# Patent

## Title
Ex vivo method for determining potential GLP-2 receptor modulators

## Abstract

Disclosed herein is a method for measuring the contractility of intestinal tissue upon treatment with GLP-2 or a GLP-2 ligand. Also disclosed is an assay which directly measures the activity of GLP-2 or GLP-2 ligands ex vivo and permits the screening of putative GLP-2 ligands in native tissue.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application is a divisional application of U.S. patent application Ser. No. 13/154,071, filed on Jun. 6, 2011, which issued as U.S. Pat. No. 8,257,983 on Sep. 4, 2012, which is a divisional application of U.S. patent application Ser. No. 12/358,123 filed on Jan. 22, 2009, which issued as U.S. Pat. No. 7,977,061 on Jul. 12, 2011, which is a divisional application of U.S. patent application Ser. No. 11/607,030, filed on Dec. 1, 2006, which issued as U.S. Pat. No. 7,498,141 on Mar. 3, 2009 which claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application Ser. No. 60/741,075 filed on Dec. 1, 2005, now expired, the entire contents of which are hereby incorporated by reference.


BACKGROUND OF THE INVENTION
Glucagon-like peptide-2 (GLP-2) is a 33-amino acid proglucagon-derived peptide secreted by the endocrine L-cell primarily in the lower gastrointestinal tract in response to luminal nutrients. Plasma levels have been shown to significantly increase within an hour of ingesting a meal, in particular following the ingestion of carbohydrates or fat (1). GLP-2 has been shown to be responsible for the regulation of proliferation and apoptosis of the intestinal epithelium (2,3). These changes in part result in an increase in mucosal surface area, enhanced absorptive efficiency and barrier function in the small intestine (4-6). GLP-2 also decreases gastric motility, inhibits gastric acid secretion, increases nutrient transport activity and acutely increases intestinal and portal blood flow (7-11).
GLP-2 and GLP-2 analogues promote the growth and repair of the intestinal epithelium in models of disease, including enhanced adaptation and nutrient absorption following small bowel resection and alleviation of TPN-induced hypoplasia in rodents (12-15). GLP-2 analogues have demonstrated decreased mortality and improvement of disease-related histopathology in animal models of intestinal damage such as indomethacin-induced enteritis, dextran sulfate-induced colitis and chemotherapy-induced mucositis (16-19).
The intestinotrophic effects of GLP-2 are mediated by the GLP-2 receptor (GLP-2R), a member of the superfamily of G-protein coupled receptors and most closely related to the GLP-1 and glucagon receptor gene subfamily (20). The GLP-2R is a high-affinity, ligand-specific functional receptor coupled to the G-protein Gs. Studies of the activation of the cloned GLP-2R by GLP-2 analogues show a correlation of in vitro activity with in vivo intestinotrophic efficacy (20).
To date, characterization of GLP-2R function has been limited to heterologous cell line expression, mucosal fractions and primary cell cultures. GLP-2 selectively stimulates cAMP production in recombinantly expressing GLP-2R cell lines, isolated intestinal mucosal fractions containing enteroendocrine and neural cells and primary hippocampal cultures (21-24). The study of native GLP-2R biological activity has focused on in vivo animal models and ex vivo model systems that require exogenous application of GLP-2 to the whole animal prior to tissue isolation (2-19, 25-31). There is no ex vivo method for characterizing putative GLP-2 ligands directly; such a method would have obvious utility, for example in the screening and characterization of GLP-2 ligands of pharmaceutical interest.
SUMMARY OF THE INVENTION
Disclosed herein is a method for measuring the contractility of intestinal tissue upon treatment with GLP-2 or a GLP-2 ligand. Thus, one aspect of the invention is an assay which directly measures the activity of GLP-2 or GLP-2 ligands. Another aspect of the invention is the direct characterization of the GLP-2 receptor. A further aspect of the invention is a method for screening compounds for activity at the GLP-2 receptor, such as putative GLP-2 agonists, antagonists, modulators or the like.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows the organ bath trace of the effect of 1 uM GLP-2 (1-33) on rat contractility in duodenum, jejunum, ileum and colon.
FIG. 2 shows the organ bath trace of the effect of GLP-2 (1-33) on rat colon contractility.
FIG. 3 Shows the organ bath trace of the effect of [gly2]GLP-2 on rat colon contractility.
FIG. 4 shows the inhibitory effects of GLP-2 analogues on spontaneous contractility in rat colon.
FIG. 5 shows the effect of GLP-2 analogues on cAMP accumulation in EBNA293 cells stably expressing rat GLP-2R.
FIG. 6 shows the effect of GLP-2 (3-33) on GLP-2 (1-33)-induced inhibition of colon contractility.
FIG. 7 shows the effect of GLP-2 (3-33) on teduglutide-induced inhibition of rat colon contractility.
FIG. 8 shows the effect of a small molecule antagonist on GLP-2 (1-33)-induced inhibition of colon contractility.
FIG. 9 shows the effect of a small molecule positive modulator on GLP-2 (1-33)-induced cAMP accumulation.
FIG. 10 shows the effect of a small molecule positive modulator on GLP-2 (1-33)-induced inhibition of colon contractility.



