Measurement of fasted state gastric antral motility before and after a standard bioavailability and bioequivalence 240 mL drink of water: Validation of MRI method against concomitant perfused manometry in healthy participants.

OBJECTIVE: The gastrointestinal environment in which drug products need to disintegrate before the drug can dissolve and be absorbed has not been studied in detail due to limitations, especially invasiveness of existing techniques. Minimal in vivo data is available on undisturbed gastrointestinal motility to improve relevance of predictive dissolution models and in silico tools such as physiologically-based pharmacokinetic models. Recent advances in magnetic resonance imaging methods could provide novel data and insights that can be used as a reference to validate and, if necessary, optimize these models. The conventional method for measuring gastrointestinal motility is via a manometric technique involving intubation. Nevertheless, it is feasible to measure gastrointestinal motility with magnetic resonance imaging. The aim of this study was is to develop and validate a magnetic resonance imaging method using the most recent semi-automated analysis method against concomitant perfused manometry method.
MATERIAL AND METHODS: Eighteen healthy fasted participants were recruited for this study. The participants were intubated with a water-perfused manometry catheter. Subsequently, stomach motility was assessed by cine-MRI acquired at intervals, of 3.5min sets, at coronal oblique planes through the abdomen and by simultaneous water perfused manometry, before and after administration of a standard bioavailability / bioequivalence 8 ounces (~240mL) drink of water. The magnetic resonance imaging motility images were analysed using Spatio-Temporal Motility analysis STMM techniques. The area under the curve of the gastric motility contractions was calculated for each set and compared between techniques. The study visit was then repeated one week later.
RESULTS: Data from 15 participants was analysed. There was a good correlation between the MRI antral motility plots area under the curve and corresponding perfused manometry motility area under the curve (r = 0.860) during both antral contractions and quiescence.
CONCLUSION: Non-invasive dynamic magnetic resonance imaging of gastric antral motility coupled with recently developed, semi-automated magnetic resonance imaging data processing techniques correlated well with simultaneous, 'gold standard' water perfused manometry. This will be particularly helpful for research purposes related to oral absorption where the absorption of a drug is highly depending on the underlying gastrointestinal processes such as gastric emptying, gastrointestinal motility and availability of residual fluid volumes. CLINICAL TRIAL: This trial was registered at ClinicalTrials.gov as NCT03191045.

Introduction

The gastrointestinal (GI) environment in which drug products need to disintegrate prior to dissolution and absorption of the drug has not been studied in detail due to limitations, especially invasiveness of existing techniques. Minimal in vivo data is available on undisturbed GI motility. This information would be extremely helpful to improve the relevance of predictive dissolution models and computational software tools that are frequently applied in preclinical drug development to predict the systemic outcome of a drug. Classic studies of GI transit time of pharmaceutical dosage forms as a function of GI motility showed substantial gastric variability as a function of dosage form size and (unmeasured) motility state of the stomach [1]. Large variability of gastric emptying as a function of fasted motility state (phase, I, II, III) has been shown for fluids [2, 3] and particles [4-7]. Recent advances in magnetic resonance imaging (MRI) methods could provide novel data and insights [8-11]. On-going studies using MRI techniques have already shown that GI fluid (water) volumes can be measured [8, 11] though the dynamic motility environment remains to be studied in more detail, particularly in an undisturbed GI tract.

Intraluminal pressure recording (manometry) is considered the gold standard method to measure the contractile activity of the GI tract [12, 13]. This technique is, however, invasive and at times poorly tolerated by patients and this may disturb motility patterns. Parts of the bowel are inaccessible to conventional perfused manometry catheters, which are best suited to study the more proximal and distal part of the GI tract only. Moreover, the method may have low sensitivity to contractions that do not occlude the lumen [14-17].

Magnetic resonance imaging (MRI) provides an important potential alternative to manometry with some key advantages; these include the ability to image the entire bowel, acquire cross-sectional images, and the non-invasive nature of the technique. A number of recent publications describe MRI assessment of GI motility, including increased automation of analysis and quantification of GI motility biomarkers [18-23]. MRI particularly could address difficulties encountered with other techniques, such as collecting measurements in the presence of food or in children and frailer patients [15].

Dynamic MRI has been used to measure the frequency and amplitude of antral contractions in previous work [19, 20]. De Zwart et al. compared the use of MRI and the barostat to evaluate gastric motility and emptying disorders [24]. They assessed gastric accommodation to liquid and solid meals, at rest and also after infusion of glucagon and erythromycin, which alter gastric volume and motility respectively. The data showed that these methods significantly correlated with each other [21, 22]. It has also been shown that MRI can assess gastric motion and quantify the effects of metoclopramide and scopolamine on gastric motility [23]. Furthermore, the use of MRI to assess gastric motility and emptying has been validated against gamma scintigraphy [25-28].

Over the last two decades, MRI has been used to provide quantitative detailed information on gastric motor function. An early study compared MRI assessment of antro-duodenal motility with simultaneous water perfused manometry [29] and noted that perfused manometry under-detected gastric contractions compared to MRI. In another study, a combination of MRI and manometry was used to investigate antropyloroduodenal motor activity and its relation to gastric emptying [24, 30]. These results showed that manometry missed approximately 20% of the contraction waves which were detected by MRI [24]. Neither of the studies used semi-automated analysis methods.

Recently, a new method of making semi-automated, Spatio-Temporal Motility MRI (STMM) measurements of gastric and colonic contractions, based on changes in bowel lumen diameter, has been introduced [31]. This has the potential to speed up data analysis and make it less operator dependent. However, the combination of MRI and STMM analysis has not yet been tested in vivo against simultaneous conventional manometry.

In this study, we aimed to validate automated MRI motility measures using the new STMM mapping technique [31] against simultaneous water perfused manometry in healthy adult participants.

Materials and methods

Participants

The study was conducted at the Sir Peter Mansfield Imaging Centre (SPMIC) at the University of Nottingham. The study was approved by the University of Nottingham Faculty of Medicine and Health Sciences Research Ethics Committee (A14112016) and by the US Food and Drug Administration Research Involving Human Participants Committee (16-073D). All participants gave written, informed consent before joining the study.

For inclusion, the participants had to be healthy and aged 18–60 years old. Health checks included a medical history questionnaire, blood pressure and arterial pulse check. A 10 mL venous blood sample was taken from the forearm for a comprehensive metabolic panel (CMP) and complete blood count (CBC) analysis. Exclusion criteria encompassed use of any medication which interferes with gastrointestinal motility, working night shifts, strenuous exercise for > 10 h/week, known alcohol dependency, contraindications for MRI scanning as assessed by a standard MRI safety questionnaire, pregnancy and inability to lie flat.

Eighteen healthy volunteers were recruited (9 males and 9 females). The mean age was 29 years old (with a standard deviation (SD) of 10, ranging from 19 (minimum) to 55 (maximum) years old) and the mean body mass index was 24 ± 2 kg/m2. They took part in two identical fasted state MRI study visits with a minimum of 1 week between visits. The study was registered with ClinicalTrials.gov with identifier NCT03191045.

Experimental design

This was a single-centre, open-label design study that consisted of two separate identical study days up to 4 weeks apart. Participants consumed their habitual diet between each visit. Each study visit lasted approximately 8 hours and it was divided into 5 sequential parts as shown in Fig 1 with a focus on the fasting state periods of motility, as the fasting state is of particular interest for oral solid drug delivery products. On arrival, the participants completed a study day eligibility questionnaire to confirm eligibility and document adherence to overnight fasting.

Fig 1

Study diagram.

