Methods for measuring intestinal permeability and gastric emptying rate

Abstract

A method for measuring intestinal permeability in a subject includes orally administering to the subject a solution containing a fluorescent contrast agent that is absorbed by healthy intestine; using a light source to illuminate a location on the skin of a body part of the subject with optical radiation, such that the optical radiation causes at least a portion of the solution that leaks out of the subject's intestine and into the subject's bloodstream to fluoresce; using a transcutaneous sensing device to periodically detect the fluorescent intensity of the solution at the location to obtain fluorescent data of the intensity as a function of time; normalizing the fluorescent data to obtain normalized data of the intensity as a function of time; and analyzing the normalized data to measure the intestinal permeability of the subject by calculating one or more of: (a) a first peak value of intensity; (b) an integral of the intensity with respect to time; (c) the product of the first peak value of intensity and the time at the peak value; (d) the time at which the first peak value of intensity occurs; or (e) the first peak value of intensity divided by the time at which the peak value occurs, to measure the intestinal permeability of the subject.

Description

[Technical Field]

[0001] The present disclosure relates to methods for measuring intestinal permeability in a subject and methods for measuring gastric emptying rate in a subject. [Background technology]

[0002] Gastrointestinal function plays a crucial role in numerous health disorders, including celiac disease, inflammatory bowel disease (IBD), human immunodeficiency virus (HIV), fatty liver disease, sepsis, chronic liver disease, environmentally induced gut dysfunction (EED), and malnutrition. In particular, disruption of the intestinal barrier plays a key role in these health conditions, leading to increased intestinal permeability (also known as "leaky") and the translocation of enteric bacteria and pathogen-associated molecular peptides into the systemic circulation, resulting in an inflammatory response.

[0003] For example, increased intestinal permeability (also known as "leaky gut") occurs when the intestinal barrier is compromised, allowing bacteria and other pathogens to escape from the intestine into the lymphatic system and blood vessels. This creates a cycle of infection and inflammation that can have additional consequences and further exacerbate the problem of increased intestinal permeability. This condition is associated with many of the diseases mentioned above, significantly impacting quality of life and limiting the physical and cognitive development of malnourished children.

[0004] Furthermore, the role and impact of the gut and gut barrier function in many of the health conditions highlighted above is poorly understood.

[0005] Similarly, alterations in gastric emptying rate (the rate at which food is emptied from the stomach into the intestine) are observed in many diseases such as functional dyspepsia, gastroparesis, gastric cancer, etc. Furthermore, gastric emptying rate is important in human nutrition as it determines the rate at which nutrients can be absorbed.

[0006] Current techniques for assessing intestinal and gastrointestinal function (especially intestinal permeability and gastric emptying rate) are cumbersome, expensive, invasive (e.g., requiring endoscopic biopsy and histopathological or x-ray examination), unreliable (e.g., using urine and / or blood samples), and difficult to perform in infants.

[0007] Thus, there is a need for new technologies that can improve our understanding of gastrointestinal function in the pathogenesis of disease and help provide earlier diagnosis and improved monitoring of the many conditions it plays a role in. In particular, there is a need for improved means to assess and quantify intestinal permeability and improved means to assess and quantify gastric emptying rates.

[0008] WO 2015 / 070256 discloses compositions and methods for assessing intestinal function. A composition for assessing intestinal function is disclosed, which comprises a fluorescent challenge molecule, where the fluorescent challenge molecule is not substantially absorbed by a healthy intestine. A composition for assessing intestinal function is also disclosed, which comprises at least two fluorescent challenge molecules, where one fluorescent challenge molecule is not substantially absorbed by a healthy intestine and another fluorescent challenge molecule is substantially absorbed by a healthy intestine.

[0009] WO 2010 / 020673 discloses a sensor plaster for transcutaneously measuring organ function, particularly kidney function. The sensor plaster comprises at least one flexible support element with at least one adhesive surface, which can be attached to a body surface. The sensor plaster also comprises at least one radiation source, particularly a light source, configured to illuminate the body surface with at least one test light. The sensor plaster also comprises at least one detector configured to detect at least one response light irradiating from the direction of the body surface.

[0010] The present disclosure seeks to at least partially alleviate and / or at least partially address the problems associated with the prior art. Summary of the Invention [Means for solving the problem]

[0011] According to a first aspect of the present disclosure, there is provided a method for measuring intestinal permeability of a subject, the method comprising: orally administering to the subject a solution comprising a fluorescent contrast agent that is absorbed by a healthy intestine; using a light source to irradiate a location on the skin of a body part of the subject with optical radiation, such that the optical radiation causes at least a portion of the solution that leaks out of the subject's intestine and into the subject's bloodstream to fluoresce; using a transcutaneous sensing device to periodically detect the fluorescent intensity of the solution at the location to obtain fluorescent data of intensity as a function of time; normalizing the fluorescent data to obtain normalized data of the intensity as a function of time; and analyzing the normalized data by calculating one or more of a first peak value of the intensity, an integral of the intensity with respect to time, a product of the first peak value of the intensity and the time at the first peak value, a product of the first peak value of the intensity and the time past the time of the first peak value, the time at which the first peak value of the intensity occurs, or a value obtained by dividing the first peak value of the intensity by the time at which the peak value occurs, to measure the intestinal permeability of the subject.

[0012] Advantageously, such methods can provide a minimally invasive, reliable, and quantifiable method for assessing intestinal permeability in a subject.

[0013] In particular, the use of a fluorescent contrast agent that is absorbed by healthy intestine advantageously provides a baseline measurement, allowing even subtle changes in permeability to be detected without the need to use multiple different fluorescent contrast agents with varying molecular properties. Therefore, the use of a fluorescent contrast agent that is absorbed by healthy intestine allows diagnostic measurements to be made in all individuals, regardless of whether their intestine is healthy or unhealthy. Advantageously, this means that all pathologies can be assessed, not just those that exceed any threshold above which the fluorescent contrast agent is initially absorbed, as would be the case if a fluorescent contrast agent that cannot be absorbed by healthy intestine were used instead. Therefore, such methods can be advantageously used to assess mildly damaged intestine rather than severely damaged intestine, and can provide a quantitative indication of the degree of intestinal damage, rather than simply a binary indication of whether the intestine is damaged or not.

[0014] Optionally, the fluorescence data is normalized with respect to acquisition time and the intensity of the backscattered light emission, ie the backscattered excitation light.

[0015] Optionally, the normalized data is analyzed to measure the intestinal permeability of the subject by calculating the integral of said intensity with respect to time up to the time when the first peak value of said intensity occurs.

[0016] Furthermore, normalizing the fluorescence data with respect to the intensity of the backscattered excitation light (i.e., the intensity of the signal from the light source (such as a laser, light-emitting diode, or other light source) used to excite the fluorescence) and analyzing the normalized data by calculating one or more of the following to measure the intestinal permeability of the subject: a first peak value of the intensity; an integral of the intensity with respect to time (a selected time point: for example, the time of the first peak, or the sum of the time of the first peak plus a selected percentage of the time of the first peak (i.e., at a time point past the time of the first peak, for example, 5, 10, or 20%); a product of the first peak value of the intensity and the time at which the first peak value occurs; a product of the first peak value of the intensity and the time past the time of the first peak value; a time at which the first peak value of the intensity occurs; or a value obtained by dividing the first peak value by the time at which the peak value occurs. Thus, continuous measurements of intestinal permeability can be obtained using a single fluorescent contrast agent.

[0017] The first peak intensity value can provide a readout of the peak concentration of the fluorescent contrast agent in the subject's bloodstream and can therefore be used to measure the subject's intestinal permeability. The peak value can represent the time when the concentration of the fluorescent contrast agent in the subject's bloodstream is greatest, and typically occurs before significant excretion of the solution from the body (e.g., via the kidneys or liver) occurs.

[0018] Overall, such methods can therefore provide quantification of intestinal permeability based on normalized fluorescence intensity data, as well as indicators of other aspects of gastrointestinal (GI) function, without the need to collect urine, blood, or other samples. This allows for quantification of dye leakage through even healthy intestinal barriers, potentially providing meaningful clinical data for a wide range of patients (i.e., subjects), not just those with highly permeable / damaged intestines. Furthermore, quantification / calculation in such methods can provide a continuously changing measure of intestinal permeability, rather than a simple binary marker (as in the prior art) that can only provide a rough clinical outcome of whether the intestine is "permeable" or "impermeable." This allows for monitoring patient response to treatments or other interventions (e.g., nutritional interventions) and also allows for comparison of values ​​measured in different individuals (i.e., for screening / diagnostic applications). This is made possible by normalizing the fluorescence data (to the backscattered excitation signal) and by periodic data collection.

[0019] Furthermore, such methods demonstrate the potential for non-invasive sensing of intestinal permeability and may provide for the development of miniaturized, low-cost, wearable sensors suitable for field-deployable use.

[0020] Optionally, the normalized data is analyzed to measure the intestinal permeability of the subject by calculating the integral of the intensity with respect to time up to a selected time point after the first peak value of the intensity.

[0021] For example, the selected time point may be equal to the sum of the time value at which the first peak intensity value occurs and a selected percentage of that time value. The selected percentage may be between approximately 5 and 25 percent, e.g., approximately 5, 10, or 20 percent. For example, if the first peak intensity value is observed at a time value of 60 minutes, the data may be integrated to times 63, 66, or 72 minutes, with selected percentage values ​​for time adjustment being 5%, 10%, and 20%, respectively. Advantageously, by integrating the data to a time slightly past the time of the first peak intensity value, the resulting analysis can account for the removal of contrast agent from the subject's bloodstream. Such correction is more important and advantageous when the first peak occurs later, since the later the first peak occurs (in time), the greater the clearance of contrast agent from the body. Advantageously, integrating to a time slightly past the peak corrects for this.

[0022] Optionally, the solution contains only a single fluorescent contrast agent that is absorbed by healthy intestine, i.e. the solution contains said fluorescent contrast agent that is absorbed by healthy intestine and no other fluorescent contrast agents.

[0023] Optionally, the following equation may be used to calculate the first peak intensity value:

[0024]

number

[0025] where GP1 represents the quantifier for intestinal permeability and I(t peak ) is the time of the first peak in the intensity data (t peak ) represents the normalized intensity data values ​​as a function of time.

[0026] The integral of the intensity with respect to time (i.e., the area under the curve of the intensity plotted against time) can be used to measure the intestinal permeability of the subject, taking into account the effect of gastric emptying rate. Optionally, the integral can be calculated up to a first peak in the intensity. Optionally, the integral can be calculated up to a selected time point past the first peak in the intensity. This can provide a quantitative measure of the total amount of fluorescent contrast agent that leaked from the intestine into the subject's bloodstream up to the time of the first peak (or up to another selected time point, such as a selected time point past the time of the first peak). Identifying the time of peak concentration from the data allows for accurate quantification of the total recovery of the fluorescent contrast agent in the subject's bloodstream.