DETAILED DESCRIPTION OF THE INVENTION
Definitions
hGLP-2R is the human GLP-2 receptor; rGLP-2R is the rat GLP-2 receptor. hGLP-2 is human GLP-2 (also known as Teduglutide); rGLP-2 is rat GLP-2.
A “GLP-2 ligand” is defined as any molecule which interacts with the GLP-2 receptor; such molecules may be peptides (such as analogues of naturally occurring GLP-2) or small molecules.
A “modulator” is defined as a molecule which modulates the activity of a GLP-2 agonist.
“Intestinal tissue” is broadly defined as intestine from the pylorus to the rectum. “Small intestine” is defined as intestine from the pylorus to the ileo-cecal valve (it may also be defined as duodenum, jejunum and ileum). “Large intestine” is defined as intestine distal from the cecum (it may also be defined as colon tissue).
Disclosed herein is an assay which, for the first time, demonstrates the ability of GLP-2 receptor ligands to directly affect native GLP-2R-mediated effects in colon tissue ex vivo. As a result, it is possible to directly screen and pharmacologically characterize GLP-2 ligands, such as peptidic analogies of GLP-2, as well as small molecule agonists, antagonists and modulators, against whole tissue.
Such tissue is of mammalian origin; in one aspect it is of rat origin, in others it is of mouse or guinea-pig origin.
The assay method comprises measuring the contractility of segments of intestinal tissue ex vivo. The fact that this assay is useful for the stated purpose is not obvious, based upon the known expression levels of the GLP-2 receptor in this tissue. Further, previous studies have shown that direct application of GLP-2 ex vivo to this tissue has no effect.
One aspect of the invention is a method for characterizing the GLP-2 receptor; for example in an endeavour to better understand the physiological role of GLP-2 and the GLP-2 receptor in intestinal tissue or to elucidate the role of putative mediators related to GLP-2 activation and physiology.
Another aspect of the invention is a method for determining whether a compound is active on the GLP-2 receptor comprising the step of measuring contractility of intestinal tissue segments ex vivo in the presence and absence of compound, wherein a difference in contractility in the presence of the compound is indicative of activity on the GLP-2 receptor.
Yet another aspect of the invention is a method for determining whether a compound acts as a GLP-2 receptor agonist comprising the step of measuring contractility of intestinal tissue segments ex vivo in the presence and absence of compound, wherein inhibition of contractility in the presence of the compound is indicative of GLP-2 receptor agonist activity.
Yet another aspect of the invention is a method for determining whether a compound acts as a GLP-2 receptor antagonist comprising the step of measuring the effect of a GLP-2 receptor agonist on contractility of intestinal tissue segments ex vivo in the presence and absence of the compound, wherein a decrease of agonist inhibition of contractility in the presence of the compound is indicative of GLP-2 receptor antagonist activity.
Another aspect of the invention is a method of assaying compounds for activity at the GLP-2 receptor comprising the steps of:
i. Obtaining a segment of intestinal tissue,
ii. Suspending the segment in an organ bath,
iii. Placing the segment under tension,
iv. Incubating the segment with a compound of interest and
v. Measuring the contractility of the tissue; wherein an effect on contractility of the segment is indicative of activity on the GLP-2 receptor.
