PCT
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`(51) International Patent Classification 6 :
`A61K 38/26, 38/30, 38/27, 35/38, GOlN
`33/50, C12N 5/06, 5/08 If (A61K 38/30,
`38:26) (A61K 38/27, 38:26) (A61K 38/26,
`38:18)
`
`Al
`
`(11) International Publication Number:
`
`WO 98/52600
`
`(43) International Publication Date:
`
`26 November 1998 (26.11.98)
`
`(21) International Application Number:
`
`PCT/CA98/00497
`
`(22) International Filing Date:
`
`15 May 1998 (15.05.98)
`
`(30) Priority Data:
`60/046,754
`9715481.9
`091059,504
`
`16 May 1997 (16.05.97)
`23 July 1997 (23.07.97)
`13 April 1998 (13.04.98)
`
`us
`GB
`us
`
`(71) Applicant: 1149336 ONTARIO INC. [CAJCA]; 19 Femwood
`Road, Toronto, Ontario M6B 3G3 (CA).
`
`(72) Inventor: DRUCKER, Daniel, J.; 19 Femwood Road, Toronto,
`Ontario M6B 3G3 (CA).
`
`(74) Agent: AITKEN, David, W.; Osler, Hoskin & Harcourt, Suite
`1500, 50 O'Connor Street, Ottawa, Ontario KIP 6L2 (CA).
`
`(81) Designated States: AL, AM, AT, AU, AZ, BA, BB, BG, BR,
`BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE,
`GH, GM, GW, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ,
`LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW,
`MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ,
`TM, TR, TI, UA, UG, UZ, VN, YU, ZW, ARIPO patent
`(GH, GM, KE, LS, MW, SD, SZ, UG, ZW), Eurasian patent
`(AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European patent
`(AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT,
`LU, MC, NL, PT, SE), OAPI patent (BF, BJ, CF, CG, CI,
`CM, GA, GN, ML, MR, NE, SN, TD, TG).
`
`Published
`With international search report.
`Before the expiration of the time limit for amending the
`claims and to be republished in the event of the receipt of
`amendments.
`
`(54) Title: METHODS OF ENHANCING FUNCTIONING OF THE UPPER GASTROINTESTINAL TRACT
`
`(57) Abstract
`
`The invention relates to glucagon-related peptides and their use for the prevention or treatment of disorders involving the upper
`gastrointestinal tract including the esophagus and stomach. In particular, it has now been demonstrated that GLP-2 and peptidic agonists
`of GLP-2 can cause proliferation of the tissue of the upper gastrointestinal tract. Thus, the invention provides methods of proliferating the
`upper gastrointestinal tract in a subject in need thereof. Further, the methods of the invention are useful to treat or prevent inflammatory
`conditions of the upper gastrointestinal tract, including inflammatory diseases. GLP-2 stimulates the growth of upper gastrointestinal tissue
`when administered in conjunction with other peptide hormones. The invention further provides pharmaceutical compositions of GLP-2
`with at least one other peptide hormone, methods of enhancing the growth of upper gastrointestinal tissue and of gastrointestinal disorders
`by increasing serum levels of GLP-2 and at least one other peptide hormone, an kits for performing the methods of the invention.
`
`CFAD Exhibit 1028
`
`1
`
`

`

`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.