[0027] If desired, the following equation may be used to calculate the integral of the intensity over time:

[0028]

number

[0029] where GP2 represents the quantifier for intestinal permeability, I(t) represents the normalized data of the intensity as a function of time, and t peak represents the time point of the first peak in the fluorescence data. Alternatively, the equation for GP2 may be modified as follows:

[0030]

number

[0031] In the formula, t peak+represents a selected time point after the first peak in the fluorescence data. For example, the selected time point may be equal to the sum of the time values ​​at which the first peak occurred in the fluorescence data plus a selected percentage of that time value. The selected percentage may be between about 5 percent and 25 percent; for example, the selected percentage may be about 5 percent, 10 percent, or 20 percent.

[0032] The product of the first peak intensity value and the time at the peak value can be used to measure the intestinal permeability of a subject and advantageously represents a simplified analytical approach for calculating the integral of the intensity over time.

[0033] Alternatively, the product of the first peak intensity value and the time past the peak intensity value can be used to measure the subject's intestinal permeability. This can also advantageously represent a simplified analytical approach for calculating the integral of the intensity over time. The time past the peak intensity value is defined as the sum of the time of the first peak and a selected percentage (e.g., 5, 10, or 20 percent) of the time of the first peak (i.e., the time is defined as the time past / after the time of the first peak). For example, if the first peak intensity value is observed at a time value of 60 minutes, the time can be selected to be 63 minutes, 66 minutes, or 72 minutes for a selected percentage of the time adjustment (5%, 10%, and 20%, respectively). Advantageously, by taking the product of the intensity at the first peak intensity value and the time just past the time of the first peak intensity value, the resulting analysis can account for the removal of the contrast agent from the subject's bloodstream. Such a correction is more important and advantageous when the first peak occurs later (in time), since the later the first peak occurs, the greater the clearance of contrast agent from the body. Advantageously, this is corrected for by taking the product of the first peak intensity value and the time just past the peak.

[0034] Optionally, the following equation may be used to calculate the product of the first peak intensity value and the time at the peak value:

[0035]

number

[0036] where GP3 represents the quantifier for intestinal permeability, and I(t peak ) is the time of the first peak in the intensity data (t peak ) represents the normalized data value of the intensity as a function of time at t peak represents the time of the first peak in the fluorescence data. Alternatively, the above formula for GP3 can be modified to calculate the product of the first peak intensity value and the time past the time of the first peak value as follows:

[0037]

number

[0038] In the formula, t peak+ represents a selected time point after the first peak in the fluorescence data (eg, 5, 10, or 20 percent past the time of the first peak).

[0039] The time at which the first peak value of the intensity occurs, or the value obtained by dividing the first peak value of the intensity by the time at which the peak value occurs, can be used to measure the intestinal permeability of a subject; the smaller the value at which the peak value occurs, the higher the permeability.

[0040] Optionally, the following equation may be used to calculate the time at which the first peak value of the intensity occurs:

[0041]

number

[0042] where GP4 represents the intestinal permeability quantifier, and t peak represents the time of the first peak in the fluorescence data.

[0043] Optionally, the following equation may be used to calculate the first peak value of the intensity divided by the time at which the peak value occurs:

[0044]

number

[0045] where GP5 represents the quantifier for intestinal permeability, and I(t peak ) is the time of the first peak in the intensity data (t peak ) represents the normalized data value of the intensity as a function of time at t peak represents the time of the first peak in the fluorescence data.

[0046] Advantageously, GP2 and GP3 can provide some degree of correction to account for gastric emptying rate and therefore can provide a more quantitative assessment of permeability.

[0047] Optionally, the step of using a transdermal sensing device to periodically detect the fluorescence intensity of the solution at the location to obtain fluorescence data of the intensity as a function of time includes using the transdermal sensing device to measure the fluorescence intensity of the solution and to measure the backscattering intensity of the optical radiation used to irradiate the location on the skin of the body part.

[0048] Optionally, the step of using the transdermal sensing device to periodically detect the fluorescence intensity of the solution at said location to obtain fluorescence data of said intensity as a function of time includes measuring backscattered light simultaneously with detecting said fluorescence.

[0049] Optionally, said measurement of backscattered light is made at the same location where the fluorescence intensity of the solution is periodically detected, i.e. at said location on the skin of said body part.

[0050] Optionally, the measurement of the backscattered light is made at a different location from the location where the fluorescence intensity of the solution is periodically detected, for example at another location close to but distant from said location on the skin of said body part.

[0051] Optionally, the different locations are locations on the subject's skin. Optionally, this is provided by a fiber optic probe with a filter wheel at its proximal end to provide switching between fluorescence and laser (excitation) measurements, which offers the advantage that the measurement locations are the same but the measurement times are different. Optionally, this is alternatively provided by a wearable sensor with one excitation source (e.g., an LED) and two closely spaced detectors (one to detect the fluorescence signal and one to detect the excitation signal), which offers the advantage that the measurement locations are nearly the same and the measurement times are the same.

[0052] Advantageously, this allows the data to be properly normalized and takes into account variability between patients and / or between repeated measurements in the same patient, etc.

[0053] Advantageously, by recording the intensity of the backscattered excitation signal at a location on the skin rather than at the light source itself and normalizing with respect to the excitation power, variations due to skin tone, skin thickness, light source movement, etc. can be corrected for.

[0054] Optionally, the solution comprises water or juice.

[0055] Advantageously, the fluorescent contrast agent can be delivered to the subject as a solution in water or juice, so that other components of the drink do not affect intestinal permeability. For young patients who find the solution too bitter, the solution can be dissolved in orange juice.

[0056] Optionally, the imaging agent comprises a dye, for example, fluorescein, methylene blue, fluorescein isothiocyanate-conjugated dextran, salts thereof, or combinations thereof.

[0057] Advantageously, fluorescein is a clinically approved dye that is rapidly taken up by the small intestine after stomach emptying, and thus the rate of fluorescein uptake into the bloodstream (and the other permeability quantifiers mentioned above) can be used to assess intestinal permeability.

[0058] Advantageously, methylene blue is a clinically approved dye that is rapidly taken up by the small intestine after emptying from the stomach, and thus the rate of methylene blue uptake into the bloodstream (and the other permeability quantifiers mentioned above) can be used to assess intestinal permeability.

[0059] Optionally, the solution comprises a dose of about 100-500 mg of contrast agent.

[0060] Advantageously, such a dose of contrast agent can ensure a good signal-to-noise ratio in the fluorescence data while remaining within clinically acceptable levels.

[0061] Optionally, the solution comprises fluorescein as a fluorescent imaging agent in a dose of at least 25 mg.

[0062] Optionally, the solution comprises a dose of about 100 mg of the contrast agent.

[0063] Optionally, the solution contains a 100 mg dose of fluorescein or methylene blue as a fluorescent contrast agent.

[0064] Optionally, the solution comprises fluorescein as a fluorescent contrast agent at a dose of about 100-500 mg in about 100 ml of water or juice.

[0065] Optionally, the solution contains a dose of contrast agent between 25-500 mg.

[0066] Optionally, the solution contains fluorescein as a contrast agent in a dose of up to 500 mg.

[0067] Optionally, the solution contains methylene blue as a contrast agent in a dose of up to 100 mg.

[0068] The dose of fluorescent contrast agent in solution is selected to find the optimum balance between minimizing dose and maximizing signal-to-noise ratio.

[0069] Optionally, the transdermal sensing device has an acquisition time, the light source has an excitation power, and the fluorescence data is normalized based on said acquisition time and said excitation power.

[0070] Advantageously, by normalizing the fluorescence intensity values ​​for each time point by the acquisition time and excitation power used in each case, all time points can be compared with each other. Advantageously, this allows quantitative comparison of measurements from different days or times, or in different subjects. Furthermore, this allows the fluorescence values ​​to provide meaningful quantification of variations in intestinal permeability (e.g., variations between different patients, or changes over time in a single patient), rather than simply providing a binary assessment of whether a contrast agent is used.

[0071] Optionally, the transcutaneous sensing device has an acquisition time of between 100 milliseconds and 15 seconds.

[0072] Optionally, the transcutaneous sensing device has an acquisition time of at least 100 milliseconds.

[0073] Optionally, the transdermal sensing device is configured such that the acquisition time for each measurement can be determined in an automated manner by the transdermal sensing device itself. That is, optionally, the transdermal sensing device is configured to determine the optimal acquisition time for each time point based on the current level of the fluorescence and / or backscatter excitation signal. Optionally, the intensity fluorescence data as a function of time may be normalized by the acquisition time used to collect both the fluorescence data and the backscatter excitation data.

[0074] Advantageously, this ensures that good signal levels are obtained at all time points, even when the fluorescence level is low (e.g., at the beginning or end of the measurement / detection process). This also ensures that there are no issues related to detector saturation when the fluorescence signal is at its maximum. Advantageously, this allows measurements to be performed reliably across patients with different skin tones without requiring high doses of fluorescent contrast agent in darker-skinned subjects. Instead of requiring higher doses or higher excitation powers, which may be clinically unacceptable, the transdermal sensing device can obtain adequate signal levels by automatically acquiring data with longer integration times.

[0075] Optionally, the transdermal sensing device is configured to detect the intensity of said fluorescence using fluorescence spectroscopy.

[0076] Optionally, the transdermal sensing device begins periodic measurements before the solution is administered to the subject, such that periodic detection of the fluorescence intensity of the solution begins before the solution is administered to the subject to obtain a background signal to be used in the step of normalizing the fluorescence data.

[0077] Optionally, background signal is subtracted from the intensity fluorescence data as a function of time for each time point (ie for each periodic measurement detected / obtained).

[0078] Optionally, the fluorescence data is normalized based on the backscatter intensity of said optical radiation.

[0079] Advantageously, such normalization of the fluorescence data makes the fluorescence data comparable across different measurements and across different subjects using only a single fluorescent imaging agent.

[0080] Optionally, the light source comprises a light emitting diode or a laser.

[0081] Optionally, the light source is configured to be worn on and / or around said body part of the subject.

[0082] Optionally, the transcutaneous sensing device includes one or more photodiodes, phototransistors, and / or fiber optic probes and is configured to be worn on and / or around said body part of the subject.

[0083] Optionally, in the step of periodically detecting said fluorescence intensity using a transdermal sensing device, measurements are recorded by said transdermal sensing device at least once every five minutes.

[0084] Optionally, in the step of periodically detecting said fluorescence intensity using a transdermal sensing device, measurements are recorded by said transdermal sensing device at least once every two minutes.

[0085] Optionally, in the step of periodically detecting said fluorescence intensity using a transdermal sensing device, measurements are recorded by said transdermal sensing device at least once per minute.

[0086] Advantageously, the frequency of the measurements is selected to ensure that the collected data has sufficient time resolution. This can be ensured by recording measurements at least once every 5 minutes. This helps to accurately identify the first peak value of the intensity, which represents the peak concentration of the fluorescent contrast agent in the subject's bloodstream.

[0087] Furthermore, if measurements are taken at a sufficiently high frequency, for example once every 5 minutes, 2 minutes, or 1 minute, the data obtained can more specifically indicate the location in the intestine where the leak is occurring, compared to less frequent, regular measurements.

