United States Patent c191
`Drucker et al.
`
`[54) GLUCAGON-LIKE PEPTIDE-2 ANALOGS
`
`[75)
`
`Inventors: Daniel J. Drucker; Anna E. Crivici.
`both of Toronto; Martin
`Sumner-Smith. Bolton. all of Canada
`
`(73) Assignees: Allelix Biopharmaceutical Inc ..
`Mississauga; 1149336 Ontario Inc ..
`Toronto. both of Canada
`
`[21) Appl. No.: 669,791
`Jun. 28, 1996
`
`Filed:
`
`[22)
`
`[63)
`
`(51)
`
`[52)
`
`(58]
`
`[56)
`
`Related U.S. Application Data
`
`Continuation of Ser. No. 631,273, Apr. 12, 19%, abandoned,
`Ser. No. 632,533, Apr. 12, 1996, and Ser. No. 422.540, Apr.
`14, 1995.
`Int. CI.6
`
`............................. A61K 38100; C07K 5100;
`C07K 7/00; C07K 17/00
`U.S. CI. ..................................... 514112; 514/2; 514/3;
`530/324; 530/303; 530/308; 435/366; 435/371;
`935152; 935155; 935166; 935no; 935n 1
`Field of Search ..................... 51412. 3, 12; 530/324.
`5301303. 308; 935/52. 55. 66. 70, 71; 435/366.
`371
`
`References Cited
`
`FOREIGN PATENT DOCUMENTS
`
`612531
`
`8/1994 European Pat. Off ..
`
`CYrHER PUBLICATIONS
`
`Barragan. J.M.; Rodriguez. R.E.; and Blazquez, E. Changes
`in arterial blood pressure and heart rate induced by glu(cid:173)
`cagon-like peptide-1-(7-36) amide in rats. American Jour(cid:173)
`nal of Physiology. 266 (3 Pt 1), pE459-66. Mar. 1994.
`Bloom. S.R. Gut Hormones in adaptation. Gut. 28. SL pp.
`31-35. 1987.
`Brubaker, Patricia L. Regulation of Intestinal Proglucagon(cid:173)
`-Derived Peptide Secretion by Intestinal Regulatory Pep(cid:173)
`tides. Endocrinology. vol. 128. No. 6. pp. 3175-3182, 1991.
`Buhl. Thora; Thim. Lars; Kofod. Hans; Orskov. Catherine;
`Harling. Henrik; and Holst. Jens J. Naturally Occuring
`Products of Proglucagon 111-160 in the Porcine and Human
`Small Intestine. The Journal of Biological Chemistry. vol.
`263. No. 18. pp. 8621-8624. Issue of Jun. 25. 1988.
`Calvo. J.C., Yusta. B; Mora. F; and Blazquez. E. Structural
`characterization by affinity cross-linking of glucagon-like
`peptide-I (7-36) amide receptor in rat brain. J. Neurochem.
`64(1). pp. 299-306. Jan. 1995.
`Cheeseman. Chris L; and Raymand Tsang. The effect of
`gastric inhibitory polypeptide and glucagon like peptides on
`intestinal basolateral membrane hexose transport. The
`American Physiological Society. APSracts 3:0071G. Apr.
`16, 1996.
`Drucker. Pancreas. 1990. 5(4):484.
`Ehrlich. Peter; Tucker. Davin; Asa. Sylvia L.; Brubacker.
`Patricia L.; and Drucker. Daniel J. Inhibition of pancreatic
`proglucagon gene expression in mice bearing subcutaneous
`endocrine tumors. American Journal of Physiology. pp.
`E662-E671. 1994.
`
`11111111111111 ~111111 11111111111111111111111111111 1111111111111111111
`5,789,379
`Aug. 4, 1998
`
`US005789379A
`[llJ Patent Number:
`(45) Date of Patent:
`
`George. S.K.; Uttenthal. L.0.; Ghiglione. M.; and Bloom.
`S.R. Molecular forms of glucagon-like peptides in man.
`FEBS letters. vol. 192. No. 2. pp. 275-278. Nov. 1985.
`
`Hoosein. Naseema M.; and Gurd. Ruth S. Human glucagon(cid:173)
`-like peptides 1 and 2 activate rat brain adenylate cyclase.
`FEBS letters. vol. 178. No. L pp. 83-86. Dec. 1984.
`
`Irwin. David M.; and Wong. Jaffe. Trout and Chicken
`Proglucagon: Alternative Splicing Generates mRNA Tran(cid:173)
`scripts Encoding Glucagon-Like Peptide 2. Molecular
`Endocrinology. 9:267-277. 1995.