`
`AL
`AM
`AT
`AU
`AZ
`BA
`BB
`BE
`BF
`BG
`BJ
`BR
`BY
`CA
`CF
`CG
`CH
`CI
`CM
`CN
`cu
`CZ
`DE
`DK
`EE
`
`Albania
`Armenia
`Austria
`Australia
`Azerbaijan
`Bosnia and Herzegovina
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`Cote d'Ivoire
`Cameroon
`China
`Cuba
`Czech Republic
`Germany
`Denmark
`Estonia
`
`ES
`FI
`FR
`GA
`GB
`GE
`GH
`GN
`GR
`HU
`IE
`IL
`IS
`IT
`JP
`KE
`KG
`KP
`
`KR
`KZ
`LC
`LI
`LK
`LR
`
`Spain
`Finland
`France
`Gabon
`United Kingdom
`Georgia
`Ghana
`Guinea
`Greece
`Hungary
`Ireland
`Israel
`Iceland
`Italy
`Japan
`Kenya
`Kyrgyzstan
`Democratic People's
`Republic of Korea
`Republic of Korea
`Kazakstan
`Saint Lucia
`Liechtenstein
`Sri Lanka
`Liberia
`
`LS
`LT
`LU
`LV
`MC
`MD
`MG
`MK
`
`ML
`MN
`MR
`MW
`MX
`NE
`NL
`NO
`NZ
`PL
`PT
`RO
`RU
`SD
`SE
`SG
`
`Lesotho
`Lithuania
`Luxembourg
`Latvia
`Monaco
`Republic of Moldova
`Madagascar
`The former Yugoslav
`Republic of Macedonia
`Mali
`Mongolia
`Mauritania
`Malawi
`Mexico
`Niger
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Singapore
`
`SI
`SK
`SN
`sz
`TD
`TG
`TJ
`TM
`TR
`TT
`UA
`UG
`us
`uz
`VN
`YU
`zw
`
`Slovenia
`Slovakia
`Senegal
`Swaziland
`Chad
`Togo
`Tajikistan
`Turkmenistan
`Turkey
`Trinidad and Tobago
`Ukraine
`Uganda
`United States of America
`Uzbekistan
`Viet Nam
`Yugoslavia
`Zimbabwe
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`2
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`WO 98/52600
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`PCT/CA98/00497
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`METHODS OF ENHANCING FUNCTIONING
`OF THE UPPER GASTROINTESTINAL TRACT
`
`5
`
`Field of Invention
`This invention relates to glucagon-related peptides and
`their use, either alone or in combination with other peptide
`hormones, for the prevention or treatment of disorders
`involving the upper gastrointestinal tract.
`
`10
`
`Background of the Invention
`Glucagon-like peptide-2 (GLP-2) is a 33 amino acid
`peptide expressed in a tissue-specific manner from the
`pleiotropic glucagon gene. GLP-2 shows remarkable homology
`in terms of amino acid sequence to glucagon and Glucagon-Like
`15 Peptide-1 (GLP-1). Further, different mammalian forms of
`GLP-2 are highly conserved. For example, the human GLP-2 and
`degu (a south American rodent) GLP-2 differ from rat GLP-2 by
`one and three amino acids respectively. When given
`exogenously, GLP-2 can produce a marked increase in the
`20 proliferation of small intestinal epithelium of test mice,
`apparently with no undesirable side effects (Drucker et al.,
`1996, PNAS:USA .2]_:7911-7916). Subsequently it was shown that
`peptide analogs of native GLP-2 with certain modifications to
`the peptide sequence possess enhanced trophic activity at the
`25 small intestine (see co-pending application U.S. Serial No.
`08/669,791, filed June 28, 1996, incorporated herein by
`reference) .
`It has further been demonstrated that GLP-2 can
`proliferate the tissue of the large intestine (co-pending
`applications U.S. Serial No. 08/763,177, filed December 10,
`30 1996, and U.S. Serial No. 08/850,664, filed on May 2, 1997,
`and Litvak et al., 1997, Gastroenterology, vol. 112 (4
`Suppl.), page Al455, all of which are incorporated herein by
`reference). Moreover, GLP-2 has also been shown to increase
`D-glucose maximal transport rate across the intestinal
`35 basolateral membrane (Cheeseman and Tseng, 1996, American
`Journal of Physiology 271:G477-G482).
`A number of peptide hormones, structurally unrelated to
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`GLP-2, have been demonstrated to have varying degrees of
`trophic activity. For example, Insulin-Like Growth Factor-2
`(IGF-2) has been shown to promote mitosis of the crypt cells
`of the small intestine in vivo (U.S. Patent No. 5,482,926).