[0088] Optionally, said body part is a finger, wrist, arm, or earlobe.

[0089] Advantageously, when measurements are taken using the transcutaneous sensing device at skin locations on the finger, wrist, arm, or earlobe, clear fluorescence of the fluorescent contrast agent is observed even with short acquisition times.

[0090] Optionally, said body part is a fingertip.

[0091] Advantageously, performing measurements using the transcutaneous sensing device at the fingertip position allows for clear fluorescence of the fluorescent contrast agent to be observed even with short acquisition times, and the intensity of the fluorescence data can be enhanced due to the proximity of blood vessels to the surface of the fingertip skin. Measuring at the fingertip also minimizes the effect of dye leakage from blood vessels.

[0092] According to a second aspect of the present disclosure, there is provided a method for measuring a subject's gastric emptying rate, the method comprising: orally administering to the subject a test meal comprising a fluorescent contrast agent that is absorbed by a healthy intestine; using a light source to irradiate a location on the skin of a body part of the subject with optical radiation, such that the radiation causes at least a portion of the fluorescent contrast agent in the test meal that leaks from the subject's stomach and enters the subject's bloodstream to fluoresce; using a transdermal sensing device to periodically detect the fluorescence intensity of the fluorescent contrast agent in the test meal at the location to obtain data about the intensity as a function of time; normalizing the fluorescence data to obtain normalized data of the intensity as a function of time; and analyzing the normalized data to calculate the percentage of the test meal remaining in the subject's stomach as a function of time based on the intensity as a function of time divided by the peak intensity value.

[0093] Advantageously, such methods can provide a minimally invasive, reliable, and quantifiable method for assessing the rate of gastric emptying in a subject.

[0094] In particular, by using a fluorescent contrast agent that is absorbed by a healthy intestine, measurements can be advantageously obtained in all participants (i.e., subjects with normal intestinal permeability or subjects with increased intestinal permeability). This allows for the detection of even small changes in gastric emptying rate, enabling measurements to be made in all participants, regardless of their gastrointestinal health status. It also allows for the use of a single fluorescent contrast agent in all subjects, rather than the need to use multiple different fluorescent contrast agents with varying molecular properties. Therefore, using a fluorescent contrast agent absorbed by a healthy intestine allows for measurements to be made in all individuals, regardless of whether their intestine is healthy or unhealthy. Advantageously, this means that all pathologies can be assessed, not just those above any threshold above which the fluorescent contrast agent is first absorbed, as would be the case if a fluorescent contrast agent that cannot be absorbed by a healthy intestine were used instead. Therefore, such methods can be advantageously used to assess mildly damaged rather than severely damaged intestines, and can provide a quantitative indication of the degree of intestinal damage, rather than simply a binary indication of whether the intestine is damaged or not. In particular, by using a fluorescent contrast agent that is absorbed by healthy intestine, rather than using a fluorescent contrast agent that is absorbed only by damaged intestine, such methods advantageously allow for the rate of gastric emptying to be measured regardless of whether the subject has other intestinal health issues.

[0095] Furthermore, normalizing the fluorescence data and analyzing the normalized data to calculate the percentage of the test meal remaining in the subject's stomach as a function of time can provide the advantage of being able to obtain a quantitative assessment of gastric emptying rate by using only a single fluorescent contrast agent (i.e., without requiring a test meal containing two or more different fluorescent contrast agents, such as two different fluorescent contrast agents having different molecular weights, and without requiring the use of different contrast agents in subjects with different intestinal health states). Thus, continuous measurements of gastric emptying rate can be obtained by using a single fluorescent contrast agent.

[0096] Overall, such a method can therefore provide quantification of gastric emptying rate based on normalized fluorescence intensity data, as well as indicators of other aspects of gastrointestinal (GI) function, without the need to collect urine, blood, or other samples. This allows for quantification of dye leakage across even healthy intestinal barriers, potentially providing meaningful clinical data across a wide range of patients (i.e., subjects), not just those with highly permeable / damaged intestines. Furthermore, quantification / calculation in such a method can provide a continuously changing measure of gastric emptying rate. This allows for patient monitoring of treatments and other interventions (e.g., nutritional interventions) and allows for comparison of values ​​measured in different individuals (i.e., for screening / diagnostic applications). This is made possible by the normalization of fluorescence data and periodic data collection.

[0097] Furthermore, such methods demonstrate the feasibility of non-invasive sensing of gastric emptying rate and may provide for the development of miniaturized, low-cost, wearable sensors suitable for use in field deployment.

[0098] Optionally, the peak intensity value is the intensity value at a first peak or a second peak of the fluorescence data, or is the maximum intensity value of the fluorescence data, which is selected based on the particular calculation method used.

[0099] Optionally, the peak intensity value is the intensity value at a first peak in the fluorescence data.

[0100] Optionally, the test meal contains only a single fluorescent contrast agent that is absorbed by the healthy intestine, i.e., the test meal may contain said fluorescent contrast agent that is absorbed by the healthy intestine and no other fluorescent contrast agents.

[0101] Optionally, the step of using a transdermal sensing device to periodically detect the intensity of fluorescence of the test meal at said location to obtain fluorescence data of said intensity as a function of time includes the step of using said transdermal sensing device to measure the intensity of fluorescence of the test meal and to measure the intensity of backscattered intensity of said optical radiation.

[0102] Optionally, the step of using a transdermal sensing device to periodically detect the intensity of fluorescence of the test meal at the location to obtain fluorescence data of the intensity as a function of time includes the step of using the transdermal sensing device to measure the intensity of fluorescence of the test meal and to measure the backscattering intensity of the optical radiation used to irradiate the location on the skin of the body part.

[0103] Optionally, the step of using the transdermal sensing device to periodically detect the fluorescence intensity of the test meal at said location to obtain fluorescence data of said intensity as a function of time includes the step of measuring backscattered light simultaneously with detecting said fluorescence.

[0104] Optionally, said backscattered light measurements are made at the same locations where the intensity of the fluorescence of the test meal is periodically detected, i.e. at said locations on the skin of said body part.

[0105] Optionally, the measurement of the backscattered light is made at a different location from the location where the intensity of the fluorescence of the test meal is regularly detected, for example at another location close to but distant from the location on the skin of the body part.

[0106] Optionally, said different locations are locations on the subject's skin.

[0107] Advantageously, by recording the intensity of the backscattered excitation signal at a location on the skin rather than at the light source itself and normalizing with respect to excitation power, variations due to skin tone, skin thickness, light source movement, etc. can be corrected for.

[0108] Optionally, a fluorescent contrast agent is dispersed within the test meal.

[0109] Optionally, the test meal comprises a solution.

[0110] Optionally, the test meal comprises a liquid test meal or a solid test meal.

[0111] Optionally, the test meal includes a milkshake or scrambled eggs on toast.

[0112] Optionally, the imaging agent comprises a dye, for example, fluorescein, methylene blue, fluorescein isothiocyanate-conjugated dextran, salts thereof, or combinations thereof.

[0113] Advantageously, fluorescein is a clinically approved dye that is rapidly taken up by the small intestine after emptying from the stomach, and therefore the rate of uptake of fluorescein into the bloodstream can be used to assess the rate of gastric emptying.

[0114] Advantageously, methylene blue is a clinically approved dye that is rapidly taken up into the small intestine after emptying from the stomach, and therefore the rate of uptake of methylene blue into the bloodstream can be used to assess the rate of gastric emptying.

[0115] Optionally, the test meal includes a dose of about 100-500 mg of contrast agent.

[0116] Advantageously, such a dose of contrast agent can ensure a good signal-to-noise ratio in the fluorescence data while remaining within clinically acceptable levels.

[0117] Optionally, the test meal includes a dose of at least 25 mg of fluorescein as a fluorescent contrast agent.

[0118] Optionally, the test meal includes a dose of about 100 mg of contrast agent.

[0119] Optionally, the test meal contains a 100 mg dose of fluorescein or methylene blue as a fluorescent contrast agent.

[0120] Optionally, the test meal contains fluorescein as a fluorescent contrast agent at a dose of about 100-500 mg in about 100 ml of water or juice.

[0121] Optionally, the test meal contains a dose of contrast agent between 25-500 mg.

[0122] Optionally, the test meal contains fluorescein as a contrast agent at a dose of up to 500 mg.

[0123] Optionally, the test meal contains methylene blue as a contrast agent at a dose of up to 100 mg.

[0124] The dose of fluorescent contrast agent in the test meal is selected to find the optimum balance between minimizing dose and maximizing signal-to-noise ratio.

[0125] Optionally, the transdermal sensing device has an acquisition time, the light source has an excitation power, and the fluorescence data is normalized based on said acquisition time and said excitation power.

[0126] Advantageously, by normalizing the fluorescence intensity values ​​for each time point by the acquisition time and excitation power used in each case, all time points can be compared to each other. Advantageously, this allows for quantitative measurement of gastric emptying rate and quantitative comparison of measurements made on different days or times, or in different subjects. Furthermore, this allows for meaningful quantification of variability in gastric emptying rate (e.g., variability between different patients or change over time in a single patient), rather than simply providing a binary assessment of whether the contrast agent remains in the stomach.

[0127] Optionally, the transcutaneous sensing device has an acquisition time of between 100 milliseconds and 15 seconds.

[0128] Optionally, the transcutaneous sensing device has an acquisition time of at least 100 milliseconds.

[0129] Optionally, the transdermal sensing device is configured such that the acquisition time for each measurement can be determined in an automated manner by the transdermal sensing device itself. That is, optionally, the transdermal sensing device is configured to determine the optimal acquisition time for each time point based on the current level of the fluorescence and / or backscatter excitation signal. Optionally, the intensity fluorescence data as a function of time may be normalized by the acquisition time used to collect both the fluorescence data and the backscatter excitation data.

[0130] Advantageously, this ensures that good signal levels are obtained at all time points, even when the fluorescence level is low (e.g., at the beginning or end of the measurement / detection process). This also ensures that there are no issues related to detector saturation when the fluorescence signal is at its maximum. Advantageously, this allows measurements to be performed reliably across patients with different skin tones without requiring high doses of fluorescent contrast agent in darker-skinned subjects. Instead of requiring higher doses or higher excitation powers, which may be clinically unacceptable, the transdermal sensing device can obtain adequate signal levels by automatically acquiring data with longer integration times.

[0131] Optionally, the transdermal sensing device is configured to detect the intensity of said fluorescence using fluorescence spectroscopy.

[0132] Optionally, the transdermal sensing device begins periodic measurements before the test meal is administered to the subject, such that periodic detection of the fluorescence intensity of the fluorescent contrast agent in the test meal begins before the test meal is administered to the subject, in order to obtain a background signal to be used in the step of normalizing the fluorescence data.

[0133] Optionally, background signal is subtracted from the intensity fluorescence data as a function of time for each time point (ie for each periodic measurement detected / obtained).

[0134] Optionally, the fluorescence data is normalized based on the backscatter intensity of the optical radiation used to excite the fluorescence.

[0135] Advantageously, such normalization of the fluorescence data makes the fluorescence data comparable across different measurements and across different subjects using only a single fluorescent imaging agent.