Schematic diagram of the study day timeline.

The study day began with a naso-duodenal catheter intubation which took place in a seated position. A local anaesthetic (Xylocaine® spray, AstraZeneca Ltd, UK) and a small amount of water-soluble lubricant were used to ease passage through the nose (Optilube 5 g sachets, Optimum Medical Solutions, Leeds UK). The naso-duodenal catheter was a custom design by Mui Scientific (Mississauga, ON, Canada) made of clear polyvinyl chloride (PVC) plastic extrusion tubing. It was a 16 channel, single-use, MRI compatible, water perfused catheter with an external diameter of 12F (4.0 mm) and luminal diameter of 0.3 mm and a core of 1.0 mm with side holes for pressure measurements placed at 5 cm intervals.

The perfused manometry system pressure to drive the water through the catheter was set at 1 bar resulting in a catheter perfusion rate of 1 mL/minute. The total length of the tube was 180 cm plus an additional 100 cm of pigtails. The tip of the tube had a small non-latex balloon attached to allow for inflation with 5–10 mL water for MRI localization. After intubation and resting, the participants were positioned in the MRI scanner. The latex balloon was inflated to determine that the catheter had passed the pylorus and was positioned correctly with recording ports in the stomach. The perfused manometry ports tend to record primarily antral contractions whilst more proximal sensors in the wider fundal region do not record contractions well, as such the assessment here was focused on the antral region of the stomach.

The balloon was then deflated and after a short period of adaptation, the catheter was connected to the MRI compatible water-perfused manometry system (Biomedical Engineering Department, The Royal Melbourne Hospital, Melbourne, Australia). The pressure was converted to electrical signal just outside the scanner bore using transducers connected to the pigtails and secured to a MRI-compatible trolley placed next to the scanner bed. Electric cables and pipes went through a wave guide opening and connected the transducers box to the electronics and pressure gas cylinder placed outside the scanner room. To set reference pressure for the catheter ports, the manometry machine was calibrated manually by gravity immediately before and after every study session and the calibration recorded on the study session’s traces so that malfunction or drift could be detected. An acquisition sample frequency of 25 Hz was used. Each of the differential input lines to the pressure amplifier unit was decoupled using 470 pF capacitors to the reference ground of the amplifier. This effectively trapped otherwise obtrusive 64 MHz RF pulses picked up from the scanner without affecting low-frequency motility information. The pressure data was stored in a digital format data logger (Trace 1.3, Biomedical Engineering Department, The Royal Melbourne Hospital, Melbourne, Australia). At the end of each manometric study, the data log was extracted to be subsequently analysed using common commercial software (MMS, Medical Measurement Systems B.V., Enschede, The Netherlands).

The second part of the study day consisted of one hour of baseline MRI measurements with simultaneous water-perfused manometry data acquisition. MRI scanning was carried out supine using a 1.5T General Electric HDX MRI scanner (General Electric Healthcare, Little Chalfont, Buckinghamshire, UK). Stomach motility was assessed at intervals using a cine-MRI acquisition set at coronal oblique planes through the abdomen, aligned to include the antral region of the stomach. Each dynamic motility MRI scan acquired 60 sets of 4 slices over a time window of approximately 3.5 min. Each dynamic scanning set was acquired across the abdomen whilst the participant breathed gently. The data were acquired using a FIESTA (TrueFISP) sequence (TR 3, TE 0.9, Flip angle 45, Slice thickness 8 mm, field of view 40 mm, matrix size 256, and pixels 1.56 mm × 1.56 mm × 8 mm) at a repetition time for a given plane of 3.7 sec for a total duration of 224 sec.

During the third part of the study day, the participants were allowed to rest for up to an hour outside the scanner room (during this period the manometry system was disconnected). None of the participants wanted a long break, they took only advantage of the rest time to stretch their legs and visit the toilet before continuing and the average time including reconnection was 15 min. Afterwards, they returned back to the scanner, the perfused manometry system was reconnected and the participants drank a standard water challenge of 240 mL while they were sitting up on the scanner bed. They were not instructed to drink the water at a given speed, but all of the participants drank the glass of water quickly, within approximately one minute. A drink of 240 mL of water is the current volume of water recommended by the US Food and Drug Administration for bioavailability/bioequivalence (BA/BE) studies in the fasted state [32, 33]. For the fourth part of the study, the participants laid in the scanner for up to 3 hours of MRI and manometry data were collected concurrently with an optional 20 minutes comfort break towards the end. Throughout each part of the study, the cine-MRI acquisition blocks were interleaved with T2-weighted MRI scans carried out to visualise the state of fluid volumes in the abdomen and also catheter positioning by inflating temporarily the tip balloon with a small volume of fluid.

After the data collection period, the participants moved to the clinical room to remove the tube, they were provided with refreshments and were then discharged.

Data analysis

Magnetic resonance imaging (MRI)

The time series ‘cine’ data were corrected for respiratory motion using Dual Registration of Abdominal Motion (DRAM) GIQuant (Motilent, London, UK) to provide information on the GI tract wall motion which allowed subsequent automatic image analysis [34]. The data were then analysed using a Spatio-Temporal Motility MRI technique (GIQuant, Motilent, London, UK) as recently described [31]. Briefly, the stomach wall boundaries were first identified manually on one of the images. The boundaries were then propagated automatically through the data set to collect wall boundary coordinates on all images. The axis of the stomach was then drawn manually along the visible part of the lumen as indicated by the green lines in Fig 2. Once done, the software automatically measured the lumen diameter as a function of time at defined node points along the stomach axis as shown by the yellow lines perpendicular to the stomach axis in Fig 2. This was performed for each time point so that plots of the stomach diameter versus time were generated. The area under the curve (AUC) was then calculated over the entire 3.5 min acquisition block. The output was AUC in mm × second [34].

Fig 2

Image analysis.

Example of MRI data analysis. The stomach wall boundaries and the axis of the stomach are shown in green. The yellow lines represent the lumen diameter perpendicular to the stomach axis. AC, ascending colon; TC, transverse colon; GB, gall bladder.

Water-perfused manometry

The water perfused manometry data were analysed using commercial MMS software (Laborie, Mississauga, ON). Respiratory artefacts were removed. The manometry traces from the gastric ports were segmented to synchronise with each 3.5 min MRI acquisition. The output was the AUC in mmHg × second [35].

Statistical analysis

The study design included power analysis considerations. Briefly, for correlation analyses involving data that are summarized to one number per subject, the standard error for the estimated correlation coefficient over 15 subjects is 0.28. However, our main analyses use time-resolved data with an overall sample size of 421 values over the 15 subjects. Using a Kronecker sum model to capture within-modality autocorrelation (for MRI or manometry) and between-modality correlation (the parameter of interest), much smaller standard errors are obtained. For example, if the within-modality autocorrelation is 0.5, the between-modality correlation coefficient is estimated with a standard error of 0.049. Thus, the confidence intervals for the correlation parameter of interest will be reported as the estimate +/- 0.1, giving us excellent power to resolve weak from strong correlation. The standard error depends on the within-modality autocorrelation, which was not known at the time of study design. The actual data obtained showed high precision of the estimated correlation coefficient, indicating that the a priori study design was successful.