`5 Insulin-Like Growth Factor-1 (IGF-1), which shares 64%
`sequence identity with IGF-2, and peptide analogs thereof
`have also been shown to increase the growth of gut tissue in
`vivo (WO 91/12018) . Growth Hormone (GH) has been shown to
`have a number of physiological effects, including increasing
`10 proliferation of the intestinal mucosa (see, for example,
`Willmore, U.S. Patent No. 5,288,703), thereby enhancing the
`absorptive capacity of the gut. However, none of the above
`peptide hormones possess the efficacy or specificity of GLP-2
`in promoting proliferation of the tissue of the lower
`15 gastrointestinal tract.
`
`25
`
`Summary of the Invention
`The invention is based, in part, on the discovery that
`GLP-2 receptor agonists act to enhance functioning of the
`20 upper gastrointestinal tract. Specifically, it has been
`demonstrated that GLP-2 can proliferate the tissue of the
`esophagus and stomach.
`It is accordingly a general object of
`the present invention to exploit GLP-2 receptor agonists for
`therapeutic and related purposes.
`In particular, it has been demonstrated that GLP-2 and
`peptidic analogs of GLP-2 can cause proliferation of the
`tissue of upper gastrointestinal tract. Thus, one aspect the
`invention provides a method of proliferating the tissue of
`the upper gastrointestinal tract in a subject in need thereof
`30 comprising delivering to the upper gastrointestinal tract of
`the subject an upper gastrointestinal tract proliferating
`amount of GLP-2 or a GLP-2 analog.
`In addition, it has been demonstrated that GLP-2 can
`ameliorate nonsteroidal anti-inflammatory drug (NSAID)
`35 induced gastrointestinal toxicity. Thus, the invention
`provides methods of therapeutically or prophylactically
`treating a subject with or at risk of an inflammatory
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`5
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`condition of the gastrointestine involving the upper
`gastrointestinal tract, comprising delivering to the upper
`gastrointestinal tract an effective amount of GLP-2 or a GLP-
`2 analog.
`More particularly, and according to one aspect of the
`invention, there is provided a method of treating a subject
`suffering from a condition involving the upper
`gastrointestinal tract, wherein GLP-2 or a GLP-2 analog is
`delivered to the upper gastrointestinal tract in an amount
`10 capable of ameliorating the condition.
`In a related aspect of the invention, there is provided
`a method of treating a subject having a damaged, partially
`resected, eroded or inflamed esophagus comprising the step of
`delivering to the subject a upper gastrointestinal tract
`15 damage or inflammation ameliorating amount of GLP-2 or an
`analog of GLP-2 in a pharmaceutically or veterinarily
`acceptable carrier.
`In a further aspect, GLP-2 or a GLP-2
`analog is provided in a pharmaceutically or veterinarily
`acceptable form in an amount effective to cause proliferation
`20 of the upper gastrointestinal tract.
`In a further aspect of the invention, there is provided
`a method of treating a subject having a damaged, atrophic or
`inflamed stomach comprising the step of delivering to the
`subject a stomach damage or inflammation ameliorating amount
`25 of GLP-2 or an analog of GLP-2 in a pharmaceutically or
`veterinarily acceptable carrier. In a further aspect, GLP-2
`is provided in a pharmaceutically or veterinarily acceptable
`form in an amount effective to cause proliferation of the
`tissue of the stomach.
`In another aspect, the invention provides a method of
`prophylactically treating a subject at risk of developing an
`inflammatory condition of the gastrointestine involving the
`upper gastrointestinal tract comprising the steps of:
`a)
`identifying a subject at risk of developing an
`35 inflammatory condition involving the upper gastrointestinal
`tract; and
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`administering to the subject an amount of GLP-2 or
`b)
`a GLP-2 analog effective to inhibit onset of the inflammatory
`condition.
