PCT
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`WO 98/03547
`
`(51) International Patent Classification 6 :
`C07K 14/605, A61K 38126, GOIN 33/68
`
`Al
`
`(11) International Publication Number:
`
`(43) International Publication Date:
`
`29 January 1998 (29.01.98)
`
`(21) International Application Number:
`
`PCT/CA97/00521
`
`(22) International l<'iling Date:
`
`18 July 1997 (18.07.97)
`
`(30) Priority Data:
`08/683,890
`
`19 July 1996 (19.07.96)
`
`us
`
`(71) Applicants (for all designated States except US): 1149336
`ONTARIO INC. [CA/CA]; 19 Femwood Road, Toronto,
`Ontario M6B 3G3 (CA). ALLELIX BIOPHARMACEUTI(cid:173)
`CALS INC. [CA/CA]; 6850 Goreway Drive, Mississauga,
`Ontario L4V I V7 (CA).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): DRUCKER, Daniel, J.
`[CA/CA]; 19 Femwood Road, Toronto, Ontario M6B 3G3
`(CA). CRIVICI, Anna, E. [CA/CA]; 64 Havelock Street,
`Toronto, Ontario M6H 3BS (CA). SUMNER-SMITH, Mar(cid:173)
`tin [CA/CA]; 110 Kingsview Drive, Bolton, Ontario L7E
`3V4 (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, Fl, GB, GE,
`GH, HU, 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,
`TT, UA, UG, US, UZ, VN, YU, ZW, ARIPO patent (GH,
`KE, LS, MW, SD, SZ, UG, ZW), Eurasian patent (AM, AZ,
`BY, KG, KZ, MD, RU, TJ, TM), European patent (AT, BE,
`CH, DE, DK, ES, Fl, 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: ANTAGONISTS OF INTESTINOTROPHIC GLP-2 PEPTIDES
`
`(57) Abstract
`
`Antagonists of glucagon-like peptide 2 have been identified. Their effects on the growth of gastrointestinal tissue are described. Its
`formulation as a pharmaceutical, and its therapeutic and related uses in treating bowel tissue, are described. Also described are methods of
`identifying antagonists of glucagon-likc peptide 2.
`
`CFAD Exhibit 1028
`
`

`

`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
`Annenia
`Auscria
`Auscralia
`Azerbaijan
`Bosnia and Herzegovina
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`Ce.te d'Ivoire
`Cameroon
`China
`Cuba
`Czech Republic
`Germany
`Denmark
`Estonia
`
`ES
`Fl
`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
`
`Lesorho
`Lithuania
`Luxembourg
`Lacvia
`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 l'ederation
`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 Scates of America
`Uzbekistan
`Viet Nam
`Yugoslavia
`Zimbabwe
`
`

`

`W098/03547
`
`PCT/CA97/00521
`
`ANTAGONISTS OF INTESTINOTROPHIC GLP-2 PEPTIDES
`
`I.
`
`FIELD OF THE INVENTION
`This invention relates to glucagon-related peptides
`5 which are functional antagonists of glucagon-like peptides-2,
`and to their use therapeutically to counter hyperplasia or
`induce hypoplasia particularly in intestinal tissue.
`
`10
`
`II. BACKGROUND TO THE INVENTION
`Expression of the glucagon gene yields a tissue-
`determined variety of peptide products that are processed
`from the 160 residue proglucagon product. The organization
`of these peptides within the proglucagon precursor was
`elucidated by the molecular cloning of preproglucagon cDNAs
`15 from the anglerfish, rat, hamster and bovine pancreas. These
`analyses revealed that preproglucagon contains not only the
`sequence of glucagon and glicentin, but also two additional
`glucagon-like peptides (GLP-1 and GLP-2) separated from
`glucagon and each other by two spacer or intervening peptides
`20 (IP-I and IP-II). These peptides are flanked by pairs of
`basic amino acids, characteristic of classic prohormone
`cleavage sites, suggesting they might be liberated after
`posttranslational processing of proglucagon (Drucker,
`Pancreas, 1990, 5(4) :484). Analysis of the peptides
`25 liberated from proglucagon in the pancreatic islets of
`Langerhans, for instance, suggests the primary pancreatic
`peptide liberated is the 29-mer glucagon, whereas glicentin,
`oxyntomodulin, IP-II and the glucagon-like peptides are more
`prevalent in the small and large intestines. This
`30 demonstration that the glucagon-like peptides are found in
`the intestine has prompted research into the precise
`structure and putative function{s) of these newly discovered
`gut peptides. Most studies have focussed on GLP-1, because
`several lines of evidence suggested that GLP-1 may be an
`35 important new regulatory peptide.
`Indeed, :t has been
`determined that GLP-1 is the most potent known peptidergic
`stimulus for insulin release, an accion mediated in a
`
`- 1
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`