[0136] Optionally, the light source comprises a light emitting diode or a laser.

[0137] Optionally, the light source is configured to be worn on and / or around said body part of the subject.

[0138] Optionally, the transcutaneous sensing device includes one or more photodiodes, phototransistors, and / or fiber optic probes and is configured to be worn on and / or around said body part of the subject.

[0139] Optionally, in the step of periodically detecting said fluorescence intensity using a transdermal sensing device, measurements are recorded by said transdermal sensing device at least once every five minutes.

[0140] Optionally, in the step of periodically detecting said fluorescence intensity using a transdermal sensing device, measurements are recorded by said transdermal sensing device at least once every two minutes.

[0141] Optionally, in the step of periodically detecting said fluorescence intensity using a transdermal sensing device, measurements are recorded by said transdermal sensing device at least once per minute.

[0142] Advantageously, the frequency of the measurements is selected to ensure that the collected data has sufficient time resolution. This can be ensured by recording measurements at least once every 5 minutes. This helps to accurately identify the first peak value of the intensity, which represents the peak concentration of the fluorescent contrast agent in the subject's bloodstream.

[0143] Furthermore, if measurements are taken at a sufficiently high frequency, for example once every 5 minutes or once every minute, the data obtained can more specifically indicate the location in the intestine where the leak is occurring, compared to less frequent, regular measurements.

[0144] Optionally, in the step of periodically detecting said fluorescence intensity using a transdermal sensing device, measurements are recorded by said transdermal sensing device for at least 30 minutes.

[0145] Optionally, in the step of periodically detecting said fluorescence intensity using a transdermal sensing device, measurements are recorded by said transdermal sensing device for at least 60 minutes.

[0146] Optionally, in the step of periodically detecting said fluorescence intensity using a transdermal sensing device, measurements are recorded by said transdermal sensing device for at least four hours.

[0147] Optionally, said body part is a finger, wrist, arm, or earlobe.

[0148] Advantageously, when measurements are taken using the transcutaneous sensing device at skin locations on the finger, wrist, arm, or earlobe, clear fluorescence of the fluorescent contrast agent is observed even with short acquisition times.

[0149] Optionally, said body part is a fingertip.

[0150] Advantageously, performing measurements using the transcutaneous sensing device at the fingertip position allows for clear fluorescence of the fluorescent contrast agent to be observed even with short acquisition times, and the intensity of the fluorescence data can be enhanced due to the proximity of blood vessels to the surface of the fingertip skin. Measuring at the fingertip also minimizes the effect of dye leakage from blood vessels.

[0151] Optionally, analyzing the normalized data comprises fitting the following function to the normalized data using a numerical fitting procedure such as least squares:

[0152]

number

[0153] During the ceremony, t represents time, I(t) represents the normalized fluorescence intensity data as a function of time; B maxrepresents the maximum intensity contribution from the fluorescent contrast agent in the subject's bloodstream as a function of time, L max represents the maximum intensity contribution from the fluorescent contrast agent leaked into the epithelium of the subject's skin as a function of time; t B1 / 2 represents the time point at which the intensity contribution from a fluorescent contrast agent in the subject's bloodstream reaches half of its maximum value as a function of time; t L1 / 2 represents the time point at which the intensity contribution from the fluorescent contrast agent leaked into the epithelium of the subject's skin reaches half of its maximum value as a function of time; C represents the rate at which the dye is eliminated from the subject's body, k B is a constant representing the logistic growth rate of the intensity contribution from the fluorescent contrast agent in the subject's bloodstream as a function of time; k L is a constant that represents the logistic growth rate of the intensity contribution from the fluorescent contrast agent leaked into the epithelium of the subject's skin as a function of time.

[0154] Optionally, analyzing the normalized data further comprises calculating the amount of dye emptied from the subject's stomach as a function of time, S(t); where S(t)=B(t)+B(t)Ct; where B(t) represents the intensity contribution from the fluorescent contrast agent in the bloodstream of the subject as a function of time.

[0155] Optionally, analyzing the normalized data comprises quantifying the percentage of the test meal remaining in the subject's stomach as a function of time, R pc The method further includes the step of calculating:

[0156]

number

[0157] In the formula, S(t peak ) represents the value of S(t) at the time when the final peak is observed in the normalized data of the intensity as a function of time.

[0158] Optionally, analyzing the normalized data alternatively comprises calculating the percentage of the test meal remaining in the subject's stomach as a function of time R pc The method includes a step of calculating as follows:

[0159]

number

[0160] where B(t) represents the intensity contribution from the fluorescent contrast agent in the subject's bloodstream as a function of time.

[0161] Optionally, analyzing the normalized data comprises quantifying the percentage of the test meal remaining in the subject's stomach as a function of time, R pc The method includes the step of calculating as follows:

[0162]

number

[0163] where I(t) represents the normalized data of the intensity as a function of time, and t peak1 represents the time at which the first peak is observed in the intensity as a function of time.

[0164] Optionally, either of the above methods according to the first aspect of the present disclosure or the second aspect of the present disclosure is performed on the fluorescence intensity data without first normalizing the fluorescence data. In other words, optionally, in the method of measuring intestinal permeability of a subject according to the first aspect of the present disclosure, the step of normalizing the fluorescence data to obtain normalized data of the intensity as a function of time may be omitted. Similarly, optionally, in the method of measuring gastric emptying rate according to the second aspect of the present disclosure, the step of normalizing the fluorescence data to obtain normalized data of the intensity as a function of time may be omitted. In either case, the data of the intensity as a function of time may be analyzed as described above without being normalized before the analysis is performed. [Brief explanation of the drawings]

[0165] The present disclosure may be carried out in various ways and embodiments of the disclosure will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0166] [Figure 1] 1 is a flow chart illustrating a method for measuring intestinal permeability in a subject. [Figure 2] FIG. 1 shows the chemical structure and molecular weight (MW) of sodium fluorescein. [Figure 3] FIG. 1 shows exemplary data that are plots of fluorescence intensity versus wavelength for solutions containing different doses of fluorescein obtained using a method for measuring intestinal permeability in a subject. [Figure 4A] FIG. 1 shows a wearable probe attached to a subject's fingertip. [Figure 4B] FIG. 1 shows a wearable probe attached to a subject's forearm. [Figure 5A] FIG. 4B shows a base for positioning the wearable probe of FIG. 4A in contact with a fingertip. [Figure 5B] FIG. 4C shows a base for positioning the wearable probe of FIG. 4B in contact with the forearm. [Figure 6]FIG. 10 shows an exemplary plot of fluorescence intensity data plotted against wavelength for test measurements taken on the fingers, arms, and wrists of test subjects injected with fluorescein. [Figure 7] FIG. 1 shows confocal endoscopic images of the distribution of fluorescein measured at locations on a subject's arm, wrist, and fingers after oral ingestion of fluorescein. [Figure 8] Schematic showing the portable dual-channel fiber optic fluorescence spectrometer optical system. [Figure 9] FIG. 9 shows the optical system of FIG. 8 mounted on a wheeled dolly. [Figure 10] Figure 1 shows a plot of normalized fluorescence intensity as a function of time. [Figure 11] FIG. 1 shows two plots of normalized fluorescence intensity as a function of time recorded in the same subject on different days. [Figure 12] 1 is a flow chart illustrating a method for measuring gastric emptying rate in a subject. [Figure 13] The plots in Figure 10 are supplemented with exemplary fitting curves applied to normalized fluorescence intensity data. [Figure 14A] FIG. 1 shows an exemplary plot of normalized fluorescence intensity as a function of time along with an exemplary fitting curve, with a subplot showing the residual difference between the fluorescence data and the fitting curve. [Figure 14B] FIG. 1 shows an exemplary plot of normalized fluorescence intensity as a function of time along with an exemplary fitting curve, with a subplot showing the residual difference between the fluorescence data and the fitting curve. [Figure 14C] FIG. 1 shows an exemplary plot of normalized fluorescence intensity as a function of time along with an exemplary fitting curve, with a subplot showing the residual difference between the fluorescence data and the fitting curve. [Figure 14D]FIG. 1 shows an exemplary plot of normalized fluorescence intensity as a function of time along with an exemplary fitting curve, with a subplot showing the residual difference between the fluorescence data and the fitting curve. [Figure 15A] Figure 1 shows plots of normalized fluorescence intensity and paracetamol concentration as a function of time for data obtained using fluorescein- and paracetamol-based measurement methods, respectively. [Figure 15B] Plot of retention as a function of time based on the data shown in Figure 15A. DETAILED DESCRIPTION OF THE INVENTION

[0167] With reference to FIG. 1, the steps of a method for measuring intestinal permeability in a subject and an exemplary procedure for carrying out said method are described below.

[0168] In a first step 1, a solution containing a fluorescent contrast agent that is absorbed by a healthy intestine is orally administered to a subject 13 (i.e., a patient). That is, the fluorescent contrast agent is delivered in a solution (e.g., water or juice) that the subject drinks. The chemical structure and molecular weight of an exemplary suitable contrast agent, sodium fluorescein (which is a dye that is absorbed by a healthy intestine), are shown in FIG. 2. However, it is contemplated that any other suitable fluorescent contrast agent may be used, such as, for example, fluorescein, methylene blue, fluorescein isothiocyanate-conjugated dextran, salts thereof, or combinations thereof.

[0169] In the example described below, the fluorescent contrast agent is fluorescein, delivered as a solution in 100 ml of water at a dose of 100-500 mg. However, the solution may contain other liquids, such as orange juice or other juices, instead of water, and it is contemplated that other doses of any other suitable fluorescent contrast agent and other amounts of solution may be used. For example, a dose of 100 mg of methylene blue in 100 ml of orange juice may be used.

[0170] Figure 3 shows exemplary data obtained using the method depicted in Figure 1 (subsequent steps are outlined below), showing plots of fluorescence intensity (y-axis) versus wavelength (x-axis) for a solution containing 5 mg of fluorescein as a fluorescent contrast agent (spectrum recorded 29 minutes after ingestion) and a solution containing 25 mg of fluorescein as a fluorescent contrast agent (spectrum recorded 37 minutes after ingestion). The plots show that the fluorescence from 5 mg of fluorescein was barely detectable compared to the background spectrum, while the fluorescence from 25 mg of fluorescein was clearly observable above the background spectrum. These results indicate that the detection limit for fluorescein using the exemplary hardware described herein is less than 25 mg. Preferably, a dose of 500 mg of fluorescein can be used to find the optimal balance between minimizing dose and maximizing signal-to-noise ratio.

[0171] Referring again to FIG. 1, in a second step 2, a light source is used to illuminate a location on the skin of the subject's body part with optical radiation such that the optical radiation causes at least a portion of the solution that has leaked from the subject's intestine and entered the subject's bloodstream to fluoresce.

[0172] Still referring to FIG. 1 , in a third step 3, a transdermal sensing device is used to periodically detect the fluorescence intensity of the solution at the location to obtain fluorescence data of intensity as a function of time. In the illustrated example, the fluorescence data is spectral data. However, it is contemplated that the fluorescence data may be in other formats (i.e., the fluorescence data need not necessarily be a spectrum for each time point (i.e., for each periodic measurement taken)). For example, the fluorescence data could instead include a single intensity measurement for each time point (i.e., for each periodic measurement taken).