`
`Lee. Ying C.; Asa. Sylvia L.; and Drucker. Daniel J. Glu(cid:173)
`cagon Gene 5'-Flanking Sequences Direct Expression of
`Simian Virus 40 Large T Antigen to the Intestine. Producing
`Carcinoma of the Large Bowel in Transgenic Mice. The
`Journal of Biological Chemistry. vol. 267. No. 15. pp.
`10706-10708. May 25, 1992.
`
`Lund. P. Kay; Hoyt. Eileen; Simmons. James G.; and
`llishen. Martin H. Regulation of Intestinal Glucagon Gene
`Expression during Adaptive Growth of Small Intestine.
`Digestion . 54:371-373. 1993.
`
`Mojsov. Svetlana; Heinrich. Gerhard; Wilson. Ira B.; Ravaz(cid:173)
`zola, Mariella; Orci. Lelio; and Habener. Joel F. Preproglu(cid:173)
`cagon Gene Expression in Pancreas and Intestine Diversifies
`at the Level of Post-translational Processing. The Journal of
`Biological Chemistry. vol. 261. No. 25. pp. 11880--11889,
`Sep. 5. 1986.
`
`Mommsen. Thomas P.. Andrews. P.C.; and Plisetskaya.
`Erika M. Glucagon-like peptides activate hepatic gluconeo(cid:173)
`genesis. FEBS letters. vol. 219. No. 1, pp. 227-232. Jul.
`1987.
`
`Nishi and Steiner. Mal. Endocrinol., 1990. 4:1192-8.
`
`Orskov, C.; Buhl, T.; Rabenhoj. L.; Kofod. H.; and Holst.
`J .J .. Carboxypeptidase-B-like processing of the C-terminus
`of glucagon-like peptide-2 in pig and human small intes(cid:173)
`tine. FEBS letters. 247(2), pp. 193-196. Apr. 24. 1989.
`
`Orskov. C.; Holst. J.J.; Pouisen. S. Seier; and Kirkegaard. P.
`Pancreatic and Intestinal processing of proglucagon in man.
`Diabetologia. 30:874-881. 1987.
`
`Orskov. C.; and Holst. J.J. Radio-irnmunoassays for glu(cid:173)
`cagon-like peptides 1 and 2 (GLP-1 and GLP-2). Scand. J.
`Clin. Lab. Invest. 47(2). pp. 165-174. Apr. 1987.
`
`(List continued on next page.)
`
`Primary Emminer-Sheela Huff
`Attorney, Agent, or Firm-Pennie & Edmonds LLP
`
`[57)
`
`ABSTRACT
`
`Analogs of glucagon-like peptide 2. a product of glucagon
`gene expression, have been identified as intestinal tissue
`growth factors. Their formulation as pharmaceutical. and
`therapeutic use in treating disorders of the small bowel. are
`described.
`
`23 Claims, No Drawings
`
`CFAD Exhibit 1029
`
`1
`
`

`

`5,789,379
`Page 2
`
`OTHER PUBLICATIONS
`
`Orskov. Catherine; Holst. Jens J.; Knuhtsen. Svend; Bald(cid:173)
`issera, Furio G.A.; Poulsen. Steen S.; and Nielsen. 0. Vagn.
`Glucagon-Like Peptides GLP-1 and GLP-2. Predicted
`Products of the Glucagon Gene. Are Secreted Separately
`from Pig Small Intestine but Not Pancreas. Endocrinology.
`vol. 119. No. 4. pp. 1467-1475. 1986.
`Ruiz-Grand. C.. Pintado. J.; Alarcon. C.; Castilla. C.; Val(cid:173)
`verde. I; Lopez-Novoa. J.M. Renal catabolism of human
`glucagon-like peptides 1 and 2. Can. J. Physiol. Pharrmicol.
`68(12). pp. 1568-1573. Dec. 1990.
`
`Shennan. K.LJ.; Holst. J.J.; and Docherty. K. Proglucagon
`expression. posttranslational processing and secretion in
`SV40-transfromed islet cells. Molecular and Cellular Endo(cid:173)
`crinology. 67(1989). pp. 93-99.
`
`Watanabe. Nobuaki; Matsuyama. Tatsuo: Namba. Mitsuy(cid:173)
`oshi; Miyagawa. Jun-ichiro; Itoh. Hidehiko; Komatsu.
`Ryoya; Kono. Norio; and Tarui. Seiichiro. Trophic Effect of
`Glucagon-(1-21)-Peptide on the Isolated Rat Deal Mucosal
`Cells. Biochemical and Biophysical Research Communica(cid:173)
`tions. vol. 152, No. 3. pp. 1038-1044. May 16. 1988.