`W098/03547
`
`PCT/CA97 /00521
`
`glucose-dependent manner through interaction with receptors
`on pancreatic E cells. GLP-1 and its derivatives are in
`development for use in the treatment of diabetics.
`With respect to the biological role of GLP-2, co-pending
`5 U.S. Application Serial No. 08/422,540 (PCT Publ. No. WO
`96/32414), incorporated in its entirety herein by reference,
`discloses that mammalian GLP-2 acts as a trophic agent, to
`promote growth of intestinal tissue. The effect of GLP-2 is
`marked particularly by increased growth of the small
`10 intestine. Furthermore, co-pending U.S. Application Serial
`No. 08/631,273 and PCT Application No. PCT/CA 97/00252, both
`of which are incorporated in its entirety herein by
`reference, disclose that analogs of vertebrate GLP-2 can have
`enhanced intestinotrophic activity.
`
`15
`
`III. SUMMARY OF THE INVENTION
`It has now been discovered that alteration of GLP-2
`peptide structure can yield peptides capable of inhibiting
`the intestinotrophic activity of GLP-2. More particularly,
`20 and according to one aspect of the invention, there are
`provided antagonists comprising an amino acid sequence
`corresponding to that of a first reference mammalian GLP-2
`which has been mutated so that from one to four of any of the
`first four N-terminal residues are deleted.
`In another
`25 aspect of the invention, the antagonists correspond to a·
`reference mammalian GLP-2 that has been mutated so that at
`least one amino acid selected from the amino acid positions
`corresponding to the amino acid positions of human GLP-2 at
`Asp::, Phe 22
`, Thr 2
`", Thr 32 and Asp 33 is substituted with an amino
`30 acid which does not naturally occur at that position in the
`reference GLP-2.
`In another aspect of the invention,
`position Ala 2 is substituted with an amino acid selected from
`the group consisting of Leu, Cys, Glu, Arg, Trp, and P0 3 -Tyr;.
`In yet another aspect of the invention, the antagonist
`35 corresponds to a polypeptide with any combination of the
`above substitutions and deletions mutated relative to the
`reference mammalian GLP-2.
`
`-
`
`2
`
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`

`

`W098/03547
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`PCT/CA97 /00521
`
`Also provided as an aspect of the invention are methods
`of producing and identifying GLP-2 antagonists.
`For use in medical or veterinary treatment, there is
`further provided by the present invention a pharmaceutical or
`5 veterinary composition comprising an amount of a GLP-2
`antagonist effective to antagonize GLP-2 activity in vivo,
`and a pharmaceutically or veterinarily acceptable carrier.
`The GLP-2 antagonist activity of the present GLP-2
`antagonists is manifest in vivo as a reduction in the mass of
`10 small bowel tissue or as an ability to inhibit the
`intestinotrophic activity of GLP-2 or intestinotrophic
`analogs thereof. Accordingly, there is provided, in another
`aspect of the invention, a method for reducing the mass or
`suppressing the proliferation of small bowel tissue in a
`15 subject, including an animal or a human, which comprises the
`step of delivering to that subject an amount of a GLP-2
`antagonist of the invention effective to cause a reduction in
`the mass of small bowel tissue.
`Subjects for whom such treatment would be useful include
`20 those suffering from hyperplastic conditions of the small
`intestine, for example, as a result of GLP-2 overdose or of
`GLP-2 overproducing tumors, and conditions wherein
`prophylactic inducement of small bowel hypoplasia would be
`useful, for example, in the treatment of clinical obesity as
`25 a non-surgical alternative to resection of the small
`intestine.
`
`IV. DETAILED DESCRIPTION OF THE INVENTION
`The present invention relates to therapeutic and related
`30 uses of a novel class of GLP-2 antagonists, particularly for
`decreasing the growth rate of gastrointestinal tissue, mos:
`particularly small bowel. The biological effect of the
`present GLP-2 antagonists manifests as a decrease in small
`bowel weight, relative to a mock treated control or as an
`35 ability to inhibit the intestinotrophic activity of GLP-2 er
`an intestinotrophic analog of GLP-2, relative to a control
`
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`