[0173] The light source and transcutaneous sensing device are part of a wearable probe 6. As shown in Figures 4A and 4B, respectively, the wearable probe 6 is worn on the fingertip 14 or forearm 15 of the subject 13. It is also envisioned that the wearable probe 6 may be worn on or around other body parts, such as the finger, wrist, arm, or earlobe. Because the thickness of the subject's 13's skin can affect the signal and the behavior of the fluorescent signal over time can depend on the body position, the body part should be selected to optimize the signal acquired by the transcutaneous sensing device. This may be due to the transfer of fluorescent contrast agent from blood vessels to the skin epithelium. Since we are interested in the concentration of fluorescent contrast agent in the bloodstream, we need to select a body position that minimizes the effects of leakage into the epithelium. Because the blood vessels are close to the surface of the skin at the fingertip and earlobe, the detected fluorescent signal is less affected by residual dye that has leaked into the epithelium. If the wearable probe 6 is attached to a body part other than the fingertip or earlobe (or if any leakage effects are observed even when the probe is attached to the fingertip or earlobe), correction of the data analysis to account for epithelial leakage is required.

[0174] To illustrate why some body parts are more suitable measurement locations than others, Figure 6 shows an exemplary plot of fluorescence intensity data plotted against wavelength for test measurements made on the finger, arm, and wrist of a test subject injected with fluorescein. Background fluorescence is also plotted. Figure 6 shows that the fluorescence intensity is greater in the finger than in the arm or wrist, likely due to the proximity of blood vessels to the surface of the finger skin, and therefore a stronger, more easily observable signal is obtained in the finger than in the arm or wrist.

[0175] Further data demonstrating that the finger is a preferred location over the arm or wrist is shown in Figure 7. Figure 7 shows exemplary confocal fluorescence endoscopic images of fluorescein distribution measured at the arm, wrist, and finger locations of a test subject after oral ingestion of fluorescein. Labels indicate the time points in minutes after ingestion for the various confocal fluorescence endoscopic images. All images show a 240 μm diameter field of view. As shown in Figure 7, compared to the background signal, only a diffuse fluorescence signal was detected when images were recorded at the arm and wrist, and no discernible structures were observed. However, on the finger, clear structures with fluorescence that appeared to emanate from cellular structures and / or blood vessels were observed. This indicates that the finger is the optimal location for placing probe 6.

[0176] The wearable probe is configured to be placed in contact with a body part by mounts 7, 8. Exemplary mounts 7, 8 are shown in FIGS. 5A and 5B, respectively, and may be fabricated, for example, by additive manufacturing. Each of the exemplary mounts 7, 8 includes a cylindrical portion 9 for receiving the end of the wearable probe 6, a substantially flat portion 10 positioned adjacent to the associated body part, and adjustment means 11 for securing the wearable probe 6 inside the cylindrical portion 9. The shape and / or dimensions of the mounts 7, 8 are selected depending on the size and shape of the wearable probe 6 and the body part to which the wearable probe 6 is to be attached. As shown in FIGS. 4A and 4B, the mounts 7, 8 and the wearable probe 6 can be secured to the associated body part (fingertip 14, forearm 15) of the subject 13 using securing means 12, such as tape, straps, and / or hook-and-loop fasteners. Alternatively, it is envisioned that the probe 6 may be manually held in contact with the subject's 13's skin to perform transcutaneous fluorescence measurements.

[0177] The light source, which may be, for example, a laser or an LED, is selected in one example described herein to be a laser having a center wavelength in the range of 450-490 nm. The transdermal sensing device is configured to detect both the backscattered excitation signal (i.e., the excitation power of the light source) and the fluorescent signal from the fluorescent contrast agent in solution in the bloodstream of the subject 13, which can be accomplished, for example, by using two detectors with appropriate optical filters (more suitable for small LED / photodiode-based sensors) or by using a single detector and a rotating filter wheel (more suitable for use with laser or fiber optic-based sensors, as described in the examples herein).

[0178] The transdermal sensing device is designed to allow measurement of the excitation signal and the fluorescence signal at the same location on the subject 13 (e.g., by placing multiple detectors in close proximity to each other or by using the same detector in combination with a rotating filter wheel). That is, it is envisioned that the transdermal sensing device may be configured to allow measurement of the excitation signal and the fluorescence signal at the same location on the subject 13, or alternatively at different locations on the subject 13, where the different locations are in close proximity to each other.

[0179] By measuring the excitation power at the same (or very similar / nearby) location as the fluorescence signal, the fluorescence data can be normalized to correct for factors including fluctuations in excitation intensity, wearable probe movement, skin tone, skin absorption characteristics, and / or skin scattering characteristics. This allows the detected fluorescence signal (corrected for excitation power) to be quantitatively analyzed as a function of time. This is particularly important because this method uses a single (i.e., only one) fluorescent contrast agent (fluorescein in the example described herein) and does not allow for the calculation of a ratio of detected intensities for two or more dyes (which is inherently true for intensity fluctuations and other issues highlighted above). Therefore, by properly normalizing the fluorescence signal and recording the data as a function of time, quantitative assessments of the fluorescence intensity and the time it takes for the fluorescent contrast agent to enter the subject's bloodstream can be made (two of which are of interest when calculating intestinal permeability and / or gastric emptying rate).

[0180] The excitation power (i.e., backscattered excitation signal) can be measured by passing the light through an appropriate optical filter (i.e., a 500 nm short-pass filter or a 10-20 nm band-pass filter centered at the wavelength of the excitation light source), which allows measurement of only the excitation light by filtering out the fluorescence signal. Alternatively, the excitation power can be simply measured by detecting the unfiltered light. Because the excitation power is orders of magnitude stronger than the fluorescence, measurements of the unfiltered signal can serve as an appropriate approximation of the excitation light intensity. Subsequently, the fluorescence signal can be measured by passing the light through a 500 nm long-pass filter. This cuts out the excitation signal, allowing for highly sensitive detection of fluorescein fluorescence.

[0181] A schematic diagram of an exemplary portable dual-channel fiber optic fluorescence spectroscopy optical system 22 suitable for use in the second step 2 and third step 3 of the method is shown in FIG. 8. The inset in FIG. 8 shows the distal and proximal fiber optic arrangements in a bifurcated fiber-based wearable probe 6, with the excitation fiber 16 shown in blue and the collection fiber 17 shown in yellow. "ND" in FIG. 8 stands for "neutral density." The exemplary spectrometer includes two laser sources 18a, 18b (488 nm and 785 nm, respectively) for exciting fluorescence, a commercially available spectrometer 19 for reliably detecting the fluorescence signal, optical excitation / emission filters 20, an automated filter wheel (including an emission filter), and a bifurcated fiber optic wearable probe 6 for delivering light to and collecting light from the skin of the subject 13.

[0182] In this example, 488 nm and 785 nm laser sources were selected to excite fluorescence from fluorescein (or fluorescein isothiocyanate) and ICG (indocyanine green), respectively, for experimental purposes (and from other dyes with comparable spectral properties). It is contemplated that laser sources (and optical filters) may alternatively be selected to have other wavelengths as needed.

[0183] The optical system 22 is housed in a light-tight anodized aluminum box 21 for laser safety and is controlled by a laptop computer running LabVIEW software. As shown in Figure 9, the entire optical system 22 can be mounted on a wheeled dolly 23 to enable use within a clinic.

[0184] For each individual measurement, the transdermal sensing device can be programmed to integrate until an appropriate signal level is obtained. Typical acquisition times range from 100 milliseconds to 15 seconds. Programming the transdermal sensing device in this manner ensures that a good signal level is obtained at all time points (i.e., for all periodic measurements), even when the fluorescence level is low (e.g., at the beginning or end of the measurement process). It also ensures that problems related to detector saturation do not occur when the fluorescence signal is at its maximum. This allows for reliable measurements across subjects / patients with different skin tones without requiring high doses for darker-skinned subjects. Instead of requiring higher doses or higher excitation powers (which may be clinically unacceptable), the transdermal sensing device can automatically acquire data over a longer integration time to obtain an appropriate signal level.

[0185] Referring again to FIG. 1, in a fourth step 4, the fluorescence data is normalized to obtain normalized data of the intensity as a function of time.

[0186] In this example, the fluorescence intensity values ​​for each time point (i.e., each periodic measurement) are normalized by the acquisition time and excitation power used in each case, allowing all time points to be compared with each other. This allows for quantitative comparison of measurements from different days / time points or different participants. Furthermore, this means that the fluorescence values ​​can be used to provide meaningful quantification of permeability variability (between different subjects / patients or over time in a single subject / patient) rather than simply providing a binary assessment of whether the contrast agent crossed the intestinal barrier.

[0187] In the examples described herein, this normalization is achieved by first subtracting the background signal from the fluorescence signal at each time point. For spectrally resolved data (e.g., data collected using the fiber-optic fluorescence spectroscopy optical system 22 shown in Figure 8), the background is calculated as the average intensity over the wavelength range of 350-450 nm (the range where no signal is observed). The background-subtracted spectra are then summed over the wavelength range containing the spectral peak of the fluorescence signal (500-580 nm for fluorescein). This integrated fluorescence value is then normalized by both the integration time and the laser power (by dividing by the product of the two). The excitation (laser) power value is also calculated based on the excitation (laser) spectrum (recorded immediately before / after each fluorescence spectrum). To obtain the laser power value, the laser spectra are summed over the range of 485-492 nm after background subtraction as described above. These summed values ​​are then normalized by the integration time (typically 1 ms) used to collect the laser spectra. Therefore, the normalized integrated fluorescence intensity value for fluorescein can be calculated according to the following equation:

[0188]

number

[0189] In the formula, I norm is the integrated normalized fluorescence intensity, λ is the wavelength, and t F and t L are the integration times for the fluorescence spectrum and the laser spectrum, respectively, I(λ) is the fluorescence spectrum, L(λ) is the laser spectrum, and B F and B L represent the background values ​​for the fluorescence spectrum and the laser spectrum, respectively.

[0190] When LED- and photodiode-based wearable probes are used (where the collected data is not spectrally resolved), background measurements can be made by recording measurements with the fluorescence detector with the excitation light (i.e., light source) switched off. Additionally, the excitation power can be measured using a second photodetector. This allows background subtraction by recording the signal level with the excitation LED turned off. Normalized fluorescence intensity can be calculated as in the previous equation above, but cannot be summed (because the wavelength ranges are defined by the optical filters placed in front of the photodetector and the data are effectively summed across those ranges as they are collected). Therefore, the normalized intensity can be shown as follows:

[0191]

number

[0192] In the formula, I F and I E represent the fluorescence and excitation intensity values, respectively, and B F and B E are the background values ​​of the fluorescence and excitation photodetectors, respectively, and t F and t E represent the integration times used to measure the fluorescence and excitation light, respectively. Note that if different integration times are used to record the fluorescence / excitation and background levels, this must be incorporated into the above equation. That is, B E and B F must be normalized by the respective acquisition times, in which case the previous equation above becomes:

[0193]

number

[0194] In the formula, t BF and t BErepresent the integration times used to record background measurements for the fluorescence detector and the excitation photodetector, respectively.

