CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 62/758,098, filed November 9, 2018, the contents of which are incorporated by reference in its entirety.

FIELD OF THE DISCLOSURE

[0002] The disclosure relates generally to the field of gut integrity. More specifically, the disclosure relates to the field of methods to measure gut integrity.

BACKGROUND

[0003] Poor gut integrity, or “leaky gut”, allows bacterial products and other undesirable compounds to be absorbed into the bloodstream of the animal. Leaky gut occurs in the context of declining health in animals for a variety of reasons. The progression of non alcoholic fatty liver disease (NAFLD) and particularly non-alcoholic steato-hepatitis (NASH) is associated with and partially driven by“leaky gut”.

[0004] Measuring gut integrity in preclinical animal research is important, particularly when screening substances that may improve leaky gut and associated co morbidities like NASH. For example, in mice, a current method for assessing gut integrity is to orally gavage the animal with an amount of 4 kDa fluorescein isothiocyanate-dextran polymer (or“FITC-dextran”), wait 4 hours to allow absorption of the FITC-dextran by the gut, then sacrifice and exsanguinate the mouse. One hundred microliters of serum is collected and placed in a cuvette, and the FITC fluorescence is measured in a spectrophotometer. A high the fluorescence signal indicates a leaky gut. Because such a large volume of serum is needed for this technique, serial measurements on the same animal over time are impossible.

[0005] Mice usually contain about 1 ml of whole blood depending on size and age. In order to get 100 microliters of serum, at bare minimum of 200 microliters of whole blood must be obtained from the mouse and spun down. Researchers must either kill the mouse outright and exsanguinate it or perform a retro-orbital bleed. Retro-orbital bleeds have a high mortality rate in order to extract enough blood. Even if the Institutional Animal Care and Use Committee (or IACUC) approves the retro-orbital bleed protocol (and many will not), and the mouse survives the retro-orbital bleeding procedure, the stress of losing such a large blood volume repeatedly can alter the physiology of the mouse and skew experimental results. Thus, repeat measurements on the same animal in a longitudinal study is impossible. Moreover, since large cohorts of animals must thus be sacrificed at various timepoints, variability is introduced into longitudinal studies because the same animals are not tested repeatedly.

[0006] Protocols may call for dosing about 200 microliters of 4 kDa FITC-dextran at 600 mg/kg body weight by gavage. Serum is typically collected 4 hours later when the dextran has been absorbed by the mouse and is in the bloodstream. The FITC fluorescence of serum degrades rapidly with time and exposure to light, meaning that the animals must be exsanguinated and serum spun in dark or low light conditions. The fluorescence must be measured very quickly and in the dark to avoid quenching or photobleaching. Because exposure to light can render the fluorescence undetectable, samples must be kept in the dark and read very quickly. Differences in time delay and light exposure while samples are being handled and prepped for measurement on fluorometer can lead to significant variability and measurement error over the course of an experiment. Sensitivity and limit of detection (LOD) for this varies depending on the instrument used and FITC quenching can be a problem resulting in loss of signal and therefore underestimating the amount of FITC-dextran present in serum, and by extension, under-estimating the leakiness of the gut of the dosed animal. Additionally, hemolysis (free hemoglobin in a serum or plasma sample) can also interfere with measurement of fluorescence.

[0007] Further, the current testing protocol uses nearly the entire serum sample extracted from the mouse, limiting the number of other analytes that can be tested in the sample. For instance, most researchers would also perform a lipid panel (total cholesterol, LDL, HDL, triglycerides), a hepatic function panel (AST, ALT, ALP, ALB, GGT, T bil, etc.), plus glucose and insulin measurements. Thus, large cohorts of mice are needed for every experiment. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Illustrative embodiments of the present disclosure are described in detail below with reference to the attached drawing figures and wherein:

[0009] FIG. 1 is a flowchart outlining the steps of a method for measuring FITC- dextran in an animal;

[0010] FIG. 2 is a calibration curve of FITC-dextran in buffered saline; and

[0011] FIG. 3 is a calibration curve of FITC-dextran in mouse serum.

DETAILED DESCRIPTION

[0012] The present approach now will be described more fully in reference to the accompanying figures, in which embodiments are shown. However, this approach should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the approach to those skilled in the art. In the drawings, like numbers refer to like elements throughout. Thicknesses and dimensions of some components may be exaggerated for clarity.

