Gallbladder volume as a biomarker for the motilin effect in healthy volunteers and patients with functional dyspepsia

Authors


Dr J. Burggraaf, Centre for Human Drug Research (CHDR), Zernikedreef 10, 2333 CL Leiden, The Netherlands.
E-mail: KB@chdr.nl

Summary

Aim : To investigate a motilin effect on gallbladder volume in healthy volunteers and patients with functional dyspepsia.

Methods : Forty-three healthy volunteers and 10 patients with functional dyspepsia received motilin (4 pmol.min/kg) or placebo in four separate double-blind, randomized, placebo-controlled, cross-over studies. The gallbladder volume was measured by ultrasonography. Analysis of variance of the combined data of these studies was performed to investigate a motilin effect on gallbladder volume and potential differences between patients and healthy volunteers.

Results : The baseline gallbladder volume was similar for placebo and motilin treatment, as well as for patients and healthy volunteers. Motilin, compared with placebo, significantly decreased the gallbladder volume in healthy volunteers (P = 0.003) and patients (P < 0.0001). A linear concentration–response relationship was observed. The decrease in gallbladder volume by motilin was greater in patients (P = 0.03). The motilin effect was consistent between studies.

Conclusion : The interdigestive gallbladder volume is a non-invasive end-point for motilin activity, displaying a consistent response across studies, a clear response to motilin and a clear concentration–response relationship. However, it is less suitable as a biomarker for future pharmacological studies on motilin agonists or antagonists as the effect is probably indirect, and a relatively large study population of 27 subjects is required to demonstrate a 15% decrease in gallbladder volume. Further investigation is required to confirm altered gallbladder motility as a feature of functional dyspepsia.

Introduction

Motilin is a gastrointestinal peptide synthesized and released by enterochromaffin cells in the proximal small intestine.1 Motilin is involved in the regulation of interdigestive and post-prandial gastrointestinal motility.2, 3 Disturbed gastrointestinal motility is a feature observed in functional bowel disorders, such as functional dyspepsia4–6 and irritable bowel syndrome.7, 8 In healthy volunteers, motilin decreases post-prandial proximal gastric volumes, similar to the impaired post-prandial relaxation observed in patients with functional dyspepsia.3, 5 The disturbed gastrointestinal motility observed in irritable bowel syndrome patients coincides with altered motilin levels.9–11 It has therefore been suggested that disturbances in the regulatory function of motilin might be associated with these functional bowel disorders.12, 13 Motilin agonists or antagonists may have potential therapeutic value in the treatment of the different subtypes of these disorders.

As the assessment of gastrointestinal motility is cumbersome and demanding for the participants in clinical studies, the availability of a non-invasive gastrointestinal end-point would be helpful for future studies on motilin agonists or antagonists. The change in gallbladder volume may be such a marker, as it has been shown that gallbladder motility and endogenous motilin release are inter-related.14 In addition, exogenous motilin significantly reduces the gallbladder volume in healthy subjects,15 and there is some evidence that disturbances of gallbladder motility can occur in patients with functional dyspepsia.16–18 Although the exact mechanism by which motilin influences gallbladder motility remains to be elucidated, these observations suggest that the gallbladder is a suitable organ to assess motilin effects. As the gallbladder volume can be assessed easily and non-invasively using ultrasound, it appears that changes in gallbladder volume may be a potential biomarker for motilin activity.

A series of four studies was performed at our institute in healthy volunteers and patients with functional dyspepsia in order to obtain insight into the pathophysiological role of motilin and the effects of its antagonist on the gastrointestinal tract. The secondary objective of these studies was to investigate the effect of motilin on the gallbladder volume. In these separate studies, no clear significant effect of motilin on the gallbladder volume could be detected.19 The reason for the disparity in outcome between these and a previous study15 is not clear, but may be due to differences in study design or to the variability of gallbladder volume during the migrating motor complex. In addition, we found that, in patients with functional dyspepsia, a different response of gallbladder volume to motilin was observed.