`In another aspect of the invention, there is provided a
`5 method to identify peptides useful to treat inflammatory
`conditions involving the upper gastrointestinal tract
`comprising the steps of:
`a)
`obtaining an analog of a vertebrate GLP-2 peptide,
`the analog having at least one amino acid substitution,
`10 deletion, addition, or an amino acid with a blocking group;
`b)
`inducing an inflammatory condition of the upper
`gastrointestinal tract in a test animal;
`c)
`treating the test animal having an induced
`inflammatory condition of the upper gastrointestinal tract
`15 with the analog using a regimen capable of eliciting an
`amelioration of the inflammatory condition of the upper
`gastrointestinal tract when utilized for human [Gly2]GLP-2;
`and
`
`determining the effect of the analog on the health
`d)
`20 status or mortality of the test animal compared with control
`animals not receiving the peptide or determining the mass of
`the upper gastrointestinal tract of test animals compared to
`control animals not receiving peptide.
`In another aspect of the invention, there is provided a
`25 method to identify peptides useful to prevent or ameliorate
`inflammatory conditions involving the upper gastrointestinal
`tract comprising the steps of:
`a)
`obtaining an analog of a vertebrate GLP-2 peptide,
`the analog having at least one amino acid substitution,
`30 deletion, addition, or an amino acid with a blocking group;
`b)
`treating a test animal with the analog using a
`regimen capable of eliciting an amelioration of the
`inflammatory condition of the upper gastrointestinal tract
`when utilized for human [Gly2]GLP-2;
`c)
`inducing an inflammatory condition of the upper
`gastrointestinal tract in a test animal;
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`determining the effect of the analog on the health
`e)
`status or mortality of the test animal compared with control
`animals not receiving the peptide or determining the mass of
`the upper gastrointestinal tract of test animals compared to
`5 control animals not receiving peptide.
`In another aspect, the invention provides a method of
`prophylactically treating a subject at risk of developing an
`inflammatory condition of the gastrointestine involving the
`upper gastrointestinal tract comprising the steps of:
`a)
`identifying a subject at risk of developing an
`inflammatory condition involving the upper gastrointestinal
`tract; and
`b)
`administering to the subject an amount of GLP-2 or
`a GLP-2 analog effective to inhibit onset and/or ameliorate
`15 the development of the inflammatory condition.
`
`10
`
`In a related aspect of the invention, there is provided
`a method useful to identify peptides capable of proliferating
`the tissue of the upper gastrointestinal tract comprising the
`20 steps of:
`a) obtaining an analog of a vertebrate GLP-2 peptide,
`the analog having at least one amino acid substitution,
`deletion, addition, or an amino acid with a blocking group;
`b) delivering the analog to the upper gastrointestinal
`25 tract of the test animal using a regimen capable of
`proliferating the upper gastrointestinal tract when utilized
`for human [Gly2)GLP-2; and
`c)
`assessing the increase in the mass, length, or total
`protein content of the upper gastrointestinal tract or a
`30 representative portion after completion of the treatment
`regime.
`In another aspect, the invention provides a method for
`growing upper gastrointestinal tract tissue or cells
`therefrom, which comprises the step of culturing the tissue
`35 or cells in a culturing medium supplemented with a growth
`promoting amount of GLP-2 or a GLP-2 analog.
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`The invention is also based, in part, on the expectation
`that GLP-2 or a GLP-2 analog will act synergistically with
`the peptide hormones IGF-1 and/or GH to promote the
`proliferation of cells. Furthermore, coadministration of
`5 GLP-2 with either IGF-1 or GH results in cell proliferation.
`The trophic effects on the small and large intestines of this
`combination therapy are greater than that seen with any one
`of GLP-2, IGF-1 or GH alone. An additional aspect of the
`invention is the coadministration of GLP-2 with IGF-2 or GH
`10 to promote growth.
`Therefore, one aspect of the invention is a composition
`for promoting the growth of upper gastrointestinal tract
`tissue in a mammal which comprises GLP-2, or a trophic analog
`of GLP-2, in admixture with at least one other peptide
`15 hormone selected from the group consisting of IGF-1, IGF-2,
`and GH, and a pharmaceutically acceptable carrier. These
`compositions of the invention may alternatively include
`analogs of GLP-2, IGF-1, IGF-2 and/or GH which exhibit
`trophic activities.