[0013] As used herein, the term "comprising" or "comprises" is open-ended, and includes one or more stated features, integers, elements, steps, components or functions but does not preclude the presence or addition of one or more other features, integers, elements, steps, components, functions or groups thereof. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items. The term "about" means the stated value plus or minus a reasonable or conventional margin of error of measurement, or plus or minus 10% if no method of measurement is indicated.

[0014] As used herein, the common abbreviation "e.g.," which derives from the Latin phrase "exempli gratia," may be used to introduce or specify a general example or examples of a previously mentioned item, and is not intended to be limiting of such item. If used herein, the common abbreviation "i.e.," which derives from the Latin phrase "id est," may be used to specify a particular item from a more general recitation.

[0015] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms“a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. [0016] Well-known functions or constructions may not be described in detail for brevity and/or clarity.

[0017] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0018] In addition, spatially relative terms, such as "under," "below," "lower," "over," "upper," "downward," "upward," "inward, "outward" and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures is turned over, elements described as "under" or "beneath" other elements or features would then be oriented "over" the other elements or features. Thus, the exemplary term "under" can encompass both an orientation of over and under. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly.

[0019] It will be understood that when an element is referred to as being "attached," "coupled" or "connected" to another element, it can be directly attached, coupled or connected to the other element or intervening elements may also be present. In contrast, when an element is referred to as being "directly attached," directly coupled" or "directly connected" to another element, there are no intervening elements present. Words such as passageway, fluid path, or flow component, etc., are intended to communicate structure supporting fluid communication and may comprise a tube, pipe, hose, boring, channel, etc.

[0020] It is noted that any one or more aspects or features described with respect to one embodiment may be incorporated in a different embodiment although not specifically described relative thereto. That is, all embodiments and/or features of any embodiment can be combined in any way and/or combination. Applicant reserves the right to change any originally filed claim or file any new claim accordingly, including the right to be able to amend any originally filed claim to depend from and/or incorporate any feature of any other claim although not originally claimed in that manner. These and other objects and/or aspects of the present approach are explained in detail in the specification set forth below.

[0021] The method described herein provides for measuring a dextran component in an animal. In one embodiment, the method measures a dextran component having a fluorescent component. In another embodiment, the method measures FITC-dextran absorbed in the digestive tract of an animal. It is envisioned that other fluorescent compounds could also be utilized. FITC-dextran may be fluorescent, and the fluorescence of FITC-dextran may be measurable. As is shown in FIG. 1, the method 100 involves the steps of dosing or causing said animal to ingest a solution of FITC-dextran 101, extracting a biological fluid sample from the animal 102, measuring an unknown quantity of FITC-dextran in the sample by using an assay 103, measuring a known quantity of FITC-dextran by using an assay 104, and comparing and calculating the unknown amount of FITC-dextran in the sample to the known amount of FITC- dextran 105. In some embodiments, the method measures the dextran component in mouse serum. Further, the assay may be a dextran-specific enzyme-linked immunosorbent assay (or “ELISA”). Instead of measuring FITC fluorescence, the method described herein measures the dextran component in a sample.

[0022] In some embodiments, the animal may be a mammal. The animal may further be a mouse. The mouse may have a diet induced non-alcoholic fatty liver disease available under the DIAMOND™ name from Sanyal Biotechnology, Inc. (or“DIAMOND™ mouse” - Diet Induced Animal Model of Non-alcoholic fatty liver Disease). The animal may have a disease affecting permeability of the gut. Alternately, the animal may have a liver disease induced by diet. The mouse may develop NAFLD, NASH, Fibrosis, and Hepatocellular carcinoma (HCC) in response to a high fat high sugar diet. The mouse may become insulin resistant, obese, dyslipidemic, and may develop changes in gut microbiome, as well as leaky gut. Disease progression in the mouse may parallel histopathology and physiology of human disease. The mouse may have liver gene expression that is similar to humans. In a DIAMOND™ mouse, gut permeability may increase on a Western Diet. In some embodiments, the mouse may initially show low levels of FITC-dextran. However, the mouse may show increased gut permeability the longer the mouse is on a Western Diet. In other embodiments, the mouse may have NAFLD, NASH, cirrhosis, viral hepatitis, autoimmune hepatitis, hepatocellular adenoma, hepatocellular carcinoma. [0023] In step 101, the animal may be dosed or caused to ingest FITC-dextran. In some embodiments, the animal may be dosed or caused to ingest a solution of FITC-dextran. Those skilled in the art will appreciate that another type of solution containing dextran and not containing FITC may be used. The FITC-dextran can be administered in any appropriate method now known or later developed. In some embodiments, the animal may be orally gavaged as described in the background section. In mice, the animal may be orally gavaged with an amount of 4 kDa FITC-dextran polymer.