In order to better understand these findings and to evaluate gallbladder volume as a biomarker for the effect of motilin, an analysis, using the data of the subjects from the four studies together, was performed. In line with the requirements defined for biomarkers to assess drug effects on the central nervous system,20 this study specifically addressed the following issues: (i) gallbladder volume should show a clear response to motilin and a clear concentration–response relationship; (ii) there should be a consistent response across studies; and (iii) there should be a plausible relationship between the biomarker, pharmacology and pathogenesis.

The analysis also allowed differences in gallbladder volume between healthy subjects and functional dyspepsia patients to be explored.

Methods

Experimental design

All four studies were carried out using a double-blind, randomized, cross-over design. The gallbladder volume was measured under similar conditions. In three studies, the gallbladder volume was measured during motilin infusion (4 pmol.min/kg) and placebo. In the fourth study, gallbladder volume assessments on placebo were not available, and the data from this study were used as extra information.

The Medical Ethics Committee of the Leiden University Medical Centre, The Netherlands approved the study protocols. The studies were performed according to Good Clinical Practice and International Conference on Harmonization guidelines.

Subjects

Retrospective analysis was performed on the data obtained from 43 healthy volunteers (16 females, 27 males; mean age, 28 years; range, 18–45 years) and 10 patients with functional dyspepsia (eight females, two males; mean age, 41 years; range, 22–59 years) participating in four separate studies (Table 1). Healthy volunteers were eligible if they were healthy, as assessed by medical screening. The main exclusion criteria were a history of gastrointestinal symptoms and abdominal surgery and the use of medication. Patients underwent medical screening and were eligible if they had functional dyspepsia, defined as persistent or recurrent upper abdominal pain or discomfort for at least 12 weeks within the last 12 months, which did not need to be consecutive, according to the Rome II criteria.21 All patients suffered from symptoms of epigastric fullness or distension after a meal (early satiety). They were excluded if there was evidence of organic disease or a history of gastrointestinal disease (verified by recent endoscopy) or surgery that was likely to explain the symptoms. Medication for functional dyspepsia was stopped 1 week prior to the first study day.

Table 1.  Summary table of the four separate studies, including the number of subjects, mean age (and range) of the subjects (years) and duration of the motilin infusion (min)
StudyNumber of subjects
(gender)
Mean age
(range) (years)
Duration of motilin infusion (min)
  1. F, female; FD, functional dyspepsia; M, male.

Study 110 (5F/5M) (healthy volunteers)21 (18–24)60
Study 216 (11F/5M) (healthy volunteers)30 (21–43)90
Study 310 (8F/2M) (FD patients)41 (22–59)90
Study 417 (17M) (healthy volunteers)26 (18–45)90

Experimental design

The subjects reported to the research unit in the morning of each study day, after an overnight fast of at least 10 h. After preparation of the subject for the study procedure, motilin (4 pmol.min/kg) or placebo (NaCl 0.9%) was administered intravenously for 60 min in one study with healthy volunteers and for 90 min in the rest of the studies. The motilin used in the studies was synthetic human motilin, manufactured by the American Peptide Company Inc. (Sunnyvale, CA, USA) and prepared for human use by Clinalfa AG (Läufelfingen, Switzerland).

Real-time ultrasonography images (longitudinal and transverse scans; Toshiba Sonolayer SSA-250A, Toshiba Corporation, Japan; equipped with a 3.75 MHz transducer) of the gallbladder were made pre-dose and at several time points after the start of the infusion, with the subject in the supine position. The gallbladder volume was calculated by the sum-of-cylinders method.22

Blood samples for motilin analysis were drawn pre-dose and at several time points after the start of the infusion. Plasma samples were assayed using a sensitive (limit of detection, 10 pmol/L) and specific radioimmunoassay.23

Data analysis

Gallbladder.  The mean baseline gallbladder volume (measurements before drug administration) and the mean gallbladder volume between 30 min after the start of the infusion (at which steady-state motilin is reached) and the end of the infusion (60 min for one study in healthy volunteers, and 90 min for two studies in healthy volunteers and one study in functional dyspepsia patients) were calculated. To evaluate the variability of the ultrasonographic gallbladder measurements within and between study days, coefficients of variance of baseline measurements between and within study days were calculated.