`
`2
`
`

`

`1
`GLUCAGON-LIKE PEPTIDE-2 ANALOGS
`
`This application is a continuation of application Ser. No.
`08/631.273. filed Apr. 12. 1996, now abandoned. and a
`continuation-in-part of application Ser. No. 08/632.533.
`filed Apr. 12. 1996 and a continuation-in-part of Ser. No.
`08/422.540. filed Apr. 14. 1995. the disclosures of which are
`incorporated by reference herein.
`
`FIELD OF THE INVENTION
`
`This invention relates to glucagon-related peptides having
`intestinal tissue growth promoting properties. and to their
`use therapeutically to treat various medical conditions
`resulting from the impaired growth or loss of such tissue.
`
`5.789.379
`
`5
`
`2
`Biol. Chem .. 1986. 261(25):11880: Orskov et al in
`Endocrinology. 1986. 119(4):1467 and in Diabetologia.
`1987. 30:874 and in FEBS Letters. 1989. 247(2):193:
`George et al. FEBS Letters. 1985. 192(2):275). With respect
`to its biological role. Hoosein et al report (FEBS Letters.
`1984. 178(1):83) that GLP-2 neither competes with gluca(cid:173)
`gon for binding to rat liver and brain tissues, nor stimulates
`adenylate cyclase production in liver plasma membranes.
`but. enigmatically. can stimulate adenylate cyclase in both
`10 rat hyopthalamic and pituitary tissue. at 30-50 pM concen(cid:173)
`trations. An elucidation of the physiological role of GLP-2
`would clearly be desirable.
`
`SUMMARY OF THE INVENTION
`
`There have now been discovered analogs of GLP-2 which
`promote growth of small bowel tissue. It is accordingly a
`general object of the present invention to provide such
`GLP-2 analogs and to provide for their use therapeutically
`and for related purposes.
`In one aspect of the invention, the GLP-2-analogs exhibit
`intestinotrophic activity and conform to the structural For(cid:173)
`mula 1 (SEQ ID NO:l):
`
`R 1-(Y 1)m-Xl-X2-X3-X4-Ser5-Phe6-Ser7-Asp8-(P1 )(cid:173)
`Leu 14-Asp15-Asn16-Leu 17-Ala18-X 19-X20-Asp21-
`Phe22-(P2 )-Trp25-Leu26-Ile2 7-Gln-28-Thr29-Lys30-
`(P3 )-(Y2 )n-R2.
`
`wherein
`Xl is His or Tyr
`X2 is Ala or an Ala-replacement amino acid conferring on
`said analog resistance to DPP-N enzyme;
`X3 is Asp or Glu;
`X4 is Gly or Ala;
`Pi is Glu-XlO-Asn-Thr-Ile or Tyr-Ser-Lys-Tyr (SEQ ID
`N0:3);
`XlO is Met or an oxidatively stable Met-replacement
`amino acid;
`X19 is Ala or Thr;
`X20 is Arg. Lys. His or Ala;
`P2 is Ile-Asn. Ile-Ala or Val-Gin;
`P3 is a covalent bond. or is Ile. Ile-Thr or Ile-Thr-Asp;
`Rl is Hor an N-terminal blocking group;
`R2 is OH or a C-terminal blocking group;
`Yl is one or two basic amino acids selected from the
`group Arg. Lys. and His;
`Y2 is one or two basic amino acids selected from the
`group Arg. Lys. and His; and
`m and n. independently. are 0 or 1: and
`wherein at least one of XL X2. X3.X4. Pl.XlO. X19. X20.
`P2 and P3 is other than a wild type, mammalian GLP-2
`residue.
`Particularly preferred analogs according to Formula 1 are
`those which are rendered resistant to cleavage by human
`DPP-N enzyme by replacing the Ala at position X2 with an
`alternative amino acid. Other analogs of the invention are
`those which replace the oxidatively sensitive Met at position
`XlO with an amino acid residue which is oxidatively stable.
`In this manner. the analog peptides have increased stability
`compared to GLP-2 peptides with the wild-type Met residue
`at this position. Yet another preferred embodiment of the
`invention is the incorporation at position X20 of a basic
`amino acid selected from His or Lys. This substitution is
`advantageous when the GLP-2 analogs are chemically syn-
`
`15
`
`20
`
`25
`
`30
`
`BACKGROUND TO THE INVENTION
`Expression of the glucagon gene yields a tissue(cid:173)
`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 from the 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 (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. V1990. 5(4):484).