[0024] It may be advantageous to wait a predetermined time period and allow for absorption of the solution after dosing or causing an animal to ingest the solution of FITC- dextran. For example, researchers may wait four hours to allow absorption of the FITC-dextran by the gut. The sample may then be taken four hours after dosing the animal. However, it will be appreciated that the time to collection of the sample may be varied. In embodiments where the biological fluid is blood, collecting the sample earlier than four hours may be possible. The gavage bolus may pass into the small intestine after one hour. A sample collected before four hours could reflect findings wherein the bolus was located primarily in the foregut. At six to eight hours after dosing, the bolus may be located in the large intestine or hindgut. Therefore, if researchers are most concerned about permeability in the small intestine, it may be advantageous to collect the sample prior to four hours. Alternately, it may be advantageous to collect the sample after four hours if the concern is the large intestine. A time of four hours may allow for measurement of global absorption from the foregut to a hindgut.

[0025] In step 102, the sample is collected from the animal. The sample may be a blood sample, from which serum may be extracted. In the case of mice, small amounts of blood extracted from a tail vein nick, which is non-fatal, may be used in measurements of FITC- dextran. A tail vein nick may be advantageous for many reasons. The animal is not killed, the tail vein nick does not cause the animal undue stress, and serial measurements can be taken and correlated with changes in gut integrity over time in the same animal, making longitudinal studies possible. However, it is appreciated that the sample may be collected in any other appropriate method now known or later developed. Serial measurements under the previous method of exsanguination were impossible (or highly difficult and potentially inaccurate if samples were collected through retro-orbital bleeds). Because the amount of serum required for the measurement is greatly decreased, a drop of blood collected in any appropriate method may be sufficient for the sample. In some embodiments, the volume of serum may be no more than 20 microliters. In some embodiments, the volume of serum may be between 5-20 microliters. In other embodiments, the volume of serum may be more than 20 microliters but less than 100 microliters. When the animal is an animal other than a mouse, a decreased amount of serum may also be required.

[0026] In some embodiments, the sample may be delivered to or received by a researcher or other person to assess the amount of unknown FITC-dextran in the sample. In such embodiments, it is appreciated that steps 101 and 102 may be performed by a third party.

[0027] In step 103, the amount of unknown FITC-dextran in the sample may be measured in an assay. In some embodiments, the assay is a dextran-specific ELISA. In other embodiments, the assay may be a competition ELISA. Competition ELISAs are well-known to those skilled in the art of immunological detection methods. Competition ELISAs may work by coating a surface, usually the bottom of wells in a plate, with an antigen (in this case dextran), then standards with known concentrations of dextran, as well as samples with unknown concentrations of dextran may be added to the wells. A dextran-antibody-horseradish peroxidase (HRP) conjugate may be further added to the wells, and the wells may then be incubated. A competitive binding reaction may proceed, wherein low dextran samples allow more antibody- HRP conjugate to attach to the wells whereas high levels of dextran in samples may compete and less of the antibody-HRP conjugate attaches to the bottom of the wells. After washing away unbound material, tetramethylbenzidine (TMB) may be added, which may react with an enzyme to make a color change that is detectable at 450 nm in an ELISA plate reader. Many different enzymes and substrates are contemplated and could be used in the described method. In contrast to a standard ELISA, where darker color indicates higher amounts of antigen detected, in a competition ELISA, the higher the concentration of antigen (in this case dextran), the less HRP may bind and the lighter the color may change.

[0028] In step 104, the amount of a known quantity of FITC-dextran may be measured. The known quantity may be the standard. In other embodiments, the known quantity is determined by taking a standard sample from the same animal. The standard sample may be taken prior to the oral gavage or after the gavage has passed through the animal. Alternately, the standard sample may be taken from another animal in the same group that is not selected to receive an oral gavage. The extraction methods for the standard sample may be similar to those of the sample after the animal has been administered FITC-dextran. Specifically, in the case of mice, a tail nick may be sufficient to extract the standard sample. In some embodiments, the standard may already have been predetermined, and step 104 may consist of determining the amount of FITC-dextran in the sample.