Data were analysed using SPSS for Windows (Release 24.0, Chicago, IL) and Graph Pad Prism 7 (GraphPad Software, San Diego, CA). All data are presented as mean ± standard deviation unless otherwise indicated. A Pearson’s correlation was used to assess the agreement between MRI area under the curve and corresponding manometry area under the curve. In addition, mixed-effects regression was used to assess the relationship between MRI and manometry measurements of gastric antral motility, while accounting for repeated measurements within study participant and study visits. The conditional mean of the MRI 3.5 min AUC was estimated as both a linear and nonlinear function of the manometry 3.5 min corresponding AUC. There was some evidence of a nonlinear association between manometry and MRI 3.5 min AUC measurements. A natural spline basis was used to assess whether the data support a non-linear conditional mean structure. The mixed effects models included random intercepts for each subject and for each visit-within-subject, and random slopes for each participant.

Results

The study procedures were well tolerated and there were no adverse events. All the eighteen participants returned for their second visit. Data from three participants had to be excluded because of technical difficulties with either the MRI, the perfused manometry hardware or the intubations, leaving 15 participants for analysis.

The MRI motility plots showed good visual correspondence compared to the perfused manometry traces during both antral contractions and quiescence. Three examples of 3.5 min acquisition blocks are shown in Fig 3. The two top panels show gastric contraction peaks detected synchronously by MRI and perfused manometry, whilst the bottom panel shows a period of quiescence sampled by both techniques. The perfused manometry traces allowed standard quality monitoring of the antral motility in the participants as described in the materials and methods section.

Fig 3

MRI and manometry plots.

Examples of MRI gastric antral motility plots (in red) and corresponding perfused manometry traces (in blue) from different participants, during antral contractions (A and B) and quiescence (C).

Fig 4 shows the average AUC of the MRI stomach antral motility for each participant plotted against the corresponding average AUC of the water perfused manometry (n = 15). As indicated by the Pearson’s correlation coefficient, a strong and significant correlation was observed between both data sets (r = 0.860).

Fig 4

Comparison of MRI and manometry mean area under the curves.

Mean (± SEM) values of manometry AUC for each participant plotted against the corresponding mean MRI AUC (n = 15) during one of the visits (Pearson’s correlation coefficient, r = 0.860).

Having considered the average AUCs as above, it is also important to consider all individual data points of the MRI motility AUCs plotted against the corresponding perfused manometry motility AUCs across the entire study (n = 421, Fig 5). These individual points correlate directly each individual trace without averaging noise from either measurement system. A positive, strong correlation was observed between the two techniques (r = 0.843).

Fig 5

Comparison of MRI and manometry individual area under the curves.

Scatter plot of individual values of MRI antral motility for each 3.5 min imaging block AUC and the corresponding 3.5 min perfused manometry motility AUC for all participants, thereby providing n = 421 data points (Pearson’s correlation coefficient, r = 0.843).

The association between MRI and manometry measurements of gastric antral motility was strong. Evidence of moderate between subject differences in MRI AUC, after controlling for manometry AUC, was found. Controlling for manometry AUC and study subject, substantial between-visit variation in MRI AUC was not observed. This suggests that there exist stable, unobserved subject-level characteristics that affect MRI AUC after accounting for manometry AUC. In addition, we also assessed whether the association between MRI and manometry AUC measures is non-linear. The non-linear model described the observed data significantly better compared to the linear model ( = 57.2, p < 0.001).

The association between MRI and manometry measurements was not found to vary substantially between subjects. The fitted non-linear model estimates that one standard deviation increase in manometry AUC is associated with an MRI AUC increase of 100 mm x min for the average subject, when manometry AUC is within one standard deviation of its mean value. For a single subject, the model predicts that his or her expected MRI AUC increase will be within 10 mm x min of the population average increase of 100 mm x min. The non-linear model described the AUC data better than the linear model.

The AUC values can also be aggregated for all subjects at consecutive time points during the study to show the time courses of gastric antral motility during the study for both MRI and perfused manometry. These time courses are plotted together in Fig 6 to show visually the good correspondence between the two time courses.

Fig 6

Time courses of gastric antral motility.

Time courses of the area under the curve of gastric antral contractile activity observed with MRI (solid line) and water-perfused manometry (dotted line) aggregated for all subjects at consecutive intervals (mean ± SD).

Discussion

The main objective of this study was to validate MRI motility measures automated using new semi-automated MRI image analysis techniques against simultaneous water perfused manometry in healthy adult participants. The fasting state and the use of a 240 mL dose of water as a challenge were specifically chosen because of their relevance to the bioavailability/bioequivalence (BA/BE) studies in the fasted state [32, 33]. However, it should be noted that the participants here did not actually ingest a drug product. The recommended 240 mL of water was applied as this volume is also used in BA/BE studies during the clinical phase of drug product development. A water drink challenge has indeed been shown to stimulate gastric motility [36].

The study clearly demonstrated a strong correlation between the two methods. The association between MRI AUC and manometry AUC was found to be rather non-linear than linear. This may be due to the fact that MRI and manometry measure different features of gastric motility. The MRI AUC is a function of the distance between the opposing stomach walls, which cannot decrease below zero millimetres. In the case of manometry, pressure at the surface of the gut reflects the force applied to a small area element on the effective mucosal surface, by the fluid when the surface element is not in direct contact with other mucosal surface elements (intrabolus hydrodynamic pressure), or when the surface element is in direct contact with other mucosal surface elements (contact pressure) [37]. It is generally the case with peristaltic motility that when pressure exceeds 15–35 mmHg (referenced to mediastinal or abdominal pressure as appropriate), the manometric port is generally measuring mucosal contact pressure. Below those values, manometry generally measures hydrodynamic pressure at the interface with intrabolus fluid [37].

Therefore, manometry AUC is a function of the pressure, which can continue to increase while MRI measures a distance of zero mm between the gastric walls. When the stomach is active, this will cause the manometry AUC to increase more than the MRI AUC, inducing a non-linear relationship between the modalities. It should be noted that the non-linear model especially improved with respect to large manometry AUC values. In the lower range, however, a clear linear regression was observed. Conversely, Fig 6 shows a large discrepancy between MRI and manometry AUCs with manometry being lower than MRI immediately after the 240 mL drink. This is likely to reflect the inability of the perfused manometry to measure non-occlusive events when a free fluid is present to redistribute the pressure changes instantaneously.

To directly quantify the details of the contractions of the gut, an imaging modality that can resolve time changes in lumen geometry is required. Imaging and manometry are, in fact, complementary since imaging identifies space-time changes in lumen geometry but does not quantify muscle squeeze, while manometry directly measures muscle squeeze but not the geometry of the lumen. Ideally, one wishes to measure both simultaneously, however, the technological challenges for concurrent measurement are sufficiently great that a single modality data collection—pressure or imaging—is the norm. Of great interest, therefore, are the potential relationships between manometric pressure data and imaging data during contractile activity along the gut [37] and interpretations in context with muscle function [38].

The contemporary dynamic fast acquisition of MRI has the advantage of being non-invasive, safe and uses non-ionising radiation, allowing assessment of stomach contractions with good spatiotemporal resolution [20, 39, 40]. Moreover, in comparison to manometry, the MRI images provide superb soft-tissue contrast, which aids direct assessment of the luminal occlusive and non-occlusive gastric contractions. At the same time, it allows the evaluation of the surrounding anatomical structures. MRI can not only assist in evaluating the response to food or a pharmacological agent but also help in a better understanding of the pathophysiology of gastric motility disorders and drug development [21].