`Analysis of the peptides liberated from proglucagon in the
`pancreatic islets of Langerhans. for instance, suggests the
`primary pancreatic peptide liberated is the 29-mer glucagon. 35
`whereas glicentin, oxyntomodulin. IP-II and the glucagon(cid:173)
`like peptides are more prevalent in the small and large
`intestines. This demonstration that the giucagon-like pep(cid:173)
`tides are found in the bowel has prompted research into the
`precise structure and putative function(s) of these newly 40
`discovered gut peptides. Most studies have focussed on
`GLP-1. because several lines of evidence suggested that
`GLP-1 may be an important new regulatory peptide. Indeed,
`it has been determined that GLP-1 is one of the most potent
`known peptidergic stimulus for insulin release, an action 45
`mediated in a glucose-dependent manner through interaction
`with receptors on pancreatic ~ cells. GLP-1 and its deriva(cid:173)
`tives are in development for use in the treatment of diabetics.
`The physiological roles of glicentin and oxyntomodulin.
`the so-called "enteroglucagons". are also under 50
`investigation. particularly with respect to regulation of acid
`secretion and the growth of intestinal cells. Oxyntomodulin
`is capable of inhibiting pentagastrin-stimulated gastric acid
`secretion in a dose-dependent manner. The role of glicentin
`in mediating the changes of intestinal adaptation and growth 55
`of the intestinal mucosa has been investigated. and the
`intestinotrophic effect of glicentin and its therapeutic use
`have recently been reported by Matsuno et al in EP 612.531
`published Aug. 31. 1994.
`In contrast to GLP-1 and other glucagon-related peptides. 60
`the physiological role of glucagon-like peptide GLP-2
`remains poorly understood despite the isolation and
`sequencing of various GLP-2 homologues including human.
`rat. bovine. porcine. guinea pig and hamster. Using GLP-2
`antisera raised against synthetic versions of GLP-2. various 65
`groups have determined that GLP-2 is present primarily in
`intestinal rather than pancreatic extracts (see Mojsov et al. J.
`
`3
`
`

`

`5.789.379
`
`5
`
`4
`20% in small bowel weight; preferred for therapeutic use are
`those which mediate an increase in small bowel weight of
`50% or more. Intestinotrophic activity is noted most signifi(cid:173)
`cantly in relation to the jejunum. including the distal
`jejunum and particularly the proximal jejunum. and are also
`noted in the ileum.
`In addition to exhibiting intestinotrophic activity as just
`defined. the GLP-2 analogs of the present invention incor(cid:173)
`porate an amino acid substitution at one or more sites within
`a GLP-2 peptide "background". which is either a mamma(cid:173)
`lian GLP-2 species per se. or is a variant of a mammalian
`GLP-2 species in which the C-terrninus and/or the
`N-terrninus has been altered by addition of one or two basic
`residues. or 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 bio(cid:173)
`chemical attack and degradation in vivo. Thus. the present
`peptides incorporate an amino acid substitution in the con-
`20 text of any mammalian GLP-2 species. including but not
`limited to human GLP-2. bovine GLP-2. rat 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. J. Biol. Chem .. 1988.
`25 263(18):8621.
`In one aspect of the invention. the intestinotrophic ana(cid:173)
`logs of GLP-2 conform to the sequence of Formula 1 (SEQ
`ID NO: 1) as follows:
`
`15
`
`30
`
`R 1-(Y 1 )m-Xl-X2-X3-X4-Ser5-Phe6-Ser7-Asp8-(Pl )(cid:173)
`Leu 14-Asp 15-Asn 16-Leu 17-Ala 18-X 19-X20-Asp21-
`Phe22-(P2 )-Trp25-Leu26-Ile27-Gln-28-Thr29-Lys30-
`(P3 )-(Y2)n-R2.
`
`3
`thesized. The Arg residue which normally occurs at this
`position tends to strongly bind solvents used in peptide
`synthesis procedures. Substitution of the Arg allows easier
`formulation of the synthetically produced GLP-2 analogs
`into pharmaceutically acceptable compositions.