[0195] The normalization procedure described above can be performed in the optical system 22 of a fiber optic fluorescence spectrometer, meaning that the output data can be immediately used for diagnostic purposes without further analysis.

[0196] Following collection and normalization of the fluorescence data, a plot of normalized fluorescence intensity as a function of time (I(t)) can be provided. A typical example of such data is shown in Figure 10. With the normalization procedure described above, such plots can be used to quantify intestinal permeability in a variety of ways.

[0197] That is, referring again to FIG. 1 , in a fifth step 5, the normalized data is analyzed to measure the subject's intestinal permeability by calculating one or more of the following: (a) the first peak value of the intensity; (b) the integral of the intensity over time; (c) the product of the first peak value of the intensity and the time at the peak value; (d) the product of the first peak value of the intensity and the time past the time at the first peak value; (e) the time at which the first peak value of the intensity occurs; and (f) the first peak value of the intensity divided by the time at which the peak value occurs. While in the examples described herein, the analysis is performed on normalized data, it is contemplated that the analysis could alternatively be performed on non-normalized data if the reduced accuracy and reliability of the analysis is acceptable. Such analysis provides useful diagnostic information. Specifically, this includes: norm This is achieved by modifying the previous equation above for

[0198]

number

[0199] and

[0200]

number

[0201] In the formula, I int and represent the integrated, non-normalized fluorescence intensity data at each time point for the spectral data (first equation) and the non-spectral data (second equation), respectively. In the following description, the first I above will be used. norm All further analysis will be performed on the normalized data by the equation above, except that the normalized data will be used. int The same analysis can be performed on non-normalized data by starting with either of the two equations for

[0202] Continuing with the analysis of the normalized data, by way of example, the intensity of the first peak 24 of the fluorescence versus time curve 25 (see FIG. 10) can be used to read the peak concentration of the fluorescent contrast agent in solution in the bloodstream of the subject 13. The first peak 24 represents the time point at which there is a maximum concentration of the fluorescent contrast agent in the bloodstream, and typically occurs before significant elimination from the body of the subject 13 (e.g., via the kidneys or liver) occurs.

[0203] In some cases, the fluorescent signal continues to increase after the initial peak 24 due to leakage of the fluorescent contrast agent from the bloodstream into the epidermis of the skin at the measurement location. Therefore, to accurately identify the initial peak 24 (representing the peak concentration of the fluorescent contrast agent in the subject's 13 bloodstream), it is necessary to collect data with sufficient temporal resolution. By recording measurements once per minute, we can comfortably confirm that this is the case. Conversely, if the measurement interval exceeds 5 minutes, there is a risk of losing the resolution necessary to observe the initial peak 24.

[0204] Alternatively, intestinal permeability can be quantified by calculating the area under the curve 25 up to the first peak 24, which is determined using an integration method to account for the effect of gastric emptying rate (which alters the time and intensity of the first peak 24). This provides a measure of the total amount of fluorescent contrast agent that has leaked from the intestine into the bloodstream by the time of the first peak 24. This approach is similar to the measurement of total lactulose recovery used in many glucose-based urine permeation assays. However, it does not suffer from the problem of not knowing the exact time urine should be collected, and by identifying the time of peak concentration from the data, it is possible to accurately quantify the total recovery of fluorescein (or other fluorescent contrast agent) in the bloodstream.

[0205] Alternatively, intestinal permeability can be quantified by calculating the product of the peak intensity and the time at which peak 24 occurs (or, as explained below, by calculating the product of the peak intensity and the time past the time of the first peak), which is a simplification of the area under the curve approach described above.

[0206] Therefore, intestinal permeability can be quantified based on normalized fluorescence data using any of the following three equations:

[0207]

number

[0208]

number

[0209]

number

[0210] In the formula, I is (I above norm where τ represents the normalized fluorescence intensity (as calculated using the equation for τ), t represents time, and t peakrepresents the time of the first peak in the fluorescence data, and GP 1-3 represent the three proposed quantifiers for intestinal permeability (as an aside, if reduced analytical reliability is acceptable, it is possible to use non-normalized fluorescence intensity data, i.e., the I int (It is also worth noting that the equations for GP2 and GP3 may be used instead to express the calculated values.) Alternatively, the equations for GP2 and GP3 may be modified as follows:

[0211]

number

[0212]

number

[0213] In these modified equations for GP2 and GP3, t peak+ represents a selected time point after the first peak in the fluorescence data, e.g., a selected time point equal to the sum of the time value at which the first peak occurred in the fluorescence data and a selected percentage of the time value.

[0214] In addition to the quantifiers shown in the three equations above, the time of the first peak (t peak ) also, t peak The lower the value, the higher the permeability.

[0215] Furthermore, I(t peak ) to t peak Dividing by gives us another quantifier that varies with transparency (in this case, increasing with increasing transparency). We can therefore define two more quantifiers for transparency, as explained below:

[0216]

number

[0217]

number

[0218] GP4 and GP5 are confounded by the effect of gastric emptying rate, as higher emptying rates may also lead to changes in GP4 and GP5 values. Conversely, GP2 and GP3 are peak Multiply by or t peak Calculating the area under the curve from 0.01 to 0.1 can provide some correction for gastric emptying rate, thus providing a more quantitative assessment of permeability.

[0219] Overall, it is possible to quantify permeability based on normalized fluorescence intensity. This allows for quantification of leakage of fluorescent contrast agents across even healthy intestinal barriers, meaning that meaningful clinical data can be provided for a wide range of subjects / patients, not just those with highly permeable intestines. Furthermore, the above quantifiers provide a continuously varying measure of permeability, rather than simply a binary marker that provides only a rough clinical outcome of whether the intestine is "permeable" or "impermeable." This allows for monitoring patient responses to treatments and other interventions (e.g., nutritional interventions) and also allows for comparison of values ​​measured in different individuals (e.g., for screening / diagnostic applications). This is made possible by the above normalization procedure and the collection of data with sufficient temporal resolution to allow identification of the first peak in the fluorescence vs. time curve (i.e., the point of peak blood concentration).

[0220] Finally, regarding the method for measuring intestinal permeability, Figure 11 compares data obtained with and without the use of a hyperosmolar solution. The hyperosmolar solution used was a representative hyperosmolar solution containing 60 g of sugar. The plot shows normalized fluorescence intensity plotted against elapsed time in minutes, along with the results of an experiment in which participants underwent two intestinal permeability tests on separate days. In the first case, the consumed solution consisted of 500 mg of fluorescein dissolved in 100 ml of water. In the second experiment, the solution consisted of 500 mg of fluorescein and 60 g of sugar dissolved in 100 ml of water. Concentrated sugar solutions are known to have a hyperosmolar effect. That is, concentrated sugar solutions temporarily increase intestinal permeability. Therefore, the exemplary experimental data shown in Figure 11 demonstrate that changes in intestinal permeability can be assessed using a single fluorescent contrast agent (e.g., fluorescein) as described above. As shown in Figure 11, when 60 g of sugar was added to the solution, the peak value (GP1) was slightly higher compared to the experiment using a sugar-free solution. Furthermore, the area under the curve (GP2) and I(t) × t peak A significant increase in the values ​​of (GP1-GP3) was observed. Thus, the exemplary data shown in Figure 11 provides preliminary validation of the proposed permeability markers GP1-GP3 above.

[0221] Some aspects of the above-described methods and procedures for measuring intestinal permeability may also be used in methods for measuring gastric emptying rate. With reference to Figure 12, the steps of a method for measuring a subject's gastric emptying rate (i.e., how quickly the stomach empties) and an exemplary procedure for carrying out said method are described below.

[0222] Steps 101 to 104 of the method shown in FIG. 12 are similar to steps 1 to 4, respectively, of the method shown in FIG. 1 and therefore may be performed in a substantially similar manner and using substantially similar hardware as steps 1 to 4 above.

[0223] Step 101 of the method shown in Figure 12 differs from step 1 of the method shown in Figure 1 and the methods described above in that the fluorescent contrast agent is prepared as part of a liquid or solid test meal (rather than in water or juice) to enable measurement of gastric emptying rate in realistic and physiologically interesting situations. This is because liquid or solid test meals empty more slowly from the stomach than pure water or juice. An exemplary suitable liquid test meal is a milkshake, and an exemplary suitable solid test meal is scrambled eggs on toast. However, it is envisioned that any other suitable liquid or solid test meal may be employed as the test meal.

[0224] Advantageously, a milkshake is a liquid-based test meal that empties from the stomach in a manner similar to solid food (where it is physiologically important) and is designed to be comfortable / enjoyable for the subject 13 to consume. 500 mg of fluorescein is the clinically approved dose of fluorescein for intravenous injection and provides optimal signal levels (as it is the highest clinically tolerated dose), so it can be included and used in the exemplary milkshake formulation. However, lower doses (up to approximately 100 mg) can also be employed while maintaining an acceptable signal-to-noise ratio. This can reduce the dose of contrast agent and, therefore, the effects and risks posed to the subject 13. The exemplary milkshake may also include chocolate powder, protein powder, sugar, and / or milk.

[0225] Prior to ingesting the test meal, subject 13 is asked to fast for 12 hours. That is, subject 13 is asked not to consume any food or drink (except water) before the method for measuring gastric emptying rate is performed. This can be achieved by performing the measurement in the morning and asking subject 13 to fast overnight before the measurement. This ensures that the gastric emptying rate measurement is not adversely affected by food or drink remaining in subject 13's stomach or intestines.

[0226] At the start of the measurement process, the transdermal sensing device is attached to the skin of the subject 13 at a selected location (e.g., a fingertip). Fluorescence measurements are then initiated prior to ingestion of the contrast agent to allow for measurement of a baseline / background signal. In the example described herein, the fluorescence signal and excitation power are measured using the transdermal sensing device once per minute. While it is contemplated that alternative timings (i.e., measurement frequencies) can be used, measuring once per minute advantageously allows data to be collected with a good signal-to-noise ratio and sufficient temporal resolution.

[0227] After the first measurement is taken, the subject / patient is asked to drink the milkshake as quickly as possible (e.g., within the next 3 minutes). A 3-minute consumption time helps ensure that the entire test meal (milkshake) reaches the stomach quickly, but also allows the subject / patient to comfortably consume the drink. Fluorescence (and excitation power) measurements are performed once per minute over the next 4 hours. This timescale ensures that in the majority of subjects, all of the fluorescent contrast agent has been emptied from the stomach and that at least 1 hour of data is collected during which the fluorescent contrast agent is no longer entering the bloodstream as a result of gastric emptying (i.e., the fluorescent contrast agent is simply removed from the bloodstream). This is referred to as steady-state clearance. Measuring the steady-state clearance rate allows the fluorescence data to be accurately converted into a measure of the proportion of stomach contents emptied. However, it is also envisioned that different measurement timescales may be used. For example, fluorescence (and excitation power) measurements may be performed over the next 30 minutes rather than the next 4 hours. The measurement timescale may be selected based on the calculation used. Advantageously, the short measurement timescale allows results to be reported more quickly, thereby reducing the impact on the subject 13.