`In another aspect, the invention provides a method of
`promoting the growth of upper gastrointestinal tract tissue
`in a mammal, comprising treating the mammal to elevate serum
`levels of GLP-2, or an analog of GLP-2, and at least one
`other peptide hormone selected from the group consisting of
`25 IGF-1, analogs of IGF-1, IGF-2, analogs of IGF-2, GH, and
`analogs of GH.
`Such methods of the invention include co(cid:173)
`administering to a mammal an effective amount of GLP-2 and an
`effective amount of at least one other peptide hormone
`selected from the group consisting of IGF-1, analogs of IGF-
`30 1, IGF-2, analogs of IGF-2, GH, and analogs of GH, treating
`the mammal with hormones or small organic molecules which act
`to elevate serum levels of GLP-2, IGF-1, IGF-2, and/or GH,
`and performing gene therapy to induce cells in the mammal to
`endogenously produce GLP-2, IGF-1, IGF-2, and/or GH or to
`35 engineer cells to produce GLP-2, IGF-1, IGF-2, or GH, alone
`or in combination, that may then be implanted in a mammal to
`produce the desired biological effect.
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`Also provided by the invention is a method for treating
`a subject to enhance functioning of the upper
`gastrointestinal tract, comprising treating the mammal to
`elevate serum levels of GLP-2, or an analog of GLP-2, and at
`5 least one other peptide hormone selected from the group
`consisting of IGF-1, analogs of IGF-1, IGF-2, analogs of IGF-
`2, GH, and analogs of GH.
`The compositions and methods of the invention are useful
`for restoring or maintaining gastrointestinal function, for
`10 promoting the healing and regrowth of injured or
`ulcerated/inflamed intestinal mucosa, for reducing the risk
`of enteric disease, for enhancing the nutritional status of a
`mammal, and for the treatment or prevention of nutritional or
`upper gastrointestinal disorders in a mammal, particularly a
`15 human.
`Alternatively, the compositions and methods of the
`invention can be used to promote proliferation of the upper
`gastrointestinal tract in a healthy mammal, e.g., to enable
`increased absorption of nutrients in cattle allowing earlier
`20 weaning or increased milk and meat production.
`In yet another aspect of the invention, there is
`provided a use of GLP-2, or analogs of GLP-2, for the
`manufacture of a pharmaceutical or veterinary preparation for
`the enhancement of upper gastrointestinal tract tissue
`25 growth.
`In a particularly preferred aspect of the invention,
`such preparations also include another peptide hormone
`selected from the group consisting of IGF-1, analogs of IGF-
`1, IGF-2, analogs of IGF-2, GH, and analogs of GH.
`Still further, the invention provides kits comprising
`30 GLP-2, or analogs of GLP-2, and at least one other peptide
`hormone selected from the group consisting of IGF-1, analogs
`of IGF-1, IGF-2, analogs of IGF-2, GH, and analogs of GH.
`Such compositions are provided in a therapeutically effective
`unit dose or multi-dose amount.
`Also provided by the instant invention is a method for
`promoting the growth of upper gastrointestinal tract tissue
`or cells which comprises the step of culturing said tissue or
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`cells in a culturing medium containing a growth promoting
`combination of both GLP-2, or a GLP-2 analog, and at least
`one other peptide hormone selected from the group consisting
`of IGF-1, analogs of IGF-1, IGF-2, analogs of IGF-2, GH, and
`5 analogs of GH. These methods may be performed on cells in
`culture and in vivo.
`In yet another aspect of the invention, there is
`provided a method in which treatment of a patient to restore
`upper gastrointestinal tract tissue is performed by the steps
`10 of: (a) culturing tissue or cells derived from the patient
`with a tissue growth promoting amount of a combination of
`GLP-2 or an GLP-2 analog, and at least one other peptide
`hormone selected from the group consisting of IGF-1, analogs
`of IGF-1, IGF-2, analogs of IGF-2, GH, and analogs of GH, and
`15 then (b) implanting said tissue or cells in the patient to be
`treated.