`W098/03547
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`PCT/CA97/00521
`
`animal given either GLP-2 or an intestinotrophic analog of
`GLP-2 alone.
`The present GLP-2 antagonists are structural analogs of
`the intestinotrophic GLP-2 peptides. GLP-2 peptides refers
`5 collectively to the various vertebrate forms of GLP-2 and to
`modified forms (characterized by at least one addition,
`deletion, substitution, and/or incorporation of an amino acid
`residue with a blocking group) of the GLP-2 analogs which
`still retain intestinotrophic activity. However, as
`10 described herein, certain site-specific alterations of these
`intestinotrophic GLP-2 peptides can confer antagonist
`activity to the site-specifically altered analog.
`Without being limited by following explanation, it is
`believed that the site specific alterations which confer
`15 an~agonist activity interfere with one of the functional
`activities of the GLP-2 hormone peptide, but not all
`functional activities. For example, an alteration conferring
`antagonist activity to a GLP-2 analog may be one which does
`not inhibit hormone binding to its cognate receptor, but does
`20 prevent the subsequent signal transduction through the bound
`receptor. For example, the site specific alteration of the
`hormone may prevent dimerization of the hormone receptor
`which :~ necessary to transmit a signal to the interior of
`the cell. Such a mechanism for antagonistic acLivity has
`25 been observed with other hormones such as, for example, human
`growth hormone (see Fuh et al., Science, 1992, 256:1677-
`
`1680) .
`Generally, sites which are highly conserved among
`mammalian GLP-2's are candidates for modification in order to
`30 obtain an antagonist. Among mammals, at least residues 1-5,
`7, 15, and 22, 29 and 32-33 are highly conserved. Therefore,
`deletion or substitution of the residues at these sites can
`result in a GLP-2 antagonist. Additionally, certain
`modifications of sites near these conserved sites may also
`35 cause antagonist activity by disrupting local tertiary amino
`acid structure or placement of the adjacent conserved
`residues.
`
`- 4
`
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`

`

`W098/03547
`
`PCT/CA97 /00521
`
`The GLP-2 antagonists of the invention include peptide
`derivatives with a sequence derived from a vertebrate GLP-2
`in that one or more of any of the first four N-terminal amino
`acids (relative to the sequence of human GLP-2) are deleted.
`5 These analogs are ref erred to herein as the deletion class of
`GLP-2 antagonists. From the deletion class of GLP-2
`antagonists, it will be appreciated that GLP-2 antagonism can
`result from disruption of the N-terminal structure of GLP-2
`within the first four amino acids. Thus, the deletion class
`10 of GLP-2 antagonists comprises: GLP-2(2-33), GLP-2(3-33),
`GLP-2 (4-33) and GLP-2 (5-33),
`[desAla 2
`] GLP-2,
`[desAsp 3
`] GLP-2
`and [desGly 4 )GLP-2.
`Additionally, the GLP-2 antagonists of the invention
`include substitution derivatives of vertebrate GLP-2's. The
`15 substitution class of GLP-2 antagonists includes those
`antagonists which replace one of the following amino acids at
`the following positions (relative to sequence of the human
`GLP-2) with another amino acid:
`residues 15, 22, 29, 32, ana
`33. Also included in the substitution class are those
`20 incorporating certain Ala 2 substitutions.
`It is to be understood that, in embodiments of the
`invention, the GLP-2 antagonists may incorporate any
`combination of a deletion and a substitution, or may
`incorporate two or more substitutions at the sites noted.
`
`25
`
`30
`
`GLP-2 Antagonists
`A.
`The GLP-2 antagonists may accordingly be analogs of
`human GLP-2, which has the following sequence:
`His-Ala-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-
`1
`5
`10
`
`Thr-Ile-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-
`15
`20
`
`Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp.
`25
`30
`33
`
`35 Unless otherwise specified, the term "GLP-2" refers to the
`sequence of human GLP-2.
`
`-
`
`5
`
`-
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`