[0228] For each individual measurement, the transdermal sensing device is programmed to integrate until an adequate signal level is obtained. Typical acquisition times range from 100 ms to 5 s. Programming the transdermal sensing device in this way ensures that good signal levels are obtained at all time points, even when fluorescence levels are low (e.g., at the beginning or end of the measurement process). It also ensures that there are no problems related to detector saturation when the fluorescence signal is at its maximum. The fluorescence intensity values ​​for each time point are normalized by the acquisition time used in each case, allowing all time points to be compared with each other.

[0229] The resulting data consists of fluorescein fluorescence intensity (normalized by excitation power and acquisition time) as a function of time, which can be used to calculate gastric emptying rate in the fifth step 105 of the method, as described below.

[0230] The method of FIG. 12 differs from the method of FIG. 1 in that, in the fifth step 105, rather than analyzing the normalized data to measure the subject's intestinal permeability, the normalized data is instead analyzed to calculate the percentage of the test meal remaining in the subject's stomach as a function of time. This is accomplished by calculating a function of (i.e., based on) intensity as a function of time, divided by the peak intensity value. This calculation can be performed as outlined below. In the examples described herein, the analysis is performed on normalized data, but it is contemplated that the analysis could instead be performed on non-normalized data if a reduction in the precision and reliability of the analysis is acceptable. Such an analysis would still provide useful diagnostic information. In either case, the analysis described below should be preceded by the I analysis outlined in the description of measuring intestinal permeability. norm (for normalized data) or I int The intensity data is prepared according to the formula for (for non-normalized data).

[0231] A typical fluorescence versus time curve 25 has a shape as shown in the example of Figure 10. In the illustrated example, the fluorescence signal initially has an S-shaped, sigmoidal shape and increases as a function of time until it reaches a first peak 24 (as shown by the (red) dashed line starting at point (0,0)). Following the first peak 24, a more gradual increase (or possibly a plateau) is observed before a steady decrease 26 as the fluorescent contrast agent is cleared from the body (as shown by the second (yellow) dashed line).

[0232] The first increase primarily represents the uptake of the fluorescent contrast agent from the intestine into the subject's bloodstream. The second increase is likely due to leakage of the fluorescent contrast agent from the blood vessels into the epithelium (the observed fluorescence intensity increases as the contrast agent approaches the probe's sensing area). Because transcutaneous fluorescence spectroscopy does not provide measurements of the absolute concentration of the fluorescent contrast agent in the blood and the relative intensities of the fluorescent contrast agent in the blood and intraepithelium are unknown, it is difficult to directly and accurately calculate a retention curve (i.e., the percentage of contrast agent remaining in the stomach as a function of time) from the fluorescence data, as is typically done in paracetamol absorption tests and breath tests. Therefore, we present below a suitable exemplary approach based on least-squares fitting to enable the extraction of a retention curve from the fluorescence data.

[0233] The fluorescence versus time curve 25 can be represented by fitting two sigmoidal (logistic) functions representing the uptake of the fluorescent contrast agent into the bloodstream (shown by the (red) dashed line starting at point (0,0) in FIG. 10) and leakage from the blood vessels into the epithelium (shown by the second (yellow) dashed line in FIG. 10), respectively, with a linearly decreasing line representing the excretion of the fluorescent contrast agent from the body of the subject 13, as shown in the exemplary plot in FIG. 10. Thus, the fluorescence intensity I(t) as a function of time can be expressed as follows:

[0234]

number

[0235] where B(t) represents the intensity contribution of the fluorescent contrast agent in the bloodstream as a function of time, L(t) represents the intensity contribution of the fluorescent contrast agent that has leaked into the epithelium as a function of time, and E(t) represents the intensity contribution from the fluorescent contrast agent that has been cleared from the body (and therefore does not contribute to the total intensity) at time t. These functions can be defined as follows:

[0236]

number

[0237]

number

[0238]

number

[0239] B max represents the maximum intensity contribution from the fluorescent contrast agent in the bloodstream, and k B and t B1 / 2 represent the logistic growth (slope) of the B(t) curve and the time it takes for B(t) to reach half of its maximum value, respectively. Similarly, L max represents the maximum intensity contribution of the fluorescent contrast agent in the epithelium, and k L and t L1 / 2 represents the logistic growth of the L(t) curve and the half-maximum point for the L(t) curve. max and B maxdetermines the relative levels of fluorescence observed from the fluorescent contrast agent in the blood and the fluorescent contrast agent in the epithelium. This approach has the advantage that prior knowledge of these relative levels is not required because it involves fitting these parameters. C represents the clearance rate, i.e., the rate at which the fluorescent contrast agent is eliminated from the body. Thus, the intensity contribution from the total amount of fluorescent contrast agent eliminated by time t (E(t)) is defined as the product of time (t), the clearance rate (C), and the total intensity contribution from the fluorescent contrast agent in the system at that time (B(t) + L(t)).

[0240] By substitution, we can obtain the following equation for I(t):

[0241]

number

[0242] Parameters C and B max , L max , k B , k L , t B1 / 2 , and t L1 / 2 This equation can be fitted to the observed I(t) data, using, for example, least-squares fitting or another numerical fitting procedure, to extract . This can be accomplished using a variety of least-squares fitting algorithms (and using a variety of programming languages), for example, by using the "lsqcurvefit" function in MATLAB. In the example described herein, data is collected at approximately 1-minute intervals, so there are more data points than unknown values, but this means that an accurate fit can be performed. For the example data shown in FIG. 10, an example fitting curve 27 is shown in FIG. 13.

[0243] Further examples of other fluorescence versus time fitting curves are shown in Figure 14, including exemplary plots A, B, C, and D. The exemplary curves shown in Figure 14 were fitted according to the method described above. In each of Examples A, B, C, and D, a main plot (top) and subplots (bottom) are shown. In each of the main plots, the fluorescence versus time data plot is represented by multiple circular markers, and the solid line represents the corresponding fitting curve fitted according to the method described above. The circular markers in each of the corresponding subplots represent residuals. That is, each data point / circular marker in the subplot represents the difference between the actual data point (represented by the corresponding circular marker in the main plot) and the fitting curve (represented by the solid line in the main plot) at each data point / time value. As can be seen by examining each of the main plots with their corresponding subplots, e.g., for Figures 14A, B, C, and D, good fits were obtained in all four examples. This demonstrates the success of the fitting method described above.

[0244] Following the fitting procedure, the fitted parameters can be used to define the amount of fluorescent contrast agent S(t) emptied from the stomach at time t. Assuming that S(t) can be approximated as the sum of the intensity contribution from the fluorescent contrast agent in the blood vessels and the amount of fluorescent contrast agent emptied up to that point, S(t) can be shown as follows:

[0245]

number

[0246] In the formula, E B (t) represents the clearance of the fluorescent contrast agent from the blood vessel and is defined as follows:

[0247]

number

[0248] This is based on the assumption that leakage of fluorescent contrast agent from the blood vessels into the epithelium has a negligible effect on the absolute value of S(t), a valid assumption since L(t) typically follows a much slower logistic growth than B(t) and does not significantly affect the observed I(t) data until later in the time course, when the stomach is expected to be largely empty.

[0249] Next, the proportion of the fluorescent contrast agent retained in the stomach at time t, R pc can be calculated as follows:

[0250]

number

[0251] In the formula, S(t peak ) is the value of S(t) at the time when the last peak is observed in the I(t) data. This is the last time point before the steady-state elimination phase (when the fluorescent contrast agent begins to be eliminated from the body and no further increase in fluorescence intensity is observed), so at this point R pc You may choose to normalize the curve, so that t peak After this, the fluorescent contrast agent is not excreted from the stomach.

[0252] If only a single peak is observed, t peak can be set as the start of the steady-state elimination phase, where a constant linear decrease in the fluorescence signal is observed. This point is easier to identify in fluorescence data (where data points are collected approximately once per minute) than in paracetamol or breath tests (where longer measurement intervals of 10-15 minutes are typically employed). Furthermore, the transdermal sensing device can be programmed to automatically terminate data acquisition once this point is reached (e.g., when the data over the selected time interval shows a constant linear decrease and then no increase).

[0253] If the emission rate cannot be measured accurately (e.g., if the acquisition time is short), we can ignore the effect of emissions and calculate R based only on the B(t) data. pc In this case, R pc can be shown as follows:

[0254]

number

[0255] By using the above approach, we obtained R values ​​that are in good agreement with the values ​​obtained from the paracetamol absorption study. pc Therefore, advantageously, this represents a method that can provide a clear and clinically valuable readout in the assessment of gastric emptying rate. For example, by using the calculated RPC curve, it is possible to measure the time at which the percentage of fluorescent contrast agent / test meal retained in the stomach has decreased to 75%, 50%, 25%, etc. of its initial value.

[0256] Furthermore, the extracted parameters (e.g., B max , t B1 / 2 ) can also be used to quantify intestinal permeability in a similar manner to that presented above (i.e., see the GP1-GP5 equation above).

[0257] In conclusion, this approach involves fitting functions (two sigmoidal lines and a linearly decreasing line) to the observed transdermal fluorescence data. Because this approach fits a function to the data rather than performing a direct calculation, prior knowledge of the relative intensity contributions of the fluorescent contrast agent in the blood vessels and epithelium is not required. It also does not require knowledge of factors such as body weight and total body fluid volume, which are often estimated based on assumptions, as are required to assess gastric emptying rate using paracetamol absorption tests. Furthermore, while the observed fluorescence trends vary depending on the geometry of the transdermal sensing device / probe used to collect the data, this fitting-based approach is not affected by such variations. For example, if the probe geometry is adjusted to reduce sensitivity to L(t) (leakage of the fluorescent contrast agent into the skin epithelium), the fitting will reduce L. max This is simply corrected by minimizing (or setting to zero) the value of . Therefore, this approach is suitable for use with any transdermal fluorescent probe or transdermal sensing device design. Overall, it allows for the extraction of parameters for assessment of gastric emptying (and intestinal permeability) suitable for clinical use. This is possible based on non-invasive data collection, without the need to collect blood, urine, or other samples. Furthermore, the analysis can be performed in real time in an automated manner, with processing performed by the transdermal sensing device itself. A fitting-based approach can also reduce acquisition time by providing a suitable fit, eliminating the need to collect a full time course of more than four hours (typically required for analysis of gastric emptying using paracetamol absorption tests and / or carbon-13 ("C") breath tests).

[0258] As an alternative to the fitting-based approach described above, retention curves can also be calculated directly from the observed fluorescence data (i.e., directly from I(t)), where retention is defined as:

[0259]

number

[0260] In the formula, R pc_direct is the retention curve, and t peak1 represents the time at which the first peak is observed in the fluorescence data (I(t)). In this case, the first peak is used to normalize the retention curve to allow for rapid calculations. Using this approach, the transdermal sensing device can automatically detect the first maximum in the fluorescence versus time data and then quickly calculate the retention curve (from which diagnostic parameters such as the time at which retention falls to 75%, 50%, 25%, etc. can be extracted).