`More particularly. and according to one aspect of the
`invention. there are provided analogs of a GLP-2 peptide
`selected from a mammalian GLP-2 species and N- and/or
`C-terrninally modified forms thereof. the analogs having
`intestinotrophic activity and incorporating. relative to said 10
`mammalian GLP-2 peptide. at least one amino acid substi(cid:173)
`tution at a position which is conserved in mammalian
`GLP-2's. In a preferred aspect. the GLP-2 analogs incorpo(cid:173)
`rate a substitution selected from:
`1) incorporation at position 2 or at position 3 of an Ala
`replacement amino acid conferring on said analog
`resistance to Dipeptidyl Peptidase-IV (hereinafter
`referred to as DPP-IV); and
`2) incorporation at position 10 of an oxidatively stable
`Met-replacement amino acid; and
`3) incorporation at X20 of a replacement amino acid other
`than Arg.
`In another of its aspects. the invention provides a phar(cid:173)
`maceutical composition comprising a GLP-2 analog of the
`present invention in a therapeutically effective amount. and
`preferably in an intestinotrophic amount. and a pharmaceu(cid:173)
`tically acceptable carrier.
`In a further aspect. the invention provides a method for
`promoting growth of small bowel tissue in a patient in need
`thereof. comprising the step of delivering to the patient an
`intestinotrophic amount of a GLP-2 analog of the present
`invention.
`Besides promoting bowel growth. in another of its aspects
`the invention provides a method for treating a gastrointes(cid:173)
`tinal disease by administering to a patient suffering from
`gastrointestinal disease a therapeutically effective amount of
`a GLP-2 analog of the invention. together with a pharma(cid:173)
`ceutically acceptable carrier. in order to reduce a pathologi(cid:173)
`cal effect or symptom of the gastrointestinal disease.
`In still another aspect of the invention. there is provided
`a method useful to identify intestinotrophic analogs of
`GLP-2. comprising the steps of:
`1) obtaining a GLP-2 analog conforming to Formula 1
`represented above;
`2) treating a mammal with said analog using a regimen
`capable of eliciting an intestinotrophic effect when
`utilized for rat GLP-2; and
`3) determining the effect of said analog on small bowel
`weight relative to a mock treated control mammal. so
`whereby said intestinotrophic analog of GLP-2 is iden(cid:173)
`tified as an analog which elicits an increase in said
`weight.
`
`40
`
`55
`
`DErAILED DESCRIPTION OF THE
`INVENTION
`The invention relates to therapeutic and related uses of
`GLP-2 analogs. particularly for promoting growth of tissue
`of the small bowel. The effect on growth elicited by the
`present GLP-2 analogs manifests as an increase in small 60
`bowel weight. relative to a mock-treated control. In
`particular. GLP-2 analogs are considered to have "intesti(cid:173)
`notrophic" activity if. when assessed in the murine model
`exemplified herein. the analog mediates an increase in small
`bowel weight of at least 10% relative to a control animal 65
`receiving vehicle alone. Particularly suitable for therapeutic
`use are those analogs which mediate an increase of at least
`
`45
`
`35 wherein
`Xl is His or Tyr
`X2 is Ala or an Ala-replacement amino acid conferring on
`said analog resistance to DPP-IV enzyme;
`X3 is Asp or Glu;
`X4 is Gly or Ala;
`Pl is Glu-XlO-Asn-Thr-Ile or Tyr-Ser-Lys-Tyr (SEQ ID
`N0:3);
`XlO is Met or an oxidatively stable Met-replacement
`amino acid;
`X19 is Ala or Tur;
`X20 is Arg. Lys, His or Ala;
`P2 is Ile-Asn. Ile-Ala or Val-Gln;
`P3 is a covalent bond. or is Ile. Ile-Thr or Ile-Thr-Asp;
`Rl is H or an N-terminal blocking group;
`R2 is OH or a C-terminal blocking group;
`Yl is one or two basic amino acids selected from the
`group Arg. Lys. and His;
`Y2 is one or two basic amino acids selected from the
`group Arg. Lys. and His; and
`m and n. independently. are 0 or 1; and
`wherein at least one ofXl. X2. X3. X4. PL XlO. X19. X20.
`P2 and P3 is other than a wild type. mammalian GLP-2
`residue.
`Wild-type mammalian GLP-2 residues which occur at a
`specific position are determined by aligning the sequences of
`GLP-2's isolated from different mammalian species and
`comparing the sequence to the human sequence. reproduced
`below. for convenience (SEQ ID N0:2):
`
`4
`
`

`

`5
`
`6
`
`5.789.379
`
`His-Ala-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-
`1
`10
`5
`Thr-Ile-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-
`15
`20
`Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Tirr-Asp (SEQ ID N0:2)
`25
`30
`
`15
`
`The amino acid residues which, for purposes of this
`application, are known to occur at specific positions in wild 10
`type mammalian GLP-2's are the following: position X13
`may be Ile or Val; Position X16 may be Asn or Ser; position
`X 19 may be Alanine or Threonine; position X20 may be Arg
`or Lys; position X27 may be Ile or Leu; and position X28
`may be Gln or His.
