 


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Fig. 1.  Influence of erythromycin on longitudinal muscle strips of the rabbit duodenum. (a) Representative trace showing the contractile response to 10-5 mol/l carbachol and 10-8 to 3x10-5 mol/l erythromycin. R denotes rinsing of the organ bath. The non-marked arrows for erythromycin indicate 3x10-8, 3x10-7, 3x10-6, and 3x10-5 mol/l. (b) Representative trace showing the lack of response to 10-8 to 10-5 mol/l tacrolimus. R denotes rinsing of the organ bath. The non-marked arrows for tacrolimus indicate 3x10-8, 3x10-7, and 3x10-6 mol/l. (c) Concentration-response curve for erythromycin obtained in the absence ({circ}) and presence (•) of 3x10-6 mol/l tetrodotoxin (means±SEM, n=6).

 
The response to erythromycin (10-8 to 3x10-5 mol/l) in the presence of 10-8 mol/l tacrolimus, 10-7 mol/l cyclosporin or 3x10-8 mol/l sirolimus, or its solvent ethanol (n=3) did not differ from that obtained with erythromycin alone, except for a decrease of the EC50 in the presence of tacrolimus (2.2±0.4x10-7 mol/l; P<0.05).

Binding studies in antral tissue
Tacrolimus weakly displaced motilin bound to its receptor. The pIC50 was 4.97 vs 9.15 for motilin itself. The latter value corresponds well with data obtained in earlier experiments [12,13].

Electrical field stimulation of antral strips
Electrical stimulation of antral strips produced a stable twitch response of about 5 g. Addition of nle13-porcine motilin to the tissue bath to attain concentrations of 10-9, 3x10-8 and 10-8 mol/l in a cumulative manner, markedly and concentration-dependently increased the response, in accordance with previous observations [14]. For example, at 10-8 mol/l nle13-porcine motilin the response was 270±10% (n=4) of the control response. In contrast, 10-9, 10-8, 10-7, 10-6 and 10-5 mol/l tacrolimus did not affect the response except at the highest concentration (10-5 mol/l) when the amplitude declined to 43±5% (n=4) of the control response.



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The aim of this study was to investigate the interaction of tacrolimus and sirolimus with motilin receptors. In the rabbit duodenum erythromycin showed its well-known contractile effect, clearly due to interaction with muscular motilin receptors since the response was not influenced by the neuronal conductance blocker tetrodotoxin. The maximal effect was similar to that reported by Clark et al. [10], who used longitudinal muscle strips cut from the duodenum after stripping off the mucosa and the submucosa. The potency in the latter study (EC50=10-6 mol/l) was slightly lower than in ours, but this may be related to the fact that the circular muscle was still contained in the strip, and may have absorbed a part of the administered erythromycin. Notwithstanding the demonstration of the activation of motilin receptors with erythromycin, neither tacrolimus nor sirolimus, which also have a macrolide structure, showed any contractile effect. In accordance with these observations, binding studies with antral homogenates, another model for the exploration of smooth-muscle motilin receptors [15], did not reveal a significant affinity of tacrolimus for motilin receptors.

The tissues were exposed to wide concentration ranges to ensure that therapeutic plasma concentrations were investigated. For tacrolimus, 10-8 to 10-5 mol/l concentrations were studied, including the therapeutic plasma concentration (trough level approximately 10 ng/ml or 1.22x10-8 mol/l [16]). For sirolimus, 10-8 to 3x10-5 mol/l encompasses the therapeutic plasma concentrations (approximately 25–30 ng/ml or 2.7–3.2x10-8 mol/l [17]). The same applies to cyclosporin: 10-8 to 10-5 mol/l concentrations were studied; therapeutic plasma concentration of approximately 150 ng/ml or 1.25x10-7 mol/l [18].

As some agents can potentiate the post-junctional contractile effect of other compounds without having a post-junctional effect per se [19,20], the effect of erythromycin was tested in the presence of a concentration of the immunosuppressants corresponding to the therapeutic plasma concentrations. However, the effect of erythromycin was not enhanced, except for a moderate twofold increase in erythromycin potency in the presence of tacrolimus.

The absence of an effect of tacrolimus and sirolimus in the rabbit duodenum suggests that these agents, although possessing a macrolide structure, do not interact with motilin receptors. Both agents have been reported to hyperpolarize and relax smooth muscle in the micromolar range, possibly by interference with ryanodine receptors [21,22]. In smooth-muscle strips from rat jejunum, tacrolimus inhibited spontaneous and carbachol-induced contractions [23]. The difference between tacrolimus and cyclosporin on in vivo solid gastric emptying observed in renal-transplant recipients [8] may reflect an inhibitory action of cyclosporin rather than a stimulatory response to tacrolimus, as the solid gastric emptying in patients on tacrolimus and also azathioprine was similar to that in healthy volunteers. This suggests that renal transplantation per se does not inhibit solid gastric emptying. The stimulatory effect of tacrolimus and erythromycin, seen in vivo in the pig small intestine [9], is possibly also related to a mechanism other than interaction with motilin receptors, since it has been reported that pig gastrointestinal smooth muscle lacks functional motilin receptors [24].

It is known that both the rabbit antrum and the human antrum contain neural motilin receptors [14,2527]. The affinity of motilin receptor ligands at neural and muscular motilin receptors differs, suggesting the presence of motilin receptor subtypes [26,27]. We therefore tested the effect of tacrolimus on the response of the rabbit gastric antrum to electrical field stimulation. In this model, motilin enhanced the response, but tacrolimus had no effect, except at the highest concentration, when it caused an inhibition. This might correspond to its relaxing effect in smooth muscle, mentioned before [21,22]. For some motilin receptor agonists, a different affinity was found at human and rabbit neural antral binding sites, but even these differentiating agents were active in both models [27]. It is thus highly unlikely that an agent, not at all active at rabbit motilin receptors, would be active at human motilin receptors.

In conclusion, we were unable to find evidence for an interaction of tacrolimus and sirolimus with neural and smooth-muscle motilin receptors in the rabbit. It is therefore unlikely that these macrolide immunosuppressive drugs possess gastroprokinetic effects via motilin agonism in renal-transplant patients.



   Notes
 
Correspondence and offprint requests to: Dr B. Van Vlem, Renal Division, Department of Internal Medicine, Ghent University Hospital, B-9000 Ghent, Belgium. Email: bruno.vanvlem{at}rug.ac.be Back



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 7. 3.01
Accepted in revised form: 7. 2.02





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