Whilst MRI has been repeatedly proposed as an alternative to invasive manometry, only a few studies addressed the issue of validation of the MRI method, particularly with a view of assessing recent automated data processing methods like STMM. Previous studies were positive but were small scale studies that acquired data for relatively short time windows [24, 29]. There is also a feasibility study on colonic motility in which dynamic MRI has been compared to perfused manometry [39], which found a strong correlation between MRI visualized colonic movements and intraluminal pressure changes. Advances in MRI hardware including improved parallel imaging techniques have enabled rapid image acquisition over a large volume coverage, leading to reduced effects of motion artefacts due to respiration and peristalsis [17]. In addition, MRI can assess the proximal and distal stomach regions simultaneously which cannot be done by ultrasound [11, 26, 41, 42]. Also, the non-invasive characteristic of MRI opens perspectives towards screening gastric motility in different populations (e.g. elderly and paediatrics). Non-invasive monitoring of gastric motility has the potential to be performed in conjunction with MRI monitoring of other physiological parameters such as evaluation of the gastric emptying time and measurement of GI fluid volumes, which can have remarkable effects, for instance, on orally administered drug’s residence time and dissolution, respectively [43]. The fact that numerous physiological variables can be studied simultaneously, provides a unique data set that can help, for instance, computer modelling of gastric function [44] and in vitro tools to be further developed and validated. With respect to future uses of the MRI technique, a pharmacokinetic study in combination with assessment of motility could provide unprecedented insights on inter-subject variability in systemic outcome of a drug, as the underlying motility patterns are likely one dominant cause of known inter-subject differences in formulation disintegration and, subsequently, dissolution and absorption of the drug. These type of studies are the next logical step to further our understanding of to investigate the actual assessment of pharmacokinetics variables.

Our study collected a large amount of MRI data which would have been very time consuming to analyse manually with individual drawings to measure changes in the gastric luminal diameter over time. Much of the analysis was however done semi-automatically with the STMM technique, which saved considerable data processing time and was less operator dependent than a manual approach.

One limitation of the MRI technique is that it cannot continuously measure the motor response of the gut over long periods of time (hours) as ambulatory manometry does. This is not only because of cost and availability, but also because prolonged periods in the scanner bore can be uncomfortable for patients. Therefore, the MRI methods may be more suitable to assessing immediate impact of interventions such as meals and drugs over a relatively short time period. The planned analysis of the motility traces was focused here on the area under the curve of motility as primary outcomes of this study. The AUC incorporates both amplitude and frequency of contractions in a single integral. In future more advanced analysis parameters such as the Motility Index (a composite measure still incorporating both contractions frequency and amplitude and also representing the fraction of time during which gastric motility was detected) could be considered.

One may argue that the intubation of a catheter may disturb GI physiology. However, it should be noted that the impact of transpyloric tubes on gastric emptying has been explored in the eighties by Müller-Lissner and co-workers and no effect was observed [45]. Moreover, in this study we allowed the participants to rest (up to 2 h) before the start of the data collection. This gave the participant time to relax and get comfortable with the presence of the catheter. To the extent of our knowledge, it is not known how motility may be influenced by the position of a person (standing versus lying down). However, most of the motility studies performed with manometry are those where the subject/patient is lying in a supine position. A study performed by Treier and co-workers demonstrated differences in gastric emptying after eating a solid/liquid meal in a lying body position or seated position [46]. However, body position did not show any effect on gastric relaxation and initial gastric volumes. In another interesting study the same group placed participants upright or upside down in an open-design MRI scanner and concluded that the rate of gastric emptying was maintained despite the two opposite body positions [47].

Data from MRI studies will play a pivotal role in the validation of predictive in vitro and in silico tools as frequently used by formulation scientists in pharmaceutical drug development. For example, MRI data from a previous study were implemented in a physiologically-based pharmacokinetic (PBPK) simulation tool to adequately reflect the residual fluid volumes in the different compartments of the GI tract [48, 49]. The acquired data from this study can be further used to revise GI motility and transit times in these platforms to make predictions with better accuracy and precision.

Conclusions

In conclusion, cine-MRI coupled with recently developed, semi-automated STMM data processing technique is a promising method to assess gastric antral motility, which produced results that were well correlated with simultaneous, ‘gold standard’ water perfused manometry. Dynamic MRI is non-invasive and provides unique data on the undisturbed GI environment. In the field of oral biopharmaceutics, the presented data are of importance to serve as a reference for validation of existing in vitro and in silico tools that are frequently applied to predict the in vivo performance of orally administered drug products.

15 Jul 2020

PONE-D-20-12517

Measurement of fasted state gastric motility before and after a standard bioavailability and bioequivalence 240 mL drink of water: validation of spatio-temporal mapping MRI imaging method against concomitant perfused manometry in healthy participants

PLOS ONE

Dear Dr. Marciani,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The reviewers had a number of concerns about the methodology and statistical analysis in the study. They require clarification about several of the procedures/methods used, and the use of correlation coefficients. The comments can be viewed in full below.

Please submit your revised manuscript by Aug 28 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Natasha McDonald, PhD

Associate Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for including your ethics statement:

"The study was approved by the local Research Ethics Committee (A14112016) and by the US Food and Drug Administration Research Involving Human Participants Committee (16-073D). All participants gave written, informed consent before joining the study."

a) Please amend your current ethics statement to include the full name of the ethics committee/institutional review board(s) that approved your specific study.

b) Once you have amended this/these statement(s) in the Methods section of the manuscript, please add the same text to the “Ethics Statement” field of the submission form (via “Edit Submission”).

For additional information about PLOS ONE ethical requirements for human subjects research, please refer to http://journals.plos.org/plosone/s/submission-guidelines#loc-human-subjects-research.

3. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.

In your revised cover letter, please address the following prompts:

a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially identifying or sensitive patient information) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.

b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. Please see http://www.bmj.com/content/340/bmj.c181.long for guidelines on how to de-identify and prepare clinical data for publication. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.

We will update your Data Availability statement on your behalf to reflect the information you provide.

4. Thank you for stating the following in the Financial Disclosure section:

' The authors GEA, GLA and LM received for this work a grant from the U.S. Food and Drug Administration (FDA), https://www.fda.gov/home, Contract HHSF223201510157C. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This article therefore reflects the views of the authors and should not be construed to represent FDA’s views or policies.'

We note that one or more of the authors are employed by a commercial company: Motilent Ltd, and Capsugel

a. Please provide an amended Funding Statement declaring these commercial affiliations, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

Please also include the following statement within your amended Funding Statement.

“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

b. Please also provide an updated Competing Interests Statement declaring these commercial affiliations along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.

Within your Competing Interests Statement, please confirm that these commercial affiliations do not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to  PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and  there are restrictions on sharing of data and/or materials, please state these.

Please note that we cannot proceed with consideration of your article until this information has been declared.

c. Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf.

Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: AUCs are compared using MRI for gastric motility. There is not much statistical content, other than some Pearson correlations and a mixed model. So I would argue about the term "spatio-temporal", as there is no multidimensional spatial/time series analyses, but only a simple repeated measures. Sample size is small and seems to have been chosen at random, as there is no power computation. P-values on correlations are really meaningless, because they test 0 correlation against "some" correlation. In this case, a significant p-value doesn't necessarily mean the correlations are strong. The regression models need a careful residual analysis. I think there could have been a lot more done with these data, but maybe the sample size is too small.

Reviewer #2: This manuscript reports on the use of non-invasive dynamic magnetic resonance imaging coupled with semi-automated spatio-temporal data analysis for measuring fasted-state gastric motility and validation of such a method vs. intraluminal pressure recording by means of perfused manometry technique.

Human gastrointestinal motility patterns are of utmost importance to oral drug delivery. The investigated RMI-based method would represent a valuable advancement in the relevant study and allow to gain better insight into the impact of many variables. The work is very interesting, broad in scope, clearly exposed and in-depth discussed.

Please find minor comments below.