`

`W098/03S47
`
`PCT/CA97 /00S21
`
`The antagonists of the invention are polypeptides which
`comprise amino acid sequences corresponding to that of a
`first reference mammalian GLP-2 which has been mutated so
`that:
`
`5
`
`10
`
`15
`
`(i) from one to four of any of the first :our N-terminal
`residues are deleted; or
`(ii} at least one amino acid selected from the amino
`acid positions corresponding to the amino acid positions
`of human GLP-2 at Asp 10
`, Phe 22
`, Thr 29
`, Thr 32 and Asp 33 is
`substituted with an amino acid which does not naturally
`occur at that position in the reference GLP-2; or
`(iii) position Ala 2 is substituted with an amino acid
`selected from the group consisting of Leu, Cys, Glu,
`; or
`Arg, Trp, and P0 3 -Tyr 2
`(iv} a combination of (i) and (ii}, or (ii} and (iii) is
`mutated.
`In specific embodiments of the invention, for example,
`the GLP-2 antagonists of the invention which are altered at
`residue positions 1, 2, 3, 4, 22, 29, 32, and/or 33 may be
`20 derivatives of rat GLP-2 which is an Ala 1
`' variant of human
`GLP-2; degu GLP-2, ox GLP-2, porcine GLP-2, guinea pig GLP-2
`and hamster GLP-2, the sequences of which have been reported
`by many authors including Buhl et al in J. Biol. Chem., 1988,
`263 (18): 8621.
`GLP-2 residues which occur at a specific position are
`determined by aligning the sequences of GLP-2's isolated from
`different vertebrate species and comparing the sequence to
`the human sequence, reproduced above.
`Further, the GLP-2 antagonists of the invention which
`30 are altered at residue positions 1, 2, 3, 4, 22, 29, 32,
`and/or 33 may be derivatives of GLP-2 agonists such as are
`described. in co-pending U.S. Application Serial Nos.
`08/632,533 and 08/631,273, and PCT Publication No. WO
`96/32414 and PCT Application No. PCT/CA 97/00252.
`Amino acids substitutions appropriate at these sites to
`yield an antagonist can readily be determined using the
`murine model of GLP-2 antagonism herein described. That is,
`
`25
`
`35
`
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`