`Finally, the invention also provides a method for
`determining the activity of a hormone when used in
`(1)
`combination with GLP-2 which comprises the steps of:
`20 coadministering the hormone with an amount of GLP-2, or a
`GLP-2 analog, to a test mammal;
`(2) assessing the subsequent
`growth of upper gastrointestinal tract tissue in the test
`mammal; and (3) determining whether the growth of upper
`gastrointestinal tract tissue in the test mammal is enhanced
`25 relative to control mammals treated with GLP-2 alone.
`
`Brief Description of the Figures
`Figure 1 illustrates the change in the number of cells
`which stain with bromodeoxyuridine, in the mucosal epithelium
`30 and muscle layer of the esophagus, after treatment with GLP-
`2.
`
`Figure 2 illustrates the change in total protein content
`of the esophagus after treatment with GLP-2.
`Figure 3 illustrates the change in weight of the stomach
`35 after treatment with GLP-2.
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`Detailed Description of the Invention
`The invention relates to therapeutic, prophylactic, and
`related uses of GLP-2 and GLP-2 analogs, in particular for
`the amelioration of medical or veterinary conditions in which
`5 functioning of the upper gastrointestinal tract is impaired
`by disease or injury. For example, the method is usefully
`applied to treat subjects suffering from an inflammatory
`condition involving the upper gastrointestinal tract,
`subjects who have undergone resection of the upper
`10 gastrointestinal tract, or subjects whose upper
`gastrointestinal tract has been damaged by toxins and the
`like.
`As used herein the term "upper gastrointestinal tract"
`means that section of the gastrointestinal (GI) tract which
`15 comprises the esophagus and the stomach. The esophagus is
`that section of the GI tract that arises proximally at the
`pharyngoesophageal junction and continues distally through
`the posterior mediastinum to end at the gastroesophageal
`junction. The stomach is a distensible structure extending
`20 from the gastroesophageal junction to the duodenum.
`As used herein the term "subject" includes a human or
`other mammal and including livestock and pets.
`As used herein the term "GLP-2 receptor agonist" means
`any molecule which on binding to the GLP-2 receptor results
`25 in activation of the GLP-2 receptor, and includes for example
`GLP-2 or peptidic analogs of GLP-2. Recently it has been
`demonstrated that the GLP-2 receptor is a G-protein coupled
`receptor. Nucleic acid encoding the GLP-2 receptor has been
`isolated (see co-pending applications U.S. Serial No.
`30 08/767,224, filed December 13, 1996, and U.S. Serial No.
`08/845,546, filed April 24, 1997, both of which are
`incorporated herein by reference). Thus, methods commonly
`used in this field to identify G-protein coupled receptor
`agonists may be usefully applied to the GLP-2 receptor. One
`35 particularly useful methodology for assessing compounds for
`GLP-2 receptor agonist activity is disclosed in the above
`mentioned co-pending applications. Briefly, suitable cells
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`such as COS cells are transformed with GLP-2 receptor
`encoding nucleic acid such that functional receptor is
`provided at the cell surface. Thereafter agonist activity of
`a test compounds can be assessed by contacting transformed
`5 cells by the test compound; an increase in the intracellular
`level of cyclic adenosine monophosphate in response to
`binding of the test compound to the transformed cells
`indicates agonist activity.
`GLP-2 peptide analogs and selected chemical libraries,
`10 may be screened for GLP-2 receptor agonist activity using
`this approach. Guidance on the types of peptidic analogs
`that may be usefully employed in this method is given herein
`and in co-pending applications U.S. Serial Nos. 08/632,533
`and 08/631,273, both filed on April 12, 1996, which are
`15 incorporated herein by reference. Moreover, any of the
`commercially available chemical libraries may be usefully
`screened for small molecule GLP-2 receptor agonists using
`high throughput or ultra high throughput screening
`technology. Peptidic analogs of GLP-2 and small molecule
`20 agonists identified as GLP-2 receptor agonists may be
`screened for therapeutic and related utility to treat
`conditions involving the upper gastrointestinal tract using
`the models described herein and in the scientific literature.