In one aspect the tissue is intestinal tissue. In another it is small intestine tissue. In yet another it is large intestine tissue or colon tissue. FIG. 1 shows the Organ Bath Trace of the effect of 1 uM GLP-2 (1-33) on rat contractility in duodenum, jejunum, ileum and colon. FIG. 2 shows that GLP-2 (1-33) inhibits spontaneous colon contractility in a concentration-dependent manner in rat colon tissue; FIG. 3 shows the analogous effect of Teduglutide.
The assay of putative agonists may be conducted by incubation of the colon segments with a predetermined concentration of the ligand to be tested for 5 to 10 minutes. FIG. 4 shows typical results for a number of GLP-2 analogues. FIG. 5 shows the effect of GLP-2 analogues on cAMP accumulation in EBNA293 cells stably expressing rGLP-2R.
The assay of putative antagonists may be conducted by pre-incubation of the colon segments with a predetermined concentration for, for example, 5 to 10 minutes of the ligand to be tested followed by addition and incubation of a predetermined concentration of a GLP-2 agonist. FIGS. 6, 7 and 8 show typical results for a number of GLP-2 antagonists; concentration-response curves are clearly right-shifted, as expected.
The assay of putative modulators, may be conducted by pre-incubation of the colon segments with a predetermined concentration of the ligand to be tested for, for example, 5 to 10 minutes followed by the addition and incubation of a pre-determined concentration of a GLP-2 agonist. FIG. 10 show typical results for a GLP-2 positive modulator; the effect of the modulation is clearly demonstrated. FIG. 9 shows the corresponding effect on GLP-2 (1-33)-induced cAMP accumulation.
Methods & Results
It should be noted that the methods described below are illustrative in nature, and that the invention is not limited to the particular embodiments described.
Segments of colon from male Sprague Dawley rats were suspended in organ bath chambers containing Krebs' solution and maintained at 37° C. with 95% O2 and 5% CO2. Basal tone, contractility rate and peak height were used to measure the properties of spontaneous and ligand-mediated colonic contractions. Segments were initially loaded to a tension of 2 g and allowed to equilibrate for 60 minutes. During this period, segments were repeatedly washed every 10 minutes. [gly2]GLP-2 (“Teduglutide”, a GLP-2 agonist) and GLP-2 analogues were subsequently incubated with each tissue segment for 5 to 10 minutes. The tissues were allowed to re-equilibrate with repeated washes following each experiment.
Teduglutide and GLP-2 (1-33) inhibited muscle contractility in a concentration-dependent manner with an estimated IC50 of 4.9±1.9 (SE) nM (n=6) and 13.5±5.4 nM (n=9), respectively. While having no effect alone, GLP-2 (3-33) (a GLP-2 antagonist/partial agonist) blocked the inhibitory effects of Teduglutide and GLP-2. In the presence of 10 μM GLP-2 (3-33), Teduglutide and GLP-2 (1-33) inhibited colon contractility with an IC50 of 59.9±11.9 nM (n=6) and 142.7±55.9 nM (n=5) respectively. The rank orders of potency of GLP-2 peptide analogues were similar to that previously reported for their cAMP activation in cell lines recombinantly expressing the rGLP-2R.