`W098/03547
`
`PCT/CA97 /00521
`
`a GLP-2 compound incorporating a structural alteration is
`obtained and then screened in the murine model exemplified
`herein for GLP-2 antagonism activity. Those GLP-2 compounds
`which elicit a decrease in bowel growth and/or inhibit the
`5 intestinotrophic activity of GLP-2 or a GLP-2 agonist, are
`identified in this screen as GLP-2 antagonists.
`GLP-2 antagonists of the present invention are
`considered to be functional antagonists of GLP-2 if, when
`assessed in the murine model exemplified herein, the
`10 antagonist:
`(1) consistently mediates a measurable decrease
`in small bowel weight relative to a control animal receiving
`vehicle alone; and/or (2) when assessed by co-administration
`in said murine model with GLP-2 or a GLP-2 agonist (in a
`molar excess ratio of preferably 10:1, and more preferably
`15 4:1 over agonist) results consistently in a measurable
`inhibition of the intestinotrophic effect of GLP-2 or the
`GLP-2 agonist, as revealed by a reduction in the increase ir.
`small bowel weight induced by GLP-2 administered alone.
`Particularly suitable for therapeutic use are those
`20 functional antagonists of GLP-2 which mediate a bowel weighr
`decrease of at least about 10% relative to a control animal
`receiving vehicle alone; preferred for therapeutic use are
`those which mediate a decrease in small bowel weight of at
`least 15% or more.
`The small intestine mass reducing activity of the
`present GLP-2 antagonists is noted most significantly in
`relation to the jejunum, and particularly the proximal
`jejunum, and is also noted in the distal ileum.
`Additionally, the activity of GLP-2 antagonists may also be
`30 noted as a reduction in the crypt/villus height of the small
`intestine.
`Alternatively, GLP-2 antagonists can be assessed using
`the co-administration model detailed above.
`In this case,
`antagonists are considered to be useful antagonists of GLP-2
`35 if, when co-administered with GLP-2, or an intestinotrophic
`analog thereof, at a molar ratio of about 10:1, or more
`preferably at a molar ratio of about 4:1, they diminish the
`
`25
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`