- The title is rather long: is the word “imaging” actually needed when the acronym RMI already contains it?

- Lines 113-114: as the two methods correlated with each other, “both” sounds odd and can be deleted.

- Lines 123-124: “Both studies did not use semi-automated analysis methods” may better read “Neither of the studies used…”.

- Line 125: please delete the comma after “making”.

Lines 127-128: “more operator independent” may be changed into “less operator-dependent” (this also applies to line 400).

Line 143: “Exclusion criteria included..” sounds a bit strange. “Exclusion criteria were”? Or “encompassed”?

Fig. 2: the caption should be changed into a more concise form taking account of the detailed description and explanation already provided in the text. When the content of the image and the meaning of green and yellow lines are indicated, it will be enough.

Line 257: “Each subject participated in either one or two intubation studies” seems to clash with the Methods section reporting that the volunteers “…took part in two identical fasted state MRI study visits” (lines 149-150) and “This was a single-centre, open-label design study that consisted of two separate identical study days...” (lines 154-155).

Lines 297-298: please check and rephrase “The correlation for these data shows a significant positive correlation …”.

Fig. 6: please check the legend as colors are not visible in the figure whereas dotted/solid lines and different symbols are used as distinguishing features for the 2 curves.

Lines 337-340: please check and rephrase” The fasting state and the use of a 240 mL dose of water as a challenge were specifically chosen because of their relevance to the bioavailability/bioequivalence (BA/BE) studies in the fasted state”, as it is implied that fasting volunteers take part in fasted-state bioavailability/bioequivalence studies. It seems that only 240 ml of water was chosen because of relevance to those studies.

Lines 354-355: “When the stomach is active, this will cause the manometry AUC to increase more quickly than the MRI AUC….” does not appear strictly related to the previously-mentioned circumstance. Could this be an additional factor differing between the two methods?

Line 364: should “complimentary” read “complementary”?

Line 382: “a relative short time window” (or without “a”).

Line 393-394: “an oral administered” should read “an orally administered”

Line 396: as no statistical optimization study is concerned, “optimized” could be replaced by a different verb. The same applies to lines 418 and 438 (“optimization”).

Lines 315-319: this concept is already mentioned at lines 390-394. Please revise or synthesize the two similar passages in order to avoid repetitions.

Lines 431: “gastric motility” can be deleted as it is specified again at line 432, same sentence.

Reviewer #3: The manuscript is interesting and well written. It is not the first time that MRI has been shown to be a valid method for the measurement of gastric emptying, secretion, motility and intragastric distribution of gastric contents. This study, however, adds value to the use of MRI that assess gastric motility by using a new spatio-temporal MRI mapping technique. Results of this study validate this new MRI assessment by comparing its measurements to simultaneous water-perfusion manometry after ingestion of 240 ml of water with the aim to study pharmacokinetics in humans.

However, I have some remarks.

1. The aim of the study is a bit confusing. In the introduction, justification for this new technique is done by explaining the lack of methods for the assessment of predictive drug dissolution models in humans. This aim seems to change in the discussion and it highlights the use of this new method mainly and rather as a gastric motility assessment technique.

As the initial aim was to validate this new technique as a method to study pharmacokinetics and therefore dissolution of drugs, this should be further addressed more clearly in the discussion section. Additionally, a proof of concept study should be suggested as a next step to investigate the actual assessment of pharmacokinetics.

I do not agree with some section in the discussion addressing focus to the validity and accuracy of this new method for the assessment of gastric motility with a broader spectrum, including assessment of general GI function in GI disorder or the impact of drugs to GI motility. The ingestion of 240 ml of water studied in the present study is not enough to make such a statement. Therefore, careful should be taken in the discussion and it should be indicated for such cases further validation studies should be needed.

2. Where are the sensors of the water-perfusion manometry located? Is this antral and duodenal assessment? Please, specify in methods.

3. Following up the previous comment, if sensors are only located in the antral section of the stomach, and thus assessing antral contractions, this should also be specified throughout the entire manuscript: validation of this technique is for ANTRAL motility assessment and not general gastric motility assessment.

4. What was the drinking speed of the volunteers? Do they had to drink all water within 5 minutes or less? This was done in the MRI in supine position. How was this managed? Specify in methods.

5. In the methods it is discussed a “the manometer was connected to the MRI compatible water-perfused manometry system”. What does this means? That the manometry device is all-plastic? Is this the research’s group own developed system or was this bought from a company? In this last case, from which company?

6. Include reference and explain possible limitation regarding supine positioning and its effect on GI motility (Might have an effect on the gastric emptying rate): Treier R, Steingoetter A, Weishaupt D, Fried M, Boesiger P, Schwizer W. Gastroenterology 2003; Gastric motor function and emptying in the right decubitus and seated body position as assessed by magnetic resonance imaging

7. I think in the figures 4 and 5 the “AUC” for MRI is missing.

8. Figure 6. Can you include in the figure the moment the water was ingested? Why is there a gap between -15 and 0? Why is the pressure so low at time 0?

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

25 Aug 2020

RESPONSE TO REVIEWERS ALSO UPLOADED AS WORD DOVUMENT

PONE-D-20-12517 REVISION R1: Measurement of fasted state gastric antral motility before and after a standard bioavailability and bioequivalence 240 mL drink of water: validation of MRI method against concomitant perfused manometry in healthy participants

We are grateful for the opportunity to revise our manuscript and we thank the Academic Editor and the Reviewers for their constructive comments. We believe that this process has considerably improved the paper.

We address first the Journal/Editorial points raised and secondly the Reviewers’ comments in the point-by-point the detailed answer below. All changes made to the main manuscript are tracked in the marked-up Word version enclosed. Some Editorial points are also addressed in the enclosed Cover Letter as requested.

Editorial and Journal points

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

Answer

Thank you, we have updated our disclosures as detailed below and in the Cover Letter. We have also resubmitted the figures after passing them through the PACE engine as required.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results.

Answer

The study protocol is publicly available on Clinicaltrials.gov (NCT03191045) as already referenced in the manuscript. We do not have extensive protocols to deposit in protocols.io.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Answer

We ahve checked our manuscript against the templates adn checked the file naming, the format corresponds to the requirements

2. a) Please amend your current ethics statement to include the full name of the ethics committee/institutional review board(s) that approved your specific study.

2. b) Once you have amended this/these statement(s) in the Methods section of the manuscript, please add the same text to the “Ethics Statement” field of the submission form (via “Edit Submission”).

Answer

Thank you, the full name of the University of Nottingham Faculty of Medicine and Health Sciences Research Ethics Committee is now included at line 141. The full name of the FDA ethics committee already appeared. The statement now reads “The study was conducted at the Sir Peter Mansfield Imaging Centre (SPMIC) at the University of Nottingham. The study was approved by the University of Nottingham Faculty of Medicine and Health Sciences Research Ethics Committee (A14112016) and by the US Food and Drug Administration Research Involving Human Participants Committee (16-073D). All participants gave written, informed consent before joining the study.” and it has been added to the submission form on Editorial Manager.

3. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. We will update your Data Availability statement on your behalf to reflect the information you provide.

Answer

Thank you, we had misunderstood this point. We have now saved the anonymized individual data and the time courses in an Excel file which we have uploaded to our Institution’s public repository, the Nottingham Research Data Management Repository (https://rdmc.nottingham.ac.uk/ ).