These studies demonstrate the ability of GLP-2 to reproducibly and significantly reduce spontaneous contractility of rat colon segments suspended in organ bath chambers. GLP-2-induced inhibition of colon contractility is concentration-dependent and reversible following a wash-out period. The GLP-2R antagonist/partial agonist GLP-2 (3-33) blocks the effect of GLP-2 (1-33) in a concentration-dependent manner. The rank order of potency of GLP-2 analogues on the inhibition of colon contractility was comparable to the rank order of increased cAMP accumulation in heterologous cells expressing GLP-2R.
Therefore the described assay enables native GLP-2R characterization by the direct application of GLP-2 to intestinal tissue segments ex vivo. As a result, it is possible to screen and pharmacologically characterize GLP-2 peptide analogues, GLP-2R small molecule agonist and antagonists against whole tissue.
Colon Contractility—Methods
Animals
Male Sprague-Dawley (SD) rats (Charles River Canada, Quebec), weighing 200-250 g, were used for these studies. The animals were housed on contact bedding and kept with free access to food (Purina Rodent Diet) and water in a facility with regulated temperature and humidity, under an alternate 12-hour light/dark cycle. Animals were acclimatized for 3 ̃5 days prior to harvesting of tissues.
Tissue Preparation
Rats were euthanized by decapitation. The entire intestine was quickly removed and placed in a Petri dish containing a Sylgard layer and 37° C. Krebs' solution bubbled with 95% O2 and 5% CO2. The Krebs' solution (pH 7.4) contained 118.1 mM NaCl, 25 mM NaHCO3, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4.7H2O, 2.5 mM CaCl2.2H2O and 11 mM glucose. The colon was isolated, cleaned of superficial fat and mesentery and sectioned into eight 1-cm long segments. The lumen was cleaned by carefully flushing each segment with Krebs' solution using plastic transfer pipets. Segments were transferred to a separate Petri dish containing fresh Krebs' solution and suture threads were tied to both ends of each segment. Tissues were suspended longitudinally using surgical suture, one end attached to the transducer on the top and the other to the tissue holder at the bottom of 20 ml organ bath chambers containing Krebs' solution, maintained at 37° C. with 95% O2 and 5% CO2. Isometric tensions were continuously measured using calibrated force displacement transducers and data was collected and processed by the MP100WSW system with AcqKnowledge software
Experiment Protocol
Segments of colon were initially loaded to a tension of 2 g and allowed to equilibrate for a 60 min period. During this period, segments were washed with Krebs' solution at 10 minutes intervals. When the tissues obtained regular and stable contraction patterns, GLP-2 agonist response experiments were conducted by the addition of eight concentrations of compound into eight separate organ baths for 5-10 minutes. Routinely, one-hour washes were interspersed between repeat applications of compounds to allow for recovery. The effect of GLP-2 (3-33) on GLP-2 agonist analogues were conducted by pre-incubating colon segments with 1 or 10 μM GLP-2 (3-33) for 5-10 minutes. Then agonist responses were measured at various concentrations as described above.
Data Analysis
Tension was measured by changes in peak height. Data were calculated and expressed as percentage response relative to the basal level (prior to the experiment). Graphs and IC50 determinations were plotted by the percentage response in GraphPad Prism (v 3.02). In order to assess the significance of the effect of GLP-2 and Teduglutide paired t-tests were conducted on the Log [IC50] values.
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## Claims