`W098/03547
`
`PCT/CA97 /0052 l
`
`5
`
`10
`
`15
`
`activity of GLP-2 or an intestinotrophic analog thereof by at
`least 10%, as manifest by a reduction in the increase in
`small bowel weight relative to a control animal treated with
`either GLP-2 or the GLP-2 agonist alone.
`In another aspect of the invention, there is provided a
`method useful to identify antagonists of GLP-2, such as those
`described above, comprising the steps of:
`obtaining a GLP-2 analog that incorporates an
`1)
`alteration within the peptide sequence:
`2)
`treating a mammal with said analog using a regimen
`capable of eliciting a measurable loss of the mass of
`the intestine; and
`3)
`determining the effect of said analog on small
`bowel weight and/or on the crypt/villus height of the
`crypt cells of the small intestine relative to a mock
`treated control animal, whereby a functional GLP-2
`antagonist is identified as an analog of GLP-2 which
`elicits a decrease in said weight and/or said height.
`In a related aspect of the invention, there is provided
`20 another method useful to identify functional GLP-2
`antagonists comprising the steps of:
`obtaining a GLP-2 analog which incorporates an
`1)
`alteration within the peptide sequence;
`2)
`treating a mammal with said analog in a regimen
`capable of inhibiting the intestinotrophic activity of
`GLP-2 or a GLP-2 agonist; and
`3)
`determining the effect of said analog on small
`bowel weight and/or on the crypt/villus height of the
`crypt cells of the small intestine relative to a control
`animal given GLP-2 or a GLP-2 agonist, whereby said
`functional GLP-2 antagonist is identified as an analog
`of GLP-2 which inhibits the intestinotroph~c activity of
`GLP-2 and/or the intestinotrophic activity of a GLP-2
`agonist.
`In a preferred version of the methods described above
`useful to identify functional GLP-2 antagonists, the GLP-2
`analog is chosen from the GLP-2 antagonists described here:n.
`
`25
`
`30
`
`35
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`W098/03547
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`PCT/CA97 /00521
`
`Choice of Substituting Amino Acids
`B.
`The substituting amino acids can be chosen from the wide
`variety of amino acids available to peptide chemists, and
`include the D-arnino acids as well as the L-amino acids and
`5 their numerous derivatives. Most practically, chosen amino
`acids will be amenable to incorporation by solid phase or
`solution phase synthesis, or by recombinant DNA production
`means.
`In a first screen, analogs which are candidates for
`10 antagonistic activity are identified by alanine scanning
`mutagenesis or other systematic mutagenesis method. These
`alanine substitutions are tested for antagonistic activity
`using the methods described in detail herein.
`In another aspect of the invention, more effective GLP-2
`15 antagonists may then be made by drastically changing the
`character of the naturally occuring amino acid residue that
`is important in forming structural interactions (hydrogen
`bonding, salt bridging, hydrophobic interactions, positioning
`of residues) of the GLP-2 hormone with its target molecule
`20 (e.g. receptor). With this goal in mind, it is not normally
`necessary to screen each site with replacements by all 18 of
`the other naturally occurring residues.
`Instead,
`representative members of residue groups are selected.
`Generally, these groups are:
`a.
`positively charged residues:
`b.
`negatively charged residues:
`c.
`amides: Asn and Gln
`d.
`aromatic residues: Phe, Tyr, Trp.
`e.
`hydrophobic residues: Ala, Pro, Gly, Val, Leu,
`30 Ile, and Met
`f.
`uncharged hydrophilic residues: Ser and Thr.
`When preparing these antagonist candidates, one would choose
`a residue from a group other than the type of residue which
`is naturally occuring at that position. Extreme
`35 substitutions are generated by selecting a residue from a
`group with opposed combinations of features. For example, a
`
`His, Arg and Lys
`Asp and Glu
`
`25
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`W098/03547
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`PCT/CA97 /00521
`
`negatively charged residue may be substituted by a positively
`charged residue.
`In the case of the Ala~ substitutions, the substituting
`ami~o acids are selected carefully so that antagonists of
`5 GLP-2 activity result.
`It should be noted that substitutions
`at position 2 can have the effect of enhancing the
`intestinotrophic activity of GLP-2. For instance, when Ala·
`is replaced by Gly, the result is dramatically enhanced
`intestinotrophic activity as well as resistance to digestiou
`10 of the GLP-2 peptide by DPP-IV enzyme. The GLP-2 antagonists
`of the present invention can surprisingly also be generated
`by substituting Ala 2
`In embodiments of the invention,
`•
`substituting amino acids at position 2 that are useful to
`generate GLP-2 antagonists are selected from Leu, Cys, Glu,
`15 Arg, Trp and P0 3 -Tyr. GLP-2 antagonists incorporating these
`substitutions have the added advantage that they render the
`peptide resistant to digestion by DPP-IV enzyme. Preferably,
`the Ala 2 substituting amino acid is selected from Cys, Glu,
`Leu, and Arg.
`Amino acids substituting for Asp>, Phe~:c, Thr>, ':'hr':,
`and Asp 3 ~ are desirably selected from those incorporating a
`small hydrophobic side chain, such as Ala, Gly and Val.
`In embodiments of the invention, the substitutio~ class
`of GLP-2 antagonists includes:
`[Gly 2
`, Ala'""JGLP-2,
`[Ala>]GL?-
`25 2,
`[Ala 1s]GLP-2 (2-33),
`[Ala 10 )GLP-2(3-33),
`[Ala'')GLP-2 !4-331,
`[Ala' 0 ]GLP-2(5-33),
`[Gly 7
`, Ala 22 ]GLP-2, [Gly~, Ala 2 .]GLP-2,
`[Gly=, Ala 32 ]GLP-2,
`[Gly2
`, Ala 33 )GLP-2,
`[Leu 2 ]GLP-2,
`[Glu 2 ]GLP-
`2,
`[Arg 2
`] GLP-2,
`[Trp 2
`] GLP-2,
`] GLP-2, [Cys~J GLP-2,
`[P0 3 -Tyr 2
`[Alac 5
`] GLP-2,
`[Ala 29
`] GLP-2,
`[Ala 32
`] GLP-2 and [Ala 33
`] GLP-2.
`
`20
`
`30
`
`C.
`
`Additional Modifications for Improvina the
`Properties of the Analogs of the Inventior.
`The present GLP-2 antagonists, while incorporating a
`structural alteration of the type noted, may have various
`35 amino acid sequences consistent with the sequences of GLP-2
`per se or of GLP-2 agonists. The GLP-2 antagonists may alsc
`~e analogs of vertebrate GLP-2 agonists, in which collatera~
`
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`
`modifications have been made to enhance other biochemical,
`biological or physiological properties of the peptide. Such
`modifications include, for example (in those peptides for
`which antagonism is conferred by substitution other than at
`5 position 2), the substitution of native Ala 2 by an amino acid
`that renders the GLP-2 antagonist resistant to digestion by
`the enzyme DPP-IV. An amino acid suitable for this purpose
`includes particularly Gly. Also, the Met 10 residue can be
`replaced by an oxidatively more stable amino acid, such as
`10 Leu, Nle, Ile or Ala. Such Met 10 -substituted analogs are
`accordingly more stable during synthesis, work-up, and
`storage. Another modification in this context is replacemer.:
`of the amino acid at position 20 by an amino acid other than
`Arg.
`In certain applications, particularly for the synthetic
`15 generation of pharmaceutically or veterinarily acceptable
`peptides, this modification is desirable to avoid the
`retention by the Arg residue of counterions from solvents
`such as TFA.
`Within the scope of the present invention are also
`20 molecules in which the N- or C-terminus has been modified to
`incorporate a blocking group of the type used conventionally
`in the art of peptide chemistry to protect peptide termini
`from undesired biochemical attack and degradation in vivo.
`Suitable N-terminal protecting groups include, for
`25 example, C,_ 5 alkanoyl groups such as acetyl. Also suit.able as
`N-terminal protecting groups are amino acid analogs lacking
`the amino function. Suitable C-terminal protecting groups
`include groups which form ketones or amides at the carbon
`atom of the C-terminal carboxyl, or groups which form esters
`30 at the oxygen atom of the carboxyl. Ketone and ester-forming
`groups include alkyl groups, particularly branched or
`unbranched C1 _5alkyl groups, e.g., methyl, ethyl and propyl
`groups, while amide-forming groups include amino functions
`such as primary amine, or alkylamino functions, e.g., mono-C
`3 5 ,alkyl amino and di- c, _5alkylamino groups such as methyl amino,
`ethylamino, dimethylamino, diethylamino, methylethylamino and
`the like. Amino acid analogs are also suitable for
`
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`