[0029] After the sample is collected, the serum may then be extracted. In one embodiment, the samples, as well as standards, may be spun at 1500RPM for 2 minutes at 4c. The samples may then be placed on ice. Reagents may be brought to room temperature, and a washing buffer of lx with DI water may be prepared. 20 microliters of the standards may be added to a pre-coated well in duplicates or triplicates. 20 microliters of the samples may be added to the rest of the wells in duplicates or triplicates. 20 microliters of plain mouse serum may be added to at least one well as a blank. 20 microliters of a detector-enzyme conjugate may be added to all wells, excluding blank wells. The plates may be covered with sealer and incubated on a rotator for 30 minutes at RTP. The complex may then be taken out using a pipet. In some instances, a multichannel pipet may be preferable. 300 microliters of wash buffer may be added and then discarded. The plate may then be blotted to absorb remaining wash buffer. The wash buffer may be applied to the plate three times.

[0030] In step 105, the unknown amount of FITC-dextrin in the sample may be compared to the known amount of FITC-dextrin in the standard, and the unknown amount of FITC-dextrin may be calculated. 100 microliters of TMB solution may be added to all of the wells, excluding blank wells. The plate may then be covered with the sealer and incubated in the dark for approximately 10-12 minutes. In some embodiments, samples with zero dextran may have a medium to dark blue color. After the plate has been incubated in the dark, 50 microliters of stop solution may be added to each well. The plate may then be placed briefly on the rotator, which may encourage mixing. The plate may then be read at 450nm. In other embodiments, the measured value of the dextran in the unknown may be compared to reference ranges of measured values corresponding to a disease state of an animal.

[0031] Alternately, a basic procedure for a competition ELISA may be to first add unknowns and standards to wells. Next, dextran detector HRP conjugate may be added to the wells. The wells may be incubated and washed. TMB may be added to the wells, and then the wells may be further incubated before the addition of a stop reagent. The wells may then be read at 450 nm. The binding percentage for each sample may be calculated using the following formula:

[A450(Sample) - A450(Blank)]/[A450(Zero Dextran Sulfate)- A450(Blank)] x 100

= % Binding

[0032] Using linear or nonlinear regression, a standard curve may be plotted of percent binding versus concentration of dextran standards. The levels of dextran unknowns may be determined by comparing their percentage of binding relative to the standard curve. The dextran amounts may be quantified by comparing the values of from the wells containing unknowns to the values in the standard curve. The result may then be multiplied by the dilution factor of the sample.

[0033] A plate-based competition ELISA may also be used. However, it is understood that other methods involving other solid supports such as beads of varying compositions and non-competitive methods may also be used that may possess the same utility. Additionally, it is understood that while one embodiment may employ, for example, HRP reaction and no signal booster, assay modifications employing a different enzyme or fluorescent conjugate that fluoresces at a wavelength different from FITC could be developed by one skilled in the art. The application claims these embodiments, whether now known or later developed. Further, in some embodiments, there may be an addition of signal boosters to enhance sensitivity for precise measurements of low concentrations of FITC-dextran.

[0034] Increasing concentrations of FITC-dextran that are detectable in a single mouse as the mouse ages and continues to be fed a Western Diet can be used to quantify gut permeability and correlate directly with the worsening of NAFLD or NASH in an animal model. In some embodiments, the animals may be assessed once and then assessed at least one other time after a period of time has passed. The sample taken from the first assessment may be compared to the sample taken from the second assessment. Additionally, all samples taken from the animal may be compared to one another. For example, a sample may be taken at week 12 and week 20. This had previously been impossible or extremely difficult due to the amount of serum required to measure the FITC-dextran. Indeed, if the animal was exsanguinated then a second sample was impossible. This meant that large cohorts of animals may have been required, and it may have been difficult to test the efficacy of certain treatments in decreasing gut permeability. [0035] If serial measurements may be obtained over time from the same animal, measurements as to whether the animal is improving (or declining) may be shown. Put another way, the current method may allow measurements to be taken serially and compared to one another to determine the efficacy of administered drugs that may prevent or reverse the disease process of NAFLD or NASH. In some embodiments a drug may be administered to the animal after the first measurement and before the at-least second measurement. The two measurements may then be compared. The measurements may be compared for changes in gut health, changes in gut permeability, changes in NASH status, or any other change. A higher measured value in the at-least second measurement may be interpreted as drug non-efficacy, and a lower measured value in the at-least second measurement may be interpreted as drug efficacy. A higher measured value in the at-least second measurement may be interpreted as higher gut permeability. A determination of the disease state of the animal may be made based on the comparison of the measured value to the reference ranges. The administered drug may be nonsystemic antibiotic rifaximin, methylnaltrexone, pioglitazone or any other appropriate drug now known or later developed.