The repository satisfies all major funders’ requirements for data access. The data file is now publicly available with this permanent DOI 10.17639/nott.7068 and this is noted also in the Cover Letter

4. Thank you for stating the following in the Financial Disclosure section:

' The authors GEA, GLA and LM received for this work a grant from the U.S. Food and Drug Administration (FDA), https://www.fda.gov/home, Contract HHSF223201510157C. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This article therefore reflects the views of the authors and should not be construed to represent FDA’s views or policies.'

We note that one or more of the authors are employed by a commercial company: Motilent Ltd, and Capsugel

a. Please provide an amended Funding Statement declaring these commercial affiliations, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

Please also include the following statement within your amended Funding Statement.

“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

Answer

Agreed, at point a. we have now included in the revised funding statement that co-author DMM is currently employed by company Capsugel and that co-author AM is CEO of Motilent Ltd and have specified in more detail their role in this work, also updated AM’s role more precisely in the ‘Author Contribution’ section.

We also added the suggested statement at point on the role of the funder.

REVISED FUNDING STATEMENT: ' The authors GEA, GLA and LM received for this work a grant from the U.S. Food and Drug Administration (FDA), https://www.fda.gov/home, Contract HHSF223201510157C. The author DMM is currently employed by company Capsugel and author AM is CEO of Motilent Ltd. The funders and company Capsugel had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Company Motilent provided the data analysis software as part of a subcontract on the research grant and had no role in study design, data collection and decision to publish. AM helped to with set up of the image analysis software and image data registration and with preparation of the final manuscript but had no role in the actual study data analysis. The funder provided support in the form of full salaries for authors KH, NA and fractional salary cost for CLH, JW GEA, GLA and LM, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.This article therefore reflects the views of the authors and should not be construed to represent FDA’s views or policies.'

b. Please also provide an updated Competing Interests Statement declaring these commercial affiliations along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.

Within your Competing Interests Statement, please confirm that these commercial affiliations do not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and there are restrictions on sharing of data and/or materials, please state these.

Please note that we cannot proceed with consideration of your article until this information has been declared.

Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests

Answer

Agreed. We have revised our Competing Interest statement accordingly as show below.

REVISED COMPETING INTEREST STATEMENT: I have read the journal's policy and the authors of this manuscript have the following competing interests: Authors GEA, GLA and LM were recipient of research grant # HHSF223201510157C by the U.S. Food and Drug Administration (FDA). This paid for research salaries and consumables for this work. The author AM is the CEO of Motilent Limited and the author DMM is an employee of Capsugel. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

c. Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf.

Answer

This has been included in our cover letter.

Additional changes

We have corrected affiliation 9 to the ‘Department of Statistics’ not Public Health as incorrectly stated

Response to Reviewer 1 Comments

“There is not much statistical content, other than some Pearson correlations and a mixed model. So I would argue about the term "spatio-temporal", as there is no multidimensional spatial/time series analyses, but only a simple repeated measures.”

Answer

Thank you for this comment. In the statistics literature, a spatio-temporal analysis models data both spatially and temporally. This study collected spatio-temporal data, but the planned analysis produced one number summaries of motility per subject and sampling period, thus collapsing the spatio-temporal data to two AUC statistics and losing some account of spatio-temporal qualities. As such we agree with the suggestion and have removed the “spatio-temporal” term throughout the manuscript unless specific, like for example in the case of the initial MRI raw data (STMM, reference 31) analysis technique which is published with the full spatio-temporal mapping features.

“Sample size is small and seems to have been chosen at random, as there is no power computation.”

Answer

Thank you for querying this, we agree a comment to this effect was missing. We have now included the following at Lines 255-266 (numbering from the clean version with no tracking): “The study design included power analysis considerations. Briefly, for correlation analyses involving data that are summarized to one number per subject, the standard error for the estimated correlation coefficient over 15 subjects is 0.28. However, our main analyses use time-resolved data with an overall sample size of 421 values over the 15 subjects. Using a Kronecker sum model to capture within-modality autocorrelation (for MRI or manometry) and between-modality correlation (the parameter of interest), much smaller standard errors are obtained. For example, if the within-modality autocorrelation is 0.5, the between-modality correlation coefficient is estimated with a standard error of 0.049. Thus, the confidence intervals for the correlation parameter of interest will be reported as the estimate +/- 0.1, giving us excellent power to resolve weak from strong correlation. The standard error depends on the within-modality autocorrelation, which was not known at the time of study design. The actual data obtained showed high precision of the estimated correlation coefficient, indicating that the a priori study design was successful."

“P-values on correlations are really meaningless, because they test 0 correlation against "some" correlation. In this case, a significant p-value doesn't necessarily mean the correlations are strong.”

Answer

Thank you for this, we agree with this point, we removed the p values on the correlations throughout.

We also noted we had quoted R2 values which are now replaced by correlation r values

“The regression models need a careful residual analysis.”

Answer

Agreed, a careful residuals analysis was performed but not included in the manuscript. The residual analysis led us to conclude that the non-linear model fit the data better than the linear model.

“I think there could have been a lot more done with these data, but maybe the sample size is too small.”

Answer

The additional analysis was beyond the scope of the manuscript and what had been planned at funding stage. The possibility to use these data towards optimization and validation of in vitro and in silico predictive biopharmaceutic models will be explored. The sample size has now been addressed in answer to another point raised.

Response to Reviewer 2 Comments

“This manuscript reports on the use of non-invasive dynamic magnetic resonance imaging coupled with semi-automated spatio-temporal data analysis for measuring fasted-state gastric motility and validation of such a method vs. intraluminal pressure recording by means of perfused manometry technique.

Human gastrointestinal motility patterns are of utmost importance to oral drug delivery. The investigated RMI-based method would represent a valuable advancement in the relevant study and allow to gain better insight into the impact of many variables. The work is very interesting, broad in scope, clearly exposed and in-depth discussed.”

Answer

Thank you for the positive appraisal of our work.

“The title is rather long: is the word “imaging” actually needed when the acronym RMI already contains it?”

Answer

Agreed, we deleted the word ‘imaging’ and in response to Reviewer 1 we also deleted the ‘spatio-temporal mapping’ words thus making the title shorter.

“Lines 113-114: as the two methods correlated with each other, “both” sounds odd and can be deleted.”

Answer

Agreed, this was corrected as suggested.

“Lines 123-124: “Both studies did not use semi-automated analysis methods” may better read “Neither of the studies used…”.”

Answer

Agreed, we rephrased this sentence as suggested.

“Line 125: please delete the comma after “making”.”

Answer

Agreed, the comma was deleted.

“Lines 127-128: “more operator independent” may be changed into “less operator-dependent” (this also applies to line 400).”

Answer

Agreed, both sentences were adjusted as suggested.

“Line 143: “Exclusion criteria included..” sounds a bit strange. “Exclusion criteria were”? Or “encompassed”?”

Answer

Agreed, we used the word ‘encompassed’ instead of ‘included’.

“Fig. 2: the caption should be changed into a more concise form taking account of the detailed description and explanation already provided in the text. When the content of the image and the meaning of green and yellow lines are indicated, it will be enough.”

Answer

Agreed, we revised the figure legend of Figure 2 shortening it as follows: “Fig 2. Image analysis. Example of MRI data analysis. The stomach wall boundaries and the axis of the stomach are shown in green. The yellow lines represent the lumen diameter perpendicular to the stomach axis. AC, ascending colon; TC, transverse colon; GB, gall bladder.”

“Line 257: “Each subject participated in either one or two intubation studies” seems to clash with the Methods section reporting that the volunteers “…took part in two identical fasted state MRI study visits” (lines 149-150) and “This was a single-centre, open-label design study that consisted of two separate identical study days...” (lines 154-155).”

Answer

Agreed, thank you, we deleted the sentence on line 257.