`Any subject requiring enhancement of the activity of the
`25 upper gastrointestinal tract may potentially be a candidate
`for treatment with a GLP-2 agonist according to the
`invention.
`In particular, one group of conditions that may
`be beneficially treated according to the invention are
`diseases involving the esophagus. Human patients are
`30 typically diagnosed as having such a condition after
`manifesting one or more of the following symptoms: pain or
`discomfort on swallowing, pain or discomfort on swallowing
`liquids or solids, heartburn, a bitter taste in the mouth, a
`sensation of food sticking in the throat or during swallowing
`35 (often manifest as a lump in the throat) and shortness of
`breath. Visualization of the esophagus using
`esophagogastroscopy can be used to confirm the presence of
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`damage or disease of the esophagus. Alternatively, the
`esophagus can be visualized radiologically using a barium
`contrast swallowing X-ray, or CT scan with an oral contrast
`material. The function of the esophagus can also be assessed
`5 by manometry. The presence of esophageal disease,
`particularly esophagitis, can be assessed functionally using
`the Bernstein test. For a more precise diagnosis of
`esophageal disease, biopsies can be taken of the esophageal
`mucosa for analysis of histological evidence of inflammation,
`10 and further, cultures can be made of this material to
`determine the presence of bacterial, viral or fungal
`infection and inflammation.
`Inflammatory conditions of the esophagus include:
`inflammation-vasculitis, post-infection inflammation,
`15 infiltrative disorder, e.g., scleroderma and amyloid, as well
`as conditions wherein the inflammation results from the
`action of a toxin such as alcohol, acid, and alkali, such as
`from an accidental overdose.
`Another group of conditions that may be beneficially
`20 treated, according to the invention, are diseases involving
`the stomach, e.g., peptidic ulcer. Human patients are
`typically diagnosed as having stomach disorders after
`manifesting symptoms of abdominal pain, either when at rest
`or with eating or both. However, symptoms of stomach
`25 disorder can be non-specific and it can be difficult to
`differentiate between pain from the esophagus and the
`stomach.
`Additionally, GLP-2 may be beneficially administered to
`patients who can be demonstrated to be at risk of developing
`30 a malfunctioning of the upper gastrointestinal tract to
`protect against onset of the condition or reduce the severity
`of the attack. Such patients include smokers and patients
`with blood group O which is associated with duodenal ulcer
`and prepyloric ulcer, patients receiving or about to receive
`35 one or more NSAIDS, patients undergoing or about to undergo
`chemotherapy, and patients who suffer from stress-induced
`ulceration.
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`Treatment with GLP-2 agonists has been demonstrated to
`increase tissue growth in the upper gastrointestinal tract.
`Models suitable for determining which analogs of GLP-2 have
`upper gastrointestinal tract proliferation activity are
`5 potentially therapeutically useful to treat medical or
`veterinary conditions of the upper gastrointestinal tract are
`described in example 1 and example 3.
`Animal models useful for studying conditions involving
`the esophagus are described in the literature (see, for
`10 example, Ann. Burg. Oneal. 1(3) :252-261 (1994); Ann. Emerg.
`Med. ~(2) :178-182 (1993)) and include mechanical injury or
`acid-induced injury of the esophagus. Thus, the animal
`models described in the art can be used to assess the ability
`of compounds identified as GLP-2 agonists to ameliorate
`15 inflammatory conditions involving the upper gastrointestinal
`tract.
`Animal models useful for studying conditions involving
`the stomach are described in the literature (see, for
`example, Experimental and Molecular Pathology 59:136-
`20 154(1993)). These models include administration of non
`specific anti-inflammatories to cause gastroduodenitis, and
`administration of ethanol alone or in combination with other
`agents that cause gastric damage. Thus, the animal models
`described in the art can be used to assess the ability of
`25 compounds identified as GLP-2 agonists to ameliorate
`conditions involving the stomach.