Three studies were performed in healthy volunteers and one in patients with functional dyspepsia; therefore the factor group (healthy volunteer or patient with functional dyspepsia) was taken into account. Because all patients were in one study, the factors group and study are confounded. The data were analysed using mixed model analysis of variance (anova) with the mean baseline volume as a covariate, with fixed factors group, treatment and group by treatment interaction, and with subject as a random factor. In order to investigate whether the gallbladder volume changed on placebo, an additional analysis was performed comparing the pre-infusion and post-infusion values using a mixed model anova with time and group as fixed factors and subject as a random factor.

Post hoc power analysis, based on the results of the anova, was performed using nQuery advisor 5.0 (Statistical Solutions Inc, Cork, Ireland) to calculate the group size which was required to demonstrate a 15% reduction in gallbladder volume due to motilin.

Pharmacokinetics.  The pharmacokinetics of motilin were evaluated in each separate study, as reported previously.3, 19 The pharmacokinetic parameters from only three of the four studies were combined, as in one study a serum motilin assay was employed instead of a plasma assay. For placebo treatment, the mean overall motilin concentrations were calculated. For motilin treatment, the mean expected steady-state concentrations were calculated.

Pharmacokinetics/pharmacodynamics relationship.  In order to investigate the relationship between motilin concentration and gallbladder volume, pharmacokinetics/pharmacodynamics modelling was performed. Therefore, the motilin concentration at the time of gallbladder measurement was estimated with the pharmacokinetics model described previously. The average placebo profiles indicated a clear placebo response. Placebo correction was therefore implemented by subtracting the average placebo profiles from the active treatment profiles at corresponding time points. Subsequently, the estimated motilin concentration was plotted against the placebo-corrected gallbladder volume.

Data management was performed using SAS Proc GLM (SAS version 8.2, SAS Institute Inc., Cary, NC, USA). Results are expressed as the mean ± standard deviation (s.d.). P values of < 0.05 were considered to be significant.

Results

Gallbladder volume

The baseline gallbladder volumes were similar for the motilin and placebo treatments for all studies performed. The coefficient of variation for the pre-dose gallbladder measurements was approximately 8% (8.2 ± 6.4%, 6.3 ± 7.1%, 9.4 ± 5.0% and 8.6 ± 6.6% for the separate studies). The coefficient of variation for the baseline measurements between the study days was approximately 18% (22.0 ± 12.1%, 16.0 ± 10.7%, 17.2 ± 16.0% and 18.3 ± 14.2% for the separate studies).

The baseline gallbladder volumes were similar for healthy volunteers and functional dyspepsia patients. Figure 1 depicts the time course of the gallbladder volume for the different studies and the main results are summarized in Table 2.

Figure 1.

Gallbladder volumes (mL) (mean ± s.d.) during placebo (open circles) and motilin (filled circles) infusion in four studies. (a) Study in 10 healthy volunteers. (b) Study in 16 healthy volunteers. (c) Study in 10 patients with functional dyspepsia. (d) Study in 17 healthy volunteers.

Table 2.  Baseline gallbladder volume (mL), gallbladder volume (mL) during motilin/placebo infusion (period from t = 30 min until the end of the infusion) and mean expected steady-state motilin concentrations (pmol/L) (mean ± s.d.) for healthy volunteers and patients with functional dyspepsia
TreatmentGroupNumber of
subjects
Baseline gallbladder
volume (mL)
Gallbladder volume during
motilin/placebo infusion (mL)
Steady-state motilin
concentration (pmol/L)
PlaceboHealthy2620 ± 818 ± 762 ± 27
Patient1021 ± 1023 ± 967 ± 46
MotilinHealthy4321 ± 816 ± 7507 ± 113
Patient1023 ± 1018 ± 7464 ± 99

Compared with placebo, motilin at steady-state concentration decreased the gallbladder volume in healthy volunteers (P = 0.003) and functional dyspepsia patients (P = 0.0001) (Table 3). The mean decrease in gallbladder volume from baseline by motilin was of the order of 25% in both healthy volunteers and dyspeptic patients. However, the change in gallbladder volume during placebo was different between the groups (P = 0.002) (Figure 2). In healthy volunteers, the gallbladder volume decreased during placebo treatment (P = 0.0054), whereas in the patients a slight increase in gallbladder volume occurred (P = 0.062). Therefore, the decrease in gallbladder volume due to motilin was significantly greater in the patient group than in healthy volunteers (P = 0.03).