`W098/03547
`
`PCT/CA97 /00521
`
`protecting the C-terminal end of the present compounds, for
`example, decarboxylated amino acid analogs such as agmatine.
`Embodiments of the invention specifically include such
`analogs in which the N-terminal blocking group is acetyl; and
`5 analogs in which the C-terminal blocking group is an amine,
`e.g., -NH 2 •
`
`Synthesis of the GLP-2 Antagonists
`D.
`The present GLP-2 antagonists can be synthesized using
`10 standard techniques of peptide chemistry and can be assessed
`for GLP-2 antagonist activity, all according to the guidance
`provided herein. Those GLP-2 antagonists that incorporate
`only L-amino acids can be produced in commercial quantities
`by application of recombinant DNA technology. For this
`15 purpose, DNA coding for the desired GLP-2 antagonist is
`incorporated into an expression vector and transformed intc a
`microbial, e.g., yeast, or other cellular host, which is then
`cultured under conditions appropriate for GLP-2 antagonist
`expression. A variety of gene expression systems have been
`20 adapted for this purpose, and typically drive expression of
`the desired gene from expression regulatory elements used
`naturally by the chosen host. Because GLP-2 does not require
`post translational glycosylation for its activiLy, its
`production may conveniently be achieved in bacterial hosts
`25 such as E. coli. For such production, DNA coding for the
`selected GLP-2 antagonist may usefully be placed under
`expression controls of the lac, trp or PL genes of E. coli.
`As an alternative to expression of DNA coding for the GLP-2
`antagonist per se, the host can be adapted to express GLP-2
`30 antagonist as a fusion protein in which the GLP-2 antagonist
`is linked releasably to a carrier protein that facilitates
`isolation and stability of the expression product.
`In an approach universally applicable to the production
`of selected GLP-2 antagonists, and one used necessarily to
`35 produce GLP-2 antagonist forms that incorporate non(cid:173)
`genetically encoded amino acids and N- and C-terminally
`derivatized forms, the well established techniques of
`
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`