[0036] In some embodiments, the dextran-specific ELISA may be implemented using an assay kit available under the S-7500 name from Lifespan Technologies (or“Lifespan S- 7500”). In some embodiments, the dextran-specific ELISA may be similar to an ELISA for measuring dextran contamination in sucrose for quality control during production and purification from sugar cane. The differing chain lengths of dextran occurring in cane sugar and sucrose are very large in molecular weights and vary from a few thousand to a few million Daltons. The Lifespan S-7500 ELISA was developed for agricultural use and optimized to measure very large dextran polymers of up to 10 thousand to 2 million Daltons, because larger molecular weight dextrans in sugar cane do the most damage to yield. However, the size of dextran that must be used to measure gut integrity in a mammal is much smaller, only 4 kDa, and measuring dextran conjugated to FITC in a high-protein solution like serum or plasma is very different than measuring dextran in a low-protein sugar solution.

[0037] The Lifespan S-7500 ELISA may not have been utilized to detect dextran in blood or body fluids of animals, to assess gut permeability in animals, or to compare levels of gut permeability in healthy vs. sick animals. The ELISA assay kit was not previously tested for ability to detect FITC-dextran conjugate, and it was previously unknown if the presence of the FITC would interfere with the ELISA chemistry. The present technique may not have previously been tested to determine if 4 kDa polymer size was detectable, or if the technique would work on serum because the kit was developed for agricultural contaminant testing and cell culture media testing, respectively.

[0038] As is shown in FIG. 2, accurate quantification of 4kDa FITC-dextran is possible with competition ELISA. FIG. 2 shows a calibration curve of 4kDa FITC-dextran in buffered saline with S-7500 antibody. As is shown in FIG. 3, the limit of detection is approximately 10 micrograms per mL. FIG. 3 shows a calibration curve of 4kDa FITC-dextran spiked into mouse serum.

[0039] The step 105 of measuring the amount of FITC-dextran described herein may be performed by an optimized competition ELISA which may detect 4 kDa FITC-dextran in mouse serum, and the assay may have good linearity, sensitivity, and a wide range of detection. Because the ELISA may detect dextran polymers of 4kDa, the ELISA has great utility for determining gut permeability in animal models.

[0040] The currently used method of fluorescence measurement in serum (fluorometry of FITC) shows ranges of FITC-dextran in mouse serum from 0-10 microliters/ml in healthy animals, to greater than 10 and up to about 70 microliters/ml is animals treated to cause leaky gut. The range of the technique described herein may be 1 to 1000 microliters/ml when FITC-dextran was spiked into mouse serum. The assay may be linear over that range. The animal may be dosed at 600 mgs/kg of body weight, FITC-dextran levels in serum for healthy animals and animals with increased gut permeability may fall well within the dynamic range of the assay described herein, and the competition ELISA may have utility for detection from small volumes of serum claimed.

[0041] The sensitivity of the technique described herein may be sufficient to detect FITC-dextran in mouse serum in volumes of 5 - 20 microliters, which is a much higher sensitivity than previous methods. Dextran may be stable, does not degrade, and is measurable in very small quantities of serum by non-fluorescent techniques such as competition ELISA. Further, hemolysis may not interfere with the detection of serum dextran via ELISA. As discussed above, 5 - 20 microliters of serum can be obtained from a drop of blood from a tail nick rather than exsanguinating the mice. Therefore, the method may be non-fatal and may avoid causing the animal undue stress. The technique may be used for serial measurements in the same animal, which may ensure more accurate results.

[0042] Many different arrangements of the various components depicted, as well as components not shown, are possible without departing from the spirit and scope of the present disclosure. Embodiments of the present disclosure have been described with the intent to be illustrative rather than restrictive. Alternative embodiments will become apparent to those skilled in the art that do not depart from its scope. A skilled artisan may develop alternative means of implementing the aforementioned improvements without departing from the scope of the present disclosure.

[0043] It will be understood that certain features and subcombinations are of utility and may be employed without reference to other features and subcombinations and are contemplated within the scope of the claims. Not all steps listed in the various figures need be carried out in the specific order described.