Table 3.  Analysis of variance (anova) results of differences in gallbladder volume (mL) with 95% confidence intervals (95% CI) and P values
Effect/contrastDifferences95% CIP value
  1. FD, functional dyspepsia.

Healthy volunteers–FD patients− 2.1− 3.9/− 0.280.02
Motilin treatment–placebo treatment− 4.7− 2.9/− 6.5< 0.0001
 Healthy volunteers/motilin treatment–FD patients/motilin treatment− 0.1− 2.5/2.30.94
 Healthy volunteers/placebo treatment–FD patients/placebo treatment− 4.0− 6.6/− 1.50.002
 Healthy volunteers/motilin treatment–healthy volunteers/placebo treatment− 2.7− 4.5/− 1.00.003
 FD patients/motilin treatment–FD patients/placebo treatment− 6.7− 9.8/− 3.50.0001
Treatment/group interaction  0.03
Figure 2.

Least-square mean estimates (with 95% confidence intervals) of gallbladder volume for healthy volunteers (open circles) and patients with functional dyspepsia (filled circles) during placebo and motilin treatment.

Motilin pharmacokinetics

The plasma concentration–time profiles for motilin were similar between the different studies. The steady-state concentrations, which were reached after approximately 30 min, are reported in Table 2.

Pharmacokinetics/pharmacodynamics

The average placebo-corrected gallbladder volume response to motilin (Figure 3) shows a linear concentration–effect relationship for both healthy volunteers and functional dyspepsia patients. It thus appears that high motilin concentrations are associated with low gallbladder volume. The line for healthy volunteers loops back because, in study 1, the gallbladder volumes were also assessed after stopping the motilin infusion (in all other studies, gallbladder measurements stopped at the end of infusion).

Figure 3.

Mean motilin concentration–gallbladder volume profiles (pharmacokinetics/pharmacodynamics) for healthy volunteers (open circles) and patients with functional dyspepsia (filled circles).

Discussion

The effects of motilin on the gallbladder volume are ambiguous. One study using ultrasound showed a significant gallbladder volume reduction of approximately 25% by motilin.15 This is supported by the findings that the motilin receptor agonist erythromycin reduces the gallbladder volume by approximately 40–50% in healthy volunteers.24–26 However, this is in contrast with a study in which scintigraphy was used to measure gallbladder contraction, and showed no reduction in gallbladder volume by motilin.27 However, as the latter study was carried out in five subjects only, this experiment may simply have been under-powered. With regard to our four separate investigations, only a modest, statistically non-significant reduction in the gallbladder volume by motilin was found in placebo-controlled studies. It seems that the experimental design and methodological factors are important in the assessment of the effects of motilin on the gallbladder volume.

The present analysis, including data from over 40 subjects, showed that motilin significantly reduced the interdigestive gallbladder volume, but in each separate study only a modest, statistically non-significant reduction in the gallbladder volume by motilin was observed compared with placebo. A larger group would be required to demonstrate that this effect was not due to chance. The reason why a significant motilin effect was not found in our separate studies in healthy volunteers may be explained by two observations. Firstly, the gallbladder volume, although statistically no different between the study days, showed a substantial inter-day variability (coefficient of variance, 18%) despite similar experimental conditions. This was higher than the variability observed in baseline measurements within an experimental day (coefficient of variance, 8%). More importantly, it was observed that the decrease in gallbladder volume was not only present during motilin treatment, but during placebo treatment as well. This obviously hampers the detection of the motilin effect. The explanation for this finding is most likely the occurrence of fluctuations in gallbladder volume during the interdigestive state. This cyclic emptying of the gallbladder is important to maintain the enterohepatic circulation of bile salts and to prevent supersaturation.28 In our studies, the start of the motilin/placebo infusion was unrelated to the phase of the migrating motor complex, and hence it is conceivable that gallbladder contraction occurred during placebo infusion as well. In order to detect the true effect of motilin on the gallbladder volume, placebo-controlled studies are required. Otherwise, it is possible that reductions in gallbladder volume due to physiological processes will be ascribed to motilin. In addition, if the gallbladder volume is to be used as a biomarker for motilin agonists or antagonists, this must be done in placebo-controlled experiments; based on the data from the present analysis, it is estimated that, for the demonstration of a 15% reduction in gallbladder volume, a population of 27 subjects would be required.