`The present GLP-2 analogs may incorporate desired
`amino acid substitutions into a "background" which is an
`N-terminally or C-terminally modified form of a mammalian
`GLP-2 peptide. Such analogs are represented in Formula 1
`as those in which Rl constitutes an N-terminal blocking
`group. and/or when mis 1 then Yl is one or two basic amino
`acids such as Arg or Lys; and/or R2 is a C-terminal blocking
`group; and/or when n is 1 then Y2 is independently. one or
`two basic amino acids such as Arg or Lys.
`In preferred embodiments of the invention. the GLP-2
`analog is an analog offull length GLP-2, i.e., GLP-2(1-33).
`and P3 is accordingly the sequence Ile-Thr-Asn.
`Alternatively. the GLP-2 analogs may be C-terrninally
`truncated, to yield GLP-2(1-32) forms in which P3 is
`Ile-Thr. or GLP-2(1-31) forms in which P3 is Ile. or
`GLP-2(1-30) forms in which P3 is a covalent bond.
`The "blocking groups" represented by Rl and R2 are
`chemical groups that are routinely used in the art of peptide
`chemistry to confer biochemical stability and resistance to
`digestion by exopeptidase. Suitable N-terrninal protecting
`groups include. for example, C 1.salkanoyl groups such as
`acetyl. Also suitable as N-terminal protecting groups are
`amino acid analogues lacking the amino function. Suitable
`C-terrninal protecting groups include groups which form
`ketones or amides at the carbon atom of the C-terminal
`carboxyl. or groups which form esters at the oxygen atom of
`the carboxyl. Ketone and ester-forming groups include alkyl
`groups, particularly branched or unbranched C 1.salkyl
`groups. e.g .. methyl. ethyl and propyl groups, while amide(cid:173)
`forrning groups include amino functions such as primary
`amine. or alkylamino functions. e.g .. mono-C 1_5alkylamino
`and di-C 1_5alkylamino groups such as methylamino,
`ethylarnino, dimethylarnino, diethylamino, methylethy(cid:173)
`larnino and the like. Amino acid analogues are also suitable
`for protecting the C-terminal end of the present compounds,
`for example. decarboxylated amino acid analogues such as
`agmatine.
`Embodiments of the invention specifically include such
`analogs in which m is 0 and Rl is a blocking group such as
`acetyl; and analogs in which m is 0 and R2 is a C-terminal
`blocking group such as an amine, e.g .. -NH2.
`In a preferred aspect of the invention, the GLP-2 analogs
`are analogs of either human GLP-2 or of rat GLP-2. Human
`GLP-2 is herein referred to interchangably as hGLP-2
`(l-33). Rat GLP-2 has the amino acid sequence of human
`GLP-2, but incorporates at position 19 a Thr residue instead
`of an Ala residue. Rat GLP-2 is accordingly referenced
`herein either as rGLP-2(1-33) or as the Thr 19 analog of
`human GLP-2. i.e .. as [Thr19JhGLP-2(1-33).
`In particularly preferred embodiments of the invention,
`with respect to both the Formula 1 analogs and the more
`specific human or rat GLP-2 analogs. the GLP-2 analogs
`incorporate an amino acid substitution selected from:
`
`1) incorporation at X2 and/or at X3 of a replacement
`amino acid which renders said analog resistant to
`cleavage by DPP-IV enzyme;
`2) incorporation at XlO of an oxidatively stable Met(cid:173)
`replacement amino acid; and
`3) incorporation at X20 of a replacement amino acid other
`than Arg.
`The OPP-IV-resistant class of GLP-2 analogs possess
`particularly advantageous properties. As is demonstrated
`herein. mammalian GLP-2 species have been found to be
`sensitive to cleavage by DPP-IV enzyme. It has also been
`20 found that this sensitivity to OPP-IV is the result of the
`recognition sequence Ala 2 Asp3found in all mammalian
`forms of GLP-2. There are accordingly provided by the
`present invention a class of GLP-2 analogs which incorpo(cid:173)
`rate at X2 and/or X3 a replacement amino acid which
`25 confers on the GLP-2 analog relative resistance to DPP-IV
`mediated cleavage, as determined by any convenient in vitro
`or in vivo assessment technique that is able to detect the
`presence of GLP-2 digestion products. A OPP-IV resistant
`GLP-2 analog is revealed as that GLP-2 analog which is
`30 processed or degraded at a rate that is measurably slower
`than the rate at which human GLP-2 is processed or
`degraded, under the same conditions.