`

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`PCT/CA97/00521
`
`automated peptide synthesis are employed, general
`descriptions of which appear, for example, in J.M. Stewart
`and J.D. Young, Solid Phase Peptide Synthesis, 2nd Edition,
`1984, Pierce Chemical Company, Rockford, Illinois; and in M.
`5 Bodanszky and A. Bodanszky, The Practice of Peptide
`Synthesis, 1984, Springer-Verlag, New York; Applied
`Biosystems 430A Users Manual, 1987, ABI Inc., Foster City,
`California.
`In these techniques, the GLP-2 antagonist is
`grown from its C-terminal, resin-conjugated residue by the
`10 sequential addition of appropriately protected amino acids,
`using either the Fmoc or tBoc protocols, as described for
`instance by Orskov et al, 1989, supra.
`For the incorporation of N- and/or C- protecting groups
`protocols conventional to solid phase peptide synthesis
`15 methods can also be applied. For incorporation of C-terminal
`protecting groups, for example, synthesis of the desired
`peptide is typically performed using, as solid phase, a
`supporting resin that has been chemically modified so that
`cleavage from the resin results in a peptide having the
`20 desired C-terminal protecting group. To provide peptides in
`which the C-terminus bears a primary amino protecting group,
`for instance, synthesis is performed using a p(cid:173)
`methylbenzhydrylamine
`(MBHA) resin so that, when peptide
`synthesis is completed, treatment with hydrofluoric acid
`25 releases the desired C-terminally aminated peptide.
`Similarly, incorporation of an N-methylamine protecting group
`at the C-terminus is achieved using N-methylaminoethyl(cid:173)
`derivatized DVB resin, which upon HF treatment releases
`peptide bearing an N-methylamidated C-terminus. Protection of
`30 the C-terminus by esterif ication can also be achieved using
`conventional procedures. This entails use of resin/blocking
`group combination that permits release of side-chain
`protected peptide from the resin, to allow for subsequent
`reaction with the desired alcohol, to form the ester
`35 function.
`FMOC protecting groups, in combination with DVB
`resin derivatized with methoxyalkoxybenzyl alcohol or
`equivalent linker, can be used for this purpose, with
`
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`

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`
`PCT/CA97 /00S21
`
`cleavage from the support being effected by TFA in
`dichloromethane. Esterif ication of the suitably activated
`carboxyl function e.g. with DCC, can then proceed by addition
`of the desired alcohol, followed by deprotection and
`5 isolation of the esterified peptide product.
`Incorporation of N-terminal protecting groups can be
`achieved while the synthesized peptide is still attached to
`the resin, for instance by treatment with suitable anhydride
`and nitrile. To incorporate an acetyl protecting group at
`10 the N-terminus, for instance, the resin-coupled peptide can
`be treated with 20% acetic anhydride in acetonitrile. The N(cid:173)
`protected peptide product can then be cleaved from the resin,
`deprotected and subsequently isolated.
`Once the desired peptide sequence has been synthesized,
`15 cleaved from the resin and fully deprotected, the peptide is
`then purified to ensure the recovery of a single oligopeptide
`having the selected amino acid sequence. Purification can be
`achieved using any of the standard approaches, which include
`reversed-phase high-pressure liquid chromatography (RP-HPLC)
`20 on alkylated silica columns, e.g. C,-, C"-, or C10 - silica.
`Such column fractionation is generally accomplished by
`running linear gradients, e.g. 10-90%, of increasing %
`organic solvent, e.g. acetonitrile, in aqueous buffer,
`usually containing a small amount (e.g. 0.1%) o: pairing
`25 agent such as TFA or TEA. Alternatively, ion-exchange HPLC
`ca" be employed to separate peptide species on the basis of
`their charge characteristics. Column fractions are
`collected, and those containing peptide of the
`In one
`desired/required purity are optionally pooled.
`30 embodiment of the invention, the peptide is then treated in
`the established manner to exchange the cleavage acid (e.g.
`TFA) with a pharmaceutically