1. A method for determining the ability of a compound to potentially differentially modulate glucagon-like peptide-2 (GLP-2) receptor activity in different sections of the intestine, comprising:
a) incubating a first ex vivo intestinal segment with the compound;
b) measuring contractility of the first intestinal tissue segment;
c) incubating a second ex vivo intestinal tissue segment with the compound; and
d) measuring contractility of the second ex vivo intestinal tissue segment;

wherein the first ex vivo intestinal tissue segment is taken from a different section of the intestine than the second ex vivo segment, and
wherein a difference in contractility of the first ex vivo intestinal tissue segment and the second ex vivo intestinal tissue segment indicates a different responsiveness of the two segments to GLP-2 receptor modulation.

2. A method according to claim 1, wherein the first and second ex vivo intestinal tissue segments are of mammalian origin.

3. A method according to claim 2, wherein the first and second ex vivo intestinal tissue segments are of human origin.

4. A method according to claim 2, wherein the first and second ex vivo intestinal tissue segments are of rat origin.

5. A method according to claim 1, wherein the first and second ex vivo intestinal tissue segments are selected from the group consisting of duodenum, jejunum, ileum, small intestine, and colon.

6. A method for determining the ability of a compound to potentially differentially modulate glucagon-like peptide-2 (GLP-2) receptor activity in different sections of the intestine, comprising the steps of:
a) obtaining a first ex vivo intestinal tissue segment,
b) obtaining a second ex vivo intestinal tissue segment,
c) suspending the first ex vivo intestinal tissue segment and the second ex vivo intestinal tissue segment in separate organ baths,
d) placing the first and second ex vivo intestinal tissue segments under tension,
e) incubating each ex vivo intestinal tissue segment with the compound,
f) measuring the contractility of the first and second ex vivo intestinal tissue segments, and
g) comparing the contractility of the first ex vivo intestinal tissue segment with the contractility of the second ex vivo intestinal tissue segment;

wherein the first ex vivo intestinal tissue segment is taken from a different section of the intestine than the second ex vivo segment, and
wherein a difference in contractility of the first ex vivo intestinal tissue segment and the second ex vivo intestinal tissue segment indicates a different responsiveness of the two segments to GLP-2 receptor activation.

7. A method according to claim 6, wherein the first and second ex vivo intestinal tissue segments are of mammalian origin.

8. A method according to claim 7, wherein the first and second ex vivo intestinal tissue segments are of human origin.

9. A method according to claim 7, wherein the first and second ex vivo intestinal tissue segments are of rat origin.

10. A method according to claim 6, wherein the first and second ex vivo intestinal tissue segments are selected from the group consisting of duodenum, jejunum, ileum, small intestine, and colon.

11. A method for determining the ability of a compound to differentially antagonize glucagon-like peptide-2 (GLP-2) receptor response to a GLP-2 receptor ligand in different sections of the intestine, comprising:
a) incubating a first ex vivo intestinal tissue segment with the GLP-2 receptor ligand;
b) taking a first measurement of the contractility of the first ex vivo intestinal tissue segment,
c) incubating the first ex vivo intestinal tissue with the GLP-2 receptor ligand and the compound,
d) taking a second measurement of contractility of the first intestinal tissue segment,
e) incubating a second ex vivo intestinal tissue segment with the GLP-2 receptor ligand;
f) measuring contractility of the second ex vivo intestinal tissue segment,
g) incubating the second ex vivo intestinal tissue with the GLP-2 receptor ligand and the compound,
h) taking a second measurement of contractility of the second ex vivo intestinal tissue segment,

wherein an increase in the second measurement of contractility relative to the first measurement of contractility for both the first and second ex vivo intestinal tissue segments is indicative of the compound potentially antagonizing the GLP-2 receptor response to the GLP-2 receptor ligand in that tissue segment, and
wherein a difference in potential antagonization of of GLP-2 receptor response to the GLP-2 receptor ligand in the first ex vivo intestinal tissue and the second ex vivo intestinal tissue indicates a differential responsiveness of the two regions of the intestine to the ability of the compound to antagonize the GLP-2 receptor response to the GLP-2 receptor ligand.

12. A method according to claim 11, wherein the first and second ex vivo intestinal tissue segments are of mammalian origin.

13. A method according to claim 12, wherein the first and second ex vivo intestinal tissue segments are of human origin.

14. A method according to claim 12, wherein the first and second ex vivo intestinal tissue segments are of rat origin.

15. A method according to claim 11, wherein the first and second ex vivo intestinal tissue segments are selected from the group consisting of duodenum, jejunum, ileum, small intestine, and colon.