`An assay suitable for assessing DPP-IV sensitivity and
`resistance is described below in Example 3. in the context of
`35 results actually obtained.
`The X2 class of GLP-2 analogs is preferred herein. These
`Ala2-substituted GLP-2 analogs can incorporate at X2 a
`structurally wide variety of Ala-replacement amino acids to
`achieve relative resistance to DPP-IV digestion. A similarly
`40 wide variety of Ala-replacement amino acids allow also for
`the retention by the analog of intestinotrophic activity. For
`purposes of identifying those OPP-IV-resistant X2 analogs
`that also retain intestinotrophic activity, the X2 analogs
`showing OPP-IV resistance are screened in the assay of
`45 intestinotrophic activity described below in Example 4.
`In embodiments of the present invention. the Ala 2
`replacements include stereoisomers of amino isomers that
`would otherwise be substrates for DPP-IV. for example
`D-Ala, 0-HPr and D-Pro; naturally occurring amino acids
`50 other than Ala. HPr and Pro which provide a basic or
`uncharged side chain, for example. Glu. Lys. Arg. Leu. Ile.
`Gly and Val. In specific embodiments of the invention. there
`are provided the following Ala2-substituted GLP-2 analogs:
`[D-Ala2 lrGLP-2(1-33). IGly2]rGLP-2(1-33). [Val2 )rGLP-2
`55 (1-33) and [Gly2]hGLP-2(1-33).
`The X2 GLP-2 analogs may incorporate amino acid
`replacements at other positions. In embodiments of the
`invention. such analogs include those carrying amino acid
`substitutions also at one or more of positions XL X3, X4,
`60 XlO. X19. X20 and X24, and therefore include those which,
`according to Formula 1. include at least one of the following
`substitutions: Xl is Tyr; X3 is Glu; X4 is Ala; Pl is
`Glu-XlO-Asn-Thr-Ile where XlO is other than Met or Pl is
`Tyr-Ser-Lys-Tyr; XlO is an oxidatively stable Met-
`65 replacement amino acid; X19 is Thr; X20 is Lys or Ala; P2
`is Val-Gin and P3 is a covalent bond, Ile. or Ile-Thr or
`Ile-Thr-Asn.
`
`5
`
`

`

`5.789.379
`
`7
`In embodiments of the present invention. the X2 analogs
`of GLP-2 include those which also incorporate one of the
`following substitutions: Xl is Ala; X3 is Ala; X4 is Ala; XlO
`is an oxidatively stable Met-replacement amino acid such as
`Val. Ile. Asn. Glx. Tyr. Phe. and preferably Leu. Nie. Ala. 5
`Ser. and Gly; and P2 is Ile-Ala. In specific embodiments of
`the invention. there are provided the following GLP-2
`analogs: ID-AlaTu19 lhGLP-2(1-33); IGlyTu 19 lhGLP-2
`( 1-33 ); IVal 2Thr 19 lhGLP-2(1-33); IGly 2Ala24 lhGLP-2
`(1-33); IGly 2Ala 10 lhGLP-2(1-33); IA1a 1Gly 2 1hGLP-2 10
`(1-33); 1Gly2Ala3 lhGLP-2(1-33) and IGly2Ala4 lhGLP-2
`(1-33).
`In a related embodiment. the X2 analogs include those
`which include the following substitutions: GLP-21 Gly2 Ala8 I
`hGLP-2; IGly 2Ala 11 lhGLP-2; IGly 2 Ala 21 lhGLP-2; 15
`IGly 2Ala 9 ihGLP-2; IGly 2Ala 16]hGLP-2; [Gly 2Ala 17
`]
`hGLP-2; fGly 2 Ala 28 JhGLP-2; IGly 2Ala 5 ]hGLP-2;
`IGly 2Ala 31 ]hGLP-2; [Gly 2Ala 27 ]hGLP-2; 1Gly2Ala 12
`]
`hGLP-2; IGly2Ala 13 lhGLP-2; [Gly2Ala7 1hGLP-2 and
`IGly2Ala6 lhGLP-2.
`Alternatively. in the case where X2 is Ala. hPr or Pro.
`DPP-IV resistance can be conferred by replacing Asp3 with
`an Asp-replacement amino acid. X3 which is Pro or hPr.