[0261] Using this approach, diagnostic parameters can be reported within approximately 60 minutes, and the data obtained has an R of 50-100%. pc_direct There is good agreement with paracetamol absorption studies for values ​​of . Furthermore, direct calculation minimizes the possibility of errors and discrepancies in the analytical procedure.

[0262] To most accurately calculate the retention rate, background subtraction can be performed on I(t) before using it in the above equation. This can be achieved, for example, by setting the background level as the initial intensity value (i.e., I(t = 0)) and subtracting this from each I(t) value. Alternatively, the background value can be defined as the average of the intensities recorded at, for example, the first five time points. In this approach, the precise time window selected for background subtraction is determined by the time point at which the fluorescence versus time curve (I(t)) begins to increase (the background region is defined as all time points before the onset of the initial increase). To ensure optimal results, this background subtraction should also be performed before the fitting process described above.

[0263] If excretion can be calculated, it can be included in the calculation of retention rate to further improve the accuracy of the calculation. For simplicity, in this example, the amount of fluorescent contrast agent excreted from the stomach, S direct (t) can be defined as follows:

[0264]

number

[0265] The removal of the fluorescent signal, EI(t), can be defined as:

[0266]

number

[0267] As noted above, C represents the clearance rate. This can be obtained by fitting (as described above) or by direct calculation based on data in the steady-state elimination region of the fluorescence versus time curve. In the latter case, C can be defined as follows:

[0268]

number

[0269] In the formula, t s represents the time domain where steady-state emissions are observed, and t s1 is the first time point in the removal of steady-state emissions, and t sf is the last time point in the steady-state discharge region, and Δt s is the steady-state interval (i.e., t sf -t s1 ), and

[0270]

number

[0271] represents the mean fluorescence intensity over the steady-state region.

[0272] As mentioned above, C and E I (t), and S direct Once (t) is calculated, the retention curve can be calculated as follows:

[0273]

number

[0274] In this case, t peak can be selected as the time when the first or second peak is observed in the fluorescence data, or when steady-state elimination begins (e.g., 5%, 10%, or 20% after the peak is observed), and can be determined data-wise (e.g., depending on whether enough data has been collected to observe the steady-state region). This approach also agrees well with paracetamol absorption study data. However, this approach requires data to be collected until at least the beginning of the steady-state region (to allow calculation / fitting of the clearance rate, C), which requires longer acquisition times than direct calculation based on the following equation:

[0275]

number

[0276] Overall, direct calculation of retention curves / values ​​based on fluorescence versus time curves can provide accurate assessments that are in agreement with gold standard data (i.e., paracetamol absorption studies), especially for retention values ​​in the range of 50-100%. By using the approach shown in the equations below, retention values ​​and curves can advantageously be calculated based on very short data collection times (e.g., 30-60 minutes or longer).

[0277]

number

[0278] These calculations can be performed automatically by transdermal sensing devices, allowing diagnostic parameters (e.g., time to 50% retention) to be reported much more quickly than other approaches (e.g., paracetamol absorption testing requires over four hours of data collection, plus processing of blood samples in a pathology laboratory and manual analysis of the resulting data, so diagnostic results are rarely reported within one to two days). Thus, all of the above approaches to calculating gastric emptying rate offer the potential to significantly speed up the reporting of results and significantly improve current standards of care.

[0279] Figures 15A and 15B show a comparison of gastric emptying data obtained using the fluorescein-based measurement method described above with data obtained using a clinically approved paracetamol absorption test conducted in 20 healthy volunteers. Participants each ingested a liquid test meal (milkshake) containing 500 mg of fluorescein and 1.5 g of paracetamol. Transcutaneous fluorescence intensity measurements were then taken at 1-minute intervals. Blood samples were collected at 10-15 minute intervals to assess serum paracetamol concentrations.

[0280] Figure 15A shows the fluorescence intensity and mean paracetamol concentration as a function of time. The shaded area indicates one standard deviation from the mean. As can be seen, the fluorescence and paracetamol data clearly overlap over the entire duration of the experiment (250 minutes).

[0281] Comparison of Figures 15A and 15B shows that the fluorescence versus time data (see Figure 15A) can be converted to the percentage of the test meal remaining in the subject's stomach, Rpc, as a function of time (see Figure 15B) using the gastric emptying rate calculation and fitting method described above.

[0282] Figure 15B shows the mean retention rate (Rpc) values ​​as a function of time for the paracetamol data (calculated by the method of Medhus et al., as described in detail in "Delay of gastric emptying by duodenal intubation: sensitive measurement of gastric emptying by the paracetamol absorption test," first published December 24, 2001: https: / / doi.org / 10.1046 / j.1365-2036.1999.00519.x) and the fluorescence data (calculated by the method described above). The shaded areas indicate the boundaries of one standard deviation from the mean. The "fluorescence-direct" data (represented by dots) are plotted against the fluorescence intensity data (i.e., the Rpc values ​​described above). pc_direct ) The "Fluorescence-S(t)" data (represented by triangles) represent the retention calculated based on the fitting procedure described above, using the Rpc calculated by the data in variable S(t) (above).

[0283] 15A and 15B, the above analysis can advantageously provide graphs and measurements similar to those used in the clinical diagnosis of delayed gastric emptying (e.g., gastric scintigraphy or paracetamol absorption tests), where the percentage of gastric contents retained in the stomach is plotted as a function of time and used for diagnostic assessment. Advantageously, by using the methods described herein, this can be achieved using very minimally invasive procedures.

[0284] Various modifications can be made to the described embodiments without departing from the scope of the invention, which is defined by the appended claims.

Claims

1. 1. A method of operating an apparatus for measuring gastric emptying rate in a subject to which a test meal containing a fluorescent contrast agent that is absorbed by a healthy intestine has been orally administered, the method comprising: The device comprises: a light source for illuminating the subject's skin with optical radiation such that the optical radiation causes at least a portion of the fluorescent contrast agent in the test meal that has leaked from the subject's stomach and entered the subject's bloodstream to fluoresce; a transdermal detection device for periodically transdermally detecting the fluorescence intensity of the fluorescent contrast agent at the irradiation position on the skin; an information processing device for processing fluorescence data regarding the fluorescence intensity obtained by the transdermal detection device; The method comprises: the light source illuminating a location on the skin of the subject's body part; the transdermal sensing device periodically transdermally detecting the fluorescence intensity of the fluorescent contrast agent in the test meal at the location to obtain fluorescence data about the fluorescence intensity as a function of time; normalizing the fluorescence data to obtain normalized data of the fluorescence intensity as a function of time; and the information processing device analyzes the normalized data based on the fluorescence intensity of the fluorescent contrast agent in the test meal that entered the subject's bloodstream as a function of time divided by the peak fluorescence intensity of the total amount of the fluorescent contrast agent orally administered to the subject to calculate the percentage of the test meal remaining in the subject's stomach as a function of time.

2. The method of claim 1 , wherein the peak value of the intensity is the value of the intensity at a first peak or a second peak in the fluorescence data, or the maximum value of the intensity in the fluorescence data.

3. 3. The method of claim 1 or 2, wherein the test meal comprises a liquid test meal or a solid test meal.

4. 4. The method of claim 1, wherein the fluorescent imaging agent comprises a dye comprising fluorescein, methylene blue, fluorescein isothiocyanate-conjugated dextran, a salt thereof, or a combination thereof.

5. The method of any one of claims 1 to 4, wherein the transdermal sensing device has an acquisition time, the light source has an excitation power, and the fluorescence data is normalized based on the acquisition time and the excitation power.

6. The method of any one of claims 1 to 5, wherein the periodic detection of the fluorescence intensity of the test meal is initiated before the test meal is administered to the subject, by having the transdermal sensing device initiate periodic measurements before the test meal is administered to the subject to obtain a background signal to be used in the step of normalizing the fluorescence data.

7. The method of any one of claims 1 to 6, wherein the light source comprises a light emitting diode or a laser.

8. 8. The method of any one of claims 1 to 7, wherein the transcutaneous sensing device comprises one or more photodiodes, phototransistors, and / or fiber optic probes and is configured to be worn on and / or around the body part of the subject.

9. 9. The method of claim 1, wherein in the step of using the transdermal sensing device to periodically detect the fluorescence intensity, a measurement is recorded by the transdermal sensing device at least once per minute.

10. The method of any one of claims 1 to 9, wherein the body part is a finger, a wrist, an arm, or an earlobe.

11. Analyzing the normalized data includes fitting the following function to the normalized data using a numerical fitting procedure, such as least squares: where t represents time; I(t) represents the normalized data of the fluorescence intensity as a function of time; B ｍａｘ represents the maximum intensity contribution from the fluorescent contrast agent in the bloodstream of the subject as a function of time; L ｍａｘ represents the maximum intensity contribution from the fluorescent contrast agent leaked into the epithelium of the subject's skin as a function of time; t Ｂ１／２ represents the time point at which the intensity contribution from the fluorescent contrast agent in the bloodstream of the subject reaches half of its maximum value as a function of time; t Ｌ１／２ represents the time point at which the intensity contribution from the fluorescent contrast agent leaked into the epithelium of the subject's skin reaches half of its maximum value as a function of time; C represents the rate at which the fluorescent contrast agent is excreted from the subject's body; k Ｂ is a constant representing the logistic growth rate of the intensity contribution from the fluorescent contrast agent in the bloodstream of the subject as a function of time; k Ｌ 11. The method of claim 1, wherein ρ is a constant representing the logistic growth rate of the intensity contribution from the fluorescent contrast agent leaked into the epithelium of the subject's skin as a function of time.

12. analyzing the normalized data further comprises calculating an amount of fluorescent contrast agent leaked from the stomach of the subject as a function of time, S(t); S(t) = B(t) + B(t)Ct, 12. The method of claim 11, wherein B(t) represents the intensity contribution from the fluorescent contrast agent in the bloodstream of the subject as a function of time.

13. Analyzing the normalized data includes calculating the percentage of the test meal remaining in the subject's stomach as a function of time, R ｐｃ and further comprising the step of calculating: In the formula, S(t ｐｅａｋ 13. The method of claim 12, wherein S(t) represents the value of S(t) at the time a final peak is observed in the normalized data of the intensity as a function of time.

14. Analyzing the normalized data includes calculating the percentage of the test meal remaining in the subject's stomach as a function of time, R ｐｃ and further comprising the step of calculating:

12. The method of claim 11, wherein B(t) represents the intensity contribution from the fluorescent contrast agent in the bloodstream of the subject as a function of time.

15. Analyzing the normalized data includes calculating the percentage of the test meal remaining in the subject's stomach as a function of time, R ｐｃ and calculating the value of where I(t) represents the normalized data of the intensity as a function of time, and t ｐｅａｋ１ The method of any one of claims 1 to 10, wherein σ represents the time at which a first peak is observed in the intensity as a function of time.

Images