Given this prerequisite, the present findings suggest that the gallbladder volume may serve as a biomarker for the effect of motilin. Indeed, a significant gallbladder volume-reducing effect of motilin was present and this effect was concentration-dependent. In addition, the motilin effect on the gallbladder volume was more or less constant across the studies. However, the pharmacological mechanism by which motilin exerts its effect on the gallbladder volume remains unclear. Until now, no motilin receptors have been demonstrated in the human gallbladder. It is likely that the effect is mediated indirectly by the cholinergic autonomic nervous system, as the contractile effect of motilin on the gallbladder can be blocked by atropine.25, 29, 30

The present study aimed to evaluate the gallbladder volume as a biomarker for the motilin effect, rather than to provide information about the physiological role of motilin in the regulation of the interdigestive gallbladder volume. A design with repeated motilin infusions might have been more representative of the endogenous motilin peaks that occur during the interdigestive migrating motor complex, rather than the continuous infusion employed. In addition, the systemic motilin concentrations were approximately 10-fold higher than the endogenous concentrations. However, as motilin is synthesized and released in the proximal gastrointestinal tract, and as it is likely that motilin is metabolized by the liver, it is conceivable that peripheral motilin levels do not represent the concentration at the site of action. Thus, the high peripheral motilin concentrations attained may be of physiological relevance at the site of action in the gut. This is supported by the fact that the changes in gallbladder volume were similar to those obtained in physiological situations.31 In addition, these motilin concentrations induce gastric antrum contractions within the physiological range.19 The finding that, at apparent supra-physiological serum motilin concentrations, a physiological response was obtained suggests that serum concentrations may be a good surrogate for the local effect.

Although no significant differences were observed in the basal or steady-state motilin concentrations between healthy subjects and dyspeptic patients, the effect of motilin on the gallbladder volume, compared with placebo, was greater in dyspeptic patients. However, this effect was mainly attributable to the difference in response to placebo, as the response to motilin compared with baseline was similar for the two groups. In contrast with the decrease in gallbladder volume during placebo in healthy volunteers, a modest increase was detected during placebo in dyspeptics. Although only a small number of patients was studied, this observation was consistent with previous reports which showed an altered motility of the gallbladder in functional dyspepsia patients.16–18

In conclusion, the interdigestive gallbladder volume is a non-invasive end-point for the motilin effect, as a consistent response across studies, a significant gallbladder volume-reducing effect and a clear concentration–response relationship exist. However, it is less suitable as a biomarker for future pharmacological studies on motilin agonists or antagonists as, due to the variability and spontaneous gallbladder fluctuations during the interdigestive state, a large study population is required. As a result of these gallbladder volume fluctuations, a placebo-controlled cross-over regimen is required when evaluating gallbladder volume as an effect measure.

In addition, the fact that the effect of motilin on the gallbladder is probably indirect25, 29 may hamper the interpretation of findings in future pharmacological studies with potential antagonists or agonists. Although a difference in gallbladder motility was observed between healthy volunteers and functional dyspepsia patients, further investigation is required to confirm that altered gallbladder motility is a feature of functional dyspepsia.

Acknowledgements

This report was financially supported by Johnson & Johnson Pharmaceutical Research & Development, L.L.C., Raritan, NJ, USA.

Ancillary