`The present invention also provides. as another class of
`GLP-2 analogs. those analogs in which XlO represents an 25
`oxidatively stable. Met-replacement amino acid. It has been
`found that the intestinotrophic activity of mammalian GLP-2
`is reduced when the Met in position 10 is oxidized. and also
`that intestinotrophic activity is retained when replacement
`amino acids insensitive to oxidation are substituted for 30
`Met 10 replacement. Such Met10-substituted GLP-2 analogs
`are accordingly more stable during synthesis. work-up and
`storage. In embodiments of the present invention. such XlO
`analogs of GLP-2 are analogs of human GLP-2. In a specific
`embodiments of the invention. such analogs include [Ser10
`] 35
`hGLP-2(1-33); INie 10]hGLP-2(1-33); [Ala 10JhGLP-2
`(1-33); and ILeu 10 irGLP-2(1-33).
`The XlO analogs may also incorporate amino acid sub(cid:173)
`stitutions at one or more positions other than Xl O. As noted
`above. such analogs include. for instance. those incorporat- 40
`ing substitutions at X2. and those in which Pl is is Tyr-Ser(cid:173)
`Lys-Tyr. as well as those in which any one of the X
`substituents or the P substituents represented in Formula 1
`are other than wild type residues or sequences. In specific
`embodiments. the GLP-2 analogs include [Gly2Ala 10
`] 45
`hGLP-2( 1-33) and [Tyr9Ser1°Lys11Tyr12deslle13 lhGLP-2
`(l-33).
`In other embodiments of the invention. the GLP-2 analogs
`incorporate single amino acid substitutions in the context of
`the mammalian GLP-2 peptide background in which they so
`are introduced. Such analogs include. for example. those
`mammalian GLP-2 analogs and particularly the human
`GLP-2 analogs in which Xl is Tyr; X3 is Glu; X4 is Ala; Pl
`is Tyr-Ser-Lys-Tyr-(desile); P2 is Ile-Ala or Val-Gln; and P3
`is a covalent bond. or is Ile or Ile-Thr.
`The present GLP-2 analogs can be synthesized using
`standard techniques of peptide chemistry and can be
`assessed for intestinotrophic activity. all according to the
`guidance provided herein. With respect to synthesis. the
`selected GLP-2 analog can be prepared by a variety of 60
`techniques well known for generating peptide products.
`Those GLP-2 analogs that incorporate only L-amino acids
`can be produced in commercial quantities by application of
`recombinant DNA technology. For this purpose, DNA cod(cid:173)
`ing for the desired GLP-2 analog is incmporated into an 65
`expression vector and transformed into a microbial. e.g ..
`yeast. or other cellular host. which is then cultured under
`
`8
`conditions appropriate for GLP-2 expression. A variety of
`gene expression systems have been 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 activity. its production may most con-
`veniently be achieved in bacterial hosts such as£. coli. For
`such production. DNA coding for the selected GLP-2 pep(cid:173)
`tide may usefully be placed under expression controls of the
`lac. trp or PL genes of£. coli. As an alternative to expression
`of DNA coding for the GLP-2 per se. the host can be adapted
`to express GLP-2 peptide as a fusion protein in which the
`GLP-2 is linkedreleasably to a carrier protein that facilitates
`isolation and stability of the expression product.
`In an approach universally applicable to the production of
`a selected GLP-2 analog. and one used necessarily to
`produce GLP-2 analogs that incorporate non-genetically
`encoded amino acids and N- and C-terminally derivatized
`forms. the well established techniques of automated peptide
`20 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 Chemi(cid:173)
`cal Company. Rockford. lli.; and in M. Bodanszky and A.
`Bodanszky. The Practice of Peptide Synthesis. 1984.
`Springer-Verlag. New York; Applied Biosystems 430A
`Users Manual. 1987. ABI Inc .. Foster City. Calif. In these
`techniques. GLP-2 analog is grown from its C-terminal.
`resin-conjugated residue by the sequential addition of appro(cid:173)
`priately 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- blocking groups.
`protocols conventional to solid phase peptide synthesis
`methods can also be applied. For incorporation of
`C-terminal blocking 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 GLP-2 peptide having
`the desired C-terminal blocking group. To provide peptides
`in which the C-terminus bears a primary amino blocking
`group. for instance. synthesis is performed using a
`p-methylbenzhydrylamioe (MBHA) resin so that. when pep(cid:173)
`tide synthesis is completed. treatment with hydrofluoric acid
`releases the desired C-terminally amidated peptide.
`Similarly, incorporation of an N-methylamine blocking
`group at the C-terminus is achieved using
`N-methylaminoethyl-derivatized DVB resin. which upon
`HF treatment releases peptide bearing an N-methylamidated
`C-terroinus. Protection of the C-terminus by esterification
`can also be achi