“Lines 297-298: please check and rephrase “The correlation for these data shows a significant positive correlation …”.”

Answer

Agreed, we rephrased the sentence: “A positive, significant correlation was observed between the two techniques (R² = 0.7134, p < 0.001).“

“Fig. 6: please check the legend as colors are not visible in the figure whereas dotted/solid lines and different symbols are used as distinguishing features for the 2 curves.”

Answer

Thank you for spotting this, this was a typo that slipped in from an older draft version, we now refer to the curves as solid and dotted lines.

“Lines 337-340: please check and rephrase” The fasting state and the use of a 240 mL dose of water as a challenge were specifically chosen because of their relevance to the bioavailability/bioequivalence (BA/BE) studies in the fasted state”, as it is implied that fasting volunteers take part in fasted-state bioavailability/bioequivalence studies. It seems that only 240 ml of water was chosen because of relevance to those studies.”

Answer

Thank you we checked the phrase and it is correct. During the clinical trials of oral drug product development, it is common practice (and FDA guidelines which we refer to) to ask the participants to fast overnight (hence the fasted state) and to take the drug product of interest drinking 240 mL of water. Our protocol copied this with the difference that our participant were not asked to ingest a drug product.

This was not clearly stated so we have added now at lines Therefore, we added the following sentences at Lines 354-357: “However, it should be noted that the participants here did not actually ingest a drug product. The recommended 240 mL of water was applied as this volume is also used in BA/BE studies during the clinical phase of drug product development.”

“Lines 354-355: “When the stomach is active, this will cause the manometry AUC to increase more quickly than the MRI AUC….” does not appear strictly related to the previously-mentioned circumstance. Could this be an additional factor differing between the two methods?”

Answer

We do see that the manometry AUC can increase more than the MRI AUC, but only seen for high pressure (mmHg) values. At a certain point, when the stomach completely contracts, no differences in MRI AUC will be observed but still some increase in manometry AUC can be noted as the pressure will increase (increasing force of the stomach towards the water-perfused electrodes), but distance will not change anymore. That is the reason why we see this non-linear correlation between both techniques at higher AUC values. The word ‘quickly’ however was vague and it has now been removed.

“Line 364: should “complimentary” read “complementary”?”

Answer

Thank you the spelling was corrected.

“Line 382: “a relative short time window” (or without “a”).”

Answer

Agreed we deleted “a”.

“Line 393-394: “an oral administered” should read “an orally administered””

Answer

Thank you, this was adjusted.

“Line 396: as no statistical optimization study is concerned, “optimized” could be replaced by a different verb. The same applies to lines 418 and 438 (“optimization”).”

Answer

Thank you for this comment; we deleted the word ‘optimized’ and replaced it with ‘developed’ at Line 414, also deleted ‘optimization’ at old numbering Lines 418 and 436

“Lines 315-319: this concept is already mentioned at lines 390-394. Please revise or synthesize the two similar passages in order to avoid repetitions.”

Answer

Having looked at this we think the Reviewer may have meant that lines 415-419 were repeating the concept at lines 390-394 about non-invasiveness, other parameters measurable and use for drug dissolution studies (315-319 are in the results relating to different things). We agree this was repeating itself a bit and we have now removed the lines at the second occurrence “The MRI technique is able to monitor different GI variables simultaneously (i.e. GI motility, gastric emptying and residual fluid volumes) which is of great interest for pharmaceutical purposes in order to study the impact of GI variables on drug and formulation behaviour.”

“Lines 431: “gastric motility” can be deleted as it is specified again at line 432, same sentence.”

Answer

Agreed, “Gastric motility” was deleted at the first occurrence.

Response to Reviewer 3 Comments

“The manuscript is interesting and well written. It is not the first time that MRI has been shown to be a valid method for the measurement of gastric emptying, secretion, motility and intragastric distribution of gastric contents. This study, however, adds value to the use of MRI that assess gastric motility by using a new spatio-temporal MRI mapping technique. Results of this study validate this new MRI assessment by comparing its measurements to simultaneous water-perfusion manometry after ingestion of 240 ml of water with the aim to study pharmacokinetics in humans.”

Answer

Thank you for this positive appraisal.

“However, I have some remarks.

1. The aim of the study is a bit confusing. In the introduction, justification for this new technique is done by explaining the lack of methods for the assessment of predictive drug dissolution models in humans. This aim seems to change in the discussion and it highlights the use of this new method mainly and rather as a gastric motility assessment technique. As the initial aim was to validate this new technique as a method to study pharmacokinetics and therefore dissolution of drugs, this should be further addressed more clearly in the discussion section. Additionally, a proof of concept study should be suggested as a next step to investigate the actual assessment of pharmacokinetics.”

Answer

Correct statement. During the past years, there was specific focus on validating existing in vitro and in silico models for their capacity to predict the in vivo performance of an orally administered drug product to humans. Numerous projects (e.g., OrBiTo, PEARRL, UNGAP) were designed to explore the predictive performance of existing in vitro and in silico models. However, there is still a general scarcity of data on gastric motility to help us understand the impact of gastric motility on an orally administered dosage form. With the MRI techniques one can look at undisturbed (no intubation) contractility. As a recent study by Hens et al. successfully implemented MRI-derived dynamic fluid volumes in commercially-available computational software (e.g., GastroPlus), we will be able now to do the same for motility patterns and investigate how this will impact formulation disintegration for instance.

We agree with you that something was missing in the Discussion section. We added there at Lines XXX-YYY a sentence to better link this study with pharmacokinetic studies: “With respect to future uses of the MRI technique, a pharmacokinetic study in combination with assessment of motility could provide unprecedented insights on inter-subject variability in systemic outcome of a drug, as the underlying motility patterns are likely one dominant cause of known inter-subject differences in formulation disintegration and, subsequently, dissolution and absorption of the drug. These type of studies are the next logical step to further our understanding of to investigate the actual assessment of pharmacokinetics variables”

“I do not agree with some section in the discussion addressing focus to the validity and accuracy of this new method for the assessment of gastric motility with a broader spectrum, including assessment of general GI function in GI disorder or the impact of drugs to GI motility. The ingestion of 240 ml of water studied in the present study is not enough to make such a statement. Therefore, careful should be taken in the discussion and it should be indicated for such cases further validation studies should be needed.”

Answer

Agreed, the last 5 lines of the Discussion were speculative as to the validity and accuracy of these methods beyond the data presented and were removed. The corresponding statements in the Abstract and in Conclusions have been removed accordingly.

“2. Where are the sensors of the water-perfusion manometry located? Is this antral and duodenal assessment? Please, specify in methods.”

Answer

Good point, agreed, we have clarified at Lines 180-182 that “The perfused manometry ports tend to record primarily antral contractions whilst more proximal sensors in the wider fundal region do not record contractions well, as such the assessment here was focused on the antral region of the stomach.”

“ 3. Following up the previous comment, if sensors are only located in the antral section of the stomach, and thus assessing antral contractions, this should also be specified throughout the entire manuscript: validation of this technique is for ANTRAL motility assessment and not general gastric motility assessment.”

Answer

Agreed, we have specified ‘antral motility’ in the title and several times throughout the manuscript.

“4. What was the drinking speed of the volunteers? Do they had to drink all water within 5 minutes or less? This was done in the MRI in supine position. How was this managed? Specify in methods.”

Answer

Agreed, we now specify at Lines 206-208 that the participants drank while they were sitting up on the scanner bed and that they were not instructed to drink the water at a given speed, but all of the participants drank the glass of water quickly, within approximately one minute.