`The various vertebrate forms of GLP-2 include, for
`example, rat GLP-2 and its homologous including ox GLP-2,
`porcine GLP-2, degu GLP-2, bovine GLP-2, guinea pig GLP-2,
`30 hamster GLP-2, human GLP-2, rainbow trout GLP-2, and chicken
`GLP-2. The sequences of these forms of GLP-2 have been
`reported by many authors including Buhl et al. in J. Biol.
`Chem., 1988, 263(18) :8621, Nishi and Steiner, Mol.
`Endocrinol., 1990, ~:1192-8, and Irwin and Wong, Mol.
`35 Endocrinol., 1995, 2(3) :267-77. The sequences reported by
`these authors are incorporated herein by reference.
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`Analogs of vertebrate GLP-2 can be generated using
`standard techniques of peptide chemistry and can be assessed
`for trophic activity at the upper gastrointestinal tract, all
`according to the guidance provided herein. Particularly
`5 pref erred analogs of the invention are those based upon the
`sequence of human GLP-2, as follows:
`His-Ala-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr(cid:173)
`Ile-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn(cid:173)
`Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp
`10 wherein one or more amino acid residues are conservatively
`substituted for another amino acid residue, as long as the
`analog still maintains its trophic activity at the upper
`gastrointestinal tract as measured by an increase in at least
`one of the following parameters: upper gastrointestinal tract
`15 length, protein content, mass or rate of mitosis/cell
`division of cells of the upper gastrointestinal tract,
`measured, for example, by the use of an indirect indicator of
`mitosis such as bromodeoxyuridine(BrDU).
`Conservative substitutions in any naturally occurring
`20 GLP-2, preferably the human GLP-2 sequence, are defined as
`exchanges within any of the following five groups:
`I. Ala, Ser, Thr, Pro, Gly
`II. Asn, Asp, Glu, Gln
`III. His, Arg, Lys
`IV. Met, Leu, Ile, Val, Cys
`V. Phe, Tyr, Trp.
`The invention also encompasses non-conservative substitutions
`of amino acids in any vertebrate GLP-2 sequence, provided
`that the non-conservative substitutions occur at amino acid
`30 positions known to vary in GLP-2 isolated from different
`species. Non-conserved residue positions are readily
`determined by aligning all known vertebrate GLP-2 sequences.
`For example, Buhl et al., J. Biol. Chem., 1988, 263(18) :8621,
`compared the sequences of human, porcine, rat, hamster,
`35 guinea pig, and bovine GLP-2's, and found that positions 13,
`16, 19, 27 and 28 were non-conserved (position numbers refer
`to the analogous position in the human GLP-2 sequence) .
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`Nishi and Steiner, Mol. Endocrinol., 1990, ~:1192-8, found
`that an additional position within the sequence encoding GLP-
`2, residue 20 in the above human sequence, also varied in
`degu, a rodent species indigenous to South America. Thus,
`5 under this standard, the amino acid positions which vary in
`mammals and which preferable may be substituted with non(cid:173)
`conservative residues are positions 13, 16, 19, 20, 27 and
`28. The additional amino acid residues which vary in
`vertebrates and which also may be substituted with non-
`10 conserved residues occur at positions 2, 5, 7, 8, 9, 10, 12,
`17, 21, 22, 23, 24, 26, 29, 30, 31, 32 and 33.
`Alternatively, non-conservative substitutions may be
`made at any position in which alanine-scanning mutagenesis
`reveals some tolerance for mutation in that substitution of
`15 an amino acid residue with alanine does not destroy all
`activity at the upper gastrointestinal tract. The technique
`of alanine scanning mutagenesis is described by Cunningham
`and Wells, Science, 1989, 244:1081, and incorporated herein
`by reference in its entirety. Since most GLP-2 sequences
`20 consist of only approximately 33 amino acids (and in human
`GLP-2 alanine already occurs at