“5. In the methods it is discussed a “the manometer was connected to the MRI compatible water-perfused manometry system”. What does this means? That the manometry device is all-plastic? Is this the research’s group own developed system or was this bought from a company? In this last case, from which company?”

Answer

Agreed, some detail was missing, thank you for pointing this out, we now added at lines 183-187 the system manufacturer (the Biomedical Engineering Department at The Royal Melbourne Hospital in Australia) and describe that the transducers connecting to the catheter were secured to a MRI-compatible trolley placed next to the scanner bed. Electric cables and pipes went through a wave guide opening and connected the transducers box to the electronics and pressure gas cylinder placed outside the scanner room.

“6. Include reference and explain possible limitation regarding supine positioning and its effect on GI motility (Might have an effect on the gastric emptying rate): Treier R, Steingoetter A, Weishaupt D, Fried M, Boesiger P, Schwizer W. Gastroenterology 2003; Gastric motor function and emptying in the right decubitus and seated body position as assessed by magnetic resonance imaging”

Answer

Thank you for raising this point. We added a comment with reference to that paper and also to the other paper from the same group whereby they put people upside down in a vertical scanner at lines 438-443 with the two references.

“I think in the figures 4 and 5 the “AUC” for MRI is missing.”

Answer

Thank you for spotting this, this was added to both figures.

“8. Figure 6. Can you include in the figure the moment the water was ingested?

Answer

Agreed, the drink ingestion time has been indicated with an arrow and text in Fig 6.

Why is there a gap between -15 and 0?

Answer

As described at Lines 207-208 the participants were allowed a rest outside the scanner room before the drink part of the study. To allow them to move we disconnected the perfused manometry system (Line 208). None of the participants wanted a long break, they took only advantage of the rest time to stretch their legs and visit the toilet before continuing and the average time including reconnection was 15 minutes corresponding to the gap. We have clarified the timing at liens 208-211

Why is the pressure so low at time 0?”

Answer

We have a comment to this effect at Lines 365-368 of the Discussion where we indicate that is likely to reflect the inability of the perfused manometry to measure non-occlusive events when a free fluid is present to redistribute the pressure changes instantaneously.

Figures Check

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Answer

Agreed, we have passed the figures through the PACE engine and uploaded the PACE Corrected files

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

29 Sep 2020

PONE-D-20-12517R1

Measurement of fasted state gastric antral motility before and after a standard bioavailability and bioequivalence 240 mL drink of water: validation of MRI method against concomitant perfused manometry in healthy participants

PLOS ONE

Dear Dr. Marciani,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Nov 13 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Florencia Carbone

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

After carefully reviewing the manuscript no main issues were found.

However, I only have two comments that might need some work:

1. It is consented that for the assessment of antral motility, the frequency and amplitude of contractile waves are measured in order to define a motility index (See reference: PMID: 19019032). The motility index (MI) represents the fraction of time during which gastric motility was detected and is normally calculated as an average of the individual detected contractions in a specific time window taking into account the relative amplitude of each contractile wave. Motility index is then a composite parameter that incorporates both contraction frequency and amplitude. May I suggest, in order to add value and credibility to your study, to think about incorporating the term of MI in your analysis. MI of both techniques will also be easier to correlate. I think this is in some way what you are doing by assessing AUCs, but in this way, the acquired information with MI will be cleaner and easier to understand for the reader.

2. Methods regarding the intragastric manometry are a bit vague. From the manuscript it seems as the original method to assess intragastric pressure has been adapted to be used in a MRI setting. Do I understand correctly that the total length of the manometry tube was 280 cm (180+100 pigtails) in order to allow the pressure to be measured from outside the MRI room? This is quite a distance. As the water-perfusion manometer is normally not validated to acquire pressure information at such a distance, how was the pressure on the tip of the manometer (water drop) double-checked to be correct? Have you performed any validation of this method to verify accurate assessment of values? This is very important as you are using this data to validate another technique (the MRI). You should include this information on your methods.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

12 Oct 2020

We are grateful for Reviewer #1’s acknowledgement that we have addressed all the comments from the first round of reviews, and for the Additional Editor’s appraisal that the manuscript does not have any main issues. We welcome the opportunity to answer the two remaining Additional Editor’s comments as detailed below.

All changes made to the main manuscript version R2 are tracked in the marked-up Word version enclosed and a separate clean unmarked version is also uploaded.

1. It is consented that for the assessment of antral motility, the frequency and amplitude of contractile waves are measured in order to define a motility index (See reference: PMID: 19019032). The motility index (MI) represents the fraction of time during which gastric motility was detected and is normally calculated as an average of the individual detected contractions in a specific time window taking into account the relative amplitude of each contractile wave. Motility index is then a composite parameter that incorporates both contraction frequency and amplitude. May I suggest, in order to add value and credibility to your study, to think about incorporating the term of MI in your analysis. MI of both techniques will also be easier to correlate. I think this is in some way what you are doing by assessing AUCs, but in this way, the acquired information with MI will be cleaner and easier to understand for the reader.

Answer: Thank you for this comment. We agree with you that the motility index is a common and accepted parameter in the field of gastrointestinal motility, indeed one of our co-authors here is also a co-author of the consensus statement that you reference. For our study, after much deliberation, we had chosen to use the area under the curve (AUC), which is another one of the ‘classic’ contratile parameters used to analyze motility manometry traces. The AUC incorporates both the strength and frequency of contractions in an integral fashion providing a single non-composite number. The AUC outcome was then stated on the contract with the FDA and declared as primary outcome of the study on the protcol registration. For the above reasons we would very respectfully ask to retain the AUC data analysis. We have however added a comment at lines 432-435 to acknowledge the possible limitation and consider future use of the motility index.

2. Methods regarding the intragastric manometry are a bit vague. From the manuscript it seems as the original method to assess intragastric pressure has been adapted to be used in a MRI setting. Do I understand correctly that the total length of the manometry tube was 280 cm (180+100 pigtails) in order to allow the pressure to be measured from outside the MRI room? This is quite a distance. As the water-perfusion manometer is normally not validated to acquire pressure information at such a distance, how was the pressure on the tip of the manometer (water drop) double-checked to be correct? Have you performed any validation of this method to verify accurate assessment of values? This is very important as you are using this data to validate another technique (the MRI). You should include this information on your methods.

Answer: Thank you this comment, we agree some details were unclear. We have now clarified at lines 184 – 191 that the pressure was transduced to electrical signal inside the scanner room, right at the bore of the scanner. We also clarify that we used the old manual, gravity method to calibrate the manometry system, before and after the study, with the claibrations recorded on the traces so that we could detect drifts or malfunctions. We agree that the total 280 cm from tip to end pigtail is long, this was to allow for the catheter to reach comfortably the transducers from inside the bore of the scanner. One typical concern with long catheters is lag of response but the system was responding rapidly to gravity changes, with no visible lag. The same system had been used and published by our colleagues in Zurich.

15 Oct 2020

Measurement of fasted state gastric antral motility before and after a standard bioavailability and bioequivalence 240 mL drink of water: validation of MRI method against concomitant perfused manometry in healthy participants

PONE-D-20-12517R2

Dear Dr.,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Florencia Carbone

Guest Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Thank you for the clarifications. Everything is very clear now.

21 Oct 2020

PONE-D-20-12517R2

Measurement of fasted state gastric antral motility before and after a standard bioavailability and bioequivalence 240 mL drink of water: validation of MRI method against concomitant perfused manometry in healthy participants

Dear Dr. Marciani:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Florencia Carbone

Guest Editor

PLOS ONE